WO1999053099A1 - Proteines recombinees de treponeme pale et leur utilisation pour former un vaccin contre la syphilis - Google Patents

Proteines recombinees de treponeme pale et leur utilisation pour former un vaccin contre la syphilis Download PDF

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WO1999053099A1
WO1999053099A1 PCT/US1999/007886 US9907886W WO9953099A1 WO 1999053099 A1 WO1999053099 A1 WO 1999053099A1 US 9907886 W US9907886 W US 9907886W WO 9953099 A1 WO9953099 A1 WO 9953099A1
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pallidum
seq
dna
msp
vaccine
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PCT/US1999/007886
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English (en)
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Wesley C. Van Voorhis
Sheila A. Lukehart
Glaber A. Centurion-Lara
Caroline E. Stebeck Cameron
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University Of Washington
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Priority to AU35533/99A priority Critical patent/AU3553399A/en
Priority to JP2000543645A priority patent/JP2002511275A/ja
Priority to EP99917401A priority patent/EP1071819A1/fr
Priority to CA002325576A priority patent/CA2325576A1/fr
Publication of WO1999053099A1 publication Critical patent/WO1999053099A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • syphilis is characterized by a painless primary ulcerative lesion called a "chancre" that generally develops at the site of inoculation after sexual contact with an infected person.
  • the chancre is the site of proliferation of the spirochete Treponema pallidum subspecies pallidum (T. p. pallidum), which causes syphilis.
  • the chancre gradually resolves, and weeks to months later a rash characteristic of secondary syphilis usually develops.
  • Syphilis also can be transmitted congenitally.
  • T. p. pallidum establishes a lifelong chronic infection. Approximately 30% of patients in late stages of the disease develop tertiary neurologic, bony, hepatic, or circulatory system manifestations which may occur decades after the primary infection event.
  • Pathogenic members of the genus Treponema include at least, four natural human pathogens and one natural rabbit pathogen. Based in part upon saturation reassociation kinetics assays (Miao, R.M., and A.H. Fieldsteel, J. Bacteriol. 141:427- 429, 1980) three of the human pathogens are currently classified as subspecies of Treponema pallidum. These are Treponema pallidum subspecies pallidum, Treponema pallidum subspecies per pneumonia, and Treponema pallidum subspecies endemicum, which, respectively, cause venereal syphilis, yaws, and bejel.
  • Treponema carateum causes a disease called pinta.
  • Yaws and bejel occur primarily in warm, humid, tropical areas of the world, primarily in children, and are transmitted by direct non-sexual contacT. Like syphilis, these diseases are characterized by primary lesions that heal within days or weeks, followed by a more serious secondary phase that is systemic. Some cases of bejel exhibit tertiary symptoms as well.
  • poorly characterized spirochetes have been isolated in plaque associated with gingivitis and periodontal lesions, and are believed to be etiologic agents of that condition.
  • T pallidum pathogenic varieties of T pallidum, including subspecies pallidum, and endemicum, have remained refractory to being propagated in culture for more than a few passages, a circumstance that has hampered efforts to fully characterize these organisms and their pathology.
  • these bacteria all can be propagated by serial inoculation of rabbit testes.
  • the rabbit provides a good experimental model for treponemal disease, in that rabbits develop primary chancres much like humans and also develop persistent infection in their lymph nodes and central nervous systems (Turner, T.B., and D.H. Hollander, Biology of the Treponematoses, World Health Organization, Geneva, 1957). Rabbits, however, do not manifest secondary or tertiary syphilis.
  • a syphilis vaccine clearly is needed due to a recent upsurgence worldwide in the frequency of occurrence of this disease.
  • the number of reported syphilis cases in the United States increased from 27, 131 to 50,578 (Rolfs, R.T., MMWR 42: 13-19, 1993).
  • syphilis infections appear to increase the risk of acquisition and transmission of human immunodeficiency virus (HIV) (Greenblatt, R.M., et al., AIDS 2:47-50, 1988; Simonsen, J.N., et al., N. Engl. J. Med.
  • T. p. pallidum One of the central paradoxes of syphilis is the induction of a rapid humoral and cellular immune response that is capable of eliminating millions of treponemes from primary syphilitic lesions, but incapable of eradicating the few organisms that remain during latency. Macrophages are believed to be responsible for this rapid clearance of T. pallidum from early lesions, presumably through antibody-mediated treponemal opsonization and subsequent phagocytosis and killing by macrophages (e.g., see Lukehart and Miller, J. Immunol. 121:2014-2024, 1978; Baker-Zander and Lukehart,
  • T. p. pallidum antigens considered as vaccine candidates have been selected simply on the basis of their reactivity with immune rabbit serum (IRS), i.e., the serum of rabbits that are immune to syphilis by virtue of having been previously infected with T. p. pallidum.
  • IFS immune rabbit serum
  • T. p. pallidum is a highly motile spirochete containing an outer membrane, a periplasmic space, a peptidoglycan-cytoplasmic membrane complex, and a protoplasmic cylinder. Proteins associated with the outer membrane are more likely to be exposed to the host immune system, and thus are more likely than other treponemal proteins to elicit an immune response by the infected hosT. However, studies have indicated that T. p. pallidum has about 100-fold fewer trans-membrane proteins than does a typical gram negative bacterium (Radolf, J.D., et al., Proc. Natl. Acad Sci.
  • T. p. pallidum outer membrane proteins a special name, "T. pallidum rare outer membrane proteins," or "TROMPS.”
  • TROMPS include 65-, 31- (basic and acidic pi forms), and 28- kDa proteins that are found in the outer membrane fraction (Blanco, D. R., et al., J. Bacteriol, 176:6088-6099, 1994; Blanco, D. R., et al., Emerg. InfecT. Dis. 3:11- 20, 1997).
  • TROMP no other T.
  • p. pallidum protein has definitively been identified as being located in the outer membrane, nor has any candidate outer membrane protein been shown to induce a protective immune response (Radolf 3D, et al., InfecT. Immun. 56:490-498, 1988; Radolf et al., InfecT. Immun. 56:1825-1828, 1988; Cunningham et al., J. Bacteriol, 170:5789- 5796, 1988;[?]11; Blanco et al., J. Bacteriol. 176:6088-6099, 1994; Cox et al., Molec. MicrobioL, 15:151-1164, 1995; Radolf, J. D., Molec.
  • SEQ ID NO:l encodes a 356 amino acid protein (SEQ ID NO:2) that is a glycerophosphodiester phosphodiesterase (hereafter called "Gpd”), a glycerol metabolizing enzyme previously identified in other bacteria, e.g., Haemophilus influenzae, Escherichia coli, Bacillus subtilis and Borrelia hermsii (Janson, H., et al., InfecT. Immun., 59:119-125, 1991; Munson, R.S., et al., J.
  • Gpd glycerophosphodiester phosphodiesterase
  • the invention provides another protein believed to be associated with the outer membrane, and that has homology with the surface- exposed D15 protein from Haemophilus influenzae (Flack, F.S., et al., Gene, 156:97- 99, 1995), and Oma87 from Pasteurella multocida (Ruffolo and Alder, InfecT. Immun., 64:3161-3167, 1996).
  • This protein is herein referred to as the "D15/Oma87 homologue” and is encoded by the nucleic acid molecule having the sequence set forth in SEQ ID NO:3.
  • the amino acid sequence of the D15/Oma87 homologue is set forth in SEQ ID NO:4.
  • SEQ ID NO:5 sets forth the nucleic acid -6-
  • Msp major outer sheath protein
  • the members of this gene family are divided into several subfamilies, and present within each subfamily are regions that are highly conserved as well as variable regions that are far less conserved. Analysis of their amino acid sequences suggests that many of these molecules are likely to be outer surface exposed. Furthermore, injection of rabbits with several of these proteins has resulted in partial protective immunity of the rabbits upon challenge with a large dose of T. p. pallidum, thus these proteins are useful as vaccine antigens.
  • Mspl SEQ ID NO:7
  • Mspl protein SEQ ID NO:8
  • Msp2 SEQ ID NO:9
  • Msp2 protein SEQ ID NO: 10
  • Msp3 SEQ ID NO: 11
  • Msp3 protein SEQ ID NO: 12
  • Msp4 SEQ ID NO: 13
  • Msp4 protein SEQ ID NO: 14
  • Msp5 SEQ ID NO: 15
  • Msp5 protein SEQ ID NO:16
  • Msp6 SEQ ID NO: 17
  • Msp6 protein SEQ ID NO: 18
  • Msp7 SEQ ID NO: 19
  • amino acid sequence of a highly conserved amino acid motif found within all of the Msp genes of T. p. pallidum is set forth in SEQ ID NO:32.
  • the nucleic acid sequence encoding the conserved amino acid sequence motif disclosed in SEQ ID NO:32 is set forth in SEQ ID NO:33.
  • T. p. permur Msp genes The nucleic acid sequences of cloned T. p. permur Msp genes, and the proteins encoded by the T. p. permur Msp genes, are disclosed in the following sequence listing entries: T. p. permur Msp homologue 1 (SEQ ID NO:34), Msp homologue 1 protein (SEQ JD NO:35); T. p. permur Msp homologue 2 (SEQ ID NO:36), Msp homologue 2 protein (SEQ ID NO:37); T. p. permur Msp homologue 3 (SEQ ID NO:38), Msp homologue 3 protein (SEQ ID NO:39); T. p.
  • Msp homologue 4 (SEQ ID NO:40), Msp homologue 4 protein (SEQ ID NO:41).
  • the amino acid sequence of a highly conserved amino acid motif found within all of the Msp genes of T. p. permur is set forth in SEQ ID NO: 42.
  • SEQ ID NO:43 The nucleic acid sequences of a cloned T. p. pallidum Msp gene (T.P. 1.6) is disclosed in SEQ ID NO:43, and the protein encoded by the nucleic acid sequence disclosed in SEQ ID NO:43 is disclosed in SEQ ID NO:44.
  • SEQ ID NO:45 shows the nucleotide sequence of a subportion of the T.P. 1.6 DNA fragment (SEQ ID NO:43) that was expressed to obtain a polypeptide (SEQ ID NO:46) to be tested for efficacy in eliciting a protective immune response against T. p. pallidum (see Example 10).
  • SEQ ID NO:47 shows a highly conserved motif present in the amino acid sequence of SEQ ID NO.43.
  • This invention relates to isolated nucleic acids, polypeptides and methods that are useful for preparing vaccines to protect against infection by Treponema spp., particularly Treponema pallidum subspecies pallidum, Treponema pallidum subspecies per pneumonia, and Treponema pallidum, subspecies endemicum.
  • the term "isolated” refers to a biological molecule that is separated from its natural milieu, i.e., from the organism or environment in which it is normally presenT.
  • the invention provides isolated polypeptides capable of inducing a protective immunologic response to T. p. pallidum, T. p. permur, and T.
  • polypeptides include those whose amino acid sequences are shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 21, 23, 25, 27, 29, 31, 32, 35, 37, 39, 41, 42, 44 and 46.
  • the invention provides representative examples of nucleic acid molecules capable of encoding these polypeptides in SEQ ID NOS.l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 22, 24, 26, 28, 30, 33, 34, 36, 38, 40, 43 and 45.
  • Isolated polypeptides and nucleic acids according to the invention maybe prepared by use of recombinant DNA techniques, or may be synthesized using widely available technology.
  • the use of recombinant methods to prepare the subject vaccines provides the advantage that the immunogenic components of the vaccines can thus be prepared in substantially purified form free from undesired contaminants.
  • the invention in one aspect, provides isolated nucleic acids capable of encoding the polypeptides whose amino acid sequences are disclosed herein.
  • the invention provides a nucleic acid molecule (SEQ ID NO:l) encoding a newly identified T. p. pallidum protein (SEQ ID NO:2) that has glycerophosphodiester phosphodiesterase activity (Gpd), and functional equivalents thereof.
  • SEQ ID NO:1 a polypeptide encoded by the nucleic acid of (SEQ ID NO: 1), and whose amino acid sequence is shown in (SEQ ID NO:2).
  • the term "functional equivalent,” as used herein, is intended to include all immunogenically active substances capable of evoking an immune response in animals, including humans, to which the equivalent polypeptide or nucleic acid has been administered, wherein the resulting antibody has immunologic reactivity with the indicated polypeptide.
  • equivalents of T. p. pallidum Gpd may include mutant or recombinantly modified forms of the protein, or subportions of the Gpd molecule that retain sufficient epitopic similarity to the native protein (SEQ ID NO: 2) to evoke an antibody response similar to that evoked by the epitope when present in the native protein.
  • the invention further provides nucleic acids (such as that shown in SEQ ID NO:3) that encode a protein that has significant homology both with the D15 protein previously identified in H. influenzae and with the Oma87 protein previously identified in Pasteurella multocida.
  • This T. p. pallidum protein hereafter is referred to as the "D15/Oma87 homologue"
  • D15/Oma87 homologue This T. p. pallidum protein hereafter is referred to as the "D15/Oma87 homologue”
  • SEQ ID NO:4 This T. p. pallidum protein hereafter is referred to as the "D15/Oma87 homologue”
  • SEQ ID NO:5 which encodes a subportion of the amino acid sequence shown in SEQ ID NO:4.
  • the polypeptide encoded by the nucleic acid molecule of SEQ ID NO: 5 encodes the polypeptide of SEQ ID NO:6, which is useful as a vaccine against syphilis.
  • the invention encompasses
  • SEQ ID NOS:7, 9, 11, 13, 15, 17, 19, 22, 24, 26, 28 and 30 depict nucleic acids encoding portions of 12 different I p. pallidum polypeptides (having amino acid sequences set forth in SEQ ID NOS:8, 10, 12, 14, 16, 18, 20, 21, 23, 25, 27, 29 and 31) that have homology with the previously described major sheath protein of T. denticola.
  • T. p. pallidum Msp homologues hereafter are referred to as "T. p. pallidum Msp proteins (or "homologues" or polypeptides)," whether the reference is to the full-length protein, or to a subportion of the protein.
  • the invention therefore provides the polypeptides having the amino acid sequences shown in SEQ ID NOS:8, 10, 12, 14, 16, 18, 20, 21, 23, 25, 27, 29 and 31, and functional equivalents thereof.
  • T. p. pallidum genome project (posted at httpJ/utmmg.med.uth.tmc.edu/treponema/docs/update.html) refers to the Msp genes as "treponemal pallidum repeats” rather than "Msp” genes, and designates them as "TPRA-L".
  • the nomenclature used herein refers instead to Tpr A-L as Msp 1-Msp 12.
  • Msps 1-12 correspond, respectively, to Tgr G, F, E, D, C, B, A, L, K, J, I and H.
  • Tgr G, F, E, D, C, B, A, L, K, J, I and H The full-length open reading frames for these 12 genes, according to the present version of the T. p.
  • Msp 1, 756 amino acids encode proteins of the following sizes: Msp 1, 756 amino acids; Msp 2, 364 amino acids; Msp 3, 762 amino acids; Msp 4, 598 amino acids; Msp 5, 598 amino acids; Msp 6, 644 amino acids; Msp 7 (ORF A), 253 amino acids; Msp 7 (ORF B), 389 amino acids; Msp 8, 443 amino acids, Msp 9, 480 amino acids; Msp 10, 758 amino acids; Msp 11, 609 amino acids; Msp 12, 693 amino acids. All of the T. p.
  • pallidum Msp homologues contain a highly conserved peptide motif encoded by the nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO:33, and whose amino acid sequence is shown in SEQ ID NO.32. In view of its high degree of conservation, this conserved peptide (SEQ ID NO: 32) may be important in eliciting antibodies that will cross-react with all of the T. p. pallidum Msps.
  • the invention further provides the PCR primers shown in Table 1, in which "S” indicates the sense primer, and “AS” indicates the primer binding to the opposite strand, i.e., the antisense primer.
  • Each of the primer pairs in Table 1 can be used to specifically amplify a portion of the T. p. pallidum Msp gene(s) as indicated in the last column of the table.
  • the invention provides a PCR primer pair having the following nucleotide sequences: 5'-ACCAGTCCTTCCTGTGTGGTTAA (sense) (SEQ ID NO:60), and 5'-ACTCCTTGGTTAGATAGGTAGCTC (antisense) (SEQ JD NO:61).
  • This primer pair is useful for amplifying not only one of the Msp genes of T. p. pallidum, i.e., TP 1.6 (SEQ ID NO:43), but also for amplifying a portion of at least four different T. p. permur Msp genes, thus defining four genes in the T. p. per pneumonia genome that are highly related to the T. p.
  • T. p. per pneumonia Msp DNA fragments have the nucleotide sequences shown in SEQ ID NOS:34, 36, 38 and 40, and the predicted amino acid sequences translated from these four amplicons are shown, respectively, in SEQ ID NOS:35, 37, 39 and 41.
  • Three of these amplicons (SEQ ID NOS:36, 38 and 40) contain the same number of nucleotides, but differ somewhat in nucleotide sequence, thus appear to represent fragments from different Msp homologues.
  • the primer pairs shown in Table 1 as well as the primer pair 5'-ACCAGTCCTTCCTGTGTGGTTAA (sense) (SEQ ID NO:60), and 5'-ACTCCTTGGTTAGATAGGTAGCTC (antisense) (SEQ ID NO:61) can be used in accord with this invention to amplify portions of the T. p. pallidum genome.
  • the resulting amplified DNA (amplicons) can be expressed as recombinant proteins in E. coli or another suitable host, and the recombinant proteins thus derived used to formulate vaccines useful for eliciting a protective immune response against syphilis, yaws, bejel, or other treponemal diseases.
  • the primers designated as "Set 1" in Table 1 are useful for amplifying portions of at least three Msp genes found in the genome of T. p. per pneumonia, and three Msp genes in the genome of T. p. endemicum (Example 7).
  • the invention provides two novel methods for identifying T. p. pallidum proteins useful as vaccine candidates.
  • the first of these methods involves the identification of T. p. pallidum proteins that are immunologically reactive with an opsonizing serum against T p. pallidum but that are immunologically unreactive with a non-opsonizing serum (Stebeck et al., FEMS Microbiol. Lett., 154:303-310, 1997).
  • Such proteins are likely to elicit protective immunity, hence are vaccine candidates, i.e., useful for vaccine trials and for eventual inclusion in a vaccine.
  • a suitable host i.e., one susceptible to infection with T. p. pallidum, for their ability to elicit an immune response that is protective against challenge by this organism.
  • Rabbits for example, can provide a suitable host for this purpose. Proteins that prove to be capable of eliciting such an immune response are determined to be vaccine candidates. This method for selecting vaccine candidates can be applied to identify polypeptides capable of eliciting a protective immune response against yaws, bejel, or any other disease caused by a subspecies of T. pallidum that is susceptible to opsonizing antibodies.
  • opsonizing antibodies are known to be involved in treponeme clearance during primary syphilis, thus a vaccine containing antigens capable of eliciting opsonizing antibodies should produce resistance or immunity against infection with T. p. pallidum.
  • the disclosed method for identifying T. p. pallidum proteins that are targets for opsonizing antibody requires the use of both opsonic and non-opsonic antisera.
  • One means of preparing opsonic serum is to use the rabbit model system. To prepare opsonic rabbit serum (ORS) using this system, serum from rabbits infected with T. p.
  • NORS Non-opsonic rabbit serum
  • Plaques that interact with ORS but not with NORS are isolated and the proteins they express are tested to determine whether they are capable of eliciting protective immunity in a susceptible hosT.
  • the application of this method has identified three different T. p. pallidum proteins, the above-described Gpd (four independent clones), the D15/Oma87 homologue, and one member of the T. p. pallidum Msp family. Because of the method by which they were obtained, each of these three proteins appears to be a target for opsonizing antibodies, and all three likely are to be exposed on the surface of T. p. pallidum cells and capable if included in a vaccine of eliciting a protective immune response against syphilis.
  • the invention further provides another method for obtaining vaccine candidates that involves identifying proteins that are expressed by genes that are present in the genome of T. p. pallidum but that are not present in the genome of the closely related treponeme, T. paraluiscuniculi, a pathogen that causes syphilis in rabbits but that does not infect humans.
  • the genes thus isolated are presumed to provide some function that enables T.
  • genes present in T. p. pallidum but absent from T. paraluiscuniculi are considered to be effective as a vaccine for syphilis, because antibodies directed against them are expected to protect against infection by T. p. pallidum.
  • This method is applicable for identifying pathogenicity-related genes present in the genomes of other treponemes that infect humans but not rabbits, e.g., the genomes of T p. permur and T. p. endemicum.
  • Genes identified by either of the aforementioned methods are tested to determine whether their gene products are capable of eliciting in an animal host an immune response that is protective against challenge with T. p. pallidum.
  • This test may be performed by any convenient means, for example, by inoculating rabbits intradermally or intramuscularly according to standard immunologic procedures with the protein being tested, then challenging the rabbit with a dose of T. p. pallidum that is capable of causing syphilis in an uninoculated rabbiT.
  • RDA representation difference analysis
  • SEQ ID NO:44 The protein encoded by the nucleotide sequence shown in SEQ ID NO:43 is set out in SEQ ID NO:44. Both are included within the scope of this invention. Sequence analysis of TP 1.6 (SEQ ID NO:44) indicated that it shared a significant degree of homology with Mspl (SEQ ID NO:8) and Msp2 (SEQ ID NO: 10) of the -14-
  • T. p. pallidum Msp gene family T. p. pallidum Msp gene family.
  • Msp 9 SEQ ID NO:25
  • members of the T. p. pallidum Msp family have been identified by two independent methods designed for isolating syphilis vaccine candidates.
  • the subject invention provides a vaccine that includes a physiologically acceptable carrier together with an effective amount of an isolated T. p. pallidum polypeptide capable of inducing a protective immunologic response to T. p. pallidum when administered to a suitable host, the isolated polypeptide being immunologically reactive with an opsonizing serum against T. p. pallidum but immunologically unreactive with a non-opsonizing serum against T. p. pallidum.
  • a rabbit model was used to test the capacity of these newly identified
  • T. p. pallidum proteins to elicit protective immunity against T. p. pallidum because proteins that elicit protective immunity in rabbits are expected to have a similar effect in humans.
  • reactive IgM becomes detectable within days after the appearance of clinical disease, and declines after clearance, while IgG responses rise somewhat later, peak at about the time of clearance, and persist for a long period thereafter at relatively high levels (e.g., see Baker-Zander et al., J. InfecT.
  • T. p. pallidum, Nichols strain, or from human syphilis patients infected with unknown strains both were observed here to contain antibodies against several members of the Msp family, and both exhibited especially high levels of activity against Msp 9 (SEQ ID NO:25) and the D15/Oma87 homologue (SEQ ID NO:4).
  • immune rabbit serum (IRS) was observed to react with Gpd (SEQ ID NO:2).
  • T. p. pallidum proteins to be tested in rabbits for their protective capacity were expressed in E. coli, and the corresponding recombinant molecules were purified and used as immunizing antigens. In all cases, rabbits were immunized three times with 200 ⁇ g of the recombinant antigen. The rabbits were subsequently challenged with 10 3 or 10 5 T. p. pallidum at multiple dermal sites three weeks after the last boost, and lesion development was monitored by comparison to a control group of rabbits that had received no immunization prior to challenge.
  • Typical red, indurated ulcerating lesions appeared in the control unimmunized animals at days 5-7 post-challenge in animals that had received 10 5 treponemes, or at days 12 to 14 post-challenge for animals that had received 10 3 treponemes (Gpd challengers).
  • the rabbits immunized with four of the Msp proteins were protected from challenge and did not exhibit typical development of progressive lesions at the corresponding time points.
  • the mild lesions that did develop in the immunized rabbits healed very quickly compared to control animals, and T. p. pallidum could not be detected by darkfield analysis in most of these atypical lesions.
  • the term "vaccine” as used herein is understood to refer to a composition capable of evoking a specific immunologic response that enables the recipient to resist or overcome infection when compared with individuals that did not receive the vaccine.
  • the immunization according to the present invention is a process of causing increased or complete resistance to infection with Treponema species.
  • the vaccines of the present invention involve the administration of an immunologically effective amount of one or more of the polypeptides described above, i.e., the entire proteins, or a functional equivalent thereof, in combination with a physiologically acceptable carrier.
  • This carrier may be any carrier or vehicle usually employed in the preparation of vaccines, e.g., a diluent, a suspending agent, an adjuvant, or other similar carrier.
  • the vaccine will include an adjuvant in order to increase the immunogenicity of the vaccine preparation.
  • the adjuvant may be selected from Freund's complete or incomplete adjuvant, aluminum hydroxide, a saponin, a muramyl dipeptide, an immune-stimulating complex (ISCOM) -16-
  • an oil such as vegetable oil, or a mineral oil, though other adjuvants may be used as well.
  • the immunogenicity of the immunogenic protein may be coupled to a macromolecular carrier, usually a non-toxic biologically compatible polysaccharide or protein, e.g., bovine serum albumin.
  • a macromolecular carrier usually a non-toxic biologically compatible polysaccharide or protein, e.g., bovine serum albumin.
  • an effective vaccine optimally will prime the immune response at mucosal surfaces to recognize T. p. pallidum.
  • Strategies that may be used to administer the subject vaccines in order to elicit a mucosal immune response include using E. coli heat labile enterotoxin as an adjuvant, expression of immunogenic antigens by plasmids carried in attenuated Salmonella spp., microsphere or liposome delivery vehicles, ISCOMS, or naked DNA encoding antigenic proteins (Staats et al., Curr. Opin. Immunol, 6:572-583, 1994).
  • DNA vaccines stimulate strong CTL responses, as well as helper T cell and B cell responses. Since CTL are known to be present in syphilis primary and secondary lesions, and since infection with T. p. pallidum itself is known to be associated with the generation of protective immunity, a DNA vaccine thus is a preferred embodiment of the subject vaccine compositions.
  • genes encoding the vaccine polypeptides of the present invention may be inserted into the genome of a non-pathogenic organism to provide a live vaccine for administration of the vaccines of the subject invention.
  • a live vaccine for administration of the vaccines of the subject invention.
  • vaccinia viruses have been employed for this purpose, as well as attenuated Salmonella spp.
  • Efficient vaccines can be prepared by inserting a variety of immunogenic genes into the same live vaccine, thus providing immunity against several different diseases in a single vaccine vehicle, e.g., a vaccine against many different sexually transmitted diseases.
  • a particularly advantageous live vaccine is one that is engineered to express one or more of the subject immunogens on the outer surface of the bacteria expressing the vaccine proteins, thus maximizing the recipient's exposure to the immunogens in an orientation likely to resemble that found in the treponemal pathogen, thereby eliciting an appropriate immune response.
  • the amount of immunogenically effective component used in the vaccine will of course vary, depending on the age and weight of the vaccine recipient, as well as the immunogenicity of the particular vaccine componenT.
  • a suitable dose will be in the range of 1-1000 ⁇ g of each immunogen, and more preferably, 5-500 ⁇ g of each immunogen. -17-
  • the present invention provides vaccines that include the T. p. pallidum glycerophosphodiester phosphodiesterase, D15/Oma87 homologue, and the members of the Msp family, each to be administered alone or in various combinations in amounts sufficient to induce a protective immunologic response to infection by T. p. pallidum in a host animal that is normally susceptible to syphilis.
  • the vaccine of the subject invention may contain one or more of the aforementioned proteins, as well as additional T. p. pallidum proteins identified by the above described methods.
  • the vaccine may include T. p. pallidum glycerophosphodiester phosphodiesterase in combination with one or more of the Msps, or may include in addition the D15/Oma87 homologue.
  • T. p. pallidum glycerophosphodiester phosphodiesterase this may be provided by expressing in a suitable expression vector system a nucleic acid having the nucleotide sequence shown in SEQ ID NO:l.
  • the isolated T. p. pallidum D15/Oma87 homologue may be obtained by expressing in a suitable vector system a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:4.
  • the isolated T. p. pallidum Msp may be derived by expressing in a suitable vector the full-length T. p.
  • pallidum Msp genes as their positions in the genome are now known, or alternatively, may be derived by PCR from the variable portions of the Msp genes, as set out in the Examples below.
  • the variable regions of Msps 1, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 are shown in SEQ ID NOS:7, 11, 13, 15, 17, 19, 22, 24, 26, 28 and 30, respectively, and polypeptides corresponding to these sequences can be obtained by standard recombinant technology, i.e., by expression in a suitable bacterium, yeast, or other expression system.
  • the Msp polypeptide for use in a vaccine of the subject invention may be provided by the nucleic acid molecules shown in SEQ ID NO:43 or SEQ ID NO:45, or their polypeptide products, shown in SEQ ID NO:44 and SEQ ID NO:46, respectively.
  • the vaccine includes several different Msps or may even include all of the Msps.
  • the vaccine includes Msps 2 (SEQ ID NO: 10), 9 (SEQ ID NO:25) and 11 (SEQ ID NO:29).
  • the vaccine may consist of a polypeptide that includes both conserved and variable regions of one or more Msps. For vaccines including the D15/Oma87 homologue, this may be provided by expressing in a suitable host a nucleic acid molecule having the nucleotide sequence as shown in SEQ ID NO:3 or SEQ ID NO:5.
  • the present invention provides vaccines to protect against yaws, which is caused by the treponeme T. p. permur. -18-
  • This vaccine contains an effective amount of at least one isolated Msp capable of inducing a protective immunologic response when administered to a suitable host; and a physiologically acceptable carrier as described above.
  • the yaws vaccine includes one or more Msp homologues derived from the T. p. permur genome, and may be obtained in isolated form by expressing in a suitable vector one of the nucleic acid sequences shown in SEQ ID NOS:34, 36, 38 or 40.
  • Other polypeptides useful for yaws vaccines may be identified by applying the RDA method described in Example 5, wherein T. p. permur DNA is used as tester DNA.
  • polypeptides for a bejel vaccine can be identified by using T. p. endemicum DNA as tester. The efficacy of polypeptides so identified can be tested for their ability to elicit protective immunity by using a rabbit model as described in Example 10 for testing syphilis vaccine candidates.
  • the invention further encompasses vaccines against bejel, the disease caused by Treponema pallidum subspecies endemicum, and pinta, caused by Treponema carateum.
  • T. p. pallidum and T. p. permur both contain Msp genes related to those present in T p. pallidum, by analogy, the closely related T. carateum must also contain Msp genes useful for vaccines, and these can be identified and isolated according to the methods disclosed herein.
  • the invention provides vaccines that provide protective immunity against the T. p. pallidum-xt ⁇ s Q ⁇ treponemes that cause gingivitis and periodontal disease.
  • the Msp genes of the oral pathogen treponemes are amplified using the primers disclosed herein (e.g., the primers of Table 1), and polypeptides expressed from the resulting amplicons are expressed and tested for their capacity to elicit protective immunity in a suitable animal host.
  • the subject invention includes methods of inducing a protective immune response against T. p.
  • any of the aforementioned treponemal vaccines e.g., the polypeptides whose amino acid sequences are shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NOS:8, 10, 12, 14, 16, 18, 20, 21, 23, 25, 27, 29 and 31, or any polypeptide whose coding region is amplifiable by one or more of the primer pairs of Table 1, or the primer pair 5'-ACCAGTCCTTCCTGTGTGGTTAA-3 * (sense) (SEQ ID NO:60) and 5'-ACTCCTTGGTTAGATAGGTAGCTC-3' (antisense) (SEQ ID NO:61), or functional equivalents thereof.
  • the vaccines may be administered by any of the methods well known to those skilled in the art, e.g., by intramuscular, subcutaneous, intraperitoneal, intravenous injection, orally, or -19-
  • the invention further provides a PCR-based method for analyzing a sample of treponemal genomic DNA to determine whether it originated from T p. subspecies pallidum, T. p. subspecies per pneumonia or T. p. subspecies endemicum.
  • DNA is isolated from the treponeme whose identity is at issue, or Chancre DNA is isolated, and this DNA is amplified using the PCR sense primer 5'-ACCAGTCCTTCCTGTGTGGTTAA-3' (SEQ ID NO:60) and antisense primer 5'-ACTCCTTGGTTAGATAGGTAGCTC-3' (SEQ ID NO:61), and the size of the resulting DNA fragments, e.g., by gel electrophoresis, or by some other method. It is determined that the treponemal genomic DNA originated from T. p. pallidum if the size analysis of the restriction products reveals a single DNA fragment having a size of about 1.7 kb, or that the treponemal genomic DNA originated from T.
  • the treponeme DNA is determined to have originated from T. p. subspecies endemicum.
  • this test can be applied to quickly determine whether the patient suffers from syphilis, yaws, or bejel. It is disclosed herein that sufficient variation exists within the Msp gene family among various clinical isolates of I p.
  • the invention provides a method of RFLP analysis for determining whether clinical isolates of T. p. pallidum from different syphilis patients are the same or differenT. This method utilizes PCR to amplify samples of genomic DNA from the clinical isolates, followed by restriction digestion and subsequent length analysis of the resulting DNA fragments.
  • RFLP restriction fragment length polymorphism
  • variable domains of six alleles of the Msp gene family were amplified using the following primers that bind to two short conserved regions that flank a highly variable region within the central portion of several members of the Msp family (see Example 7).
  • the nucleotide sequences of the primers used in this example were 5'-CGACTCACCCTCGAACCA-3' (sense) (SEQ ID NO:48), and 5'-GGTGAGCAGGTGGGTGTAG-3' (antisense) (SEQ ID NO:49).
  • the amplified DNA is digested with one or more restriction endonucleases that recognize a four-base cleavage site, and the resulting restriction fragments are analyzed on a gel.
  • the restriction endonucleases used for differentiating individual isolates of T. p. pallidum are BstUl, Alul, Hhal and Nl ⁇ I, as these enzymes yielded 15 distinct patterns among 18 tested T. p. pallidum strains.
  • the RFLP method described here can be applied to clinical specimens without any need for the technically difficult and expensive isolation of the organism prior to analysis.
  • this method can provide a means for a public health entity to be able to identify a single strain of T. p. pallidum as responsible for a high proportion of incident cases versus the multiple strains causing a background level of syphilis in a community, or to trace the parties involved in spreading clusters of the disease.
  • nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO:45, and the polypeptide it encodes which is shown in SEQ ID NO:46.
  • This polypeptide represents the amino terminal portion of the TP 1.6-encoded polypeptide (SEQ ID NO:44) that is described in Example 5, and the portion of the TP 1.6 polypeptide shown in SEQ ID NO:46 matches a portion of Msp 2 (SEQ ID NO: 10).
  • Msp 2 (SEQ ID NO: 10) lacks a variable region, yet vaccine testing with the polypeptide shown in SEQ ID NO:46 provided protective immunity in rabbits, thus indicating that conserved as well as variable region epitopes of Msp proteins are useful in vaccine compositions.
  • antiserum was prepared from rabbits that had been injected with live infectious T. p. pallidum. Sera were collected at various times following infection, and were pooled.
  • Adsorbed opsonic antiserum Two rabbits infected with T p. pallidum (Nichols strain) for three months were boosted intradermally and intraperitoneally with 2 x 10 8 T pallidum one month prior to blood collection. Sera from the two animals were pooled and shown to have opsonic activity. The antiserum was sequentially adsorbed with the following antigens that do not induce opsonizing antibodies or have been shown not to elicit immune protection against syphilis: T. phagedenis, biotype Reiter (Lukehart, S.A, et al., J.
  • VDRL Venereal Disease Research Laboratory
  • opsonic antiserum opsonic antiserum
  • Non-opsonic antiserum was prepared by immunization of a seronegative rabbit with 6 x 10 7 T. p. pallidum, Nichols strain, that had been heated at 63°C for 1 h, followed by two boosts of 2-8 x 10 7 heat-killed organisms. All immunizations were performed using incomplete Freund's adjuvanT. The resulting antiserum was weakly reactive in the VDRL test, 4+ reactive at 1:1000 dilution in the FTA-ABS test, and non-opsonic in the phagocytosis assay. This antiserum is hereafter termed "non- opsonic antiserum," or "NORS.” -22-
  • Anti-i ⁇ . coli antibodies present in the opsonic and non-opsonic antisera were removed using standard techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989, which is hereby incorporated by reference in its entirety). Briefly, eight nitrocellulose filters were incubated with an E. coli lysate prepared from 50 ml of OD 1.0 bacteria , then air dried. Following blocking of non-specific sites, four of the E. coli lysate-impregnated filters were incubated with the antiserum.
  • T. p. pallidum lysate was subjected to SDS-PAGE and the separated proteins were tested by immunoblot analysis for reactivity with the T. pallidum- specific ORS.
  • Total T. pallidum lysate was separated by SDS-PAGE, immunoblotted onto nitrocellulose, and exposed to ORS that had not yet been adsorbed, to post- adsorption ORS, or to NORS. Results of these analyses indicated that fifteen molecules with approximate molecular masses of 70, 68, 60, 55, 45, 43, 41, 39, 38, 35, 33, 32, 31, 29 and 13 kDa reacted with the adsorbed ORS. Of these fifteen, those with approximate sizes of 68, 43, 41, 39, 38, 35, 31 and 29 kDa exhibited minimal immunoreactivity with the non-opsonic antiserum, thus seemed likely to encode proteins exposed on the surface of J. pallidum.
  • T. p. pallidum in vitro using antiserum from T. p. pallidum- fected rabbits, i.e., IRS as a source of opsonizing antibody (Lukehart and Miller, J. Immunol, 121:2014- 2024, 1978; Baker-Zander and Lukehart, J. InfecT. Dis., 165:69-74, 1992).
  • IRS has been shown to block T. p. pallidum adherence to host cells (Fitzgerald et al., InfecT.
  • antigens exhibiting reactivity with IRS may have additional functional roles in cytoadherence and immune protection.
  • Opsonic antibodies generally recognize bacterial peptidoglycan, lipopolysaccharide, capsular polysaccharides or proteins, and since T. pallidum does not have an accessible peptidoglycan layer nor does it contain either lipopolysaccharide or capsular material, the opsonic targets are likely to be surface-exposed outer membrane proteins.
  • T. p. pallidum genomic DNA was isolated from approximately 10 10 organisms using the QIAamp Tissue Purification Kit (Qiagen, Chatsworth, CA) and a genomic expression library was constructed using the Lambda ZAP ® IJ EcoRI/CIAP cloning kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Briefly, 2 ⁇ g of T.
  • pallidum genomic DNA were partially digested with Tsp509I and DNA fragments in the size range of 0.5 to 4.0 kb were gel-purified using standard techniques (Sambrook et al., 1989).
  • Tsp509I size-selected Tsp509I-digested DNA preparation
  • EcoRI predigested Lambda ZAP II vector arms were ligated to EcoRI predigested Lambda ZAP II vector arms and the ligated DNA was packaged using the Gigapack ll packaging extract (Stratagene).
  • the resulting bacteriophage library had a titer of 4.7 x 10 6 pfu/ml.
  • E. coli XL-1 Blue (Stratagene, La Jolla, CA) was used as the host strain to plate approximately 50,000 plaques (12,500 pfu plate) using established methods (Sambrook et al., 1989). The plates were incubated for 5.5 h at 37°C, overlaid with 10 mM isopropylthiogalactopyranoside (D?TG)-impregnated nitrocellulose filters and incubated for a further 4 h at 37°C. Duplicate lifts were prepared by removing the filters and overlaying the plates with fresh IPTG-impregnated filters prior to a second overnight incubation at 37°C.
  • Immunoreactive plaques were converted to pBluescript SK(-) phagemids by in vivo excision in the E. coli host strains XL-1 Blue and SolR according to the manufacturer's instructions. Both strands of insert DNA were sequenced by a combination of single-stranded and double-stranded DNA sequencing using the Sequenase" Version 2.0 and the Applied Biosystems dye terminator sequencing kits and the ABI 373A DNA sequencer according to the manufacturer's instructions. In all cases both universal sequencing primers and internal primers designed from DNA sequences were used.
  • a Lambda ZAP II T. P. pallidum genomic expression library was constructed and screened in duplicate with the ORS as well as with the NORS. Ten clones were identified that were immunoreactive exclusively with the opsomc antiserum. As discussed in more detail in the examples to follow, nucleotide sequence analysis has been performed for six of these clones.
  • Example 5 Ten clones that specifically reacted with ORS were selected for DNA sequence analysis. Of these, four proved to encode the same protein (see Example 3), while one encoded a putative outer membrane protein (see Example 4), and the remaining positive encoded one member of a 12-member gene family (see Example 5). Nucleotide sequences were analyzed using the SeqApp 8 software (Gilbert, D.G. (1992) SeqApp", which is published electronically on the Internet, and which is available via anonymous ftp from ftp.bio.indiana.edu. IUBio archive of molecular and general biology software and data). Database searches were performed using the basic local alignment search tool (BLAST) algorithm (Altschul et al., J. Mol. Biol, 215:4673-4680, 1990) and either the BLASTN, BLASTX or BLASTP programs. Alignments of the protein sequences encoded by the clones were performed using the Clustal W general purpose multiple alignment program -25-
  • Example 2 The ten ORS-specific plaques described in Example 2 were subjected to tertiary screening to obtain well-isolated plaques and to verify positivity. Analysis of one of these plaques has been reported previously in Stebeck et al., FEMS Microbiol Letters, 154:303-310, 1997, which is hereby incorporated by reference in its entirety. In vivo excision of the plaque described in Stebeck et al., 1997, produced a pBluescript phagemid containing a 3.5 kb inserT. Nucleotide sequence analysis of the 3.5 kb insert revealed a 1071 bp open reading frame (SEQ ID NOT) encoding a 356 amino acid translated product (SEQ ID NO:2).
  • Sequence analysis of three more of the ten positive plaques described in Example 2 revealed nucleotide sequences encoding this same 41 kDa protein.
  • the polypeptide shown in SEQ JD NO:2 has a predicted isoelectric point at pH 9.13 and a predicted molecular mass of 41,014 kDa.
  • Putative -35 (TGCACG) and -10 (TATAA) promoter regions and a ribosome binding site (GAGGAG) were noted in the nucleotide sequence encoding this protein, upstream from the ATG initiation codon.
  • the 41 kDa protein of SEQ ID NO:2 contains a two- amino acid signal peptide characteristic of previously identified prokaryotic membrane lipoproteins, including an amino-terminal basic residue, a hydrophobic core and a putative Leu-Val-Ala-Gly-Cys signal peptidase II cleavage site (Hayashi and Wu, J. Bioenerg. Biomembr., 22:451-471, 1990), strongly indicating that this protein itself is a membrane lipoprotein.
  • Another group of investigators using a different gene isolation approach reported the isolation of a gene encoding this same 356 amino acid protein from T. p. pallidum, but reported that the protein was anchored to the periplasmic leaflet rather than being part of the outer membrane. (Shevchenko et al., InfecT. Immun., 65:4179-4189, 1997).
  • Sequence database analysis of the 356 amino acid translated sequence identified glycerophosphodiester phosphodiesterase (Gpd) from a variety of bacterial species as the optimal scoring protein, the closest match being with the Gpd of Haemophilus influenzae.
  • the T. p. pallidum Gpd homologue (SEQ ID NO:2) exhibited about 72.2% sequence similarity with the corresponding H. influenzae protein (Janson et al., InfecT. Immun., 59:119-125, 1991; Munson and Sasaki, J. Bacteriol, 175:4569-4571, 1993), as well as 70.5% amino acid sequence homology with an E.
  • pallidum protein (SEQ ID NO: 2) is within the range of masses reported for Gpds from other bacterial species, and closely matches the 40-kDa T. pallidum immunoreactive antigen identified by Shang et al. using rabbit an -B. hermsii glycerophosphodiester phosphodiesterase antiserum (Shang et al., J. Bacteriol, 179:2238-2246, 1997). Taken together, these results indicated that the 356 amino acid translated sequence (SEQ ID NO:2) is a Gpd encoded by T. p. pallidum.
  • the T. pallidum D15/Oma87 homologue (SEQ ID NO:4) is predicted to have a type I cleavable signal sequence (using rules devised by von Heinje, et al. (Nucleic Acids Res., 14:4683-4690, 1986) and McGeoch, et al. (Virus Res., 3:271-286, 1985).
  • the protein was shown to have an 85% probability of being an outer membrane protein by the pSORT program which takes into account hydrophobic domains and secondary structure (see http://psort.nibb.ac.jp/).
  • the Borrelia burgdorferi homologue of this clone has been identified from the B. burgdorferi genome project (Vugt et al., Nature, 390:580-586, 1997) and has been classified as a probable outer membrane protein.
  • Example 10 As described in Example 10, this protein has been expressed mE. coli and the recombinant protein used to immunize rabbits.
  • Example 5 Identification of a family of T. pallidum major sheath protein homologue
  • RDA representational difference analysis
  • tester-tester hybrid molecules are separated from tester-driver hybrids as follows. Prior to the first hybridization step, short adapter oligonucleotides are ligated to the tester DNA. After the tester DNA has been hybridized with the driver DNA, the adapter sequences are annealed with PCR primers that bind to the protruding adapter sequences, and the tester-tester hybrids are thus selectively amplified.
  • the organisms used were T. p. pallidum,
  • Nichols strain and T. paraluiscuniculi, Cuniculi A strain.
  • the bacteria were extracted from infected rabbit testes in sterile saline, collected in DNAse RNAse-free 1.7 ml microfuge tubes, and spun immediately in a microfuge at 12,000 X G for 30 minutes at 4°C.
  • Bacterial pellets were resuspended in 200 ⁇ l of IX lysis buffer (10 mM Tris pH 8.0, 0.1M EDTA, 0.5% SDS), and DNA was extracted using the Qiagen Kit for genomic DNA extraction (Qiagen Inc., Chatsworth, CA) using the manufacturer's instructions.
  • the DNA was treated with RNAse A.
  • RDA was carried out using the CLONTECH PCR- Select Subtraction Kit (Clontech, Palo Alto, CA) following the manufacturer's protocol beginning from the section describing the restriction digestion step.
  • T. paraluiscuniculi was used as a driver DNA because this relative of T. p. pallidum, unlike its virulent cousin, cannot infect humans. Thus, it was surmised that genes present in T. p. pallidum but absent from T. paraluiscuniculi would be involved in pathogenicity, and would provide likely candidates for vaccine testing.
  • Rabbit genomic DNA was included in the driver to remove any traces of rabbit DNA that co-purified with the bacterial DNA.
  • Adapter 2.5'-GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCG AGGT-3' SEQ IDNO:63
  • tester DNA For the first hybridization, two aliquots of tester DNA (tester- 1 and tester-2) were heat denatured in separate reaction tubes in the presence of an excess of driver and allowed briefly to reanneal. During this time, low abundance DNA fragments that are unique to the tester remained as single-stranded DNA, and common DNA fragments annealed with the driver to form double stranded DNA.
  • both of the first hybridization mixtures were pooled and hybridized again with additional excess denatured driver DNA.
  • This second hybridization step permitted further removal of common sequences, and permitted the single-stranded DNA fragments unique to the tester populations to form hybrids with one another, these latter hybrids including tester-tester duplexes having different adaptors at each end, i.e., tester- l/tester-2 duplexes.
  • the adapter sequences were single-stranded, forming overhangs at each end of the duplex molecules. These overhangs were filled in with DNA polymerase, yielding unique double-stranded molecules having different primer binding sites on their 5' and 3' ends adaptor sequences.
  • PCR primer No. 1 which binds to both adaptors 1 and 2, followed by a nested PCR (nested primer 1, 5'-TCGAGCGGCCGCCCGGGCAGGT (SEQ ID NO:64), and nested primer 2, 5'-AGCGTGGTCGCGGCCGAGGT (SEQ ID NO:65)), to further enrich unique sequences, to reduce the background, and to increase the specificity of the amplification.
  • Secondary PCR products were then cloned directly into the PCR 3.1 T/A cloning vector (Invitrogen, Sorrento, CA), and the cloned inserts subjected to DNA sequence analysis. -30-
  • Double-stranded plasmid DNA was extracted with the Qiagen Plasmid Kit (Qiagen, Chatsworth, CA), and 500 ng of each DNA was used for fully automated sequencing by the dye terminator method (Perkin Elmer, Foster City, CA) according to the manufacturer's instructions but with the addition of 1 ⁇ l of molecular grade dimethylsulfoxide (Sigma, ST. Louis, MO) per reaction, giving a final concentration of 5% vol/vol.
  • Cloned DNAs were sequenced in both directions using the T7 and reverse sequencing primers homologous to plasmid regions flanking the cloned inserts. The cloned inserts were found to range in size from 100 bp to 500 bp. Two clones of particular interest were obtained, clones 3 and 33, each of which was isolated from an independently constructed subtraction library made as described above.
  • the sequences obtained from clones 3 and 33 were used to do Blast searches in the nucleotide and protein databases. No significant homologies were found at the nucleotide sequence level, but the predicted amino acid sequences encoded by both clones indicated that these polypeptides were related to the Msp protein of T. denticola, an oral treponeme associated with periodontal disease (Genbank accession No. U29399). Alignment using the Clustal W program indicated that the inserts of clones 3 and 33 aligned, respectively, with regions near the amino and carboxyl ends of the T. denticola Msp protein. These clones were subsequently used as described below for hybridization with Southern blots of the T. p. pallidum genomic DNA, and to design oligonucleotides for PCR amplification of longer pieces of the T.p. pallidum Msp homologue from which they appeared to be derived.
  • T. p. pallidum To determine the specificity of the cloned unique sequences for T. p. pallidum, as well as their hybridization patterns to digested genomic DNA, approximately 3 ⁇ g each of T. p. pallidum (Nichols strain) and rabbit DNA were digested with Eco Rl, Pstl, and Bam HI, then separated in 1% TBE agarose gels, denatured with 0.5 M NaOH and transferred to Hybond N membrane (Amersham Laboratories, Arlington Heights, IL). The inserts of clones 3 and 33 were labeled as follows to use as hybridization probes.
  • the inserts were PCR amplified from the cloning vectors using the nested primers described above under the same conditions as for the nested PCR during the subtraction experiments, and purified using the Qiaquick PCR Purification Kit (Qiagen, Chattsworth, CA). Fifty ng of the purified amplicons were then labeled by random priming with ⁇ - 32 P using the Random Priming labeling Kit (Boehringer Manheim, Indianapolis, IN) according to the manufacturer's protocol. -31-
  • the labeled inserts of clones 3 and 33 were hybridized under high stringency conditions to the above-described Southern blots. Each probe was allowed to bind the PCR products on a separate filter for 12 hours at 37°C in hybridization solution (50% formamide, 5X SSC, 50 mM NaPO 4 , 1% SDS, 5X Denhardt's solution). The blots were then subjected to stringent washes at 65°C in buffers containing 2X SSPE, 0.1% SDS, and 0.2X SSPE, 0.1% SDS, for 20 minutes each (SSPE: 150 mM NaCl, 10 mM aPO4, 1 mM NaEDTA, p 7.4). Hybridization was detected by autoradiography.
  • primers were designed to amplify that portion of the T. p. pallidum Msp homologue that presumably lay between the two clones.
  • Primers used were the S-3 sense primer corresponding to the 5' end of the insert of clone 3 and having the sequence 5'-ACCAGTCCTTCCTGTGTGGTTAA (SEQ ID NO:66), and the antisense primer As-33, corresponding to the 3' end of the insert of clone 33, and having the sequence 5'-ACTCCTTGGTTAGATAGGTAGCTC (SEQ ID NO:67).
  • a hot start PCR amplification was performed as described above using as templates approximately 1 pg of genomic DNA of T. p. pallidum, Nichols strain.
  • the DNA was amplified in a total volume of 100 ⁇ l per tube, each containing 200 ⁇ M dNTPs, 50 mM TRIS-HCl (pH 9.0 at 200° C), 200 mM ammonium sulfate, 1 ⁇ M each primer and 2.5 units of Taq polymerase (Promega, Madison, WI).
  • MgCl 2 beads (Invitrogen, San Diego California) were added giving a final MgCl 2 concentration of 1.5 mM.
  • the following cycling conditions were used: an initial step of 4 minutes denaturation at 94°C followed by 4O cycles at 94°C for 1 minute, 65°C for 2 minutes, 72°C for 1 minute, and a final elongation step of 10 minutes at 72°C.
  • PCR products were then kept at 4°C and directly cloned into T/A cloning vectors for sequencing and for further analysis on agarose gels.
  • the PCR was repeated several times, and each time yielded one band that proved to contain 1687 bp (TP 1.6)(SEQ ID NO:43).
  • TP 1.6 (SEQ ID NO:43) was found later to have high homology with a newly released T p. pallidum Msp-like sequence in Genbank (TPU88957), and to at least 10 different ORFs that were present in the initial release on June 24, 1997 of the TIGR T. p. pallidum genome project (posted at http://med.uth.tmc.edu rre 70wewa/tpall.html ).
  • the TIGR T. p. pallidum sequence was not annotated, i.e., the locations of open reading frames were not indicated.
  • pallidum sequence at positions 73,979 - 75,665 (based on the August 18, 1997 version) that is 90.21% identical to the sequence of TP 1.6 (SEQ ID NO:43).
  • the aligned sequences contained 55 amino acid mismatches spread throughout the 5' end from amino acid positions 1 through 123. Beyond this point to the 3' end, the identity of both amino acid sequences is 100%.
  • Msp genes are arranged into five regions on the T. p. pallidum chromosome. There are three major subfamilies of Msps as defined by homology of their predicted amino acid sequences. Subfamily I includes Msps 2 (SEQ ID NO: 9), 4 (SEQ JD NO: 13), 5 (SEQ ID NO: 15), and 11 (SEQ ID NO:28), which are highly homologous to one another at their 5' and 3' termini. Msps 4 (SEQ ID NO: 13) and 5 (SEQ JD NO: 15) and 11 (SEQ ID NO:28) have central variable regions of about 600 bp, while Msp 2 (SEQ ID NO:9) lacks any variable region.
  • Msp 4 (SEQ ID NO: 13) and 5 (SEQ ID NO: 15) are identical.
  • Subfamily II includes Msps 1 (SEQ JD NO:7), 3 (SEQ JD NO: 11) and 10 (SEQ JD NO:26), and has larger variable regions of about 1000 bp. This subfamily shares significant homology at the 5' and 3' ends with the Subfamily I.
  • Subfamily HI includes Msps 6 (SEQ JD NO: 17), 7 (SEQ ID NO: 19), 8 (SEQ ID NO:22), 9 (SEQ JD NO:24) and 12 (SEQ JD NO:30), all of whose sequences are comparatively distinct from the two other groups and from one another.
  • Msp 7 (SEQ ID NO: 19) appears to have a premature termination, in that at the termination of ORF A (SEQ JD NO:20), in another reading frame, there is another -33-
  • ORF B SEQ JD NO:21
  • the TP 1.6 sequence (SEQ ID NO:43) was found by comparison to the TIGR Tpr sequences to be a hybrid gene.
  • One Msp gene is predominantly transcribed by T. p. pallidum Nichols strain:
  • T. p. pallidum Nichols that was isolated on days 5, 7, and 15 after infection transcribes predominantly Msp 9 (SEQ JD NO:24) mRNA, as determined by reverse transcriptase PCR (RT-PCR), a procedure that amplifies cDNA synthesized from total
  • RNA including mRNA, found in the bacteria, thus reflecting transcribed genes.
  • a group of oligonucleotide primers were prepared that are specific to the variable regions of Msps 1 (SEQ JD NO:7), 3 (SEQ JD NO: 11), 4 (SEQ JD NO: 13), 5 (SEQ JD NO: 15), 6 (SEQ JD NO: 17), 7 (SEQ ID NO: 19), 8 (SEQ JD NO:22), 9 (SEQ JD NO:24), 10 (SEQ JD NO:26), 11 (SEQ JD NO:28), and 12 (SEQ JD NO:30)(see Table 1), thus providing specific amplification of transcripts of those Msps.
  • T T.
  • RNA extracted from infected rabbit testes RT-PCR analysis of the Msp transcription pattern was conducted beginning at day 5 after infection. At day 5, a strong signal for Msp 9 (SEQ JD NO:24) was evident with a weak signal for Msps 6 (SEQ JD NO: 17) and 11 (SEQ JD NO:28). Transcripts from Msps 1 (SEQ JD NO:7) or 12 (SEQ JD NO:30) mRNA were detected, but signals were weak and variable. After 5 more PCR cycles, signal was discernible for all the Msps, indicating that transcripts from all of them were present, but at relatively low levels.
  • Msp 9 (SEQ JD NO:24) product was not due to an overly efficient Msp 9 (SEQ JD NO: 24) PCR, because when these same primers were used to amplify T. p. pallidum genome DNA, it was found that the primers for Msp 9 (SEQ ID NO:24) were less efficient than the primers for Msp 7 (SEQ JD NO: 19) or 4 (SEQ ID NO: 13) or 5 (SEQ JD NO: 15).
  • the PCR products obtained from the RT-PCR RNA likely reflected mRNA and not contaminating T. p. pallidum genome DNA because the RNA preparation was extensively pre-treated with DNAse before the cDNA synthesis step. Furthermore, omitting reverse transcriptase from the reactions led to no producT. -34-
  • the primers described above for amplification of TP 1.6 were used to amplify a fragment of DNA from the closely related treponeme, T. p. permur, the etiologic agent of yaws.
  • a hot start PCR amplification was performed as described above using as templates approximately 1 pg of genomic DNA of T. p. pallidum, Nichols strain, and T. p. permur, Gauthier strain, using the same cycling conditions described in Example 5 for these primers.
  • the PCR products were then kept at 4°C and directly cloned into T/A cloning vectors for sequencing and for further analysis on agarose gels.
  • T. p. pallidum DNA fragment (TP 1.6)(SEQ JD NO:43) has 1687 bp, thus predicting a peptide sequence of 562 amino acids (frame +1).
  • the long homologue of T p. permur (17Ty) had a DNA sequence of 1705 bp, and encodes a putative polypeptide of 568 amino acids (SEQ JD NO:35).
  • the shorter amplicons 13Ty 238, 13Ty5, and 13Ty7 all were 1291 bp long, and predicted polypeptides having the same length, 438 amino acids, but differing at their carboxyl termini (SEQ JD NOS:37, 39 and 41).
  • the deduced peptide sequences of amplicons identified in both subspecies were aligned, i.e., TP 1.6, 17 Ty and 13 Ty, it was found that the T. p. permur Msp homologue, like those of T. p.
  • the pallidum have highly conserved regions located at the amino and carboxyl terminal ends, separated by a central variable region.
  • the amino terminal conserved regions extend from amino acid positions 1 through 153, the carboxyl terminal conserved regions from positions 444 through 592, and the internal variable region from positions 154 through 443.
  • the central variable portions of the 438 amino acid polypeptides lack the 161 amino acids present at positions 241 through 400 of the 17 Ty polypeptide.
  • T. pallidum The various subspecies of T. pallidum, including the etiologic agents of human syphilis, yaws, and bejel, possess very small and highly-related genomes, yet all are able to produce lifelong infection in untreated patients.
  • the past inability to differentiate subspecies and strains of T. p. pallidum using serologic methods has led some investigators to hypothesize that these pathogens actually are identical, with only environmental factors dictating different clinical manifestations (Hudson, E.H., Treponematosis Perspectives Bull, WHO 32:735-748, 1965). However, this view is contraindicated, e.g., by differences in the pathogenesis of the infections, and by the -36-
  • T. p. pallidum TP 1.6 the proteins predicted from all four of the T. p. permur DNA fragments described above were found to have significant homology to the Msp protein of T. denticola.
  • TmPred program Hofman and Stoffel, A Database of Membrane Spanning Protein Segments, Biochem. Hoppe-Seylor 348, 166.
  • the T. p. pallidum Msp homologue described by another laboratory was similarly analyzed to determine whether it also has a predicted transmembrane topology to the sequences disclosed here.
  • the three transmembrane regions in the proteins encoded by the 1.6 kb clone of T. p. pallidum and the 1.7 kb clone of T. p. permur, and the three in the 1.3 kb homologues of T. p. per pneumonia were found to overlap extensively with the corresponding predicted transmembrane regions of the GenBank Msp homologue.
  • the differences found between the syphilis and yaws Msp proteins are located in the variable, middle portion of the protein, which is relatively hydrophilic, and thus may be exposed to the extracellular environmenT.
  • the pathogenic treponemes are classified based upon the distinct clinical infections they produce, as well as their host specificity and very limited genetic studies.
  • the syphilis and the yaws treponemes have been classified as subspecies of -37-
  • T. pallidum based upon saturation reassociation assays, methods of low sensitivity to detect small differences. All attempts to show species or subspecies-specific signatures had failed until it was recently shown that these two organisms differ in the 5' and 3' untranslated regions of their 15 kDa lipoprotein genes.
  • the 15 kDa lipoprotein gene is neither a protective antigen or a molecule related to differential pathogenesis because the open reading frame is identical in T. p. pallidum and in T p. per pneumonia.
  • immunization of rabbits with recombinant 15 kDa lipoprotein has failed to provide any evidence of protection against virulent challenge.
  • TP 1.6 SEQ JD NO:43
  • Msp 1 and 2 may indicate that homologous recombination may be occurring between two homologues so that the 5' region corresponds to one gene in which the downstream portion has been replaced by the corresponding piece of another gene, creating a hybrid molecule with different antigenic characteristics.
  • PCR was performed using a 100 ⁇ l reaction containing 200 ⁇ M dNTPs, 50 mM TRIS-HCl (pH 9.0 at 20°C), 1.5 mM MgCl 2 , 200 mM N ⁇ SO ⁇ 1 ⁇ M of each primer, and 2.5 units of Taq polymerase (Promega, Madison, WI).
  • the cycling conditions were as follows: denaturation at 94°C for 3 minutes, then 40 cycles of 94°C for 1 minute, 60°C for 1 minute and 72°C for 1 minute.
  • Amplicons were purified away from primer-dimers using the QuiaQuick Kit extraction (Qiagen Inc., Chatsworth, CA), and the purified DNAs were quantitated by spectrophotometry. Restriction digests of amplicons were performed with 10 ⁇ g of purified PCR product from each treponemal strain, according to the manufacturer's instructions (New England Biolabs, Beverly, MA), using the following 13 restriction endonucleases, all of which recognize four base cleavage sites: BstUl, Alul, J ⁇ 509I, Mse ⁇ , Nhe ⁇ , Taq* ⁇ , Hhal, Nldm, Bfal, Rsa ⁇ , Mspl, Mbo ⁇ , and Acil. The resulting DNA fragments were separated by electrophoresis in 2.5% T ⁇ E/ethidium bromide NuSieve agarose gels. PCR amplification was optimized so that no smearing of bands was detected on the gels.
  • each recognized four groups Taq a I, five; Hha I, Tsp 5091, BstU I, six; and Alu I and NLA JJJ, seven groups.
  • Combining the data from these enzyme digests permitted the division of the 18 strains into 15 distinguishable groups, based upon RFLP differences. Further analysis of the restriction patterns of the T. p. pallidum strains showed that digestion with only four individual enzymes, BstUI, Alul, Hhal, and NlaHI, was sufficient to differentiate the 15 groups.
  • Group I comprises the strains Bal 9, Sea 81-8, and Sea 84-2; group TJ, Nichols and Yobs strains, and group HI includes the Bal 2 and Bal 8 strains.
  • the strains in each subgroup do not represent unique geographical areas, year of isolation or tissue tropism.
  • the other 11 T. p. pallidum strains tested showed distinct, specific patterns.
  • Some strains, such as Sea 81-1 and 81-3, were collected in the same city, year, and from the same site in the body, yet showed very different RFLP profiles.
  • the RFLP patterns demonstrate that there is marked heterogeneity in the variable regions of the different strains of T. p. pallidum.
  • Table JU shows the distribution the variability of Msp variable domains amongst the different strains and restriction enzymes tested to date.
  • One of the strains appeared identical to T. p. pallidum Nichols strain, but the other 16 differed from Nichols in their variable domains.
  • these results demonstrate that the variable domains differ in different strains of T. p. pallidum and this may be the basis for the lack of complete protection of infected animals after heterologous strain challenge. Accordingly, a fully effective vaccine may require a combination of several or all of the Msp proteins.
  • the primer pair used to amplify the DNA fragments for these RFLP analyses appears to be useful for identifying Msps from many or perhaps all species of Treponema, including pathogens associated with gingivitis and periodontitis.
  • this primer pair was used with DNA from Treponema denticola (an oral pathogen not reactive with antibodies for the 47 kDa protein of T. p. pallidum; Riviere et al, 199L> or from Treponema phagedenis (not considered a pathogen)
  • bands of about 1 and 0.6 kb were obtained.
  • Example 8 Expression va.E. coli of Recombinant Gpd and D15
  • Methods expected to ultimately obtain expression of the remaining clones will involve minimal bacterial growth times to prevent accumulation of the toxic protein, lowering the growth temperature to 30°C instead of the standard 37°C to prevent bacterial overgrowth, immediate purification of recombinant proteins from recently transformed bacterial constructs rather than purification from previously frozen bacterial construct stock cultures, and additional experimental approaches.
  • T. p. pallidum homologue of D15/Oma 87 (SEQ JD NO:3) was expressed in E. coli with the pRSET expression vector system.
  • the expressed D15 homologue was used to immunize rabbits, as described below in Example 10. Antibodies to this protein are being prepared. -42-
  • the T. p. pallidum Gpd protein (SEQ JD NO:2) was expressed in E. coli BL21 (DE3) pLysS using the pET-3a expression system by inserting the entire coding region of Gpd (SEQ JD NO:l). This yielded a full-length, 41 kDa recombinant protein molecule.
  • T. p. pallidum Gpd SEQ JD NO:2
  • Gpd activity was measured in crude lysates of E. coli that were expressing the recombinant molecule. (Larson et al., J. Biol. Chem., 258, 5428-5432, 1983).
  • a glycerophosphodiester phosphodiesterase functions by hydrolyzing glycerophosphodiesters from phospholipid and triglyceride metabolism to glycerol 3 -phosphate.
  • the assay used here measures the conversion of the substrate glycerophosphocholine, a glycerophosphodiester, to dihydroxyacetone phosphate (DHAP) via glycerol 3 -phosphate with the concomitant reduction of NAD to NADH. This reduction of NAD is followed spectophotometrically by measuring the increase in absorbance at 340 nm.
  • Inclusion bodies containing recombinant T. p. pallidum Gpd (SEQ JD NO:2) were recovered from transformed E. coli and used as an immunogen to generate polyclonal antiserum. This antiserum failed to induce opsonization of T. p. pallidum appreciably compared to nonimmune rabbit serum.
  • Gpd is not involved in opsonization, but alternatively, it may be that Gpd -43-
  • opsonic target antigen is an opsonic target antigen, but that for opsonization to occur addition opsonic target antigens must also be present.
  • a 1:1000 dilution of the rabbit anti-Gpd antiserum was used to develop Western blots containing lysates of T. p. pallidum before and after washing by centrifugation. The washes are know to partially remove the bacterium's outer membrane. Blots were developed with 1:3000 dilution of goat anti-rabbit IgG (peroxidase-conjugated Fab fragment, Amersham), using the chemiluminescence protocol provided by Amersham. An immunoreactive band was observed that had a size of 41 kDa, the approximate molecular weight predicted for Gpd from the open reading frame identified in the cloned DNA.
  • the polyclonal antiserum to Gpd was used in further studies to analyze the surface disposition of Gpd using a previously described immunofluorescence assay (Cox et al., Mol. Microbiol, 15:1151-1164, 1995). Because of the fragility of the T. pallidum outer membrane, special precautions to preserve this membrane were employed (Cox et al., 1995) Briefly, virulent T. pallidum were encapsulated in gel microdroplets to preserve the treponemal molecular architecture prior to immunofluorescence analysis, thus ensuring an accurate cellular localization for Gpd within T. pallidum. Preliminary results using the anti-Gpd antiserum showed uniform surface immunofluorescence on both intact and detergent-treated T.
  • H. influenzae Gpd homologue has been reported to have IgG-binding capability (Janson et al., InfecT. Immun., 59:119-125, 1991; Sasaki and -44-
  • the antibody pairs used were: (i) human IgA followed by goat F(ab') 2 anti-human IgA ( ⁇ -chain specific); (ii) human IgD followed by goat F(ab') 2 anti- human IgD ( ⁇ -chain specific); (iii) human IgG followed by goat F(ab') 2 anti-human IgG ( ⁇ -chain specific); and (iv) human IgM followed by goat F(ab') 2 anti-human IgM ( ⁇ -chain specific).
  • the primary incubation was conducted in the absence of any primary immunoglobulin. As expected, no signal was observed for the control blots.
  • T. pallidum Gpd The IgG binding of T. pallidum Gpd was further characterized by IgG fractionation studies.
  • Fab and Fc fragments of human IgG were prepared by papain digestion, and purified using a standard procedure (Harlow and Lane, Eds., Antibodies: A Laboratory Manual, Cold Spring Harbor, NY, 1988, which is hereby incorporated by reference in its entirety). Immunoblots were incubated with either the Fab or Fc fragment, then developed with horseradish peroxidase/goat anti-human IgG (F(ab')2 fragment) and the Enhanced Chemiluminescense Reagent (Amersham, Cleveland, OH). Results of binding assays with these IgG fragments revealed that the T. p.
  • pallidum Gpd specifically binds the Fc fragment of human IgG with an intensity similar to that observed for intact IgG, while no binding to either the Fab fragment of human IgG or the secondary antibody was detected.
  • Gpd In H. influenzae, the Gpd homologue has been linked to pathogenesis, as Gpd knockout mutants for that organism have been shown to be 100-fold less virulent in animal models (Janson et al., InfecT. Immun., 62:4848-4854, 1994). Similarly, Gpd may be relevant to the pathogenesis of T. pallidum. It has been proposed that the coating of T. pallidum by host IgG is a factor in long-term treponeme survival in the host (Alderete and Baseman, InfecT.
  • Gpd contributes to treponemal evasion of the host immune system
  • introduction of excess high affinity Gpd-specific antibodies through recombinant Gpd vaccination may provide protective immunity to T. p. pallidum infection.
  • the protection afforded by immunization with Gpd was tested in the rabbit syphilis model in two separate experiments. In the first experiment, one rabbit was immunized with inclusion bodies purified from£. coli expressing the pET-3a-Gpd construct emulsified in RLBI® adjuvant prior to intradermal challenge. A control rabbit received no prior immunization and served as a comparison animal for intradermal challenge.
  • test rabbit was immunized intramuscularly, subcutaneously, and intradermally three times at three-week intervals with RJJ3I adjuvant using 200 ⁇ g recombinant Gpd per immunization.
  • immunized and unimmunized control rabbits were challenged intradermally at each of six sites with 10 3 T. pallidum Nichols strain per site.
  • the Gpd immunized rabbit developed atypical pale, flat, slightly-indurated and non-ulcerative lesions within several days of challenge at two out of the six challenge sites, with no lesions observed at the remaining four challenge sites.
  • the control rabbit developed typical red, raised, highly-indurated and ulcerative lesions at five of six challenge sites at 12 to 14 days post-challenge.
  • T p. pallidum infected rabbits anti-D15 antibodies were observed to develop between days 13 and 17, and to peak at about day 30 after infection, after which time the level of anti-D15 activity decreased slightly and plateaued.
  • the appearance of antibodies to the T. p. pallidum D15 corresponds to the appearance of antibodies that opsonize and block cytoadherence of the organism, and to the time of treponemal clearance from the syphilis lesions in these animals.
  • immunization with the D15/Oma87 homologue is likely to elicit protective immunity, especially given that D15 of H.
  • influenzae and the Oma87 protein of Pastuerella multocida are protective against infection by those organisms (Flack et al., Gene, 156:97-99, 1995; Loosmore et al., InfecT. Immun., 65:3161-3167, 1996; Ruffolo and Alder, InfecT. Immun., 64:3161-3167, 1996).
  • D15/Oma87 homologue SEQ JD NO:3
  • SEQ JD NO:3 The amino acid sequence of this portion of the D15/Oma87 homologue is shown in SEQ ID NO: 6.
  • the T. p. pallidum recombinant D15 was purified using Ni-NTA matrices according to the manufacturer's instructions (Qiagen, Valencia, CA).
  • TP 1.6 The 785 bp at the 5' end of TP 1.6 (SEQ JD NO:45), which corresponds to the 5' half of Msp 2 (SEQ JD NO: 9), was expressed with a 6-histidine tag in the pRSET system (Kroll et al., DNA & Cell Biol, 12:441-453, 1993) to yield a polypeptide having 261 amino acids (SEQ JD NO:46).
  • Recombinant protein was purified by nickel chromatography and a rabbit was immunized subcutaneously, intramuscularly and intradermally with RLBI adjuvant and 200 ⁇ g recombinant TP 1.6 protein. Injections were given three times at three-week intervals, as for Gpd immunization.
  • the rabbit that was immunized with the polypeptide corresponding to TP 1.6 was challenged with 10 5 T. p. pallidum, Nichols strain, intradermally in eight sites on the back.
  • a control rabbit that was not immunized was also challenged.
  • the TP 1.6-immunized rabbit developed small, slightly indurated patches which cleared in seven days. These lesions were not typical of syphilis chancres, but rather resembled delayed type hypersensitivity responses.
  • the control rabbit developed red, indurated nodules at the sites of inoculation at 5 days. These persisted and reached a maximum size of 2 cm and ulcerated at approximately 20 days.
  • the VDRL (Venereal Diseases Research Laboratory cardiolipin-antibody test) serology of the TP 1.6-immunized animal remained negative, but the VDRL serology of the control rabbit was positive at a 1:2 dilution.
  • the TP 1.6-immunized animal was sacrificed, and its lymph -48-
  • Treponemes were found on dark field examination, implying that the TP 1.6 immunization was only partially protective.
  • PCR primers were devised to specifically amplify the central variable region present in all of the Msps that contain a variable region. Because some of the Msp variable regions share short stretches of identity even within their variable regions, it was possible to amplify all of the variable regions using primers sets shown in Table 1.
  • the amplified variable region DNAs were prepared from a T. p. pallidum genomic DNA template using the primers in Table 1 to amplify all of the Msps (except for Msp 2), and each of the DNAs thus obtained was expressed inJE. coli, and the recombinant polypeptides recovered in order to test their capacity to induce protective immunity against T. p. pallidum.
  • nucleotide sequences of these amplified DNA fragments are shown in SEQ JD NO:7 and SEQ JD NOS: 11, 13, 15, 17, 19, 22, 24, 26, 28 and 30, and the amino acid sequence of each of the corresponding variable region recombinant polypeptides are shown in SEQ JD NO: 8 and SEQ JD NOS:12, 14, 16, 18, 20, 21, 23, 25, 27, 29 and 31.
  • variable region polypeptides corresponding to Msps 1 (SEQ JD NO:8), 9 (SEQ JD NO:25) and 11 (SEQ ID NO:29) have been used to immunize a single rabbit as described above for the first test conducted with the TP 1.6 amino terminus polypeptide (SEQ JD NO:46).
  • immunization with Msp 9 (SEQ ID NO:25) and Msp 11 (SEQ JD NO:29), but not Msp 1 (SEQ ID NO:8) were found to have conferred protective immunity as compared with controls.
  • Msp 1 (SEQ JD NO: 8) failed to yield positive results in this preliminary trial, it cannot be ruled out that the single rabbit inoculated here with Msp 1 (SEQ ID NO: 8) was unusually susceptible to syphilis, or that Msp 1 (SEQ JD NO: 8) could contribute to immunity if injected in combination with other Msp antigens.
  • antiserum was withdrawn from rabbits immunized as described above with Msp polypeptides 1 (SEQ JD NO:8), 9 (SEQ JD NO:25), 11 -49-
  • TP 1.6 SEQ JD NO:46
  • rabbit macrophages were mixed with the test antiserum, treponemes added, then incubated for 4 hours. At that time, the cells were fixed and stained using an immunofluorescent tag specific for T. p. pallidum. Macrophages containing ingested treponemes were scored by microscopy. All four test antisera were found to have promoted opsonization over negative control serum from unimmunized rabbits. IRS provided a positive control.
  • the 90 percentages of macrophages containing ingested treponemes were: unimmunized control, 16.9%; JRS, 45.3%; Msp 1 antiserum, 67.9%; Msp 9 antiserum, 47.4%; Msp 11 antiserum, 33.5%; and TP 1.6 32.7%. These values are the averages of triplicate plates for each antiserum.
  • pallidum proteins including Tp47, Tp37, Tp34.5, Tp33, Tp30, Tpl7, Tpl5, Tpl90 (4D), Tp44.5 (TmpA), Tp34 (TmpB), Tp37 (TmpC), Tp 29-35 (TpD)
  • Tp terminology refers to MW consensus according to Norris et al., 54), and TROMP1 (Blanco et al., J. Bacteriol. 178:? 199?).
  • the Msp family provides a group of antigens useful for vaccination against syphilis.
  • a vaccine is made whose administration to a suitable animal host confers protective immunity to syphilis, yaws and bejel.
  • a vaccine may include the T. p. pallidum Gpd (SEQ JD NO:2) and D15/Oma87 homologues (SEQ JD NO:4) disclosed above, and may further include Msp genes from pathogenic -50-
  • a vaccine comprising at least one Msp from any one of the three subspecies should confer at least partial protection against infection with either of the other two.
  • Gpd glycerophosphodiester phosphodiesterase
  • the Gpd coding sequence was PCR amplified from genomic DNA isolated from a variety of treponemal strains. All strains were propagated in New Zealand white rabbits as previously described (Lukehart, S. A., S. A. Baker-Zander, and S. Sell. 1980. Characterization of lymphocyte responsiveness in early experimental syphilis. I. In vitro response to mitogens and Treponema pallidum antigens. J. Immunol. 124:454-460). T. pallidum subsp. pallidum, Nichols strain, was originally sent to the University of Washington by James N. Miller (University of California, Los Angeles) in 1979, and T. pallidum subsp.
  • T. pallidum subsp. pallidum, Bal-3, Bal-7 and Bal 73-1 strains; T. paraluiscuniculi, Cuniculi A strain; T. pallidum subsp. permur, Haiti B strain; T. pallidum subsp. endemicum, Iraq B strain; and the Simian isolate were supplied by Paul Hardy (John Hopkins University, Baltimore, MD).
  • T. pallidum subsp. pallidum, Sea 81-3 and Sea 83-1 strains were isolated by Sheila A. Lukehart from the cerebrospinal fluid of untreated syphilis patients.
  • primers were designed from the 5' (5'-TGCACGGTGACGATCTGTGC-3')(SEQ JD NO:70) and 3' (5'-GGTACCAGGCGACACTGAAC-3')(SEQ JD NO:71) non-coding regions flanking the gpd gene (Fraser, C. M.,et al., 1998, Science 281:375-388). These primers are located 48 bp upstream and 51 bp downstream, respectively, of the gpd open reading frame.
  • PCR amplification of the gpd gene was performed using a 100 ⁇ l reaction containing 200 ⁇ M dNTP's, 0.25 ⁇ M of each primer, lx Taq polymerase buffer (50 mM Tris-HCl, pH 9.0 at 20°C, 1.5 mM MgCl2, 20 mM NH4SO4), and 1 ⁇ l of genomic DNA containing 5,000-10,000 treponeme equivalents for each strain.
  • the PCR reaction conditions were 30 cycles of 1 minute denaturation at 94°C, -51-
  • RFLP Restriction fragment length polymorphism
  • Nucleotide accession numbers The nucleotide sequences of the gpd genes from the Nichols, Bal-3, Bal-7, Bal 73-1, Sea 81-3, Sea 83-1, Mexico A, Haiti B, Gauthier, Iraq B, Simian, and Cuniculi A strains have been assigned GenBank accession numbers AF004286 and AF127415-AF127425, respectively, each of which nucleotide sequences, accorded the foregoing GenBank accession numbers, are incorporated herein by reference.
  • Table 2 Summary of Gpd sequence conservation between T. pallidum subsp. pallidum ( " Nichols strain) and various pathogenic treponeme strains.
  • T. paraluiscuniculi (the only different species represented) has 5 additional base pair changes, one of which (base pair 263) results in a conservative -53-
  • the base pair change at position 579 in the non-syphilis strains introduces a Plel restriction site that creates different RFLP patterns between the T. pallidum subsp. pallidum strains and the other human and animal pathogens.
  • Plel digestion of the T. pallidum subsp. pallidum strains generates three restriction fragments of sizes 766, 241 and 163 base pairs.
  • the presence of the additional Plel site in the non- syphilis strains generates four restriction fragments of sizes 635, 241, 163 and 131 base pairs.
  • Gpd Homologues of Gpd from other bacterial species also demonstrate remarkable conservation of amino acid sequence.
  • the corresponding molecule from the relapsing fever spirochete Borrelia hermsii, GlpQ exhibits a range of 96.5% to 100% amino acid sequence similarity among 26 B. hermsii isolates (Schwan, T. G., and S. F. Porcella. Personal communication).
  • results reported here show Gpd is highly conserved among twelve strains that encompass a total of five pathogenic treponemes. The invariant nature of the Gpd, combined with the -54-
  • Tp92 The T pallidum D15/Oma87 homologue protein is referred to as Tp92 in the present example.
  • Tp92 is protective against challenge with T pallidum.
  • the predicted Tp92 amino acid sequence from a variety of different strains of T pallidum is almost identical. This observation suggests that immunization with Tp92 should protect against many strains of syphilis.
  • Tp92 is a target of opsonizing antibodies for T pallidum, and thus Tp92 is likely to be a surface antigen.
  • T. pallidum subsp. pallidum tpa.92 gene was identified using the previously published method of differentially screening a T. pallidum genomic expression library (Stebeck, C.E., et al. FEMS Microbiol. Lett. 154:303-310 (1997)). Briefly, the library was prepared using the Lambda ZAP® JJ cloning kit (Stratagene) according to the manufacturer's instructions. Approximately 200,000 plaques (12,500 pfu/plate) were plated and duplicate lifts prepared and screened using established methods (Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • Filters were differentially screened with a T. pallidum-spec ⁇ c immune rabbit serum depleted of activity against the major known treponemal antigens but still retaining its opsonic capacity (termed opsonic rabbit serum; ORS), and a non-opsonic antiserum prepared using heat-killed T. pallidum (termed non-opsonic rabbit serum; NORS).
  • the ORS was prepared by sequential adsorption of pooled syphilitic rabbit serum with T. phagedenis, biotype Reiter, recombinant T. pallidum 47, 37, 34.5, 33, 30, 17 and 15 kDa molecules (as designated in Table 3 in Norris, S.J.
  • plaques showing consistent differential reactivity were screened a third time with ORS and converted to pBluescript SK(-) phagemids by in vivo excision in the E. coli strains XL-1 Blue and SolR according to the manufacturer's instructions.
  • Double-stranded plasmid DNA was extracted using the Qiagen Plasmid Mini Kit (Qiagen, Chatsworth, CA) and both strands of insert DNA were sequenced using the Applied Biosystems dye terminator sequencing kit (PE Applied Biosystems, Foster City, CA) and the ABI 373A DNA sequencer in accordance with the manufacturer's instructions. In all cases both universal sequencing primers and internal primers designed from the insert sequence were used.
  • DNA and Protein Sequence Analyses Nucleotide sequences were translated and analyzed using the SequencherTM Version 3.1RC4 sequence analysis software (Gene Codes Corporation, Ann Arbor, MI).
  • Tpa92 coding sequence was PCR amplified from genomic DNA isolated from a variety of T. pallidum subspecies and strains. To obtain the entire open reading frame, primers were designed from the 5' (5'-GGGTGTCGTGGAGTTTTGCG- 3'XSEQ ID NO:72) and 3' (5'-CTTGCCTGGTGGACGCAGC-3')(SEQ JD NO:73) non-coding regions flanking the tpa.92 gene. These primers are located 55 bp upstream and 49 bp downstream, respectively, of the tpa.92 open reading frame.
  • PCR amplification of tpa.92 was performed using a 100 ⁇ l reaction containing 200 ⁇ M dNTP's, 0.25 ⁇ M of each primer, lx Taq polymerase buffer (50 mM Tris-HCl, pH 9.0 at 20°C, 1.5 mM MgCl2, 20 mM NH4SO4), and 1 ⁇ l of genomic DNA containing 5,000-10,000 treponeme equivalents for each T. pallidum subspecies and strain.
  • the PCR reaction conditions were as follows: 30 cycles of 1 minute denaturation at 94°C, 1 minute annealing at 60°C, 2 minutes extension at 74°C for T.
  • Tpa92 The open reading frame encoding Tpa92 was PCR amplified from T. pallidum subsp. pallidum (Nichols strain) genomic DNA using primers designed from the 5' (5'-CGGGATCCACAATTGGTACGAGGGAAAGCC- -57-
  • the 2457 bp amplification product was digested with BamHl and Ec ⁇ B , ligated to a similarly digested pRSETc T7 expression vector (Invitrogen, Carlsbad, CA) and transformed first into E. coli XL-1 Blue and then into the E. coli expression strain BL21 (DE3) pLysS.
  • the reading frame and sequence of the expression construct was verified by DNA sequencing using the T7 promoter primer (Pharmacia, Piscataway, NJ) and internal primers designed from the tpa.92 DNA sequence, the Applied Biosystems dye terminator sequencing kit and the ABI 373A DNA sequencer according to the manufacturer's instructions. Expression of the recombinant T.
  • pallidum Tpa92 was performed using 500 ml of LB broth seeded with 50 ml of OD 0.6 E. coli transformed with the Tpa92-pRSETc construct. Cells were grown for 3 hours at 30°C prior to induction of protein expression from the T7 promoter by the addition of 0.4 mM JJPTG and a further 4 hour incubation at 30°C. Cells were harvested by centrifugation, and the histidine-tagged recombinant Tpa92 protein was purified from the bacterial pellet according to the manufacturer's instructions (Invitrogen).
  • Immune rabbit serum was collected from rabbits that had been chronically infected with T. pallidum for >90 days.
  • Anti-Tpa92 polyclonal antiserum was raised in four New Zealand white rabbits (#5061, #5200, #5202, and #5207) by immunizing three times with 100 ⁇ g each of the purified recombinant Tpa92 emulsified in the Ribi adjuvant MPL + TDM + CWS (Monophosphoryl lipid A + Trehalose dicorynomycolate + Cell wall skeleton; Sigma, St. Louis, MO).
  • Immunizations were administered intradermally (JD), subcutaneously (SC), intramuscularly (JM) and intraperitoneally (JJ?) at three week intervals as outlined by the Ribi adjuvant system, and antiserum was collected one week after the final immunization.
  • IRS Opsonization Assay.
  • IRS anti-Tpa92 polyclonal antiserum collected from rabbit #5061, and the corresponding control pre-immune serum were tested in three -58-
  • the level of immunoreactivity of anti-Tpa92 polyclonal antiserum on purified recombinant Tpa92 was assayed by electrophoresis and blotting of 2 ⁇ g of purified recombinant protein, and probing with a 1:200 dilution of anti-Tpa92 polyclonal rabbit serum followed by a 1:3000 dilution of alkaline phosphatase-labeled goat anti- rabbit IgG (Fc; Promega).
  • T. pallidum was extracted from infected testes as previously described (Lukehart, S.A.
  • the immunized rabbits and two unimmunized control rabbits were intradermally challenged at each of eight sites on their shaved backs with 10 ⁇ T. pallidum subsp. pallidum (Nichols strain) per site.
  • the rabbits were examined daily to monitor the development, morphological appearance and progression of lesions appearing at the challenge sites. Lesion development was designated for each individual rabbit as typical if lesions were red, raised, indurated and generally progressed to ulceration, and atypical if lesions were pale, flat, only slightly indurated and generally non-ulcerative.
  • a Lambda ZAP JJ T. pallidum subsp. pallidum genomic expression library was constructed and screened with a T. pallidum-spe ⁇ Rc, antigen-adsorbed opsonic antiserum preparation.
  • T. pallidum-spe ⁇ Rc antigen-adsorbed opsonic antiserum preparation.
  • immunoreactivity against known T. pallidum antigens had been adsorbed from this preparation, although the opsonic capability of the antiserum was retained as demonstrated by phagocytosis assays (data not shown).
  • duplicate plaque lifts were differentially screened with a T. pallidum-spe ⁇ Rc non-opsonic antiserum. Plaques exhibiting consistent immunoreactivity with the opsonic antiserum but no immunoreactivity with the non-opsonic antiserum on the primary and secondary screens were selected for further study and subjected to tertiary screening to obtain well isolated plaques.
  • Tpa92 T. pallidum antigen, 92 kDa
  • the DNA sequence of Tpa92 is incorporated herein by reference and is available from EMBL/Genbank/DDBJ under accession number AF042789. Sequence Analyses.
  • sequence database analysis using the blastp algorithm revealed the T. pallidum Tpa92 shares the highest degree of sequence similarity with a putative outer membrane protein identified by genome sequencing of the related spirochete, Borrelia burgdorferi (28.1% identical, 44.7% similar; Fraser, CM. et al., Nature 390:580-586 (1997)).
  • the T. pallidum Tpa92 also shares approximately equal levels of sequence similarity with high molecular weight outer membrane proteins identified from a large variety of bacterial species (18.6-22.1% identical, 35.1-40.9% similar). The observed sequence similarity within this group of bacterial proteins is evenly distributed throughout the coding sequence of Tpa92, with the exception of a stretch of serine residues at the C-terminal end of the translated protein that is unique to the T. pallidum Tpa92. The presence of transmembrane segments within Tpa92 was analyzed using the TMPred program, resulting in the prediction of three transmembrane helices (data not shown). In this putative model, the C-terminal serine-rich stretch of Tpa92 is predicted to be located within an external loop on the outer face of the outer membrane.
  • T. pallidum subsp. pallidum strain DNA sequence protein sequence open reading frame size divergence divergence (# of amino acid residues) base pair change residue change
  • T. pallidum subsp. pallidum strain DNA sequence protein sequence open reading frame size - I ⁇ . divergence divergence (# of amino acid residues) O v ⁇ base pair change residue change
  • Table 8 Summary of Tpa92 sequence conservation between T. pallidum subsp. pallidum (Nichols strain) and various VO VO i T. pallidum subspecies and strains. o
  • Tpa92 The amino acid sequence of Tpa92 is highly conserved, with a range of 95.5- 100% identity and 96.8-100% similarity shared between the Nichols Tpa92 sequence and that of the various other T. pallidum strains.
  • several of the amino acid sequence changes that do exist are of particular interest.
  • First, parallel sequence divergence is observed between Bal-2 and Sea 81-3 strains and again with Gauthier and Simian strains, thus suggesting a common strain origin for each of these groups.
  • the tpa.92 genes of the Gauthier and Simian strains have base pairs 2336-2350 deleted (data not shown), which corresponds to deletion of the amino acids that comprise the end of the T. pallidum Tpa92 signature serine stretch, residues 780-784.
  • the tpa92 gene sequence of the Cuniculi A strain possesses an additional complexity, in that base pairs 2293-2352, which encode the characteristic serine stretch comprising amino acid residues 765-784, are deleted. This DNA sequence is replaced with 30 base pairs that encode an alternative 10 amino acids that, although serine-rich, represents a minimal serine content compared to that of the same stretch of amino acids in the other I pallidum strains.
  • the 70 kDa molecular mass of the recombinant protein is unexpectedly lower than the 97 kDa molecular mass predicted for the histidine-tagged recombinant molecule (92 kDa for the T. pallidum Tpa92 plus 5 kDa extra for the N-terminal hexa-histidine tag).
  • the recombinant 70 kDa molecule represented the major protein in the resulting preparation (approximately 90% of the total protein). Proteins of a smaller molecular mass present in the nickel-purified preparation represent breakdown products of the 70 kDa recombinant Tpa92.
  • the recombinant T. pallidum Tpa92 was used to generate polyclonal antiserum, and subsequent immunoblot analysis showed an immunoreactive 70 kDa protein in both the nickel-purified recombinant protein preparation and lysates of E. coli expressing the Tpa92-pRSETc construct. No corresponding immunoreactive protein was observed using either control pre-immune serum on the nickel-purified recombinant protein preparation or the anti-Tpa92 antiserum on preparations of E. coli expressing the pRSETc vector alone.
  • the anti-Tpa92 antiserum was also investigated for its ability to opsonize T. pallidum in three separate experiments using a standard phagocytosis assay.
  • the level of opsonic activity observed for anti-Tpa92 approximated that observed with serum collected from rabbits chronically infected with T. pallidum (immune rabbit serum; p ⁇ .0001).
  • mice were intradermally challenged at eight independent sites with 10 ⁇ T. pallidum per site. Two control rabbits received no prior immunization but underwent the same intradermal challenge. Table 9 summarizes the post-challenge analyses performed on the rabbits to determine the degree of protection provided by immunization with the T. pallidum recombinant Tpa92.
  • Tpa92 5061 atypical 0/8* 1/8* 1 1+
  • Tpa92 5202 atypical 2/8* 6/8 Tpa92 5207 typical 8/8 6/8
  • the control animals developed typical red, raised and highly-indurated lesions, the majority of which progressed to ulceration.
  • the rabbits immunized with the T. pallidum recombinant Tpa92 prior to challenge all demonstrated alteration of lesion development.
  • the degree of protection varied amongst the immunized rabbits, with the highest levels of protection observed for those rabbits exhibiting strong anti-Tpa92 immunoreactivity in immunoblot analysis.
  • Significant attenuation of lesion development was observed in rabbits #5061 and #5200, with atypical pale, flat, slightly-indurated and non-ulcerative lesions appearing at the sites of challenge.
  • rabbit #5207 developed lesions that were paler, flatter and less indurated than those of the control rabbits, all lesions progressed to ulceration and therefore were designated as typical.
  • C. trachomatis Although the majority of these proteins have been identified through genome sequencing of the bacterial species in which they are found, and thus are hypothetical, six have been independently isolated using molecular biological or protein immunochemical approaches. These include an unknown protein from E. coli (genbank accession number P39170), OMPl from B. abortus (genbank accession number U51683), Omp85 proteins from N. meningitidis and N gonorrhoeae (Manning, D.S., D.K. Reschke, and R.C. Judd. 1998.
  • Omp85 proteins of Neisseria gonorrhoeae and Neisseria meningitidis are similar to Haemophilus influenzae D-15- Ag and Pasteurella multocida Oma87. Microb. Pathog. 25:11-21), Oma87 from P. multocida (Ruffolo, C.G., and Adler, B. 1996. Cloning, sequencing, expression, and protective capacity of the oma87 gene encoding the Pasteurella multocida 87- kilodalton outer membrane antigen. Infect. Immun. 64:3161-3167) and D15 from fi influenzae (Flack, F.S., S. Loosmore, P. Chong, and W.R. Thomas. 1995.
  • Omp85 proteins of Neisseria gonorrhoeae and Neisseria meningitidis are similar to Haemophilus influenzae D-15-Ag and Pasteurella multocida Oma87. Microb. Pathog. 25:11-21.; Thomas, W.R., M.G. Callow, R.J. Dilworth, and A.A. Audesho. 1990. Expression in Escherichia coli of a high-molecular weight protective surface antigen found in nontypeable and type b Haemophilus influenzae. Infect. Immun. 58:1090-1913), and passive immunization of antiserum against Oma87 and D15 has been shown in animal models to be protective against P. multocida and H.
  • a 20-kilodalton ⁇ -terminal fragment of the D15 protein contains a protective epitope(s) against Haemophilus influenzae type a and type b. Infect. Immun. 66:3349-3354; and 32.Loosmore, S.M., Y. Yang, -77-
  • Tpa92 in T. pallidum evidence for the surface location of Tpa92 in T. pallidum comes from the observation that antibodies directed against Tpa92 have significant opsonic activity for living T. pallidum, thus demonstrating that this protein is accessible on the surface of intact treponemes.
  • Indirect evidence for the presence of Tpa92 in T pallidum outer membranes was obtained by immunoblot analysis using the anti-Tpa92 antiserum on T pallidum lysate preparations. A loss of immunoreactivity was observed in lysates prepared from treponemes whose outer membranes had been partially removed by washing prior to lysis, compared to lysates prepared from unwashed treponemes in which the fragile outer membrane and its constituent proteins remain intact prior to lysis.
  • Tpa92 Analysis of the amino acid sequence of Tpa92 also provides supporting evidence for the presence of Tpa92 on the bacterial surface.
  • the first 21 amino acid residues at the N-terminus of Tpa92 comprise a cleavable signal sequence that is characteristic of proteins translocated across the bacterial inner membrane (Von Heijne, G. 1983. Patterns of amino acids near signal-sequence cleavage sites. Eur. J. Immunol 133:17-21).
  • Tpa92 is associated with the T. pallidum outer membrane, and additional biochemical studies are currently underway to investigate the potential cell surface disposition of this molecule.
  • Tpa92 The potential significance of the serine-rich sequence present in Tpa92 becomes apparent when one considers similar serine-rich sequence stretches are observed in proteins involved in attachment to cells or cellular substances, including the Saccharomyces cerevisiae A-agglutinin attachment subunit precursor (Roy, A. et al., Mol. Cell. Biol. 11:4196-4206 (1991)) and the Candida albicans chitinase 3 precursor (McCreath, K.J. et al., Proc. Natl. Acad. Sci. USA. 92:2544-2548 (1995)). Numerous studies have shown T. pallidum attaches to host cells (Fitzgerald, T.J., J.N.
  • Tpa92 could be hypothesized to constitute one such attachment ligand.
  • the stretch of serine residues present in the C-terminal end of the Tpa92 sequence which have been predicted to reside within an external loop on the outer face of the outer membrane, could act as potential sites for hydrogen bonding to carbohydrates present on the surface of host cells.
  • Tpa92-specific antiserum can inhibit T. pallidum attachment to rabbit epithelial cells ( S. Sun, unpublished observations). Studies are currently underway to further investigate this putative functional role of Tpa92 as a T. pallidum adhesion. The immunoprotective potential of the T.
  • pallidum Tpa92 was also investigated in this study for several reasons.
  • antiserum raised against the analogous proteins Oma87 and D15 from P. multocida and H. influenzae, respectively, have been shown to induce protection in animal models (Ruffolo, C.G., and Adler, B., Infect. Immun. 64:3161-3167 (1996); Thomas, W.R. et al., Infect. Immun. 58:1090-1913 (1990); Yang, Y., W.R. et al., Infect. Immun. 66:3349-3354 (1998); Loosmore, S.M. et al., Infect. Immun. 65:3701-3707 (1997)).
  • Tpa92 the invariant nature of Tpa92 among various T. pallidum subspecies and strains makes it an attractive candidate for design of a universal subunit vaccine against T. pallidum infections.
  • the level of protection achieved strongly corresponded to the antibody response generated in the immunized rabbit, with rabbits exhibiting the highest level of Tpa92-specific immunoreactivity demonstrating significant protection upon challenge.
  • These rabbits developed atypical small, pale, flat, slightly indurated and non-ulcerative lesions at the sites of challenge. Darkfield examination of aspirates collected from the sites of challenge in these rabbits showed a lower number of lesions contained viable treponemes compared to control unimmunized animals.
  • Alternative methods of antigen delivery will be investigated in an attempt to generate higher levels of anti- Tpa92 reactivity and, correspondingly, more significant protection against T. pallidum challenge. -80-
  • T. pallidum Tpa92 represents a target of opsonic antibodies and an invariant, immunoprotective antigen. Further studies will be performed to determine whether co-vaccination of Tpa92 with other promising immunoprotective antigens, such as glycerophosphodiester phosphodiesterase (Cameron, C.E., et al., Infect. Immun. 66:5763-5770 (1998)) and Tpr K (Centurion-Lara, A., C, et al., J. Exp. Med In Press (1999)), can achieve complete immunity against T. pallidum challenge.
  • immunoprotective antigens such as glycerophosphodiester phosphodiesterase (Cameron, C.E., et al., Infect. Immun. 66:5763-5770 (1998)
  • Tpr K Centurion-Lara, A., C, et al., J. Exp. Med In Press (1999)
  • Example 13 DNA-mediated vaccination with a vector expressing Gpd is partially protective against challenge with T. palladium.
  • a Gpd DNA vaccine based on the high-expression CMV promoter vector pCR3.1 (Invitrogen, San Diego, CA) expressing Gpd.
  • pCR3.1 High-expression CMV promoter vector
  • Sf-1 Ep American Type Culture Collection
  • transfected with pCR3.1-Gpd expresses Gpd detectable by Western blot (data not shown).
  • the two DNA-injected rabbits and one control (uninjected) rabbit were challenged intradermally with 10 5 T. palladium Nichols strain at each of eight separate sites on their shaved backs.
  • the DNA-injected rabbits developed only small papules at the sites of challenge which cleared before the control rabbit developed ulcerated lesions.
  • variable domains of the msp-homologues have been expressed in E. coli as 6 his-fusion proteins, purified and used to immunize rabbits before intradermal challenge with 10 5 T pallidum per site.
  • Table 10 describes immunizaion of single animals with recombinant variable domains from msp 3, 4/5, 6, 10, and 12; of these, msp 4/5 showed evidence of protection, as measured by lesion appearance and lack of treponemes on darkfield microscopy of lesion aspirates.
  • the recombinant carboxyl-terminal conserved domain from Subfamily II appears to confer significant protection.
  • tprK sequence The sequence differences are limited to defined "hypervariable" regions. Given the nature of the sequence diversity, it is highly unlikely that these differences are due to PCR-induced errors. It is particularly interesting that this heterogeneity is seen in msp 9, which is a protective and opsonic antigen in the Nichols strain, and is the msp-homologue that is predominantly transcribed and expressed.
  • SEQ JD NO:76 (strain IN); SEQ ID NO:77 (strain 1-n); SEQ JD NO:78 (strain 1-1-Bal2); SEQ ID NO:79 (strain 2-l-Bal2); SEQ JD NO:80 (strain 1-1-Bal3); SEQ JD NO:81 (strain 1-1-Bal7); SEQ JD NO:82 (strain l-2-Bal7); SEQ ID NO:83 (strain 2-3-Bal7); SEQ JD NO:84 (strain 1-1-Bal8); SEQ JD NO:85 (strain l-2-Bal8); SEQ ID NO:86 (strain l-3-Bal8); SEQ ID NO:87 (strain 1-1-Bal73-1); SEQ JD NO:88 (strain l-2-Bal73-l); SEQ JD NO:89 (strain 1-3

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Abstract

L'invention concerne des molécules d'acides nucléiques, des polypeptides et des procédés utiles pour former des vaccins contre la syphilis et d'autres maladies tréponémiques.
PCT/US1999/007886 1998-04-10 1999-04-09 Proteines recombinees de treponeme pale et leur utilisation pour former un vaccin contre la syphilis WO1999053099A1 (fr)

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AU35533/99A AU3553399A (en) 1998-04-10 1999-04-09 Recombinant proteins of treponema pallidum and their use for a syphilis vaccine
JP2000543645A JP2002511275A (ja) 1998-04-10 1999-04-09 Ｔｒｅｐｏｎｅｍａｐａｌｌｉｄｕｍの組換えタンパク質および梅毒ワクチンのためのその使用
EP99917401A EP1071819A1 (fr) 1998-04-10 1999-04-09 Proteines recombinees de treponeme pale et leur utilisation pour former un vaccin contre la syphilis
CA002325576A CA2325576A1 (fr) 1998-04-10 1999-04-09 Proteines recombinees de treponeme pale et leur utilisation pour former un vaccin contre la syphilis

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Publication number Priority date Publication date Assignee Title
WO2006112962A2 (fr) * 2005-04-14 2006-10-26 University Of Washington Essais et trousses de diagnostic de la syphilis

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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
US5350842A (en) * 1986-09-30 1994-09-27 Board Of Regents, The University Of Texas System DNAs encoding Treponema pallidum antigens
US5681934A (en) * 1986-09-30 1997-10-28 Board Of Regents, The University Of Texas System 47-kilodalton antigen of Treponema pallidum

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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
US5350842A (en) * 1986-09-30 1994-09-27 Board Of Regents, The University Of Texas System DNAs encoding Treponema pallidum antigens
US5681934A (en) * 1986-09-30 1997-10-28 Board Of Regents, The University Of Texas System 47-kilodalton antigen of Treponema pallidum

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JANSON H., ET AL.: "PROTEIN D, AN IMMUNOGLOBULIN D-BINDING PROTEIN OF HAEMOPHILUS INFLUENZAE: CLONING, NUCLEOTIDE SEQUENCE, AND EXPRESSION IN ESCHERICHIA COLI.", INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY., US, vol. 59., no. 01., 1 January 1991 (1991-01-01), US, pages 119 - 125., XP002921477, ISSN: 0019-9567 *
SHEVCHENKO D. V., ET AL.: "IDENTIFICATION OF HOMOLOGS FOR THIOREDOXIN, PEPTIDYL PROLYL CIS-TRANS ISOMERASE, AND GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE IN OUTER MEMBRANE FRACTIONS FROM TREPONEMA PALLIDUM ,THE SYPHILIS SPIROCHETE", INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY., US, vol. 65., no. 10., 1 October 1997 (1997-10-01), US, pages 4179 - 4189., XP002921476, ISSN: 0019-9567 *
STEBECK C. E., ET AL.: "IDENTIFICATION OF THE TREPONEMA PALLIDIUM SUBSP. PALLIDUM GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE HOMOLOGUE.", FEMS MICROBIOLOGY LETTERS, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 154., 1 January 1997 (1997-01-01), GB, pages 303 - 310., XP002921475, ISSN: 0378-1097, DOI: 10.1016/S0378-1097(97)00346-7 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006112962A2 (fr) * 2005-04-14 2006-10-26 University Of Washington Essais et trousses de diagnostic de la syphilis
WO2006112962A3 (fr) * 2005-04-14 2006-12-28 Univ Washington Essais et trousses de diagnostic de la syphilis

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