WO1984001961A1

WO1984001961A1 - Recombinant dna derived antigens of treponema pallidum

Info

Publication number: WO1984001961A1
Application number: PCT/US1983/001718
Authority: WO
Inventors: Michael A Lovett
Original assignee: Univ California
Priority date: 1982-11-12
Filing date: 1983-11-04
Publication date: 1984-05-24
Also published as: GB2157696A; EP0126765A1; JPS59502131A; GB8412158D0; KR840006677A

Abstract

DNA fragments from Treponema pallidum are packaged into suitable bacteriophage vectors for propagation within E. Coli. The resulting plaques are screened for antigens reactive with antibodies to Treponema pallidum. Clones from positive plaques are further propagated and purified to provide antigens useful in the serodiagnosis and study of syphilis and as vaccines against syphilis infection. Plasmid subclones of treponemal antigen producing phage clones are propagated in E. coli to provide an alternate source of treponemal antigens. In addition to bacteriophage clones which encode for polypeptide treponemal antigens, a clone is also disclosed which encodes for a protease resistent heat labile treponemal antigen.

Description

RECOMBINANT DNA DERIVED ANTIGENS OF TREPONEMA PALLIDUM

Cross-Reference To Related Applications

This application is a Continuation-In-Part of pending United States application Serial No. 06/441,391 filed November 12, 1982, the priority for the common subject matter of which is hereby claimed.

Background of the Invention The present invention relates generally to the production of antigens which are reactive with antibodies developed in response to Treponema pallidum. More particularly, the present invention is directed to the production of such antigens by genetically engineered microorganisms.

Treponema pallidum is the known causitive agent of syphilis. Syphilis continues to cause significant morbidity throughout the world despite the ready availability of penicillin for treatment. Studies on Treponema pallidum have been severely restricted because of difficulty in culturing the organism in or on artificial media. The organism can be propagated in rabbit testes, but purification of large amounts . of T. pallidum in its motile virulent form has not yet been achieved. Thus, purified treponemal antigens are not readily available for experimental studies relating to the biology, pathogenesis, serodiagnosis, and immunity of syphilis.

An important factor in the continued high morbidity associated with syphilis is the lack of adequate sero- logical tests for detecting syphilis. Present serological tests are designed to detect antibodies developed by the host in response to infection with Treponema pallidum. The two types of tests are: the nontreponemal or screening tests and the confirmatory treponemal tests.^•■ The present screening and confirmatory tests for syphilis are flawed for several reasons. For example, the Venereal Disease Research Laboratory (VDRL) test is widely employed as a screening test for syphilis. The VDRL test gives false positive results at rates of about 2 to 10 percent. In addition to false positives, the VDRL test fails to diagnose about 30 percent of cases of primary syphilis. The VDRL test is also fasely negative in up to 1/3 of cases of late syphilis. This is especially important since late manifestations of syphilis are potentially destructive to many vital organ systems.

Due to the high false positive rates provided by screening tests, such as the VDRL test, it is necessary to use more expensive confirmatory tests to verify positive VDRL results. The Florescent Treponemal Antibody Adsorption (FTA-ABS) test is a widely used confirmatory test. The FTA-ABS test is limited to use in verification of positive VDRL tests in part due to its expense. Even if the FTA-ABS test was less expensive, it would still not be entirely adequate as a screening test since it gives up to 4 percent false positives. Further, interpretation of the FTA-ABS test can be subjective and is complicated by results termed "borderline" or "1+".

As set forth by Hanff et al. [Hanff, P.A., T.E. Fehniger, J.N. Miller, and M.A. Lovett, 1982, Humoral Immune Response in Human Syphilis to Polypeptides of Treponema pallidum, J. Immunol, Vol. 129, No. 3] a particularly useful technique for determining the presence of T. pallidum antibodies in infected hosts is to prepare treponemal antigens from T. pallidum which are reactive with the antibodies and can be used to identify their presence in the host. The contents of the above identified article is hereby incorporated by reference. At present, processes for producing such treponemal antigens are limited, as mentioned above to propagation of T. pallidum in rabbit testes followed by subsequent purification of the antigens.

As is apparent from the above, it would be highly

O H desirable to provide a new method for producing substantial quantities of specific purified treponemal antigens which is not dependent upon raising the antigens in rabbit testes. Such antigens can be used in experimental studies into the biology pathogenesis and immunity of Treponemal pallidum. Further, the treponemal antigens would be especially useful in the serodiagnosis of syphilis and potentially in construction of a vaccine against syphilis.

Summary of the Invention

In accordance with the present invention, it has been discovered that antigens reactive with antibodies to Treponema pallidum can be prepared by recombinant DNA techniques. By using recombinant DNA techniques, large amounts of purified antigens reactive with antibodies to Treponema pallidum can be prepared for use in the serodiagnosis of syphilis and experimental studies into the biology of Treponema pallidum and as vaccines to immunize individuals against Treponema pallidum. The production of Treponema pallidum reactive antigens by recombinant DNA techniques is an improvement over the prior cumbersome technique of purifying antigens from Treponema pallidum raised and separated from rabbit testes.

The present invention in its broadest sense involves cloning fragments of Treponema pallidum DNA into a suitable vector and propagating the cloned vector in a suitable host. Screening tests are conducted to identify those clones which encode for antigens reactive with antibodies to Treponema pallidum. The clones which encode for desirable antigens are separated out and further propagated in accordance with conventional techniques.

As a particular feature of the present invention, it was discovered that randomly cleaved Treponema pallidum DNA fragments may be combined with suitably cleaved DNA of the bacteriophage vector Charon 30 and packaged into viable Charon 30 particles to form bacteriophage clones. These clones, when used to infect Escherichia coli produced plaques which include antigens reactive with antibodies to Treponema pallidum. The reactive plaques are identified by screening with serum known to contain antibodies to Treponema pallidum.

The bacteriophage clones which encode for desirable reactive antigens were purified from reactive plaques and identified. A bacteriophage clone identified as Tp3A was found to be typical of cloned bacteriophages encoding for

10 polypeptide antigens. It was surprisingly discovered that in addition to the production of clones encoding for polypeptide antigens, clones were also produced which encoded for unusual polypeptide antigens which exhibit properties more commonly attributed to carbohydrates as _ well as being specifically reactive with syphilitic sera containing antibodies to Treponema pallidum. A bacteriophage clone which is exemplary of clones encoding for these unusual antigens was isolated and purified and identified as Tp4D.

„_ Since bacteriophage clones are difficult to maintain and propagate in commercial settings, the Tρ3A, and Tp4D bacteriophages were subcloned into plasmids and established in Escherichia coli by conventional techniques. The recombinant plasmids subcloned from Tp3A have been

- m. identified as pAW305 with the subcloned plasmids from Tp4D being identified as pA 329. Both the pAW305 and pA 329 plasmids are on deposit with the American Type Culture Collection and are identified as ATCC No. 39237 and ATCC No. 39238, respectively. The recombinant plasmids provide

₃₀ a convenient alternative method for producing antigens reactive with anitbodies to Treponema pallidum in accordance with the present invention.

The DNA derived antigens of a Treponema pallidum in accordance with the present invention have a wide variety

₃e of uses in the serodiagnosis of syphilis. The treponemal antigens may be used in any of a variety of forms including the coating of beads with antigens followed by th measurement of agglutination, radio immunoassay and enzym linked immunosorbent assay (ELISA) . Use of several of th treponemal antigens derived in accordance with the presen invention, singly or in combination may be employed fo optimal specific sensitive serodiagnosis of IgM or Ig syphilitic antibody.

Additionally, the treponemal antigens derived i accordance with the present invention can be used a vaccinations to immunize individuals against Treponem pallidum infection. The treponemal antigens may be use alone or in combination with Freundi complete adjuvant t subcutaneously immunize individuals against syphilis.

The above discussed and many other features an attendant advantages of the present invention will becom apparent as the invention becomes better understood b reference to the following detailed description.

Brief Description of the Drawings Figure 1 is the DNA map of an exemplary Charon 30 clone (Tp4D) and its subclone PA 329 both of which encodes for unusual protein antigens which exhibit properties commonly attributed to carbohydrates.

Figure 2 is the DNA map of three exemplary Charon 30 clones (Tp3A, TplC and Tp2D) which encode for protein antigens.

Detailed Description of the Present Invention

The present invention basically involves using conventional recombinant DNA techniques to^' insert fragments of Treponema pallidum DNA into a suitable vector to form a clone. The clone is then propagated in a suitable host. The clones which encode for treponemal antigens are then identified by screening with serums containing antibodies to Treponemal pallidum. The clones which are identified as producing Treponemal antigens are then separated and

OMPI propagated to produce treponemal antigens which may be further purified based on molecular weight and used in serodiagnosis or experimental uses for reaction with and identification of antibodies to Treponemal pallidum and as vaccines inducing immunity to syphilis.

The DNA from clones identified as producing treponemal antigens may be subcloned into suitable plasmids or vectors and propagated in suitable hosts by conventional recombinant DNA techniques. Of course the Treponema pallidum DNA fragments may be cloned directly into plasmids, cosmids, or other suitable vectors, by conventional techniques, instead of initial cloning into bacteriophages. The plasmid or other vector would then be raised in a suitable host with screening being carried out to find treponemal antigen encoding clones. The present process of identifying "positive" bacteriophage clones and then subcloning the identified bacteriophage into a plasmid for prolonged antigen production is preferred.

The Treponemal pallidum DNA may be prepared by any number of known techniques. Preferably, the Treponema pallidum DNA will be obtained and purified from Treponema pallidum raised in rabbit testes by well known techniques. The cleavage of Treponema pallidum DNA into fragments may be carried out using a number of different restriction enzymes. It is preferred that the restriction enzyme Sau 3AI and similar type restriction enzymes be used. The use of Sau 3AI as the restriction enzyme for digesting the Treponema pallidum DNA is particularly preferred.

Vectors which may be suitably used for packaging the cleaved Treponemal pallidum fragments include plasmids, cosmids, bacteriophage M13 and bacteriophage lambda. The use of bacteriophage lambda is preferred because it will not bias the specific methodology used to identify clones producing treponemal antigens by eliminating clone antigens that must exist in stable association with the host. Charon 30 is the particularly preferred bacteriophaged

OMPI lambda for use in accordance with the present invention. Charon 30 is a well known and readily available bacteriophage lambda.

Packaging of the Treponemal pallidum DNA fragment into the Charon 30 may be accomplished by any of the well known recombinant DNA techniques. Preferably, the DNA fro

Charon 30 is cleaved by the restriction enzyme Bam HI or a similar suitable restriction enzyme. The extraneous central Charon 30 DNA fragments are physically removed followed by ligation of the complementary Charon 30 DNA ends to the Treponema pallidum DNA fragments . The resulting recombinant DNA includes a central Treponema pallidum fragment inserted between the complementary arms of the Charon 30 DNA as shown in Figures 1 and 2 for exemplary Charon 30 clones.

The Charon 30 clones produced in accordance with the present invention may be propagated in any suitable host. The preferred host is Escherichia coli. The various Charon 30 clones prepared as outlined above are propagated within E. coli with the resulting plaques being screened for desired antigenic activity with known positive serums. The Charon 30 clones identified as producing antigens reactive with antibodies to Treponema pallidum are then further propagated to produce antigens for use in serodiagnosis and otherwise.

The treponemal antigens derived from the above described recombinant DNA technique can be applied to serodiagnosis in any variety of forms including, but not limited to, coating of beads with antigen and measuring agglutination, radioimmunoassay, and enzyme linked immuno sorbent assay (ELISA). Use of several treponemal antigens, singly or in combination can be employed for optimal specific sensitive serodiagnosis of IgM or IgG syphilitic antibody. Additionally, the treponemal antigens derived from the above-described recombinant DNA technique can be used as vaccines to immunize individuals against Treponema pallidum infection. The treponemal antigens can be used alone or in combination with various adjuvants including, but not limited to, Freundi complete adjuvant, to subcutaneously immunize individuals against Treponema pallidum infection.

A more detailed discussion and description of the Charon 30 clones, plasmids propagated or subcloned from the Charon 30 and their antigen products is given in the following examples. Example 1:

T. Pallidum, Nichols strain, was grown in 20 rabbit testicles and extracted from the testicles by soaking in phosphate buffered saline, and by two differential centrifugations as described by Hanff et al. CHanff, P.A., T.E. Fehniger, J.N. Miller, and M.A. Lovett, 1982 Humoral Immune Response in Human Syphilis to Polypeptides of Treponema pallidum, J. Immunol, Vol. 129, No. 3]. The treponemes were purified of rabbit tissue by centrifugation to equilibrium through a conventional Percoll density gradient. High molecular weight DNA was obtained by suspending the T. pallidum into 0.5ml of 50mM Tris-HCl pH7.8, lOmM NaCl, I MEDTA, 0.2% Sarcosyl, 200ug/ml Proteinase K and incubating overnight at room temperature. The lysed treponemes were extracted 2 times with phenol saturated with buffer (50mM Tris HCl pH7.8, lOmM NaCl) and two times with ethyl ether. The DNA was purified by equilibrium sedimentation through a CsCl density gradient. Fractions were collected from the top of the tube with a cut off pipet tip and were monitored for DNA in the lanes of a 0.6% agarose gel. Fractions from the middle of the gradient were shown to contain DNA of size greater than 50kbp (kilobase pairs). These fractions were pooled and dialyzed against lOmM Tris-HCl pH7-^*8, lOmM NaCl, ImMEDTA. For cloning, two 3ug samples were subjected to partial digestion with SAU 3AI (New England Biolabs) to

WIP produce randomly cleaved overlapping fragments as describe by Rimm et al. [Ri m, D.L., D. Horness, J. Kucera, an F.R. Blattner. 1980. Construction of coliphage lambd Charon vectors with Bam HI cloning sites. Gene, 12: 301-309.] One sample was cut with 0.3 units of Sau 3AI, the other with 0.15 units for 30 min. at 16°C. The digestions were stopped by extraction with buffer saturate phenol, followed by 2 extractions with ethyl ether. The samples were combined and the Sau 3AI digest was sized on a 0.7% agarose gel. The digest was in a range from 40 to 2 kbp. The DNA was concentrated by precipitation with ethanol.

The bacteriophage Charon 30, obtained from F. Blattner [Rimm, D.L., D. Horness, J. Kucera, and F.R. Blattner. 1980. Construction of coliphage lambda Charon vectors with Bam HI cloning sites. Gene, 12: 301-309.], was grown and the DNA prepared as previously described by alfield et al. [ alfield, A.M., U. Storb, E. Seising, and H. Zentgraf. 1980. Comparison of Different Rearranged Immunoglobulin Kappa Genes of a Myeloma by Electronmiαro- scopy and Restriction Mapping of Cloned DNA: Implications for "Allelic Exclusion." Nucl. Acids Res. 8: 4689-4707].

The Charon 30 DNA was digested with Bam HI (New England Biolabs) and the extraneous central Bam HI fragments were physically removed from the outer arms of phage DNA by a cos site annealing and sedimentation through a NaCl density gradient as described by Newell et al. [Newell, N., J.E. Richards, P. . Tucker, and F.R. Blattner. 1980. J genes for heavy chain immunoglobulins of mouse. Science, 209: 1128-1132.]

Recombinant genomes were formed by ligating 2.Oug of the annealed outer Bam HI fragments of Charon 30 to 1.2ug of the Sau 3AI partial digest of T. pallidum DNA through the enzyme generated complementary ends with 10 units (New England Biolabs) of T₄DNA ligase, at a DNA concentration of lOOug/ml. The chimeric DNA was packaged in vitro and

OMPI plated on Escherichia coli K802 on a 150mm petri dish of NZC agar and incubated overnight as described by Blattner et al. [Blattner, F.R., A.E. Blechl, K. Denniston-Thomp- son, H.E. Faber, J.E. Richards, J.L. Slightom, P.W. Tucker, and O. Smithies. 1978. Cloning human fetal globin and mouse a-type globin DNA: Preparation and screening of shotgun collections. Science, 202: 1279-1284.] The soft top agar was made with agarose for greater strength.

About 150 plague forming units were plated. These plaques were screened for T. pallidum antigens by an in situ radio-immunoassay. Screening was done by a modification of the procedure of Towbin et al. [H. Howbin, T. Staehelin, J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76. 4350 (1979)]. Nitrocellulose disks were placed over the phage plaques, and the disks were allowed to absorb protein for 10 to 30 minutes. Little protein was absorbed from the unlysed Ξ. coli of the lawn. -The nitrocellulose filters were then soaked for 10 minutes in 5 percent ovalbumin in 50mM tris-HCl. pH7.5, 150mM NaCl, and 0.15 percent sodium azide (TSA-5 percent OA) . The plaque blots were incubated overnight in either human secondary syphilitic serum or in normal human serum diluted 1:300 in TSA-1 percent OA, washed, exposed to 125I-labeled Staphylococcus aureus protein A, and washed; autoradiographs were made as described by Towbin et al. [H. Towbin, T. Staehelin, J.

Gordon, Proc. Natl. Acad. Sci. U.S.A. 76. 4350 (1979)].

One plaque designated Tp3a, which gave a strong reaction with a secondary syphilitic serum, was chosen for further study. Phage from this plaque was diluted and replated on E. coli CSH 18. When screened with three different secondary syphilitic serums, all plaques showed radioactivity, whereas control plaques of the cloning vector, Charon 30, showed little or no reaction. Secondary syphilitic serums reacted strongly with Tp3a plaques while serums from individuals without syphilis show no reaction with Tp3a plaques. The polypeptide treponemal antigens of Tp3a wer studied to determine the molecular weight of the antigen by electrophoretic transfer of total lysate proteins t nitrocellulose filters after sodium dodecyl sulfat (SDS)-polyacrylamide gel electrophoresis [H. Towbin, T Staehelin, J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76 4350 (1979)]. Tp3a was found to encode for at least seve clearly discernible polypeptides which have molecula weights of 46,000, 43,000, 38,000, 24,000, 23,000, 20,000 and 18,500 and react specifically with syphilic serums The molecular weights of these antigens correspond to thos of antigenic proteins of T. pallidum. Identical or greate amounts of Charon 30 lysate proteins do not react with th syphilitic serums.

DNA isolated from Tp3a was characterized b restriction endonuclease analysis. Bacteriophage Tp3 contains a 16-kbp insert of T. pallidum DNA. The tota molecular weight of T. pallidum antigens is well within th coding capacity of this 16-kbp insert of cloned DNA. partial restriction endonuclease map of the T. pallidum DN of clone Tp3a is shown in Fig. 2. The 16-kbp insert i circumscribed by Sau 3A1 or Bam HI sites at the insert vector junctions, indicated by X. The outer Eco RI site o the left is on the long Bam HI arm of Charon 30; th outermost Bgl II site on the right is on the short arm o Charon 30.

The remote possibility that we had cloned contaminated sequence of rabbit DNA because our original T. pallidum preparation was harvested from rabbit testicle was considered. However, the treponemal origin of the Tp3 DNA was established on the basis of the antigencity of it products with human syphilic serums. Since secondar syphilic serums do not react with protein blots of norma rabbit testicular tissue and normal rabbit serum, thes serums were specific for detection of treponemal antigens. Further, two additional clones that produce Tp3a, antigen

OMPI were identified during a subsequent screening of 500 clonees prepared as described above. Therefore, we have obtained three isolates having identical DNA sequences from a total of 750 clones, an indication that genome size of the organism from which the DNA was cloned is the prokaryotic range of 10 8 to 109 daltons [L. Clarke and J.

Carbon, Cell 9, 91 (1976)].

Example 2:

Treponema pallidum DNA fragments where packaged in Charon 30 bacteriophage and plated on Escherichia coli K802 as described in Example 1. Approximately 500 plaques were obtained. Each plaque represented a clone of T. pallidum DNA since the background level of plaques formed by packaging ligated Charon 30 arms themselves was virtually 0.

Proteins from the plaques were transferred onto nitrocellulose discs by blotting as previously. This procedure created an antigenic image of the plaques on the nitrocellulose filters. The filters were^' screened for plaques that were antigenic to T. pallidum with anti-serum from a secondary syphilitic and 125I-labeled protein A from

Staphlococcus aureus. The protein A (Pharmacia Fine Chemicals) was labeled with 125I (Amersham) by a lactoperoxidase reaction as described by Marchalonis, J.J. [Marchalonis, J.J. 1969. An enzymic method for trace iodination of immunoglobulins and other proteins. Biochem.

J. 113: 299-305]. Dilutions were made from agar plugs of the reactive plaques and the phage were plated on E_^ coli

CSH18 [Rimm, D.L., D. Horness, J. Kucera, and F.R. Blattner. 1980. Construction of coliphage lambda Charon vectors with Bam HI cloning sites. Gene, 12: 301-309] for secondary screenings.

An autoradiogram of a primary screening was taken.

The signals were labeled. The stronger signals which were labeled were identified as clones TplC, Tp3C, TplD, Tp2D,

Tp3D, Tp4D, Tp5D, Tp6D and TplΞ. All of the labeled signals could be aligned to clearly identifiable, wel isolated plaques. These recombinant clones wer synthesizing antigens specific to T. pallidum.

Secondary screenings of the above identified plaques were performed to confirm the initial results and to ensure the purity of the clones. Each of these clones was plaque-purified by replating the recombinants onto E. coli CSH18 for the secondary screenings. Every plaque on the plates from the secondary screenings displayed positive signals on autoradiograms for the reactions with syphilitic serum and 125I-labeled protein A. The strength of the signals for the secondary screenings was generally stronger than those observed for the primary screenings. This was because the Charon 30-derived recombinants formed larger plaques on E. Coli CSH18 (hsr *r*, hsm+) than on the non-restrictive K802 strain (hsr , hsm ) which was required for the primary selection. The relative intensities of the secondary screenings signals corresponded in strength to the relative intensities initially observed. The antigenicity of each of the above identified clone's is characteristic. A control of Charon 30 plaques and plaques of Tp3A, the clone previously described in Example 1, were tested along with the clones prepared in this example. It was apparent that the reaction of human syphilitic serum with the recombinant plaques is specific and does not represent antibodies directed against E. coli or Charon 30 proteins.

The recombinants of Example 2 were grown in broth on E. coli DP50/supF as described in Walfield, A.M., U. Storb, E. Seising, and H. Zentgraf. 1980. Comparison of Different Rearranged Immunoglobulin Kappa Genes of a Myeloma by Electronmiσroscopy and Restriction Mapping of Clones DNA: Implications for "Allelic Exclusion." Nucl. Acids. Res. 8: 4689-4707. Cells and debris were centrifuged out. To characterize the polypeptide products of clones specifically reactive with syphilitic serum, we

O P1 electrophorectically transferred to nitrocellulose sheets the clone polypeptides after they had been resolved by SDS-PAGΞ, and probed these electrophoretic transfers (blots) for bands of antigenic proteins. For analysis by electrophoresis through sodium dodecyl sulfatepolyacrylamide gels (SDS-PAGΞ), the lysate was concentrated 20-fold by precipitation in 2 volume of 2% sodium dodecyl sulfate (SDS), 5% -mercaptoethanol, 10% glycerol and bromophenol blue, and boiled for 5 minutes prior to loading onto gels as described by Towbin et al. [Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA. 76: 4350-4354]. The concentrates of 2ml of lysates were loaded into 5mm wells, the concentrates of 40ml of lysate were applied to

118mm wells. The lanes containing authenitc T. pallidum

9 antigens were loaded with 1x10 organisms suspended and boiled in the 2% SDS, 5% -mercaptoethanol, 10% glycerol solution. The molecular weight markers, supplied by Pharmacia Fine Chemicals or Bethesda Research Laboratories were treated similarly. The samples were run through 8-20% SDS-PAGE as described previously by Hanff et al. [Hanff, P.A., T.E. Fehniger, J.N. Miller, and M.A. Lovett. 1982. Humoral immune response in human syphilis to polypeptides of Treponema pallidum. J. Immunol, Vol. 129, No. 3].

Proteins were electrophoretically transferred to nitrocellulose sheets (pore size 0.2 micron, Sartorius) as also described in Hanff et al. [Hanff, P.A. , T.E. Fehniger, J.N. Miller, and M.A. Lovett. 1982. Humoral immune response in human syphilis to polypeptides of Treponema pallidum. J. Immunol. Vol. 129, No. 3] except that the transfer was done at 195mA for 1/2 hour followed by overnight transfer at 50mA. The electrophoretic blots were left whole when probed with one serum or were cut into 6-8mm strips for probing with individual sera. The whole blots and strips were probed with serum diluted l:100and the human sera were evaluated and obtained as described by Hanff et al. [Hanff, P.A., T.E. Fehniger, J.N. Miller, and M.A. Lovett. 1982. Humoral immune response in human syphilis to polypeptides of Treponema pallidum. J. Immunol. Vol. 129, No. 3] and the immune rabbit serum was collected from the rabbit 1 year after recovery from experimental syphilis. Where specified, sera were pre-adsorbed with a sonicated lysate of E. coli bound to polyacrylamide [Carrel, S. and S. Barandun. 1971. Protein-containing polyacrylamide gels: Their use as immunoadsorbents of high capacity. Immunochemistry, 8: 39-48].

Autoradiograms of the electrophoretic blots for the concentrated lysates of clones Tp2D and TplE were made. These electrophoretic blots were probed with a high titer secondary syphilitic serum that had been pre-adsorbed with E. coli lysate proteins . Beside each electrophoretic lane of clone lysate was run T. pallidum which gave the characteristic antigenic pattern of whole T. pallidum, as previously observed [Hanff, P.A., T.E. Fehniger, J.N. Miller, and M.A. Lovett. 1982. Humoral immune response in human syphilis to polypeptides of Treponema pallidum. J. Immunol. Vol. 129, No. 3]. The blots showed that clone Tp2D produces a cluster of antigenic bands from 16, 000 to 20,000 daltons and cluster at higher molecular weight around 43,000 daltons, and that TplE produces at least 2 strongly reactive antigens of 64,000 and 40,000 daltons.

A more detailed analysis of the antigens expressed by these two clones was accomplished by assaying the clone lysates with a spectrum of sera from different stages of syphilis. The tests were run in tandem with one strip being incubated in unadsorbed serum and the other strip being incubated in that same serum preadsorbed with E. coli lysate proteins . E. coli and bacteriophage lambda proteins are the major constituents of the recombinant phage lysates. Human sera contain antibodies to E. coli proteins, which can be adsorbed by use of the E. coli immunoadsorbent described previously. The adsorption procedures was found to improve the results of the electrophoresis by reducing interference by E. coli and bacteriphage lambda proteins.

The strips were probed with serum from an immune rabbit, with sera from 3 secondary syphilitics, and with serum from a patient in early latency. They all showed numerous consistent bands. The sera from a primary patient, a late latent syphilitic, and a late syphilitic patient showed weaker responses or lack of response. This is consistent with the low treponemal reactivity that is normally seen with patient sera from these stages. There were no responses against normal human sera, and it is noteworthy that the lysate exhibited no specific antigenicity with serum that gave a biological false positive on the VDRL test.

While examining the protein blots of the T^ pallidum-Charon 30 chimeras, it became obvious that the clones could be grouped into classes. One such class is the clones Tp3A, TplC and Tp2D. The 3 clones Tp3A, TplC, and Tp2D each produce the identical antigenic "fingerprint" with clusters of antigens at 46,000, to 37,000 daltons and from 23,000 to 18,000 daltons. A control of Charon 30 lysate proteins was included because this blot was assayed with unadsorbed secondary syphilitic serum. The results show that the 3 clones contain DNA fragments which overlap in the genome of T. pallidum. To identify the common DNA sequence of these clones, restriction endonuclease site maps of the 3 recombinant DNAs were constructed. Results of the mapping are shown in Fig. 2. The maps show that in an area of approximately 9kbp (the bold line), the 3 clones display an overlapping sequence. This common region of DNA therefore includes the segments of the T. pallidum genome which code for the antigens expressed by the 3 clones. This conclusion was confirmed by subcloning the 6.2kbp Hind III fragment of Tp2D, which includes most of the common region (Fig. 2), into the plasmid pBR322 and determining that the recombinant plasmid encoded treponemal antigens when transformed into E. coli.

It can be seen in Fig. 2 that the sequences were inserted into the bacteriophage vector in both possible orientations. Clone Tp2D was inserted in opposite orientation with respect to the bacteriophage Bam HI arms than were the other 2 clones. The direction of the DNA insert does not seem to affect expressions of the genes for T. pallidum antigens. This observation strongly suggests that the structural genes coding for the treponemal antigens were cloned along with their own promotor sequences. The DNA inserts of clones Tp3A and Tp2D both extend rightward beyond the common sequence and fail to correspond. This suggests that one or both of these DNA inserts were formed from the in vitro recombination of at least 2 smaller unrelated S u 3AI fragments of T. pallidum DNA.

The Tp3A clone was subcloned into pBR322 plasmid and propagated in E. coli. The subcloning was accomplished by ligating Hind III-digested DNA of clone Tp3A to Hind Ill-cleaved and alkaline phosphatase-treated ρBR322 at a 1:2 molar ratio, transforming into E. coli RRl, selecting for ampicillin resistant tetracycline sensitive transformants, lysing replicas of such colonies in situ (Erlich, H.A., S.N. Cohen, and H.O. McDevitt. 1978. A sensitive radioimmunoassay for detecting products translated from cloned DNA fragments. Cell, 13: 631-689), and screening for the presence of T. pallidum antigens, and screening for treponemal antigens as previously described. (Alkaline phosphatase obtained from Boehringer Mannheim and Hind III from New England Biolabs).

The subcloned plasmid encodes for antigens identical to those produced by the Tp3A type clones. The Tp3A plasmid subclone was identified as pAW305. This plasmid is now on deposit with the American Type Culture Collection (ATCC), 1230 Parklawn Drive, Rockville, Maryland 20852 and is identified as ATCC No. 39237.

A Charon 30 clone identified at Tp4D produced a particularly reactive plaque that is on the order of 100 times more reactive than the Tp3A type clones. It was surprisingly discovered that the treponemal antigens encoded for by Tp4D were unusual proteins that exhibit properties more commonly attributed to carbohydrates. More specifically, the Tp4D antigens exhibit resistance to protease treatment, relatively highly acidic isoelectric points, and stain specifically with stains felt to be selective for carbohydrates. These unexpected properties originally led to the conclusion that the Tp4D antigens were mucopolysaccharides. However, further analysis indicates that the unusual Tp4D antigens are largely protein in nature with an estimated 2-3% of their composition being neutral sugars of a standard nature and perhaps some additional carbohydrates of a more unusual nature. The treponemal antigens produced by Tp4D were purified from lysates of the Tp4D clone by lOx concentration of the lysate by precipitation with acetone, followed by chromatography on a Sephacryl S-200 column. Proteinase K treatment was used. The estimated size of the Tp4D antigen based on S-200 chromatography corresponds to that of a protein with a 150,000 molecular weight. Periodate-silver staining of SDS-polyacrylamide gels (SDS-PAGE) identifies the Tp4D antigen as a molecule on the order of 180,000-200,000 apparent molecular weight. Treatment with proteinase K decreases the size of the Tp4D antigen to a 90,000 molecular weight form, as will be discussed in more detail below. The Tp4D Charon 30 clone was subcloned into pBR322 and propogated in E. coli♦ as previously described for the

OM?I Tp3A clone. The Tp4D plasmid subclone was identified as pAW327. pAW327 was further subcloned by digestion with restriction endonuσleases Hind III and Eco RI, self-ligation with T4DNA ligase, and the ligated DNA was used to transform E. coli RRl. One class of antigenic clone isolated, represented by pAW330, contained only a 3.2 Kb Eco RI/Hind III fragment of treponemal DNA and produced an amount of antigen equivalent to pAW327. pAW330 also contained a 0.6 Kb Hind Ill/Bam HI fragment which did not direct the synthesis of T. pallidum antigen when it was ■ found in other subclones of pAW327.

Another subclone, pAW329, was isolated which apparently contains gene duplication. The pAW329 plasmid subclone was created by double digesting pAW327 with Hind IIl/Eco RI and self-ligation and contains a 1.8 kb Hind III/Eco RI fragment of treponemal DNA. The pAW329 is a super producer of the Tp4D antigen, producing more than ten times the amount of antigen produced by pAW327. The pAW329 subclone is also on deposit with the American Type Culture Collection and is identified as ATCC No. 39238.

The Tp4D antigens produced by the subcloned plasmids were purified by fractional ethanol precipitation, taking advantage of the solubility of the Tp4D antigen in 40% but not 75% ethanol, followed by Proteinase K treatment and cetavalon precipitation. The product was greater than 99% pure by densitometer tracing of the periodate-silver stained gel (Tsai, C-M. , and Frasch, C.E. (1982) Analyt Biochem 119:115). Coomassie blue staining showed 85% purity. Though primarily protein in nature, the cloned Tp4D antigen is unexpectedly hydrophillic being fully soluble in 40% ethanol.

Further purification and determination of the isoelectric point of the Tp4D antigen was achieved by preparative flat-bed isoelectric focusing, using a pH gradient of 3.5 - 10. The pi is between 4.6 - 4.8.

As stated above, the apparent molecular weight of the

OMPI Tp4D antigen is on the order of 180,000. Treatment with proteinase K decreases the size of the Tp4D antigen to a 90,000 molecular weight form which is fully resistant to further proteolysis with proteinase K in 0.1% SDS at 60° centigrade for 96 hours. There is no apparent diminution of antigenicity of the Tp4D antigen despite proteinase K treatmen .

The proteinase K treated Tp4D antigen is heat labile and is converted to a 14,000-19,000 molecular weight form upon boiling for 5 minutes without reducing agents. This 14,000-19,000 molecular weight form is no longer reactive with syphilitic sera and is susceptible to further protease degradation. Because the standard techniques for assessing both native and cloned antigens of Treponema pallidum involve a boiling step prior to analysis, earlier attempts at identifying the Tp4D antigenic gene product of Treponema pallidum were unsuccessful. It is believed that the Tp4D antigen is a multimeric association of the 14,000-19,000 molecular weight subunits whose synthesis is determined by the 1.8 Kb treponemal DNA fragment contained in the Tp4D plasmid subclones. The cloned Tp4D antigen is readily stainable with silver stain alone, but staining is several fold enhanced by prior periodate treatment as Tsai and Frasch have described for lipopolysaccharide (Tsai, C-M., and Frasch, C.E. (1982) Analyt Biochem 119:115) . It is readily stainable with Coomassie blue. Ruthenium red, toluidine blue and periodic-acid Schiff (PAS) also stain the Tp4D antigen. The staining with ruthenium red was conducted on nitrocellulose because aqueous ruthenium red avidly binds to polyacrylamide, but not nitrocellulose. Use of nitrocellulose also facilitates PAS staining. Color development with Schiff's reagent on nitrocellulose is absolutely dependent on prior periodate treatment and it is easy to wash and obtain a low background.

The cloned Tp4D antigen is reactive with human syphilitic sera from all stages of disease while false-positive and normal sera are unreactive. Both the Proteinase K treated and untreated cloned Tp4D antigen react with serum from a patient with secondary syphilis. The Proteinase K treatment approximately halves the apparent molecular weight of the Tp4D antigen; however, no loss of antigenicity upon Proteinase K treatment is evident. The cloned Tp4D antigen reacts strongly with sera from patients with early latent syphilis and does not react with sera from uninfected humans under identical conditions. The cloned Tp4D antigen also reacts with serum from a rabbit immunized with a T. pallidum sonicate but not with serum from control rabbits. Upon boiling the heat labile proteinase K treated Tp4D antigen is converted to the 14,000-19,000 molecular weight form which is no longer reactive with human syphilitic sera.

Motile, virulent, T. pallidum was purified from rabbit testicles as described and aliquots were frozen at -70°. Upon sonication, treatment with DNase, RNase, and Proteinase K, and electrophoresis, the gel was stained with the periodate-silver procedure. It was found that T. pallidum has a Proteinase K resistant molecule with size comparable to that of the cloned Tp4D antigen. The nonpathogen T. phagedentis (Reiter Treponeme) does not contain such a molecule but, unlike T. pallidum, appears to have smooth lipopolysaccharide (LPS) material as judged by similarity in size and staining properties to gram-negative LPS described by Tsai and Frasch (Tsai, C-M., and Frasch, C.E. (1982) Analyt Biochem 119:115). Upon transfer to nitrocellulose, the T. pallidum molecule, like the cloned Tp4D antigen, is detected immunologically by a secondary syphilis serum. Syphilitic serum does not react with the protease resistant components of T. phagedentis.

As previously mentioned the pattern of serological responses to the cloned antigens mimics the responses to authentic T. pallidum antigens. The anti-serum from the most serologically reactive stages of syphilis, secondary,

O PI early latent, and experimental syphilis in the rabbit, were most reactive with the cloned antigens. Furthermore, the antigenic bands in the clone lysates is roughly correlated to bands of similar electrophoretic mobility found in the antigenic fingerprint of T. pallidum.

The above characterization and identification of the antigens synthesized by the treponeme-phage recombinants and plasmid subclones show that such clones can provide a sufficient variety of antigens to be adopted into any number of the known serodiagnostic tests for all the stages of syphilis. Recombinant clones can also be a source of pure antigens for methodical studies on i munogenicity in both humoral and cellular systems. Further, the clones and their purified antigen products will be valuable in elucidating the basic biology of the organism: ultrastructure, physiology, and genetics.

The antigen products produced from the clones can be used as vaccines to immunize individuals against Treponema pallidum infection. For example, the immunogenicity of the cloned Tp4D antigen has been established. Approximately 200 micrograms and 800 micrograms of the Tp4D antigens purified by proteinase K treatment and cetavalon precipitation were used along with Freundi complete ad uvant to subcutaneously immunize two adult male rabbits. Each rabbit was bled 2 weeks after immunization. Each rabbit developed antibody levels indistinguishable from those detected in human early latent syphilis. Pre-immune serum from these rabbits showed no reactivity with the cloned Tp4D antigen.

Similarly, immunization with the cloned Tp4D antigen induces serum activity in rabbits that immobilizes virulent Treponema pallidum in vitro. Two rabbits were immunized with a single injection of proteinase K treated Tp4D antigen. The rabbits were bled at 3, 8, 10, and 12 weeks and sera were strongly reactive with the Tp4D antigen. Preimmunization sera lacked Venereal Disease Research Laboratory (VDRL) antibody and Fluorescent Treponemal Antigen (FTA) antibody using a goat anti-rabbit fluorescent conjugate as second antibody and commercially available slide preparations of Treponema pallidum (Beckman). Complement dependent immobilizing activity was present in one rabbit at 8 weeks after the single immunization while the second rabbit showed immobilizing activity in the 10-week bleed. Preimmunization sera and sera from all other time points were negative for immobilizing activity. The induction of immobilizing antibodies by the Tp4D antigen suggests that it has a surface location on Treponema pallidum.

The above examples describe the cloning in E. coli of a Treponema pallidum DNA segment on the order of 1.8 Kb and no greater in size than 3.2 Kb which determines the production of a protein molecule that unexpectedly exhibits properties associated with carbohydrates. We believe that this Tp4D molecule represents a class of treponemal antigens which has not been defined by SDS-PAGE before due to its heat labile nature. No plasmid clone lacking this approximately 1.8 Kb fragment has been shown to produce this Tp4D antigen. Because the approximately 1.8 Kb fragment of Treponema pallidum DNA encodes only peptides on the order of 18,000-22,000 molecular weight, we believe the Tp4D antigen to be a multimeric association of these smaller monomeric subunits, perhaps held together through hydrophobic interaction. The Tp4D antigen molecule contains a trypsin sensitive linkage, but is not further degraded by such proteases as Proteinase K, Pronase, and Papain, and is not at all degraded by σhymotrypsin. There is no evidence that there is any gross loss of antigenicity upon protease treatment.

Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations and

OMPI modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein.

Claims

What is claimed is:

1. A process for producing antigens reactive with antibodies to Treponema pallidum comprising: cleaving the DNA of a bacteriophage vector to produce a first DNA fragment; combining said DNA fragment with a second DNA fragment from Treponema pallidum, said first and second DNA fragments being capable of recombination to form recombinant DNA; packaging said recombinant DNA into a viable bacteriophage particle to form bacteriophage clones; infecting a suitable host with said bacteriophage clone to form plaques; and selecting the plaques having antigens which are reactive with antibodies to Treponema pallidum.

2. A process according to claim 1 wherein said first DNA fragment is provided by cleaving said bacteriophage DNA into a left arm fragment, a central stuff r fragment and a right arm fragment; and removing said central stuffer fragment from said left and right arm fragments, to provide a first DNA fragment.

3. A process according to claim 2 wherein said bacteriophage vector is Charon 30.

4. A process according to claim 3 wherein said host is Escherichia coli.

5. A process according to claim 1 wherein the antigens reactive with Treponema pallidum antibodies in said selected plaques are separated into protein antigens and protease resistant, heat labile protein antigens.

6. A process according to claim 4 wherein the DNA of said bacteriophage is cleaved with the restriction enzyme

_OMPi Ba HI.

7. A process according to claim 4 wherein said second DNA fragment is prepared by cleaving the DNA of Treponema pallidum with the restriction enzyme Sau 3AI.

8. A method for detecting the presence of antibodies to Treponema pallidum in biological fluids comprising: adding antigens reactive with antibodies to Treponema pallidum produced by recombinant DNA techniques to a sample of biological fluid; and determining whether said antigens react with antibodies to Treponema pallidum present in said sample.

9. A composition of matter comprising: antigens reactive with antibodies to Treponema pallidum, said antigens being produced by recombinant DNA techniques.

10. A bacteriophage containing therein a Treponema pallidum gene combined into the DNA of said bacteriophage, said bacteriophage being capable of producing antigens reactive with antibodies to Treponema pallidum.

11. A bacteriophage according to claim 10 wherein said bacteriophage is Charon 30.

12. A bacteriophage according to claim 11 wherein said bacteriophage is capable of producing protein antigens having molecular weights of 46,000 daltons, 37,000 daltons, 23,000 daltons, and 18,000 daltons which react specifically with antibodies to Treponema pallidum.

13. A process for modifying Charon 30 to provide a bacteriophage clone capable of producing antigens reactive with antibodies to Treponema pallidum comprising the steps of: cleaving the DNA of said charon 30 to produce a first DNA fragment; combining said first DNA fragment with a second DNA fragment from Treponema pallidum, said first and second fragments being capable of recombination to form reσominant DN ; and packaging said recombinant DNA into viable Charon 30 particles to form bacteriophage clones capable of producing antigens reactive with antibodies to Treponema pallidum.

14. A process according to claim 13 wherein the charon 30 DNA is cleaved with the restriction enzyme Bam HI.

15. A process according to claim 14 wherein said DNA fragment from Treponema pallidum is produced by cleaving Treponema pallidum DNA with the restriction enzyme Sau Al.

16. A modified Charon 30 bacteriophage capable of producing antigens reactive with antibodies to Treponema pallidum produced by: cleaving the DNA of said Charon 30 to produce a first DNA fragment; combining said first DNA fragment with a second DNA fragment from Treponema pallidum, said first and second fragments being capable of recombination to form recombinant DNA; packaging said recombinant DNA into viable Charon 30 particles to produce Charon 30 clones; infecting a suitable host with said Charon 30 clones to form plaques; and selecting said modified Charon 30 bacteriophage from said plaques having antigens which are reactive with antibodies to Treponema pallidum.

17. A modified Charon 30 bacteriophage according to

- ΕE O PI claim 16 wherein said Charon 30 DNA is cleaved by the restriction enzyme Bam HI.

18. A modified Charon 30 bacteriophage according to claim 16 wherein the DNA fragment from Treponema pallidum is prepared by cleaving Treponema pallidum DNA with restriction enzyme Sau Al.

19. A composition of matter comprising: protein antigens reactive with antibodies to

Treponema pallidum, said antigens being produced according to claim 5.

20. A composition of matter comprising: protease resistant, heat labile protein antigens reactive with antibodies to Treponema pallidum, said antigens being prepared according to claim 5.

21. A method for determining Treponema pallidum infection comprising: adding antigens produced according to claim 1 to a sample of biological fluid; and determining whether said antigens react with antibodies to said Treponema pallidum present in said sample.

22. A microorganism of the strain Eschericia coli, having the identifying characteristics of ATCC No. 39237, said microorganism being capable of producing protein antigens reactive with anitbodies to Treponema pallidum.

23. A microorganism of the strain Eschericia coli, having the identifying characteristics of ATCC No. 39238, said microorganism being capable of producing protease resistant, heat labile protein antigens reactive with antibodies to Treponema pallidum.

CMPI

24. The microorganisms of claims 22 or 23 in freeze dried form.

25. Antigens reactive with antibodies to Treponema pallidum produced by the microorganisms of claim 22 or 23.

26. A process according to claim 8 wherein said antigens are produced according to claim 1.

27. Antigens according to claim 9 produced by the process of claim 1.

28. A method for raising antibodies in an animal, said antibodies being immunoreactive with Treponema pallidum, said method comprising the step of administering an immunologically effective amount of the antigens of claim 9 to said animal to raise antibodies therein which are immunoreactive with Treponema pallidum.

29. A method according to claim 28 wherein said antigens are the antigens of claim 25.

30. A microorganism of the strain Eschericia coli, containing therein a Treponema pallidum gene combined into plasmid DNA of said microorganism, said gene encoding for antigens reactive with antibodies to Treponema pallidum.

31. A vaccination agent comprising protease resistant, heat labile protein antigens of Treponema pallidum.

32. A vaccination agent according to claim 31 including Freundi adjuvant.

33. A composition of matter comprising a protease resistant, heat labile protein antigen which is reactive with antibodies to Treponema pallidum.

34. A composition of matter according to claim 33, wherein said protease resistant, heat labile antigen has a molecular weight on the order of 90,000 daltons.

35. A composition of matter comprising protease resistant, heat labile antigens produced by microorganisms defined in claim 23.

10

36. A method for determining Treponema pallidum infection comprising: transferring antigens of Treponema pallidum to a nitrocellulose sheet;

15 probing said nitrocellulose sheet with a sample of biological fluid; and determining whether said antigens react with antibodies to Treponema pallidum present in said sample.

20

37. A method according to claim 36 wherein said antigens are the antigens of claim 33.

38. A process for producing antigens reactive with _ antibodies to Treponema pallidum comprising: cleaving the DNA of a plasmid vector to produce a first DNA fragment; combining said DNA fragment with a second DNA fragment from Treponema pallidum, said first and second DNA _₀ fragments being capable of recombination to form recombinant DNA; transforming said recombinant DNA into a suitable host; propagating colonies of said transformed host; and ₃₅ selecting the colonies producing antigens which are reactive with antibodies to Treponema pallidum.

"gtRE O PI

39. A process for producing antigens reactive with antibodies to Treponema pallidum comprising: cleaving the DNA of a suitable vector to produce a first DNA fragment; combining said DNA fragment with a second DNA fragment from Treponema pallidum, said first and second DNA fragments being capable of recombination to form recombinant DNA; transforming said recombinant DNA into a suitable host; and propagating said transformed host.