WO1990015132A2 - Gene endoflagellaire clone de treponema hyodysenteriae - Google Patents

Gene endoflagellaire clone de treponema hyodysenteriae Download PDF

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WO1990015132A2
WO1990015132A2 PCT/US1990/002908 US9002908W WO9015132A2 WO 1990015132 A2 WO1990015132 A2 WO 1990015132A2 US 9002908 W US9002908 W US 9002908W WO 9015132 A2 WO9015132 A2 WO 9015132A2
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hyodysenteriae
polypeptide
endoflagellar
antigen
clone
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PCT/US1990/002908
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WO1990015132A3 (fr
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David A. Boyden
Fred G. Albert
Charles S. Robinson
Michael Mcdonell
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Syntro Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to antigenic preparations, and more particularly to certain cloned polypeptides of a Treponema hyodysenteriae endoflagellar antigen used to induce a protective immune response in animals.
  • Such polypeptides can be used immunologically as a vaccine for dysentery caused by this organism.
  • Treponema hyodysenteriae is the etiologic agent of swine dysentery, a severe mucohemorrhagic diarrheal disease which primarily affects young pigs.
  • T. hyodysenteriae is a gram-negative anaerobic spirochete which has seven to nine endoflagella (axial filaments, periplasmic flagella) that are surrounded by an outer envelope. Seven serotypes of T. hyodysenteriae are known based on the antigenic specificity of a phenol and water-extracted lipopoly- saccharide (LPS) (Mathoper, M.E., et al . , J. Clin, Microbiol . , 22 :161, 1985). Serotypes 1 and 2, represented by T. hyodysenteriae strains B234 and B204 respectively, axe the most prevalent in the U.S.
  • LPS lipopoly- saccharide
  • Pigs convalescing from acute swine dysentery are protected from disease when subsequently reexposed to T. hyodysenteriae .
  • This natural immunity may be serotype-specific, as suggested by challenge experiments carried out in a ligated colonic loop model.
  • attempts to induce immunity to swine dysentery using whole-cell bacterins have not been successful, as these preparations typically lead to only partial protection in pigs (Glock, R.D., et al . , Am J. Vet . Res. , 39:639, 1978; Fernie, D.S.,, et al . , Res. Vet . Sci . , 35:217, 1983).
  • T. hyodysenteriae Although a major humoral response in pigs to T. hyodysenteriae is to the serotype-specific LPS (Wannemuehler, M.J., et al . , Infect . Immun . , 56:3032, 1988), there is a group of protein antigens of T. hyodysenteriae having molecular weights ranging from 25 Kd to 45 Kd that are conserved among serotypes and also appear to be important in the immune response to T. hyodysenteriae (Chatfield, S.N., et al . , Infect . Immun. , 56 : 1010, 1988).
  • T. hyodysenteriae endoflagella T. hyodysenteriae endoflagella
  • the endoflagella of the human treponemes Treponema pallidum and Trepo ⁇ ej ⁇ a phagedenis biotype Reiter are also composed of multiple protein species which are immunologically related and may be encoded by separate structural genes.
  • purified endoflagella of T. phagedenis biotype Reiter induce serum treponemicidal activity against T.
  • the present invention relates to antigenic preparations and methods of immunizing animals to induce a protective immune response to T. hyodysenteriae endoflagellar proteins.
  • an antigen preparation is produced which contains a polypeptide corresponding to an endoflagellar protein of T. hyodysenteriae using recombinant DNA techniques. This antigenic preparation can be used to immunize an animal such that antibodies are produced to the polypeptide.
  • compositions comprising the antigen preparation of the invention together with pharmacologically appropriate carriers are also included in this invention.
  • the invention comprises a polypeptide free from non- endoflagellar polypeptides of T. hyodysenteriae comprising an endoflagellar protein of T. hyodysenteriae, expression vehicles comprising a DNA sequence coding for the endoflagellar protein, prokaryotes transformed with the expression vehicle, methods of producing the endoflagellar protein in prokaryotes, and methods of inducing an immune response in an animal to T. hyodysenteriae comprising immunizing the animal with a pharmaceutical composition containing the recombinant endoflagellar protein from. T. hyodysenteriae.
  • a major advantage of the present invention is that it provides the art with a ready source of T. hyodysenteriae endoflagellar polypeptide corresponding to that purified from natural sources, while avoiding the problems associated with the isolation of naturally occurring T. hyodysenteriae endoflagellar polypeptide to separate it from other T. hyodysenteriae non-endoflagellar polypeptides.
  • This absence of other T. hyodysenteriae non-endoflagellar polypeptides is significant in that it allows the development of a well-defined vaccine.
  • T. hyodysenteriae endoflagellar polypeptide in host cells is that by so doing it is possible to obtain much larger quantities of the polypeptide than are currently practicably available from natural sources. As a consequence, not only is it possible to immunize animals more effectively with a defined vaccine, but it is also now possible to provide commercially useful quantities of endoflagellar polypeptide for use in vaccines.
  • the T. hyodysenteriae endoflagellar polypeptides of the present invention retain the ability to stimulate a protective immune response to T. hyodysenteriae even though the prokaryotic host in which the polypeptides of the invention can be cloned may degrade the expressed endoflagellar polypeptide molecule.
  • FIG. 1 shows the immunoblot analysis of various antisera used to screen the genomic libraries of T. hyodysenteriae.
  • T. hyodysenteriae B204 cells were lysed by ultrasonic treatment, the proteins separated by SDS-PAGE, and transferred to nitrocellulose.
  • Each lane represents a nitrocellulose strip reacted with a different serum: lane 1, R anti-B204; lane 2, CS anti-B204; lane 3, M anti-B204 Aq; lane 4, S anti-B204 Aq; lane 5, M anti-B204 SI; lane 6, R anti-TpREF.
  • Fig. 2 SDS-PAGE analysis of the partially-purified endoflagellar antigen expressed by clone 101.
  • the nonmembrane (NM) fraction of the E. coli clone 101 lysate was absorbed to a SEP-PAK C 18 column and fractions eluted with a step gradient of acetonitrile.
  • Fig. 3 shows the immunoblot analysis of sarcosine fractions of T.
  • hyodysenteriae B204 demonstrating that the antigens expressed by clones 66 and 101 are related to the same endoflagellar proteins of T. hyodysenteriae.
  • Membrane fractions were extracted with sarcosine, separated by SDS-PAGE, and transferred to nitrocellulose. Lanes 1, 3, 5, 7, and 8, sarcosine-insoluble fraction; lanes 2, 4, and 6, sarcosine-soluble fraction; lane 9, molecular weight markers. Nitrocellulose strips were reacted with various immune sera: lane 1, mouse anti- clone 101 antigen (SEP-PAK-purified); lanes 6 and 7, mouse anti-clone 66 extract (sarcosine-soluble). Lanes 8 and 9 were stained with Amido black.
  • Fig. 5 DNA and amino acid sequence of T. hyodysenteriae endoflagellin gene from clone 101 and clone 103 (927bp, 309aa). Restriction sites are noted above the sequence. Sequence over the solid bar denote regions corresponding to synthetic antigenic peptides .
  • the invention comprises antigen preparations derived from the recombinant expression of T. hyodysenteriae genes and methods of utilizing these antigen preparations to stimulate the immune system of an animal to T. hyodysenteriae .
  • the invention includes a method of producing the polypeptide portion of the endoflagellar protein of T. hyodysenteriae using recombinant DNA techniques. This was done by cloning the bacterial gene for the T. hyodysenteriae endoflagellar protein into a plasmid vector which was then used to transform E. coli . When the endoflagellar gene is expressed in E. coli, the cloned polypeptide that is produced has a molecular weight of approximately 25 Kd for one clone, and 36Kd for another clone, as determined by SDS-PAGE. The 36Kd antigen represents the complete endoflagellar protein.
  • T. hyodysenteriae three different genomic libraries of T. hyodysenteriae were constructed for screening with antibody probes.
  • the libraries were made using chromosomal DNA from the B204 strain (serotype 2).
  • Three different cloning approaches were used to increase the likelihood that important clones would be isolated.
  • the first library was made in the bacteriophage expression vector lambda gtll which generates transcriptional or translational fusions to the ⁇ -galactosidase gene of E. coli .
  • Fragments of T. hyodysenteriae B204 DNA of 1-8 kb were generated by partial digestion with Sau 3A and cloned into lambda gt11.
  • two plasmid libraries were made in which the T.
  • hyodysenteriae chromosomal DNA was randomly sheared by ultrasonic treatment.
  • the second library was constructed using a series of expression vectors which allow for translational fusions to be made to the tenth amino acid of gene 10 of bacteriophage T7.
  • These vectors, pSY908, pSY909, and pSY910 contain the CI857 repressor gene and heat-induc- ible lambda Pr promoter upstream of the T7 gene 10 promoter.
  • Each vector contains a Bam HI site in a different reading frame following the tenth codon of gene 10. Fragments of T.
  • hyodysenteriae DNA of 0.5 to 5 kb were generated by ultrasonic treatment and cloned into an equal mixture of the vectors pSY908, pSY909, and pSY910.
  • the third genomic library was constructed by cloning sonicated fragments of the same size into the plasmid cloning vector pUC13.
  • Fig. 1 shows the reactivity to electrophoretically separated proteins of T. hyodysen teriae lysates of each of the sera used to screen the libraries. These reagents varied in their reactivity to T. hyodysenteriae proteins, both in the number and the type of antigens recognized.
  • the rabbit anti-T. hyodysenteriae B204 serum R anti-B204
  • rabbit serum to the endoflagella of T rabbit serum to the endoflagella of T.
  • phagedenis biotype Reiter (R anti-TpREF) recognized only 3 or 4 bands (Fig. 1, lane 6), most likely the endoflagellar proteins of T. hyodysenteriae (Limberger, R.J., et al . , J.Bacteriol ., 168:1030, 1986).
  • T. hyodysenteriae B204 serum from a convalescent pig immune to rechallenge with T. hyodysenteriae B204 (CS anti-B204, Fig.1, lane 2). It was believed that this serum could help identify T. hyodysenteriae antigens responsible for natural immunity to swine dysentery.
  • a second useful reagent was a mouse antiserum raised against the sarcosine-insoluble fraction T. hyodysenteriae B204 (M anti-B204 SI). This serum recognized seven major proteins of the sarcosine-insoluble (SI) fraction, including the three endoflagellar proteins recognized by the R anti-TpREF serum (Fig. 1, lane 5).
  • the bacterial genes for the endoflagellar protein can be derived from any serotype of T. hyodysenteriae . All that is required is that the genetic sequence for the protein be expressed in the host organism. Preferred is the protein from T. hyodysenteriae serotype 2. Especially preferred are the endoflagellar genes of T.
  • hyodysenteriae serotype 2 produced by cell lines 101 or 66. Prior to May 24, 1989, these cell lines were placed on deposit for 30 years with the American Type Culture Collection, Rockville, Maryland, and have Accession Nos. ATCC 67962 for cell line 101 and ATCC 67961 for cell line 66.
  • host as used in the present invention is meant to include not only prokaryotes, but also, such eukaryotes as yeasts, filamentous fungi, as well as plant and animal cells.
  • prokaryote is meant to include all bacteria which can be transformed with the gene for the expression of the endoflagellar protein of T. hyodysenteriae.
  • a recombinant DNA molecule coding for the endoflagellar protein can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Especially preferred is the use of a plasmid containing the endoflagellar coding sequence for purposes of prokaryotic transformation. Methods for preparing fused, operably linked genes and expressing them in bacteria are known and are shown, for example, in U.S. 4,366,246. The genetic constructs and methods described therein can be utilized for expression of endoflagellar protein from T. hyodysenteriae in prokaryotic hosts. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted treponemal gene fragment are used in connection with the host.
  • the expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells.
  • the transformed prokaryotic hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.
  • the isolation and purification of the microbially expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibody.
  • promoters which can be used in the invention are: rec A, trp, lac, tac, and bacteriophage lambda p R or p L .
  • plasmids which can be used in the invention are listed in Maniatis, et al . , Molecular Cloning, Cold Spring Harbor Laboratories, 1982.
  • the invention extends to any host modified according to the methods described, or modified by any other methods, commonly known to those of ordinary skill in the art, such as, for example, by transfer of genetic material using a lysogenic phage, and which result in a prokaryote expressing the T. hyodysenteriae gene for endoflagellar protein.
  • Prokaryotes transformed with the T. hyodysenteriae gene encoding the endoflagellar protein are particularly useful for the production of polypeptides which can be used for the immunization of an animal.
  • Prokaryotic hosts may include Gram negative as well as Gram positive bacteria, such as E. coli, S. typhimurium, Serratia marcescens, and Bacillus subtilis.
  • immunologically effective amount is meant to denote that amount of T. hyodysenteriae antigen which is necessary to induce in an animal the production of an immune response to T. hyodysenteriae.
  • the endoflagellar protein of the invention is particularly useful in sensitizing the immune system of an animal such that, as one result, an immune response is produced which ameliorates the effect of T. hyodysenteriae infection.
  • Preferred is endoflagellar protein derived from cell lines of T. hyodysenteriae serotype 2.
  • serotype 2 strain B204 is especially preferred.
  • the endoflagellar protein can be administered parenterally by injection, rapid infusion, nasopharyngeal absorption, dermal absorption, and orally. Preparations for parenteral administration include sterile or aqueous or non- aqueous solutions, suspensions, and emulsions.
  • Non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Carriers for occlusive dressings can be used to increase skin permeability and enhance antigen absorption.
  • Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending the liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water.
  • compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
  • adjuvants are substances that can be used to nonspecifically augment a specific immune response. Normally, the adjuvant and the antigen are mixed prior to presentation to the immune system, or presented separately, but into the same site of the animal being immunized. Adjuvants can be loosely divided into several groups based on their composition.
  • These groups include oil adjuvants (for example, Freund's Complete and Incomplete), mineral salts (for example, AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH 4 (SO 4 ), silica, alum, Al(OH) 3 , Ca 3 (PO 4 ) 2 , kaolin, and carbon), polynucleotides (for example, poly IC and poly AU acids), and certain natural substances (for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella ).
  • oil adjuvants for example, Freund's Complete and Incomplete
  • mineral salts for example, AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH 4 (SO 4 ), silica, alum, Al(OH) 3 , Ca 3 (PO 4 ) 2 , kaolin, and carbon
  • the antigenic preparation of the invention can be administered as either single or multiple dosages and can vary from 10-1,000 ug for the T. hyodysenteriae endoflagellar antigen per dose, more preferably 50-700 ug endoflagellar antigen per dose, most preferably 50-300 ug endoflagellar antigen per dose.
  • T. hyodysenteriae strains B204 (ATCC 31212) and B234 (ATCC 31287) were grown under an atmosphere of H 2 :CO 2 (1:1) in Trypticase Soy Broth (TSB) (Becton Dickinson Microbiology Systems, Cockeysville, Md) supplemented with 4% fetal bovine serum (Gibco Laboratories, Grand Island, N.Y.). The growth conditions were derived from Kinyon, et al . (Vet . Record, 95:219, 1974). Antisera. Rabbit serum prepared against whole, formalin inactivated T. hyodysenteriae B204 (R anti-B204)
  • Plasmid and ⁇ enomic library construction The cloning vectors pSY908, pSY909, and pSY910 were constructed by replacing the Bglll-Nrul fragments of pAR2156, pAR2106, and pAR2113 (Rosenberg, et al . , Gene, 56:125, 1987), respectively, with the Clal-BamHI fragment of pCQV2 (Queen, C, J. Mol . and Appl . Gen. , 2:1, 1983), following Klenow treatment of the Clal end.
  • T. hyodysenteriae chromosomal DNA was prepared by first pelleting 500 ml cultures followed by resuspension in 60 ml of 50 mM Tris-HCL pH 7.5, 10mM NaCl, 10 mM EDTA, 0.5% SDS. Proteinase K was added to 50 ug/ml and incubated at 50°C for 2.5 hr. Phenol and chloroform extractions were performed (Maniatis, et al . , Molecular Cloning, Col Spring Harbor, 1982), before adding CsCl to 1 gram/ml.
  • Ethidium bromide was added to 100 ug/ml and the DNA purified by equilibrium centrifugation at 150,000 x g for 48 hr at 20°C in a L8-70M ultracentrifuge (Beckman Instruments, Inc., Fullerton, CA) using a Ti 50.2 rotor (Beckman). The DNA was removed from the gradient after visualizing with short-wave UV, butanol extracted, and dialyzed against Tris-EDTA buffer.
  • a lambda gtll library was constructed by first partially digesting the T. hyodysenteriae chromosomal DNA with
  • the fragments were filled-in with Klenow fragment, methylated with Eco RI methylase, and linkered using an equal mixture of 8-, 10-, and 12-mer Eco RI linkers.
  • the linkered DNA fragments were ligated using T4 DNA ligase into lambda gtll which had been digested with Eco RI and calf intestinal phosphatase.
  • the ligated DNA was packaged in vitro using Gigapack (Stratagene, La Jolla, CA).
  • Plasmid libraries were constructed by first shearing the chromosomal DNA using a Tekmar Sonic Disrupter (Tekmar
  • Fragments of 0.5-5 kb were gel purified and the ends were repaired to ensure that the ends were ligatable. In doing this, the sonicated fragments were treated with T4 DNA polymerase in 33 mM Tris-acetate pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT, 100 ug/ml
  • Klenow was added and incubated 12 hr at 16°C.
  • these fragments were then cloned directly into pUC13 which had been digested with Sma I and calf intestinal phosphatase.
  • the end-repaired sonicated fragments were methylated with BamHI methylase and ligated to BamHI linkers.
  • the fragments were then ligated into an equal mixture of the plasmids pSY908, pSY909, and pSY910 which had been digested with BamHI and calf intestinal phosphatase.
  • the ligations for both plasmid libraries were used to transform frozen competent cells of Escherichia coli DH5 alpha (Bethesda Research Laboratories, Inc.).
  • the lambda gt11 library was screened using the method of Huynh, et al . (in DNA Cloning: A Practical Approach, D.M. Glover, (ed) 1:49, 1985).
  • the plasmid libraries were screened using a chloroform vapor method (Helfman, et al . , BRL Focus, 6:1, 1984), to lyse bacterial colonies which had been lifted onto nitrocellulose.
  • the colonies were incubated at 42°C for 2 hr before lysis in order to induce the lambda Pr promoter (Queen, C., J. Mol . and Appl . Gen . , 2:1, 1983).
  • the filters were incubated 1-12 hr with serum which was diluted 1:200 in Tris-buffered saline plus 5% nonfat dry milk. The filters were then washed 4x15 min with Tris-buffered saline, followed by a 1 hr incubation with [ 125 I]-labelled staphylococcal protein A (Dupont NEN Research Products, Boston, Mass.) diluted in Tris-buffered saline plus 5% nonfat dry milk. The filters were then washed again as above and autoradiographed using Kodak X- AR5 film.
  • the immunoassay was as described above for screening the library.
  • an enzymatic detection method was sometimes used to detect the primary antibody.
  • the filters were incubated for 1 hr with a 1:1000 dilution of a protein A-horseradish peroxidase conjugate (Boehringer Mannheim Biochemicals), or a specific second antibody (goat anti-mouse IgG or goat anti-swine IgG) also conjugated to horseradish peroxidase (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD).
  • the filters were then washed with Tris-buffered saline and the color was developed 4-chloro-l-napthol.
  • antigen-specific antibodies eluted from nitrocellulose filters were sometimes used as primary antibodies in the immunoblots. These specific antibodies were affinity-purified by the method of Beall and Mitchell (Beall, J.A., et al . , J. Immunol . Method. , 86:217, 1986), using 100 mM glycine in 150 mM NaCl (pH 2.6) to elute the antibodies.
  • Triton X-114 detergent phase fractionation was used to determine the integral membrane properties of the cloned and native T. hyodysenteriae antigens. Cultures of E. coli or T. hyodysenteriae were centrifuged, the pelleted organisms washed in PBS (pH 7.2), and repelleted. The cells were then resuspended in 10 mM Tris-HCl pH 7.5 and lysed by ultrasonic treatment with a Tekmar ultrasonic disrupter (3x30 sec, pulse mode, 90% duty cycle, 40% output).
  • the lysates were cleared by spinning at 15,000 x g for 30 min at 4oC in a Sorvall RC- 5B centrifuge (Dupont Sorvall, Wilmington, Del.) using an H-B4 swinging bucket rotor (Dupont Sorvall).
  • the cleared supernatants were fractionated using Triton X-114 (Sigma Chemical Co.) into an aqueous (AQ) and detergent (TX) phase by the method of Bordier [J. Biol . Chem. , 256:1604, 1981).
  • the fractions were analyzed for the presence of specific antigens by SDS-PAGE and immunoblot.
  • T. hyodysenteriae cell membranes were also fractionated using n-lauryl sarcosine (sarcosine) (Sigma Chemical Co.). Cultures of T. hyodysenteriae were pelleted, washed, and lysed ultrasonically as above. The membranes were then separated from the nonmembrane fraction (NM) by ultracentrifugation at 160,000 x g for 1 hr at 4°C using a Ty 65 rotor (Beckman). The membrane fraction was washed with Tris buffer and resuspended in 10 mM Tris-HCl (pH 7.5) containing 1.5% sarcosine at a protein concentration of 1 mg/ml.
  • NM nonmembrane fraction
  • the sarcosine-insoluble fraction (SI) was pelleted by ultracentrifugation as above at 20°C.
  • the sarcosinesoluble supernatant fraction (SS) was precipitated with either 9 volumes of acetone or 1-2 volumes of 2-propanol with 10 mM magnesium chloride.
  • the cartridges were pre-wet with 80% acetoni trile, 0.1% trifluoroacetic acid (TFA) and equilibrated with 0.025% TFA.
  • the nonmembrane (NM) fraction of clone 101 (in 0.025% TFA) was absorbed and fractions were eluted using a step gradient of acetonitrile in 0.1% TFA (20%, 40%, 60% and 80% acetonitrile). The eluted fractions were lyophilized and resuspended in PBS. The 80% fraction was most enriched for the cloned antigen.
  • clone 101 antigen was accomplished by the addition of ammonium sulfate to the nonmembrane fraction (NM) of clone 101 to a final concentration of 45% (wt/vol) followed by stirring at 0oC for 1 hr to precipitate the proteins.
  • the precipitated proteins were isolated by centrifugation at 15,000 x g for 30 min at 4 oC using an HB-4 rotor and dialyzed against 10 mM Tris- HCl pH 7.5. The material was then separated by SDS-PAGE and transferred to Immobilon PVDF transfer membrane (Integrated Separation Systems, Hyde Park, Mass.) according to the manufacturer.
  • the 25 kd endoflagellar antigen was then eluted from the membrane with 50 mM Tris- HCl pH 9.0 containing 2% SDS and 1% Triton X-100 (Szewzyk, B. and D.F. Summers, Anal. Biochem. , 168:48, 1988).
  • T. hyodysenteriae DNA Sequencing of cloned T. hyodysenteriae DNA.
  • the sequence of the T. hyodysenteriae DNA in clone 101 is determined directly from the plasmid double-stranded DNA, or following subcloning into the single-stranded phage vectors mpl8 and mpl9.
  • Synthetic universal primers compatible with both types of vectors are obtained commercially (New England Biolabs), or custom made on a DNA synthesizer (Applied Biosystems International, Foster City, Ca.).
  • a Sequenase kit (United States Biochemicals, Cleveland, Ohio) is used in conjunction with [ 35 S]-deoxyadenosine 5'- (alpha-thio)-triphosphate (Dupont NEN Research Products) as a label, and the reactions run on 6 or 8% polyacrylamide gels containing 8M urea (Sanger, F. and A.R. Coulson, J. Mol . Biol . , 94:441, 1975). Immunization and challenge of mice. The protein concentrations of antigen preparations to be tested were determined by the method of Lowry, et al . (J. Biol . Chem. , 193:265. 1951). The mouse challenge studies were performed as described by Joens, et al .
  • mice Three-week-old female CF-1 mice (Charles River Labs, Inc.) were immunized intraperitoneally (i.p.) on days 1 and 14. Antigen preparations were emulsified with Emulsigen Plus adjuvant (Modern Veterinary Products, Ralston, Neb.) using a Virtus 23 homogenizer (American Scientific Products, McGaw Park, IL) with final adjuvant concentration of 40%. The mice were not fed for 12 hours before the first of two consecutive intragastric challenges on days 21 and 22 with 1 ml of a log phase culture of T. hyodysenteriae . The mice were necropsied 10 days later to evaluate signs of T.
  • Emulsigen Plus adjuvant Modern Veterinary Products, Ralston, Neb.
  • Virtus 23 homogenizer American Scientific Products, McGaw Park, IL
  • Cecal swabs were streaked on TSB plates containing 5% citrated sheep blood (Becton Dickinson Microbiology Systems) and 400 ug/ml spectinomycin. The plates were incubated at 42°C for two days under H 2 :CO 2 (1:1). Shedding was determined by the degree of beta- hemolysis seen on the plates.
  • the serum bactericidal assays were performed according to the method of Joens and Nuessen (Joens, L.A., et al . , Infect . Immun . , 51:282, 1986) using normal swine serum as a complement source.
  • the antigenic clones that were isolated from each library and the serum that was used to isolate each are shown in Table 1. Most of the clones isolated did not require the vector transcription or translation initiation signals for expression of their antigens. The antigens were apparently expressed in E. coli by the T. hyodysenteriae expression signals. Although antigenic clones were readily obtained from the lambda gt11 library, the ease of subsequent manipulations with the plasmid clones made the plasmid library approach more desirable. The antigenic clones shown in Table 1 were plaque-or colony-purified for further analysis of their antigens.
  • the antigens expressed by selected clones listed in Table 1 were more extensively characterized based on their reactivity with either the CS anti-B204, or the M anti- B204 SI sera.
  • the molecular weights, serum reactivities, and integral membrane properties of these cloned antigens were determined.
  • the molecular weights, integral membrane properties, and apparent cellular locations of the corresponding native T. hyodysenteriae antigens were determined using antibodies specific to the cloned antigens. These antibodies were generated either by affinity-purification from immunoblots containing the cloned antigen, or by immunization of mice.
  • CM cytoplasmic membrane
  • P periplasmic
  • EF endoflagellar
  • Tx detergent phase integrated membrane
  • Aq aqueous phase
  • the first class of clones expressed polypeptides corresponding to cytoplasmic membrane proteins of T. hyodysenteriae .
  • Antibodies specific to these cloned antigens reacted in immunoblots to proteins in the sarcosinesoluble (SS) membrane fraction of T. hyodysenteriae .
  • Cytoplasmic membrane proteins of gram-negative bacteria are selectively solubilized by sarcosine (Filip, C, et al . , J. Bacteriol . , 115:717, 1973).
  • both the cloned and native T. hyodysenteriae antigens were also shown to be integral membrane proteins based on Triton X- 114 detergent phase fractionation (Table 2).
  • the second class represented by clones 56, 86, and 89, expressed polypeptides which corresponded serologically to T. hyodysenteriae antigens found in the nonmembrane fraction of T. hyodysenteriae . It is thought that these clones may encode periplasmic proteins of T. hyodysenteriae, particularly since one of the clones, 56, accumulated a hydrophilic polypeptide in the periplasmic space of E. coli that was quantitatively released by osmotic shock. A cytoplasmic cellular location is unlikely because the sera used to isolate these clones were raised against antigens of T. hyodysenteriae which were extracted by a method that did not lyse the cells.
  • the antigens expressed in E. coli were tested for their ability to protect mice against challenge with virulent T. hyodysenteriae.
  • the CF-1 mouse provides a convenient and reproducible model for T. hyodysenteriae infection and immunity (Joens, L.A., et al . , Infect . Immun . , 25:757, 1979).
  • CF-1 mice develop cecal lesions following oral challenge with virulent T. hyodysenteriae . Since these mice can be protected from lesions caused by T. hyodysenteriae by intraperitoneal (i.p.) injection of conventional T. hyodysenteriae crude preparations, immunogenic clones were screened using the CF-1 mouse model.
  • mice with positive cecal swabs at the time of c necropsy/total number of mice in group b Number of mice with positive cecal swabs at the time of c necropsy/total number of mice in group.
  • the in vitro results provided an impetus to test a more purified preparation of the clone 101 endoflagellar antigen in the in vivo mouse model.
  • the 25 kd antigen expressed by clone 101 was partially purified from E. coli by chromatography. The purification was monitored by SDS-PAGE, as shown in Fig. 2. In contrast to the crude extract, i.p. injection of the partially-purified clone 101 antigen completely protected mice (P ⁇ 0.01) against oral challenge with T. hyodysenteriae B204 (Table 3, study 2). In addition, the cloned endoflagellar antigen induced serum bactericidal activity in these mice before challenge (>99% killing). There was no detectable shedding of T. hyodysenteriae at the time of necropsy (P ⁇ 0.01), as indicated by the lack of visible hemolysis on blood agar plates. Therefore, like the SI fraction of T. hyodysenteriae, the clone 101 antigen not only prevented cecal lesions, but also any detectable colonization by T. hyodysenteriae .
  • Study 4 examined the ability of the partially-purified clone 101 endoflagellar antigen, which was cloned from T. hyodysenteriae strain B204, to protect mice against challenge with a heterologous serotype of T. hyodysenteriae, such as strain B234. Since the clone 101 antigen apparently corresponds to endoflagellar proteins which appear to be conserved, it was believed that it would protect mice against challenge with more than one serotype. The results from study 4 (Table 3) show that this was the case. The clone 101 antigen was effective in protecting mice against challenge by the B234 strain of T. hyodysenteriae (P ⁇ 0.05). In addition, prechallenge serum from mice injected with the clone 101 antigen killed the heterologous strain B234 in vitro (>99.9% killing).
  • the 25 kd product of clone 101 as expressed in E. coli migrates faster on SDS-PAGE than the smallest T. hyodysenteriae protein of 32 kd recognized by the anti-clone serum.
  • Two possible explanations are that the cloned gene may be truncated, or the gene product may be proteolytically processed in E. coli .
  • the immunogenicity of the endoflagellar proteins of T. hyodysenteriae is demonstrated by the ability of the cloned endoflagellar antigen to protect mice against oral challenge with live, infectious T. hyodysenteriae .
  • purified endoflagella of T. phagedenis biotype Reiter do not protect rabbits against challenge with T. pallidum (Hindersson, P., et al . , Sex. Transm. Dis . , 12:124, 1985), even though rabbit serum to the T. phagedenis biotype Reiter endoflagella kills T. pallidum in vitro (Cunningham, T.M., J. Bacteriol . , 170:5789, 1988).
  • T. hyodysenteriae endoflagellar proteins not all epitopes of the T. hyodysenteriae endoflagellar proteins appear to be targets for bactericidal antibodies.
  • the heterologous R anti-TpREF serum which recognized the same T. hyodysenteriae endoflagellar proteins as the bactericidal anti-clone 101 antigen serum (Fig. 3) did not kill T. hyodysenteriae in vitro (data not shown). Therefore, the endoflagellar proteins of the human and porcine treponemes appear to have shared and unique epitopes, the latter of which give rise to and are the targets of bactericidal antibody.
  • T. hyodysenteriae the endoflagellar proteins are encoded by related, clustered genes.
  • the T. hyodysenteriae endoflagella are comprised of multiple protein bands when electrophoretically separated by SDS- PAGE (Miller, D.P., et al . , Am. J. Vet . Res. , 49:786, 1988).
  • SDS- PAGE SDS- PAGE
  • three endoflagellar proteins of apparent molecular weights 37 kd, 34 kd, and 32 kd were serologically cross-reactive as determined by immunoblotting with band-specific antibodies.
  • phagedenis biotype Reiter each contain two classes of endoflagellar proteins which may comprise different structural parts of the endoflagella, such as the sheath and core. Within each class, the proteins are serologically cross-reactive and have similar N-terminal sequences. However, because of the lack of complete sequence identity, it has been proposed that these related proteins are encoded by separate structural genes of a gene family (Norris, S.J., et al . , J. Bacteriol . , 170:4072. 1988). This is further supported by the fact that flagellin gene families have been confirmed in other bacterial species.
  • the 2.2kb T. hyodysenteriae expressing insert was sequenced directly from the pUC13 subclone as described above.
  • the DNA sequence of the 2.2kb Hindlll fragment revealed 2 large open reading frames (ORF) that are oriented in opposing directions.
  • the smaller ORF sequence 600bp is contained within a 1.4kb Sspl to Hindlll fragment, that by the methods described above expresses the T. hyodysenteriae endoflagellin specific material.
  • the small ORF of clone 101 encodes the endoflagellin gene.
  • the DNA sequence of the smaller T. hyodysenteriae ORF revealed that the polypeptide actually expressed in E. coli was in fact using the promoter and regulatory sequences of the lacZ gene of the pUC13 expression vector.
  • the antigenic T. hyodysenteriae material that was expressed in E. coli had 15 amino acids of lacZ fused to its amino terminus.
  • Clone 103 was then sequenced in the 5' direction towards the start of the gene by using a synthetic DNA primer derived from the clone 101 sequence at a position 45 bases 3' of the junction overlap of clone 103 and 101. Clone 103 DNA sequence was obtained for 546 bases upstream of the overlap junction with clone 101. This sequence contained an ORF that was coherent with the ORF of clone 101.
  • the DNA sequence for this T. hyodysenteriae endoflagellin encoding ORF is shown in Figure 5 along with the corresponding restriction endonuclease cleavage sites.
  • Figure 5 also gives the deduced primary amino acid sequence for the T. hyodysenteriae endoflagellin polypeptide encoded by the recombinant clone 103.
  • the calculated molecular weight of the protein is 36 Kd. This molecular weight is consistent with the antigenic properties ascribed to clone 101 (see. Table 2) where clone 101 specific antibodies were found to cross react with polypeptides from cultured T. hyodysenteria having molecular weights of approximately 37kD, 34kD and 32kD.
  • Each peptide was chemically synthesized on an Applied Biosysterns Inc. (Foster City, CA) model 430A peptide synthesizer essentially as described by R.B. Merrifield (J. Amer. Chemical Soc. , 85:2149, 1963) using T-boc (t- butyloxycarbonyl) chemistry with acetic anhydride capping after the addition of each amino acid. Peptides were cleaved from the support resin using hydrogen fluoride as recommended by the manufacturer. The purity of each peptide was monitored by performing HPLC analysis on C- 18 reverse-phase columns. Each peptide was judged to be greater than 90% pure.
  • the clone 103 specific peptides were initially used for generating endoflagellar specific antibodies in mice. For this purpose, approximately 20 ⁇ moles of each peptide was coupled to about 10 mg of Keyhole Limpet Hemocyanin (KLH) using glutaraldehyde as the cross-linking reagent (Kagen and Glick, Methods of Radioimmunoassay, Jaffe & Behrman, Eds. Academic Press, 328, 1979) to increase the inherent antigenicity of the peptide.
  • KLH Keyhole Limpet Hemocyanin
  • a mixture of the KLH conjugated peptide (about 20 ⁇ g) and unconjugated peptide (about 10 ⁇ g) were emulsified in 30% Emulsigen Plus (Modern Veterinary Products) prior to immunizing mice.
  • 28 day- old CF-1 mice (Example 3) were injected i.p. with the peptide mixture and then boosted i.p. 14 days later with an identical preparation.
  • Serum samples were obtained by periorbital bleeding 13 and 21 days after the initial injection of peptide antigen. The mice were sacrificed 27 days after injection and their serum was retained for use as described below. Western blot analysis of the day 13 and day 21 sera probed against ppurified T. hyodysenteriae S.I.
  • the serum reactivity of the ⁇ -peptide sera shown in Table 4 shows that 89-08 specific ⁇ -serum reacts strongly to the 3 major components of the S.I. polypeptide array ( ⁇ 37 kD, ⁇ 34 kD, ⁇ 32 kD), but fails to react with the full-length peptide made in E. coli from a derivative of clone 103.
  • Peptide 89-09 ⁇ -serum reacts strongly with the ⁇ 37 kD S.I. polypeptide, and also fails to react with the E. coli produced clone 103 antigen.
  • Peptide 89-09 ⁇ -serum reacts slightly with a higher molecular weight polypeptide ( ⁇ 39 kD) that is typically present at very low levels in purified S.I.
  • This same 89.10 ⁇ -serum reacts strongly with an ⁇ 39 kD polypeptide expressed by the clone 103 like vector in E. coli .
  • mice were injected with the S.I. purified fraction of T. hyodysenteriae B204 essentially as described previously (see PD-9457, pp10).
  • a positive control that typically reduces viability by 4 logs.
  • Peptide specific antiserum was raised by injecting a mixture of KLH coupled and uncoupled peptide i.p. in mice. The mice were boosted and serum was obtained at necropsy (see text).
  • T. hyodysenteriae normally produces an endoflagellar translational product of ⁇ 39 kD that subsequently is post translationally cleaved to generate an ⁇ 37 kD polypeptide that contains the 89.08 and 89.09 determinants.
  • This 37 kD antigen is then further modified to a ⁇ 34 kD and ⁇ 32 kD form that both contain only the 89.08 epitope.
  • the enzyme(s) required for such processing are not present and, consequently, the clone 103 related material is made as the ⁇ 39 kD form that does not become processed.
  • Unprocessed ⁇ 39 kD material can presumably react only with the 89.10 N-terminal probe; the remaining 2 epitopes are made accessible after the N- terminus of 103 is processed.
  • This interpretation of the peptide serological results is consistent with the predicted secondary structure of the antigen which indicates a "classic" prokaryotic signal sequence (peptide 89.10 is contained within this region). Cleavage of the signal sequence would generate forms of the polypeptide that are reactive with 89.08 and 89.09 specific ⁇ -sera.
  • the ⁇ -serum reacted strongly with the E. coli produced 103 antigen, but the only reactivity to S.I.
  • polypeptides was a slight recognition of the 39 kD form of the endoflagellar antigen (Table 4). These results suggest that the 89.10 epitope is a dominant region that suppresses response to the other 2 determinants.
  • the ability of CF-1 mice to immunologically respond to the predicted antigenic determinants of the endoflagellar polypeptide suggest that the peptides can provide protection. This capability was shown using the in vitro killing properties of the mouse serum from ⁇ -89.08 peptide serum. This serum was used because of the predicted peptide's antigenicity and because it is entirely contained within the clone 101 encoded ORF that had previously been shown to protect mice from T. hyodysenteriae B204 challenge (Table 2).
  • Serum from 89.08 vaccinated mice was heat inactivated, mixed with pig complement and then reacted with viable T. hyodysenteriae B204 organism (as described previously). Results show that the ⁇ -89.08 peptide serum inactivated the T. hyodysenteriae at a level that was comparable to the positive control mouse ⁇ -S.I. serum ( ⁇ 4 logs killing relative to control). Attempts to repeat the in vitro killing results with the same 89.08 peptide serum were positive, but sera specific to peptides 89.09 and 89.10 were negative. The in vitro killing ability of the 89.08 peptide ⁇ -serum suggests that the peptide itself can act as an effective vaccine.

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Abstract

L'invention concerne des préparations antigéniques utiles pour induire une réponse immune contre T. hyodysenteriae chez un animal, des compositions immunogéniques, ainsi que des procédés d'immunisation d'un animal afin de permettre la production d'une réaction immune contre T. hyodysenteriae.
PCT/US1990/002908 1989-05-24 1990-05-23 Gene endoflagellaire clone de treponema hyodysenteriae WO1990015132A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0534526A1 (fr) * 1991-09-25 1993-03-31 Duphar International Research B.V Vaccin de treponema hyodysenteriae

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4748019A (en) * 1984-03-02 1988-05-31 National Research Development Corporation Vaccine for swine dysentery
US4868118A (en) * 1986-09-30 1989-09-19 Board Of Regents, The University Of Texas System Cloning and expression of the 47-kilodalton antigen of treponema pallidum

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4748019A (en) * 1984-03-02 1988-05-31 National Research Development Corporation Vaccine for swine dysentery
US4868118A (en) * 1986-09-30 1989-09-19 Board Of Regents, The University Of Texas System Cloning and expression of the 47-kilodalton antigen of treponema pallidum

Non-Patent Citations (3)

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Title
INFECTION AND IMMUNITY, Volume 56, No. 1, issued January 1988, BLANCO et al., "Antigenic and Structural Characterization of Treponema Pallidum (Nichols Strain) Endoflagella", pages 168-175. *
INFECTION AND IMMUNITY, Volume 57, No. 12, issued December 1989, BOYDEN et al., "Cloning and Characterization of Treponema Hyodysenteriae Antigens and Protection in a CF-1 Mouse Model by Immunization with a Cloned Endoflagellar Antigen", pages 3808-3815. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 80, issued January 1983, HELFMAN et al., "Identification of Clones that Encode Chicken Tropomyosin by Direct Immunological Screening of a cDNA Expression Library", pages 31-35. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0534526A1 (fr) * 1991-09-25 1993-03-31 Duphar International Research B.V Vaccin de treponema hyodysenteriae

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