WO2006138324A2 - Treponema pallidum antigens for vaccine development and diagnostic tests - Google Patents

Abstract

The present invention relates to T. pallidum antigens. More particularly, the present invention relates to immunogenic compositions comprised of T. pallidum antigens, methods of inducing an immune response in a subject agains T, pallidum antigens, and methods to detect anti-T. pallidum antibodies in a sample.

Description

TREPONEMA PALLIDUM ANTIGENS FOR VACCINE DEVELOPMENT AND

DIAGNOSTIC TESTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 60/690,481 filed June 14, 2005, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] The work herein was supported by NIH grants AI45842 and AI49557. The United States Government may have certain rights in the invention.

TECHNICAL FIELD

[0003] The present invention relates to the field of immunology. More specifically, it relates to the use of T. pallidum antigens in syphilis vaccine development. Further, it relates to the use of T. pallidum antigens in the development of methods to diagnose and treat T. pallidum infection or syphilis, in a subject.

BACKGROUND OF THE INVENTION

[0004] Spirochetes are a family of motile, unicellular, spiral shaped bacteria. Three genera of Spirochetes are pathogenic in humans: (a) Treponema, which includes the pathogens that causes syphilis (T. pallidum), yaws (7". pertenue), and pinta (T. carateum); (b) Borrelia, which includes the pathogens that cause epidemic and endemic relapsing fever and Lyme disease; and (c) Leptospira, which includes a wide variety of small spirochetes that cause mild to severe systemic human illness (Koff and Rosen, 1993).

[0005] Syphilis, a multistage, sexually transmitted disease, is the primary manifestation of infection with T. pallidum (US Public Health Service, 1967). Syphilis is typically transmitted by sexual contact, but can also be transmitted transplacentally. In the first stage of syphilis, the infecting organism multiplies at the site of infection and within 10-60 days post infection results in a primary ulcer-like lesion termed a chancre. A small number of organisms move from the primary lesion to the lymph nodes and establish small infectious centers termed satellite buboes. Organisms from these locations enter the blood stream and result in a systemic infection.

[0006] The secondary stage of syphilis manifests itself as a widespread skin rash that begins between two and twelve weeks following the primary infection. Lesions with varying degrees of severity may develop in a number of locations, including the bone, liver, kidneys, and the central nervous system of the afflicted individual (Veeravahu, M. 1985). The infected individual usually experiences a low grade fever coupled with swollen lymph nodes. This stage of syphilis is highly contagious, but will in time spontaneously subside.

[0007] The third stage of syphilis occurs many years after the first and second stages, and is frequently called the latent stage. This stage of syphilis occurs in approximately 30% of infected, but not treated, individuals. The lesions which characterize the third stage of syphilis are minor in terms of the number of organisms, but can be severe in terms of tissue damage. These lesions may result in necrosis, scar formation, general paresis, damage to aortic valves, permanent blindness, and other extensive tissue damage. These manifestations may be due to a delayed hypersensitivity to the T. pallidum organism by the infected individual (Scheck and Hook, 1994).

[0008] T. pallidum is an invasive organism capable of colonizing virtually any tissue (Thomas et al., 1988). It is adept at evading both the humoral and cellular components of the immune system and causing a persistent infection (Radolf, 1994). Studies of the outer membranes of pathogenic spirochetes revealed that the density of their integral transmembrane outer membrane proteins is one to two orders of magnitude less than that of typical gram negative bacterial pathogens. It has been proposed that this paucity of cell surface proteins — potential antigens that could be recognized by the host immune system - allows pathogenic spirochetes to evade the immune system and cause chronic infection.

[0009] Although effective therapies have been available since the mid 20^th century with the introduction of penicillin, syphilis remains a major public health problem, with an estimated 12 million new cases per year worldwide (Gerbase et al., 1998). Syphilis is endemic in Africa and Southeast Asia, and is becoming more common in the former Soviet Union and Eastern Europe. One reason behind the continued medical threat posed by syphilis is the fact that this disease can go undiagnosed and can continue to be transmitted sexually. Moreover, by going undetected, the disease can result in extensive tissue damage which may not be resolved by therapy. An increasingly common complication of syphilis infection is co infection with the human immunodeficiency virus (HIV). Studies have indicated that ulceratous genital diseases such as syphilis may facilitate transmission of HIV (Rufli, 1989). Further, since the treatment for syphilis requires an adequate host immune system response, HIV patients infected with syphilis exhibit a highly increased occurrence of early neurosyphilis and other syphilis related symptoms (Musher, 1990). Thus, the need exists for effective vaccines to prevent the spread of syphilis, effective treatments for syphilis infection, and improved methods to diagnose the disease.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention reveals for the first time a nearly complete set of T. pallidum antigens which are antigenic in an infected human. In total, 34 T. pallidum antigens which induce an immune response in humans were identified. Further, sixteen antigens gave rise to antibody responses at all three stages of syphilis infection - primary, secondary, and early latent stages - and thus represent candidates for immunodiagnostic antigens. Three of these sixteen gave rise to strong, rapid antibody responses. Use of these three antigens in diagnostic assays may increase the sensitivity of these tests during the early stages of T. pallidum infection. Twelve antigens induced an immune response only in the early latent stage of syphilis infection. It is contemplated that any method or composition described herein can be implementd with respect to any other method or composition.

[0011] The present invention is directed to a system and method to induce an immune response in an individual against T. pallidum. Further, the present invention is directed to a system and method of treating T. pallidum infection in an afflicted individual. Yet further, the present invention is directed to a system and method for diagnosing T. pallidum infection in an individual. Yet further, the present invention can be used to stage a subject infected with T. pallidum or stage a subject suffering from or suspected of suffering from syphilis. Thus, the present invention can be used to diagnose subjects in any stage of syphilis, for example, primary, secondary or early latent stage of syphilis. Staging syphilis can be performed by detecting T. pallidum antibodies.

[0012] An embodiment of the present invention is an immunogenic composition capable of eliciting an immune response in an organism against T. pallidum. Preferably, the immunogenic composition is comprised of T. pallidum polypeptide antigens and dispersed in a pharmaceutically acceptable carrier.

[0013] A specific embodiment of the present invention is an immunogenic composition comprised of one or more T. pallidum polypeptide antigens herein described as TPOl 33, TPOl 36, TP0277, TP0327, TP0463, TP0470, TP0486, TP0625, TP0639, TP0727, TP0750, TP0769, TP0772, TP0789, TP0954, TP0956, TP0974, and TP0993 (e.g., SEQ ID NO. 1-18) and/or functionally equivalent variants thereof and/or any combination thereof. The polypeptides described as SEQ ID NO. 1-18 represent the novel T. pallidum antigens disclosed in the present invention that elicit an immune response in humans.

[0014] In a preferred embodiment, the immunogenic composition is comprised of one or more T. pallidum polypeptide antigens herein described as SEQ ID NO. 1-18 and/or functionally equivalent variants wherein at least one polypeptide is selected from the group consisting of SEQ ID NO. 3, 4, 6, 11, 14, 15, 16, and 17 and/or functionally equivalent variants thereof. The polypeptides described as SEQ ID NO. 3, 4, 6, 11, 14, 15, 16, and 17 represent those novel T. pallidum antigens which elicit an immune reaction in a human only at the early latent stage of the disease.

[0015] In another embodiment, the immunogenic composition is comprised of one or more T. pallidum polypeptide antigens herein described as SEQ ID NO. 1-18 and/or functionally equivalent variants thereof and one or more T. pallidum polypeptide antigens herein described as SEQ ID NO. 19-34 and/or functionally equivalent variants thereof and/or any combination thereof. The polypeptides herein described as SEQ ID NO. 19-34 represent the group of T. pallidum antigens that had previously been identified as antigenic in humans.

[0016] In a preferred embodiment, the immunogenic composition is comprised of one or more polypeptides herein described as SEQ ID NO. 1-18 and/or functionally equivalent variants thereof and one or more T. pallidum polypeptide antigens herein described as SEQ ID NO. 19-34 and/or functionally equivalent variants thereof wherein at least one polypeptide is selected from the group consisting of SEQ ID NO. 3, 4, 6, 11, 14, 15, 16, 17, 20, 21, 24, and 34 and/or functionally equivalent variants thereof. The polypeptides described as SEQ ID NO. 3, 4, 6, 11, 14, 15, 16,17, 20, 21, 24, and 34 represent all those T. pallidum antigens disclosed in the present invention which elicit an immune reaction in a human only at the early latent stage of the disease.

[0017] A further embodiment of the present invention is a method of inducing an immune reaction in an individual comprising administering to the individual one of the above described immunogenic compositions. Additionally, another embodiment is a method of treating an individual infected with T. pallidum comprising administering to the individual one of the above described immunogenic compositions.

[0018] Another embodiment of the present invention is a method to detect T. pallidum antibodies in a sample comprising the steps of (a) obtaining a sample from a subject; (b) mixing the sample with one or more T. pallidum polypeptide antigens; and (c) detecting the presence of an immune complex. Furthermore, this method of detecting T, pallidum antibodies can be used to stage syphilis or a person infected with T. pallidum. The T. pallidum polypeptide antigens for step (b) are chosen from the group consisting of SEQ ID NO. 1-18 and/or functionally equivalent variants thereof. In a preferred embodiment, one or more T. pallidum polypeptide antigens are from the group consisting of SEQ ID NO. 1-18 and/or functionally equivalent variants thereof wherein at least one polypeptide is selected from the group consisting of SEQ ID NO. 5, 7, 9, 12, and 13 and/or functionally equivalent variants thereof. The polypeptides herein described as SEQ ID NO. 5, 7, 9, 12, and 13 represent those novel T. pallidum antigens which elicit an immune response in a human at all stages of infection.

[0019] In yet another embodiment of detecting T. pallidum antibodies, one or more T. pallidum polypeptide antigens are chosen from the group consisting of SEQ ID NO. 1-18 and/or functionally equivalent variants thereof and one or more T. pallidum polypeptide antigens are chosen from the group consisting of SEQ ID NO. 19-34 and/or functionally equivalent variants thereof. In a preferred embodiment, one or more T. pallidum polypeptide antigens are chosen from the group consisting of SEQ ID NO. 1-18 and/or functionally equivalent variants thereof and one or more T. pallidum polypeptide antigens are chosen from the group consisting of SEQ ID NO. 19-34 and/or functionally equivalent variants thereof wherein at least one polypeptide is selected from the group consisting of SEQ ID NO. 5, 7, 9, 12, 13, 19, 22, 23, 25, 26, 27, 28, 29, 31, 32, and 33. The polypeptides herein described as SEQ ID NO. 5, 7, 9, 12, 13, 19, 22, 23, 25, 26, 27, 28, 29, 31, 32, and 33 represent all identified T, pallidum antigens disclosed in the present invention which elicit an immune response in a human at all stages of infection.

[0020] More specifically, the method may comprise detecting at least two T. pallidum polypeptide antigens are selected from the group consisting of SEQ ID NO. 27, 29, and 31. The polypeptides described herein as SEQ ID NO. 27, 29, and 31 represent those T. pallidum antigens which elicit a rapid, strong immune response in a human in the first stage of infection, and therefore it is envisioned that the use of these particular antigens in a method to detect T. pallidum antibodies will lead to a more sensitive assay.

[0021] In a specific embodiment of the above described method to detect T. pallidum antibodies in a sample, the subject is a human suspected of having a T. pallidum infection. Further, the method described above is used to diagnose syphilis in a human subject and/or stage the syphilis or T. pallidum infection. Thus, the antibodies can be used to determine or stage a person that this is in the primary, secondary and/or early latent stage of syphilis.

[0022] In another embodiment of the current invention the above described method to detect T. pallidum antibodies in a sample is used to determine the efficacy of an immunogenic composition or vaccine administered to an individual. Specifically, after an individual has been administered an immunogenic composition or vaccine comprised of T. pallidum antigens and a suitable amount of time has passed to allow the individual to mount an immune response, the above described method can detect the immune response.

[0023] In another embodiment of the above described method to detect T, pallidum antibodies in a sample, one or more T. pallidum polypeptide antigens for step (b) are selected from the group consisting of SEQ ID NO. 3, 4, 6, 11, 14, 15, 16, and 17 and/or functionally equivalent variants thereof. The T. pallidum polypeptide antigens herein described as SEQ ID NO. 3, 4, 6, 11, 14, 15, 16, and 17 are those novel antigens disclosed in the present invention which elicit an immune response in a human only at the early latent stage of infection. In a further embodiment, one or more T. pallidum polypeptide antigens for step (b) are selected from the group consisting of SEQ ID NO. 3, 4, 6, 11, 14, 15, 16, and 17 and/or functionally equivalent variants thereof further comprising one or more polypeptides antigens selected from the group consisting of SEQ ID NO. 20, 21, 24, and 34 and/or functionally equivalent variants thereof. The T. pallidum polypeptide antigens herein described as SEQ ID NO. 3, 4, 6, 11, 14, 15, 16, 17, 20, 21, 24, and 34 are all those antigens disclosed in the present invention which elicit an immune response in a human only at the early latent stage of infection. Further, this embodiment of said method can be used to diagnose the early latent stage of syphilis disease in a human subject.

[0024] In another embodiment of the above described method to detect T. pallidum antibodies in a sample the antigens of step (b) are conjugated to a solid support. In a specific embodiment, the antigens are fused to glutathione S transferase molecules (GST) and bound to a glutathione coated surface. Further, the solid surface may be a glutathione coated microplate or other suitable surface, as would be known to one skilled in the art.

[0025] In yet a further embodiment of the above described method to detect T. pallidum antibodies in a sample, the detecting step (c) is further defined as mixing the reaction of step (b) with secondary antibodies that recognize the antibodies produced by the subject conjugated to a reporter molecule. In a specific embodiment the reporter molecule is horseradish peroxidase (HRP) and the presence of a horseradish peroxidase containing immune complex is detected by chemiluminescence.

[0026] In another embodiment, components for use in the above described method for detecting T. pallidum antibodies in a sample is comprised in a kit. Specifically, components such as, but not limited to, the T. pallidum polypeptide antigens, wash solutions, secondary antibodies, detection reagents, and optionally a solid support may be each individually supplied in containers and packaged together in a convenient manner. In one embodiment, the T. pallidum antigens are fused to a GST molecule and the solid support is coated with glutathione. Further, the GST fused antigens may be supplied pre-bound to the glutathione coated solid support.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawings:

[0028] FIG. IA show immunoblot reactivity of pooled sera from three rabbits infected with T. pallidum for 28 days. T. pallidum were subjected to non- equilibrium pH gradient electrophoresis, followed by SDS-PAGE using an 8% to 20% gradient gel. Locations of some of the prominent T. pallidum polypeptides are indicated.

[0029] FIG. IB shows immunoblot reactivity of pooled sera from three rabbits infected with T. pallidum for 84 days. T. pallidum were subjected to non- equilibrium pH gradient electrophoresis, followed by SDS-PAGE using an 8% to 20% gradient gel. Locations of some of the prominent T. pallidum polypeptides are indicated.

[0030] FIG. 2 shows schematic of the ELISA assay. Glutathione coated wells on microplates capture GST fusion proteins and array potential antigens for the immunoassay. Antibodies present in sera collected from rabbits or humans previously infected with T. pallidum bind to T. pallidum antigens. Interactions between arrayed antigens and rabbit or human antibodies are detected with a secondary anti-rabbit antibody or anti-human antibody conjugated to horseradish peroxidase (HRP). Chemiluminescence is produced by the captured HRP when it reacts with a peroxidase substrate added to the wells. Chemiluminescence is measured by a plate reader and recorded as relative units.

[0031] FIG. 3 shows an immunoassay to identify antigenic proteins in the T. pallidum proteome. 882 T. pallidum proteins were arrayed and incubated with serum collected from rabbits 84 days post intratesticular inoculation with virulent T. pallidum. Interactions between rabbit antibodies and arrayed proteins were identified with a secondary anti-rabbit antibody conjugated to horseradish peroxidase. After peroxidase substrate addition, chemiluminescence was monitored with a plate reader and measured in relative light units, shown along the y-axis. The protein products of the 1039 open reading frames (ORFs) identified in the genomic sequence of T. pallidum (Fraser, 1998) have been named according to their ORF number, proteins TPOOOl -TP 1041. The proteins arrayed in this immunoassay are presented numerically along the X-axis according to their ORF number. Statistical analysis was used to identify proteins yielding a chemiluminescent signal significantly above background values; these were considered immunogenic and members of the rabbit-specific T. pallidum immunoproteome. Overall, 106 immunogenic proteins were identified by this methodology (Table 1).

[0032] FIGS. 4A-F: Development of the rabbit humoral immune response over the course of T. pallidum infection. The progressive development of the humoral immune response against T. pallidum infection in rabbits is presented. The change in reactivity of antibody against 74 antigens is depicted at six different time points: FIG. 4A: prior to infection; FIG. 4B: 7 days post intratesticular inoculation of virulent T. pallidum; FIG. 4C: 14 days post intratesticular inoculation of virulent T. pallidum; FIG. 4D: 28 days post intratesticular inoculation of virulent T. pallidum; FIG. 4E: 56 days post intratesticular inoculation of virulent T. pallidum; and FIG. 4F: 84 days post intratesticular inoculation of virulent T. pallidum. The antigens are arranged along the X-axis according to their reactivity with pooled sera collected 84 days post infection.

[0033] FIG. 5: Single point ELISA. Phage expressing individual T. pallidum proteins were screened for binding to fibronectin.

[0034] FIG. 6: Test of binding of purified TPOl 36 protein to extracellular matrix proteins by ELISA. The proteins shown on the X-axis were coated at the same concentration into wells of a microtiter plate and binding of soluble TPOl 36 was detected with an anti-TP136 antibody. SuperFN is a fragment of the fibronectin protein.

DETAILED DESCRIPTION OF THE INVENTION

[0035] It is readily apparent to one skilled in the art that various embodiments and modifications may be made to the invention disclosed in this Application without departing from the scope and spirit of the invention.

I. DEFINITIONS [0036] As used herein, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Still further, the terms "having", "including", "containing" and "comprising" are interchangeable and one of skill in the art is cognizant that these terms are open ended terms.

[0037] The term "subject" or "individual", as used herein refers to mammals. More specifically, mammals include, but are not limited to rats, mice, rabbits, cats, dogs, monkeys and humans. These terms can be used interchangeably.

[0038] The term "antigen" or "immunogen" as used herein is defined as a molecule that provokes an immune response when it is introduced into a subject. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. An antigen can be derived from organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates. Commonly, and antigen is a molecule which causes the subject in which it is introduced to produce antibodies which specifically recognize the antigen. The part of the antigen with which the antibody interacts is termed an "epitope" or "antigenic determinant". A skilled artisan realizes that any macromolecule, including virtually all proteins or peptides, can serve as antigens. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan realizes that any DNA which contains nucleotide sequences or partial nucleotide sequences of a pathogenic genome or a gene or a fragment of a gene for a protein that elicits an immune response results in synthesis of an antigen. Furthermore, one skilled in the art realizes that the present invention is not limited to the use of an entire nucleic acid sequence of a gene or an entire protein encoded by a gene. It is readily inherent that the present invention includes, but is not limited to, the use of partial nucleic acid sequences and protein fragments.

[0039] The term "antigenic" and "immunogenic" as used herein describe a structure that is an antigen. These terms can be used interchangeably.

[0040] The term "antibody" as used herein refers to an immunoglobulin molecule, which is able to specifically bind to a specific epitope on an antigen. As used herein, an antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as single chain antibodies and humanized antibodies (Harlow et ah, 1988; Bird et ah, 1988).

[0041] The term "secondary antibody" as used herein, refers to an antibody that specifically recognizes and binds to another antibody. For example, an anti-human IgG antibody would specifically bind to a human IgG antibody. Secondary antibodies are commonly used as reagents to detect an immune complex. For example, a primary antibody that becomes bound to an antigen, forming a primary immune complex, may be detected by means of a secondary antibody that has binding affinity for the primary antibody. In these cases, the second binding ligand may be linked to a detectable label to allow visualization of the complex.

[0042] The term "variant" or "variants" as used herein refers to polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide respectively. Variants in this sense are described below and elsewhere in the present disclosure in greater detail. For example, changes in the nucleotide sequence of the variant may be silent, i.e., they may not alter the amino acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide with the same amino acid sequence as the reference polypeptide. Changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Generally, differences in amino acid sequences are limited so that the sequences of the reference and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. A variant may also be a fragment of a polynucleotide or polypeptide of the invention that differs from a reference polynucleotide or polypeptide sequence by being shorter than the reference sequence, such as by a terminal or internal deletion. For example, a variant may be a result of alternative mRNA splicing. A variant may also be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. A variant of the polynucleotide or polypeptide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms, or may be made by recombinant means. Among polynucleotide variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. AU such variants defined above are deemed to be within the scope of those skilled in the art from the teachings herein and from the art.

[0043] The term "functional equivalent" as used herein is defined as a variant of a polynucleotide that retains the capacity to perform the biologic function of interest of the wild-type or reference protein. Thus, as used herein, the term functional equivalent includes mutations, truncations, deletions, insertions, fusions, fragments, or substitutions of SEQ ID NO. 1-34 which retain the antigenicity of the full-length protein. In a specific example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of antigenic capacity, such as, for example, the ability to stimulate and immune response and be recognized by antibodies. So-called "conservative" changes do not disrupt the biological activity of the protein, as the structural change is not one that impinges of the protein's ability to carry out its designed function. Alternatively, a functional equivalent may be a polypeptide comprising an immunogenic epitope-bearing portion of a polypeptide of the invention. An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention.

[0044] The term "immunologically functional equivalent" as used herein refers to a variant of a polypeptide that retains the same or nearly the same ability to induce an immune response in a subject as the reference polypeptide.

[0045] The term "immunogenic composition" as used herein, may comprise an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding an antigen (e.g., an antigen expression vector), an antibody or antibodies directed against an antigen, a cell expressing or presenting an antigen, or any combination thereof. An immunogenic composition induces an active or passive immune response in a cell, tissue, or animal (e.g., a human). In a preferred embodiment of the present invention, the immunogenic composition comprises or encodes one or more of the sequences shown in SEQ ID NO: 1-34, or immunologically functional equivalents thereof. An immunogenic composition can be a mixture that comprises additional immunostimulatory agents or nucleic acids encoding such an agent. Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell or an adjuvant.

[0046] As used herein, the term "vaccine" refers to a formulation which contains the immunogenic composition of the present invention and which is in a form that is capable of being administered to an animal. Typically, the vaccine comprises a conventional saline or buffered aqueous solution medium in which the immunogenic composition of the present invention is suspended or dissolved. In this form, the immunogenic composition of the present invention can be used conveniently to prevent, ameliorate, or otherwise treat a condition. Upon introduction into a subject, the vaccine is able to provoke an active or passive immune response including, but not limited to, the production of antibodies, cytokines and/or other cellular responses that are protective against infection. Protection may be complete or partial, ie. providing resistance to disease, attenuating symptoms of disease, or delaying onset of disease. The process of administering to an individual a vaccine is denoted "vaccination" or "immunsation". After receiving a vaccine, an individual is described as being "vaccinated" or "immunized" or "immune".

[0047] As used herein, the term "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.

[0048] The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. The carrier may not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. These terms can be used interchangeably.

[0049] The term "prophylactic" as used herein is defined as a treatment that protects from or prevents the spread or occurrence of disease or infection.

[0050] As used herein, the terms "treatment", "treat", "treated", or "treating" refer to prophylaxis and/or therapy. When used with respect to an infectious disease, for example syphilis, the term refers to a prophylactic treatment which increases the resistance of a subject to infection with T. pallidum, or in other words, decreases the likelihood that the subject will become infected with T. pallidum or will show signs of illness attributable to the infection, as well as a treatment after the subject has become infected in order to fight the infection, e. g., reduce or eliminate the infection or prevent it from becoming worse.

[0051] The term "polynucleotide" or "nucleic acid" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means. Furthermore, one skilled in the art is cognizant that polynucleotides include, without limitation, mutations of the polynucleotides, including but not limited to, mutation of the nucleotides, or nucleosides by methods well known in the art.

[0052] The term "polypeptide" as used herein is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term polypeptide is mutually inclusive of the terms "peptides" and "proteins". One of skill in the art is cognizant that polypeptides include, without limitation, mutations of polypeptides by methods that are well known in the art, i.e., site directed mutagenesis or chemical mutagenesis.

[0053] The term "immune complex" as used herein, refers to a complex formed by the interaction of an antibody with its antigen and any other associated molecules.

[0054] The term "reporter molecule" as used herein, refers to compounds and/or elements that can be detected due to their specific functional properties and/or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffmity molecules, and colored particles or ligands, such as biotin.

[0055] As used herein, the term "active immunization" refers to the process whereby a non-immune individual acquires long lasting ability to respond to an organism or its toxic products by generating his or her own protective mechanism. [0056] As used herein, the term "passive immunization" denotes the process of confering protective immunity without the need for an immune response on the part of the recipient, for example by giving injections of antibodies.

[0057] As used herein, the term "humoral immune response" refers to antibody production in response to an antigen mediated by B-lymphocytes, and all the accessory processes that accompany it: T-helper cell activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to the effector functions of antibody, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

[0058] As used herein, the term "acquired immune response" refers to resistance resulting from a previous exposure to an antigen or infectious agent. This type of immune response can be active or specific, for example, as a result of naturally acquired infection or vaccination or it may be passive as being acquired from transfer of antibodies from another person or from an animal either naturally, as from mother to fetus, or by intentional inoculation.

[0059] As used herein, the term "immune response" refers to the response made by the host to defend itself againts a pathogen or antigen.

II. T. pallidum

[0060] Despite the impact T. pallidum has on human health worldwide, no effective vaccine for syphilis has been developed (Guevara et ah, 1982; Morgan et ah, 2002). Several factors have contributed to the difficulty in developing a syphilis vaccine. First, T. pallidum is a microaerophilic bacterium that is killed by atmospheric oxygen levels. Thus, it is one of the few important human pathogens that has not been cultured continuously in vitro, making it extremely difficult to study. T. pallidum must be propagated through the intratesticular inoculation of rabbits. A second problem is the limited number of organisms that can be obtained from infected animals, and the contamination of these organisms with host tissue components. A third difficulty is the fragility of the outer membranes of spirochetes compared to other gram negative bacteria, making it difficult to separate the outer membrane from the rest of the cellular components. This has hindered the identification of cell surface proteins that may act as antigens to stimulate an immune response in an individual and thus be candidate vaccine components.

A. Polynucleotide Composition

[0061] The genome of T. pallidum comprises a single circular chromosome of ~1.14 Mb encoding 1039 open reading frames (ORFs) designated TPOOOl through TP1041 (Fraser et al., 1998; Salazar et al., 2002). The entire genome of T. pallidum has been sequenced (Fraser et al. Science. 1998 JuI 17;281(5375):375-88), and is incorporated herein by reference. The polynucleotide sequences of the present invention can be found in databases known by those of skill in the art. One such database is Genbank. The T. pallidum genome is Genbank accession number NC_000919, which is incorporated herein by reference in its entirety.

[0062] In certain embodiments, the present invention provides polynucleotides that code for T. pallidum antigens SEQ ID NO. 1-34. The polynucleotides of the present invention may be in the form of DNA, such as genomic DNA or cDNA, or RNA, such as mRNA. The DNA may be in the form of double stranded or single stranded DNA. Single stranded DNA may be the coding, or sense strand, or the non-coding, or antisense strand.

[0063] In certain aspects, a nucleic acid comprises a wild-type or a mutant nucleic acid. In particular aspects, a nucleic acid encodes for or comprises a transcribed nucleic acid. In other aspects, a nucleic acid comprises a nucleic acid segment of SEQ ID NO. 35-68, or a functional equivalent thereof. In particular aspects, a nucleic acid encodes a polypeptide.

[0064] The polynucleotides of the present invention may encode, the entire polypeptide sequence of a T. pallidum antigen (SEQ ID NO. 1-34) or any variant thereof, including homologs, mutations, truncations, deletions, and insertions. Polynucleotides that have between about 70% and/or about 79%; and/or more preferably, between about 80% and/or about 89%; and/or even more preferably, between about 90% and/or about 99%; of nucleotides that are identical to the nucleotides of that encode the polypeptides of SEQ ID NO. 1-34 are considered immunologically functional equivalent variants and are contemplated in the present invention, provided the encoded polypeptide retains its immunogenic activity. The polynucleotides of the present invention encompass those coding for immunologically functional equivalent polypeptides of SEQ ID NO. 1-34. Such sequences may arise as a consequence of codon redundancy and/or functional equivalency that are known to occur naturally within nucleic acid sequences and/or the proteins thus encoded. Alternatively, functionally equivalent proteins, polypeptides and/or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced, for example, through the application of site-directed mutagenesis techniques as discussed herein below, e.g., to introduce improvements to the antigenicity of the encoded protein.

[0065] The polynucleotides may include, but are not limited to, additional sequences such as coding sequences that would result in a fusion polypeptide, sequences encoding a leader or secretory sequence, and noncoding sequences such as introns, promoters, enhancers, and non-translated 5' and 3' sequences that play a role in RNA processing and translation such as polyadenylation signals and ribosome binding sites.

[0066] In addition to the "standard" DNA and/or RNA nucleotide bases, modified bases are also contemplated for use in particular applications of the present invention. A table of exemplary, but not limiting, modified bases is provided herein below. One or more modified base may be incorporated into the polynucleotides of the present invention.

[0067] In particular embodiments, the invention concerns isolated DNA segments and/or recombinant vectors incorporating DNA sequences that encode a T. pallidum polypeptide that includes within its amino acid sequence a contiguous amino acid sequence as set forth in SEQ ID NO: 1-34. Said vectors may be transformed or transfected into a host cell by methods well known by those of skill in the art. Alternatively, a host cell may be genetically engineered to incorporate the polynucleotides of the present invention into its genome.

B. Polypeptide Compositions [0068] In another embodiment of the present invention provides antigenic T. pallidum polypeptides comprising the amino acid sequences of SEQ ID NO. 1-34, homologs of SEQ ID NO. 1-34, and immunologically functional equivalents variants thereof. The polypeptides of the present invention may be natural polypeptides, synthetic polypeptides, or recombinant polypeptides. The polynucleotide sequences of the present invention can be found in databases known by those of skill in the art. One such database is Genbank. The polynucleotides sequences used in the present invention can be obtained under Genbank accession number NC_000919, , which is incorporated herein by reference in its entirety.

[0069] The inventors have utilized a variation of an enzyme-linked immunosorbent assay (ELISA) to systematically screen 908 of the 1039 predicted T. pallidum genes for those whose protein product elicits an antibody immune response in humans infected with syphilis. In total, 34 T. pallidum antigens which induce an immune response in humans were identified (SEQ ID NO. 1-34, see table C and table 3). Sixteen antigens gave rise to antibody responses at all three stages of syphilis infection - primary, secondary, and early latent stages - and thus may represent good candidates for immunodiagnostic antigens (SEQ ID NO. 5, 7, 9, 12, 13, 19, 22, 23, 25-29, 31-33). Three of these sixteen gave rise to strong, rapid antibody responses (SEQ ID NO. 27, 29, 31). Use of these three antigens in diagnostic assays may increase the sensitivity of these tests during the early stages of T. pallidum infection. Twelve antigens induced an antibody immune response only during the early latent stage of syphilis infection (SEQ ID NO. 3, 4, 6, 11, 14-17, 20, 21, 24, 34).

Table C: SEQ IN NO. of the T. pallidum polypeptides antigenic in humans

SEQ ED NO. [ TP No. Name/Function SEQ ID NO. I TP No. Name/Function

[0070] The polypeptides of the present invention may be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al, 1989, incorporated herein by reference. Additionally, the polypeptides can be expressed in a cell-free environment such as a rabbit reticulocyte lysate expression system. Further, the polypeptides of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and are well known to those skilled in the art.

1. Functional equivalents and/or Variants [0071] As modifications and changes may be made in the structure of an antigen of the present invention (SEQ ID NO. 1-34) and still obtain molecules having like or otherwise desirable characteristics, such immunologically functional equivalents are also encompassed within the scope of the present invention. The term "immunologically functional equivalent" is well understood in the art and is further defined in detail herein. Immunologically functional equivalents may increase the antigenicity of a polypeptide, maintain the same level of antigenicity of the reference polypeptide, or decrease the antigenicity of a polypeptide only slightly so that it maintains its usefulness as an antigen in an immunogenic composition. Examples of such changes include, but are not limited to, amino acid changes, deletions, truncations, polypeptide fragments, fusions to other polypeptides, insertions, or any combination thereof. Accordingly, sequences that have between about 70% and/or about 80%, and more preferably, between about 81% and/or about 90%, and even more preferably, between about 91% and/or about 99% of amino acids that are identical and/or functionally equivalent to the amino acids of SEQ ID NO: 1-34 will be sequences that are considered immunologically functional equivalents, provided the immunogenic activity of the protein is maintained. For the purposes of the present invention, a polypeptide that is useful as an antigen in an immunogenic composition (i.e. has sufficient "immunogenic activity") is defined by the ELISA assay described further on in this specification (see example 4 in particular). The ratio of the immune complex detected from a sample well containing the subject antigen fused to GST to a control well containing only GST must be 1.5 or greater, preferably 2.0 or greater.

[0072] As an example of modifications contemplated to be within the scope of the present invention, certain amino acids may be substituted for other amino acids in a polypeptide structure without appreciable loss of interactive binding capacity of the structure such as, for example, the epitope of an antigen that is recognized and bound by an antibody. Since it is the interactive capacity and nature of a polypeptide that defines its biological {e.g., immunological) functional activity, certain amino acid sequence substitutions can be made in a amino acid sequence (or its underlying DNA coding sequence) and nevertheless obtain a polypeptide with like properties. It is thus contemplated by the inventors that various changes may be made in the amino acid sequences of the antigens of the present invention (SEQ ID NO. 1-34) without appreciable loss of immunogenic activity.

[0073] It is understood in the art that in order to make functionally equivalent amino acid substitutions, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a polypeptide is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics; these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0074] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the immunologically functional equivalent polypeptide thereby created is intended for use in immunological embodiments, as in certain embodiments of the present invention. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a immunological property of the protein. In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[0075] While discussion has focused on functionally equivalent polypeptides of SEQ ID NO.1-34 arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA. The immunologically functional equivalent may also comprise a polynucleotide that has been engineered to contain distinct sequences while at the same time retaining the capacity to encode the "wild-type" or standard protein. This can be accomplished by the degeneracy of the genetic code, i.e., the presence of multiple codons which encode for the same amino acids.

[0076] It is well understood that where certain residues are shown to be particularly important to the immunological or structural properties of a protein or peptide, e.g., residues in binding regions or epitopes, such residues may not generally be exchanged. This is an important consideration in the present invention, where changes in the antigenic sites of 7! pallidum antigens SEQ ID NO.1-34 should be carefully considered and subsequently tested to ensure maintenance of immunological function (e.g., antigenicity), where maintenance of immunological function is desired. In this manner, functional equivalents are defined herein as those polypeptides which maintain a substantial amount of their native immunological activity. In general, the shorter the length of the molecule, the fewer changes that can be made within the molecule while retaining function. Longer domains may have an intermediate number of changes. The full-length protein will have the most tolerance for a larger number of changes. However, it must be appreciated that certain molecules or domains that are highly dependent upon their structure may tolerate little or no modification.

[0077] In particular embodiments, an antigen of the present invention may be mutated for purposes such as, for example, enhancing its immunogenicity or producing or identifying a immunologically functional equivalent sequence. Methods of mutagenesis are well known to those of skill in the art (Sambrook et al, 1987, incorporated herein by reference). In a preferred embodiment, site directed mutagenesis is used. Site directed mutagenesis is a technique useful in the preparation of an immunologically functional equivalent polypeptide through specific mutagenesis of the underlying DNA. In general, the technique of site directed mutagenesis is well known in the art. The technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site directed mutagenesis allows the production of a mutant through the use of specific oligonucleotide sequence(s) which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the position being mutated. Typically, a primer of about 17 to about 75 nucleotides in length is preferred, with about 10 to about 25 or more residues on both sides of the position being altered, while primers of about 17 to about 25 nucleotides in length being more preferred, with about 5 to 10 residues on both sides of the position being altered.

[0078] In general, site directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein. As will be appreciated by one of ordinary skill in the art, the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.

[0079] The mutagenic primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as, for example, E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. [0080] The preparation of sequence variants of the selected gene using site directed mutagenesis is provided as a means of producing potentially useful functionally equivalent species and is not meant to be limiting, as there are other ways in which sequence variants of polypeptides may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

2. Epitope bearing polypeptides [0081] In another aspect, the invention encompasses a polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope portion of a polypeptide is defined as the part of the polypeptide that elicits an antibody response when the whole polypeptide is the immunogen. An epitope bearing portion of a polypeptide often retains the immunogenic capacity of the full length polypeptide, and therefore is also an immunologically functional equivalent.

[0082] A polypeptide corresponding to one or more epitope bearing portions of the T. pallidum antigens of the present invention should generally be at least five or six amino acid residues in length, and may contain up to about 10, about 15, about 20, about 25 about 30 ,about 35, about 40, about 45 or about 50 residues or so. A peptide sequence may be synthesized by methods known to those of ordinary skill in the art, such as, for example, peptide synthesis using automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, CA).

[0083] Immunogenic epitope-bearing peptides of the invention are identified according to methods well known in the art. For instance, Geysen et ah, 1984, supra, incorporated herein by reference, discloses a procedure for rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner a peptide bearing an immunogenic epitope of a desired protein may be identified routinely by one of ordinary skill in the art. For instance, the immunologically important epitope in the coat protein of foot-and-mouth disease virus was located by Geysen et al. with a resolution of seven amino acids by synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope were synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. Thus, peptide analogs of the epitope-bearing peptides of the invention can be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen (1987), incorporated herein by reference, further describes this method of identifying a peptide bearing an immunogenic epitope of a desired protein.

[0084] Another method for determining an epitope bearing portion of a polypeptide is the SPOTs™ system (Genosys Biotechnologies, Inc., The Woodlands, TX). In this method, overlapping peptides are synthesized on a cellulose membrane, which following synthesis and deprotection, is screened using a polyclonal or monoclonal antibody. An epitope bearing portion of the polypeptide which is initially identified can be further localized by performing subsequent syntheses of smaller peptides with larger overlaps, and by eventually replacing individual amino acids at each position along the immunoreactive sequence.

[0085] Moreover, computer programs are currently available to assist with predicting an antigenic, or epitope bearing, portion of a polypeptide. Examples include those programs based upon the Jameson - Wolf analysis (Jameson & WoIf, 1988; Wolf et al, 1988, both incorporated herein by reference), the program PepPlot® (Brutlag et al, 1990; Weinberger et al, 1985, both incorporated herein by reference), and other new programs for protein tertiary structure prediction (Fetrow & Bryant, 1993, incorporated herein by reference). Another commercially available software program capable of carrying out such analyses is MacVector (IBI, New Haven, CT). Numerous scientific publications have also been devoted to the prediction of secondary structure, and to the identification of an epitope, from analyses of an amino acid sequence (Chou & Fasman, 1974a,b; 1978a,b, 1979, incorporated herein by reference). U.S. Patent 4,554,101, (Hopp) incorporated herein by reference, teaches the identification and/or preparation of epitopes from primary amino acid sequences on the basis of hydrophilicity. Through the methods disclosed in Hopp, one of skill in the art would be able to identify epitopes from within an amino acid sequence. 3. Mimetics [0086] In addition to the polypeptide compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key antigenic portions of the polypeptide structure or to interact specifically with, for example, an antibody. Such compounds, which may be termed peptidomimetics, may be used in the same manner as a polypeptide of the present invention and hence are also immunologically functional equivalents.

[0087] Certain mimetics that mimic elements of protein secondary and tertiary structure are described in Johnson et al. (1993) , incorporated herein by reference. The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and/or antigen. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.

[0088] Some successful applications of the peptide mimetic concept have focused on mimetics of β-turns within proteins, which are known to be highly antigenic. Likely β-turn structures within a polypeptide can be predicted by computer-based algorithms. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains. Methods for generating specific structures have been disclosed in the art. For example, alpha-helix mimetics are disclosed in U.S. Patents 5,446,128; 5,710,245; 5,840,833; and 5,859,184, all incorporated herein by reference.

[0089] Other approaches have focused on the use of small, multidisulfϊde- containing proteins as attractive structural templates for producing biologically active conformations that mimic the binding sites of large proteins (Vita et al. 1998, incorporated herein by reference). A structural motif that appears to be evolutionarily conserved in certain toxins is small (30-40 amino acids), stable, and highly permissive for mutation. This motif is composed of a beta sheet and an alpha helix bridged in the interior core by three disulfides.

III. Immunogenic compositions [0090] Another embodiment of the present invention is an immunogenic composition capable of inducing an immune response in a host subject to which it is administered. The immunogenic composition may comprise an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding an antigen (e.g., an antigen expression vector), a cell expressing or presenting an antigen (e.g., a cell transfected with an antigen expression vector) or an antibody or a combination thereof. In a preferred embodiment, the immunogenic composition comprises or encodes one or more of the polypeptide sequences shown in SEQ ID NO: 1-34 or an immunologically functional equivalent as discussed above (see table C). Yet further, the immunogenic composition can comprise antibodies that bind with high specificity to the T. pallidum polypeptides provided herein (SEQ ID NO. 1-34).

[0091] It is understood that an immunogenic composition of the present invention may be made by a method that is well known in the art, including but not limited to chemical synthesis of proteinaceous components by solid phase synthesis and purification away from the other products of the chemical reactions by HPLC, or production by the expression of a nucleic acid sequence (e.g., a DNA sequence) encoding a peptide or polypeptide comprising an antigen of the present invention in an in vitro translation system or in a living cell. It is further understood that additional amino acids, mutations, chemical modification and such like, if any, that are made in an immunogenic composition will preferably not substantially interfere with the antibody recognition of the epitopic sequence.

A. Antibodies

[0092] In certain embodiments the present invention provides antibodies that bind with high specificity to the T. pallidum polypeptides provided herein (SEQ ID NO. 1-34). Thus, antibodies that bind to the protein products of the isolated nucleic acid sequences of SEQ ID NO. 35-68 are provided. As detailed above, in addition to antibodies generated against the full length proteins, antibodies may also be generated in response to smaller constructs comprising epitopic core regions, including wild-type and mutant epitopes, and mimetics. The antibodies of the present invention may be monoclonal, polyclonal, any antibody-like molecule that has an antigen binding region, and antibody fragments such as Fab¹, Fab, F(ab')₂, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. "Humanized" antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, heteroconjugate antibodies, recombinant and engineered antibodies and fragments thereof. The techniques for preparing, characterizing, and using various antibody-based constructs and fragments are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). The antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, immunohistochemistry assays, immunoprecipitation assays, or therapy known to one of ordinary skill in the art. (Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158, CRC Press, Inc., 1987, incorporated herein by reference.)

[0093] Several methods can be used to generate antibodies or antibody fragments. Typically, an antigen is inoculated into an animal which will then produce antibodies against that antigen. Alternatively, antibodies can be made using a molecular cloning approach, synthesized using an automated peptide synthesizer, or by expression of a full-length gene or gene fragment in E. coil or any other suitable expression system. The following sections will discuss various means of generating antibodies.

1. Polyclonal antibodies [0094] Polyclonal antibodies generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the antigen and an adjuvant. It may be useful to conjugate the antigen or a fragment containing the antigen to a protein that is immunogenic in the species to be immunized, e.g. keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glytaraldehyde, succinic anhydride, SOCl₂, or R¹ N.dbd.C.dbd.NR, where R and R¹ are different alkyl groups. The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen including but not limited to subcutaneous, intramuscular, intradermal, intraepidermal, intravenous and intraperitoneal. The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.

[0095] A wide range of animal species can be used for the production of antisera. Typically the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice of animal may be decided upon the ease of manipulation, costs or the desired amount of sera, as would be known to one of skill in the art.

[0096] As is well known in the art, the immunogenicity of a particular antigen can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Adjuvants can be used in the production of antibodies by co- administeration to the animal with the antigen. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, chemokines, cofactors, toxins, Plasmodia, synthetic compositions or LEEs or CEEs encoding such adjuvants. In addition to adjuvants, it may be desirable to co administer biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg/m²) (Johnson/ Mead, NJ), cytokines such as γ-interferon, IL-2, or IL- 12 or genes encoding proteins involved in immune helper functions, such as B-7.

[0097] A second, booster dose of antigen (e.g., provided in an injection), may also be given to the animal. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immune response (ie. antibodies produced against the antigen) is obtained, the immunized animal can be bled and the serum isolated and stored. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots. The serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g., protein A or protein G chromatography. 2. Monoclonal antibodies [0098] Monoclonal antibodies (MAbs) are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. The invention thus provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies will often be preferred.

[0099] The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred. MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference. A brief description of these techniques follow.

[0100] Following immunization of the animal, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody- producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.

[0101] The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell line, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma producing fusion procedures preferably are non antibody producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells

(hybridomas). Any one of a number of myeloma cell lines may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984, incorporated herein by reference).

[0102] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976) , incorporated herein by reference, and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al, (1977) , incorporated herein by reference. The use of electrically induced fusion methods is also appropriate (Goding pp. 71-74, 1986 , incorporated herein by reference). Fusion procedures usually produce viable hybrids at low frequencies, about 1x10^" to 1x10^" . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.

[0103] The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The preferred myeloma celk lines are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and therefore they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.

[0104] This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

[0105] The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, producing clones that can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. First, a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. Second, the individual cell lines could be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.

[0106] DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison, et ah, Proc. Nat. Acad. Sci. 81, 6851 (1984), incorporated herein by reference, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In that manner, "chimeric" or "hybrid" antibodies may be prepared.

[0107] Human monoclonal antibodies can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor, J. Immunol. 133, 3001 (1984), and Brodeur, et ai, Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987), both incorporated herein by reference. [0108] It is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. (Jakobovits et al, 1993).

[0109] Alternatively, the phage display technology (McCafferty et al, 1990) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle.

3. Humanized antibodies [0110] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, Nature 321, 522-525, 1986; Riechmann et al, Nature 332, 323-327, 1988; Verhoeyen et al, Science 239, 1534-1536, 1988, all incorporated herein by reference), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (Cabilly, supra), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0111] It is important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three- dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. For further details see U.S. application Ser. No. 07/934,373 filed Aug. 21, 1992, which is a continuation-in-part of application Ser. No. 07/715,272 filed Jun. 14, 1991, incorporated herein by reference.

4. Heteroconjugate antibodies [0112] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (PCT application publication Nos. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0113] Antibodies produced by any of the above means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Fragments of the antibodies of the invention can be obtained from the antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer. A more thorough discussion of these techniques can be found in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference.

5. Conjugation of antibodies to effector and reporter molecules [0114] In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. As such, the antibodies of the present invention conjugated to a molecule are contemplated and are therefore encompassed by the scope of the invention. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radiolabeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or poly-nucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.

[0115] Any antibody of sufficient selectivity, specificity or affinity may be employed as the basis for an antibody conjugate. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art. The binding affinity of a monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson & Pollard, Anal. Biochem. 107:220 (1980), incorporated herein by reference.

[0116] Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and/or those for use in vivo diagnostic protocols, generally known as "antibody directed imaging".

[0117] Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patent Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference). The imaging moieties used can be paramagnetic ions; radioactive isotopes; fluorochromes; NMR-detectable substances; and radioactive isotopes for X-ray imaging. Specific imaging agents contemplated for use in the present invention are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and/or avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.

B. Immunogenic composition component purification

[0118] An immunogenic composition component (e.g., an antigenic polypeptide, nucleic acid encoding an antigenic polypeptide, or cell expressing an antigenic polypeptide) may be isolated and/or purified from the chemical synthesis reagents, cell or cellular components. In a method of producing the immunogenic composition component, purification is accomplished by any appropriate technique that is described herein or well-known to those of skill in the art (e.g., Sambrook et al, 1987, incorporated herein by reference). Although preferred for use in certain embodiments, there is no general requirement that an immunogenic composition of the present invention always be provided in their most purified state. Indeed, it is contemplated that a less substantially purified immunogenic composition component, which is nonetheless enriched in the desired compound relative to the natural state, will have utility in certain embodiments, such as maintaining the activity of an expressed protein.

[0119] The present invention also provides purified, substantially purified, and in certain embodiments, immunogenic composition components purified to homogeneity. In preferred embodiments, the immunogenic composition components comprise SEQ ID NO. 1-34 and immunologically functional equivalents thereof. The term "purified immunogenic composition component" as used herein, is intended to refer to at least one immunogenic composition component (e.g., a proteinaceous composition or nucleic acid, isolatable from cells), wherein the component is purified to any degree relative to its naturally-obtainable state, e.g., relative to its purity within a cellular extract or reagents of chemical synthesis. In certain aspects wherein the immunogenic composition component is a proteinaceous composition, a purified component also refers to a wild-type or mutant protein, polypeptide, or peptide free from the environment in which it naturally occurs. The term "substantially purified" refers to a composition in which the specific compound (e.g., a polypeptide) forms the major component of the composition, such as constituting about 50% of the compounds in the composition or more. In preferred embodiments, a substantially purified immunogenic composition component will constitute more than about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or even more of the compounds in the composition. In certain embodiments, an immunogenic composition component may be purified to homogeneity. As applied to the present invention, "purified to homogeneity," means that the immunogenic composition component has a level of purity where the compound is substantially free from other chemicals, biomolecules or cells. For example, a purified polypeptide will often be sufficiently free of other protein components so that degradative sequencing may be performed successfully. Various methods for quantifying the degree of purification of an immunogenic composition component will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific protein activity of a fraction (e.g., antigenicity), or assessing the number of polypeptides within a fraction by gel electrophoresis.

[0120] Various techniques suitable for use in chemical, biomolecule or biological purification, well known to those of skill in the art, may be applicable to preparation of an immunogenic composition component of the present invention. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; fractionation, chromatographic procedures, including but not limited to, partition chromatograph (e.g., paper chromatograph, thin-layer chromatograph (TLC), gas-liquid chromatography and gel chromatography) gas chromatography, high performance liquid chromatography, affinity chromatography, supercritical flow chromatography ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity; isoelectric focusing and gel electrophoresis (see for example, Sambrook et al. 1989; and Freifelder, Physical Biochemistry, Second

Edition, pages 238-246, incorporated herein by reference). [0121] In certain aspects, a nucleic acid may be purified on polyacrylamide gels, and/or cesium chloride centrifugation gradients, or by any other means known to one of ordinary skill in the art (see for example, Sambrook et al. 1989, incorporated herein by reference). In further aspects, a purification of a proteinaceous sequence may be conducted by reconϊbinantly expressing the sequence as a fusion protein. Such purification methods are routine in the art. This is exemplified by the generation of an specific protein-glutathione S-transferase fusion protein, expression in E. coli, and isolation to homogeneity using affinity chromatography on glutathione-agarose or the generation of a polyhistidine tag on the N- or C-terminus of the protein, and subsequent purification using Ni-affinity chromatography. In particular aspects, cells or other components of the immunogenic composition may be purified by flow cytometry. Flow cytometry involves the separation of cells or other particles in a liquid sample, and is well known in the art (see, for example, U.S. Patent Nos. 3,826,364, 4,284,412, 4,989,977, 4,498,766, 5,478,722, 4,857,451, 4,774,189, 4,767,206, 4,714,682, 5,160,974 and 4,661,913, all incorporated herein by reference). Any of these techniques described herein, and combinations of these and any other techniques known to skilled artisans, may be used to purify and/or assay the purity of the various chemicals, proteinaceous compounds, nucleic acids, cellular materials and/or cells that may comprise an immunogenic composition of the present invention. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified antigen or other immunogenic composition component.

[0122] In other embodiments, the immunogenic composition is in a mixture that comprises one or more additional immunostimulatory agents or nucleic acids encoding such an agent. Immunostimulatory agents include but are not limited to additional antigens, immunomodulators, antigen presenting cells or adjuvants.

C. Immunomodulators

[0123] It is contemplated that immunomodulators can be included in the immunogenic composition to augment a cell's or a patient's {e.g., an animal's) response. Immunomodulators can be included as . purified proteins, nucleic acids encoding immunomodulators, and/or cells that express immunomodulators in the immunogenic composition. The following sections list non-limiting examples of immunomodulators that are of interest, and it is contemplated that various combinations of immunomodulators may be used in certain embodiments (e.g., a cytokine and a chemokine).

[0124] Interleukins, cytokines, nucleic acids encoding interleukins or cytokines, and/or cells expressing such compounds are contemplated as possible vaccine components. Interleukins and cytokines, include but are not limited to interleukin 1 (IL- 1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l₅ IL-12, IL-13, IL-14, IL- 15, IL-18, β-interferon, α-interferon, γ-interferon, angiostatin, thrombospondin, endostatin, GM-CSF, G-CSF, M-CSF, METH-I, METH-2, tumor necrosis factor, TGFβ, LT and combinations thereof.

[0125] Chemokines, nucleic acids that encode for chemokines, and/or cells that express such also may be used as immunogenic composition components. Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine coding sequence in combination with, for example, a cytokine coding sequence, to enhance the recruitment of other immune system components to the site of treatment. Such chemokines include, for example, RANTES, MCAF, MlPl-alpha, MIPl-Beta, IP- 10 and combinations thereof. The skilled artisan will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines.

[0126] It may be desirable to co administer biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); low-dose cyclophosphamide (CYP; 300 mg/m²) (Johnson/ Mead, NJ), or a gene encoding a protein involved in one or more immune helper functions, such as B-7.

D. Adjuvants

[0127] Immunization protocols have commonly used adjuvants to stimulate immune responses, and as such adjuvants are well known to one of skill in the art. Some adjuvants affect the way in which antigens are presented. For example, the immune response is increased when protein antigens are precipitated by alum. Emulsification of antigens also prolongs the duration of antigen presentation. The following sections list non limiting examples of adjuvants that are contemplated by the inventors for use with the present invention.

[0128] In one aspect, an adjuvant effect is achieved by use of an agent, such as alum, used in about 0.05 to about 0.1% solution in phosphate buffered saline. Alternatively, the antigen is made as an admixture with synthetic polymers of sugars (Carbopol^®) used as an about 0.25% solution. An adjuvant effect may also be created by aggregation of the antigen in the vaccine by heat treatment with temperatures ranging between about 70°C to about 101°C for a 30 second to 2 minute period, respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cell(s) such as C. parvum, an endotoxin or a lipopolysaccharide component of Gram-negative bacteria, emulsion in physiologically acceptable oil vehicles, such as mannide mono-oleate (Aracel A), or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA®) used as a block substitute, also may be employed.

[0129] Some adjuvants, for example, certain organic molecules obtained from bacteria, act on the host rather than on the antigen. An example is muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), a bacterial peptidoglycan. The effects of MDP, as with most adjuvants, are not fully understood. MDP stimulates macrophages but also appears to stimulate B cells directly. The effects of adjuvants, therefore, are not antigen-specific. If they are administered together with a purified antigen, however, they can be used to selectively promote the response to the antigen.

[0130] Various polysaccharide adjuvants may also be used. For example, the use of various pneumococcal polysaccharide adjuvants on the antibody responses of mice has been described (Yin et al, 1989, incorporated herein by reference) . The doses that produce optimal responses, or that otherwise do not produce suppression, should be employed as indicated (Yin et al, 1989 , incorporated herein by reference). Polyamine varieties of polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin. [0131] Amphipathic and surface active agents, e.g., saponin and derivatives such as QS21 (Cambridge Biotech), form yet another group of adjuvants for use with the immunogens of the present invention. Nonionic block copolymer surfactants (Rabinovich et al, 1994; Hunter et al, 1991, both incorporated herein by reference) may also be employed. Oligonucleotides are another useful group of adjuvants (Yamamoto et al, 1988, incorporated herein by reference). Quil A and lentinen are other adjuvants that may be used in certain embodiments of the present invention.

[0132] Another group of adjuvants contemplated for use in the present invention are the detoxified endotoxins, such as the refined detoxified endotoxin of U.S. Patent 4,866,034 , incorporated herein by reference. These refined detoxified endotoxins are effective in producing adjuvant responses in mammals. Of course, the detoxified endotoxins may be combined with other adjuvants to prepare multi-adjuvant- incorporated cells. For example, combination of detoxified endotoxins with trehalose dimycolate is particularly contemplated, as described in U.S. Patent 4,435,386, incorporated herein by reference. Combinations of detoxified endotoxins with trehalose dimycolate and endotoxic glycolipids is also contemplated (U.S. Patent 4,505,899, incorporated herein by reference), as is combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and trehalose dimycolate, as described in U.S. Patents 4,436,727, 4,436,728 and 4,505,900, incorporated herein by reference. Combinations of just CWS and trehalose dimycolate, without detoxified endotoxins, is also envisioned to be useful, as described in U.S. Patent 4,520,019, incorporated herein by reference.

[0133] In other embodiments, the present invention contemplates that a variety of adjuvants which may be employed in the membranes of cells, resulting in an improved immunogenic composition. The only requirement is, generally, that the adjuvant be capable of incorporation into, physical association with, or conjugation to, the cell membrane of the cell in question. Those of skill in the art will know the different kinds of adjuvants that can be conjugated to cellular vaccines in accordance with this invention and these include alkyl lysophosphilipids (ALP); BCG; and biotin (including biotinylated derivatives) among others. Certain adjuvants particularly contemplated for use are the teichoic acids from Gram- cells. These include the lipoteichoic acids (LTA), ribitol teichoic acids (RTA) and glycerol teichoic acid (GTA). Active forms of their synthetic counterparts may also be employed in connection with the invention (Takada et ah, 1995a, incorporated herein by reference).

[0134] One group of adjuvants contemplated for use in some embodiments of the present invention are those that can be encoded by a nucleic acid (e.g., DNA or RNA). It is contemplated that such adjuvants may be encoded in a nucleic acid (e.g., an expression vector) encoding the antigen, or in a separate vector or other construct. These nucleic acids encoding the adjuvants can be delivered directly, such as for example with lipids or liposomes.

IV. Pharmaceutical compositions

[0135] The preferred embodiment of the immunogenic compositions of the present invention comprise an effective amount of one or more T. pallidum antigens or immunologically functional equivalents thereof, as disclosed herein, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one T. pallidum antigen or immunologically functional equivalent thereof and any additional active ingredients will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0136] In embodiments where the immunogenic composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof. In accordance with the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.

[0137] An immunogenic composition of the present invention may be formulated as a neutral or salt form. A pharmaceutically-acceptable salt, includes the acid addition salts (formed with the free amino groups of the peptide) and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. A salt formed with a free carboxyl group also may be derived from an inorganic base such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and combinations thereof.

[0138] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.

[0139] Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0140] Once produced, synthesized and/or purified, an antigen or other immunogenic composition component may be prepared as a vaccine for administration to a patient. The preparation of a vaccine is generally well understood in the art, as exemplified by U.S. Patents Nos. 4,608,251, 4,601,903, 4,599,231, 4,599,230, and 4,596,792, all incorporated herein by reference. In preferred embodiments, such methods may be used to prepare a vaccine comprising an antigenic composition of T. pallidum antigens or immunologically functional equivalents thereof (SEQ ID NO. 1-34) as active ingredient(s), in light of the present disclosure. In preferred embodiments, the compositions of the present invention are prepared to be pharmacologically acceptable vaccines. In other embodiments, the immunogenic composition or vaccine of the present invention can be combined with other known vaccine formulations. This would result in a "compound vaccine" in which an individual could be immunized against several diseases with one innoculum.

V. Methods of inducing an immune response in a subject

A. Active Immunization

[0141] Another embodiment of the present invention is a method of inducing an active immune response against T. pallidum in a subject by administering to the subject an immunogenic composition described above. In a preferred embodiment, the immune response in the subject provides protection agains subsequent T. pallidum infection. Protection may be complete or incomplete, for example reducing the chance of infection with T. pallidum but not completely preventing it. In a preferred embodiment, the subject is a human at risk for contracting syphilis.

[0142] Yet another embodiment of the present invention is a method to treat syphilis in an individual encompassing administering to the individual an immunogenic composition as described above subsequent to infection with T. pallidum in order to alter the course of the disease. The administration of the immunogenic composition or vaccine can be given in combination with, or instead of, conventional treatments for T. pallidum infection. Some variation in the formulation of the composition (ie. the choice of antigens to be used, the inclusion of other immunostimulatory agents, etc.) and in dosage will necessarily occur depending on the condition of the subject being treated and the stage of the disease. For example, it may be advantageous to administer to a patient in the primary stage of T. pallidum infection an immunogenic composition comprised of T. pallidum antigens that are known to elicit an antibody response in humans at the early latent stage of infection. The person responsible for administration will, in any event, determine the appropriate formulation and dose for the afflicted individual.

[0143] In certain embodiments, an immunogenic composition of the present invention may be used as an effective syphilis vaccine by inducing an anti-7¹. pallidum humoral and/or cell-mediated immune response in a subject. For an immunogenic composition to be useful as a syphilis vaccine, the immunogenic composition must induce an immune response to the antigen in a cell, tissue or animal (e.g., a human) which provides some measure of protection against infection with T. pallidum. The present invention contemplates one or more immunogenic compositions or vaccines for use in active immunization embodiments.

[0144] In a preferred embodiment, the immunogenic composition comprises one or more of the peptide antigens herein described as SEQ ID NO. 3, 4, 6, 11, 14-17, 20, 21, 24 and 34 or an immunologically functional equivalent thereof. This subset of antigens was shown to elicit an antibody immune response in humans suffering from early latent stage syphilis but not in the primary or secondary stages of the disease. The inventors contemplate that these antigens may induce a protective immune response, as the onset of the humoral immune response occurs between the secondary and early latent stages of syphilis, and therefore may be useful as a syphilis vaccine.

[0145] In certain embodiments, an immune response may be promoted by transfecting or inoculating an animal with a nucleic acid encoding an antigen. One or more cells comprised within a target animal then expresses the sequences encoded by the nucleic acid after administration of the nucleic acid to the animal. Thus, the vaccine may comprise a "genetic vaccine" useful for immunization protocols. A vaccine may also be in the form, for example, of a nucleic acid (e.g., a cDNA or an RNA) encoding all or part of the peptide or polypeptide sequence of an antigen. Expression in vivo by the nucleic acid may be, for example, by a plasmid type vector, a viral vector, or a viral/plasmid construct vector.

[0146] In preferred aspects, the nucleic acid comprises a coding region that encodes all or part of the sequences disclosed as SEQ ID NO: 1-34 or an immunologically functional equivalent thereof. Of course, the nucleic acid may comprise and/or encode additional sequences, including but not limited to those comprising one or more immunomodulators or adjuvants. The nucleotide and polypeptide encoding sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http://www.ncbi.nlm.nih.gov/). The coding regions for these known genes may be amplified, combined with the sequences SEQ ID NO. 35-68 disclosed herein (e.g., ligated) and/or expressed using the techniques disclosed herein or by any technique that would be know to those of ordinary skill in the art (e.g., Sambrook et ah, 1987 , incorporated herein by reference). Though a nucleic acid may be expressed in an in vitro expression system, in preferred embodiments the nucleic acid comprises a vector for in vivo replication and/or expression. This genetic vaccine may be administered as a prophylactic treatment prior to infection to prevent or reduce the incidence of infection, or as a therapeutic treatment after infection to attenuate symptoms or alter the course of disease.

[0147] In another embodiment, an immune response may be elicited in a subject by administering to the subject a cell expressing a polypeptide antigen of the present invention (SEQ ID NO. 1-34 and immunologically functional equivalents). Thus, the present invention contemplates a "cellular vaccine." The cell may be isolated from a culture, tissue, organ or organism and administered to an animal as a cellular vaccine. The cell may be transfected with a nucleic acid encoding an antigen to enhance its expression of the antigen. Of course, the cell may also express one or more additional vaccine components, such as immunomodulators or adjuvants. A vaccine may comprise all or part of the cell. This cellular vaccine may be administered as a prophylactic treatment prior to infection to prevent or reduce the incidence of infection, or as a therapeutic treatment after infection to attenuate symptoms or alter the course of disease. B. Passive Immunizations

[0148] In certain embodiments of the present invention, passive immunotherapy may be used. Generally, passive immunotherapy involves administering to a subject antibodies directed against a particular antigen or antigens. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect destruction of the pathogen. In human subjects, one or more human monoclonal antibodies are preferred in passive immunotherapy, as they produce few or no side effects in the patient. In certain embodiments, it may be favorable to administer more than one monoclonal antibody directed against two different antigens, two variants of same antigen (e.g., longer and shorter sequences that share an overlap of homology) or even antibodies with multiple antigen specificity. Treatment protocols also may include administration of a lymphokine or other type of an immunomodulator (Bajorin et al. 1988 , incorporated herein by reference). The development of human monoclonal antibodies is described in greater detail elsewhere in this specification.

[0149] Therefore, in a further embodiment of the present invention, a passive immune response may be elicited in a subject by administering to the subject a compostion comprising one or more antibodies against T. pallidum antigens (SEQ ID NO. 1-34) or immunologically equivalent variants thereof. This "antibody vaccine" may be administered as a prophylactic treatment prior to infection to prevent or reduce the incidence of infection, or as a therapeutic treatment after infection to attenuate symptoms or alter the course of disease.

[0150] In another specific embodiment, it is also provided a method of passive immunization against syphilis comprising administering to an expectant mother T. pallidum antigens (SEQ ID NO. 1-34).

C. Administation Parameters

[0151] The manner of administration of an immunogenic composition or vaccine may vary widely. Any of the conventional methods for administration of a vaccine are applicable. For example, a vaccine may be conventionally administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, mtratumorally, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, intrapericardially, orally, rectally, nasally, topically, in eye drops, locally, using aerosol, injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). A vaccine is administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. For example, the intramuscular route may be preferred in the case of toxins with short half lives in vivo.

[0152] The dosage of a vaccine or immunogenic composition may be varied on a patient by patient basis, taking into account, for example, factors such as the weight and age of the patient, the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the stage of disease being treated, the severity of the disease condition, previous or concurrent therapeutic interventions, the manner of administration and the like, which can be readily determined by one of ordinary skill in the art. In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein However, a suitable dosage range may be, for example, of the order of several hundred micrograms active ingredient per vaccination. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per vaccination, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

[0153] A suitable regime for initial administration and booster administrations (e.g., innoculations) are also variable, but are typified by an initial administration followed by subsequent inoculation(s) or other administration(s). In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations. The vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, may be desirable to maintain protective levels of the antibodies.

[0154] The course of the immunization may be followed by assays to detect antibodies against T. pallidum antigens in the immunized subject. The assays may be performed by techniques well known to those of skill in the art and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064, both incorporated herein by reference. Additionally, other tests can be performed, such as a challenge with T, pallidum to determine the effectiveness of the vaccine.

VI. Diagnostic Assays

[0155] In further embodiments, the present invention concerns immunodetection methods for detecting the presence of anti-7¹. pallidum antibodies in a sample. In general, the immunodetection method includes the steps of: (1) obtaining a sample suspected of containing T. pallidum antibodies, and (2) contacting the sample with a T. pallidum antigen of the present invention, under conditions effective to allow the formation of immunocomplexes, and then (3) detecting and quantifying the amount of immune complex formed. In further embodiments, this method can be used to stage the syphilis infection or T. pallidum infection.

[0156] Immunoassays, in their most simple and/or direct sense, are binding assays. A preferred immunoassay for use with the present invention is the enzyme linked immunosorbent assay (ELISA). All versions of ELISAs have certain standard features, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. It will be readily appreciated by one of skill in the art that detection is not limited to such techniques; western blotting, dot blotting, FACS analyses, radioimmunoassays (RIA), immunohistochemical detection using tissue sections and/or the like may also be used. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle MH and Ben-Zeev O, 1999; Gulbis B and Galand P, 1993; De Jager R et al, 1993; and Nakamura et al, 1987, each incorporated herein by reference.

[0157] In a non limiting example of a preferred embodiment of the present invention, an ELISA is performed wherein the known T. pallidum antigens of the invention are immobilized onto the surface of microplate wells, one antigen per well, and then contacted with the sample suspected of containing anti T. pallidum antibodies. After binding and/or washing to remove non-specifically bound immune complexes, the bound antigen and antibody is detected. The immune complexes may be detected using a second antibody that has binding affinity for the antibodies in the sample, with the second antibody being linked to a detectable label. This method is described in further detail below.

[0158] In coating a microplate or other suitable solid surface with antigen, one will generally incubate the wells of the plate with a solution of the antigen, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0159] After binding a protein to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation while reducing nonspecific background. The incubation step is performed under conditions sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C, or overnight at about 4°C or so.

[0160] Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

[0161] Detection of the immune complex then requires a labeled secondary binding ligand or antibody. In ELISAs, it is customary to use a secondary detection means rather than a direct procedure. To provide a detecting means, the secondary antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the immune complex with a urease, glucose oxidase, alkaline phosphatase or (horseradish) hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween). Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patent Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference).

[0162] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'- azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or H₂O₂, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.

[0163] In yet another embodiment, the immunodetection methods of the present invention have evident utility in the diagnosis and prognosis of syphilis in a human. Here, a biological and/or clinical sample from a subject suspected of having syphilis is used and the presence of T. pallidum antibodies in the sample is indicative of syphilis.

[0164] In certain embodiments of this method to diagnose syphilis in an individual and/or stage the syphilis infection, one or more T. pallidum antigens herein described as SEQ ID NO. 1-34 are used. In a preferred embodiment, at least one T, pallidum antigen is selected from the group consisting of SEQ ID NO. 5, 7, 9, 12, 13, 19, 22, 25-29, 31, 32, and 33. These antigens elicit an immune response in a human at all stages of syphilis disease, and can therefore be used to diagnose syphilis at all stages. In a most preferred embodiment, at least one T. pallidum antigen is selected from the group consisting of SEQ ID NO. 27, 29, and 31. These three antigens elicit a particularly strong and rapid antibody immune response in a human. In another embodiment, one or more T. pallidum antigens is selected from the group consisting of SEQ ID NO. 3, 4, 6, 11, 14-17, 20, 21, 24, and 34. These antigens elicit an antibody immune response in a human only at the early latent stage of the disease, and therefore these antigens are useful in an assay to diagnose this particular stage of syphilis.

[0165] In yet another embodiment, the immunodetection methods of the present invention can be utilized to determine the efficacy of an immunogenic composition or vaccine administered to an individual. In this case, a biological and/or clinical sample from a subject that has been inoculated with one or more T. pallidum antigens from the group of SEQ ID NO. 1-34 is used. Ideally, the antigens chosen for use in the assay should be those administered to the individual in the inoculums.

VII. Kits

[0166] Any of the reagents used in the above described immunoassay may be comprised in a kit. The kit may comprise a suitably aliquoted T. pallidum antigen or antigens, wash solutions, blocking agents, primary antibodies, secondary, or tertiary antibodies covalently linked to a reporter molecule, a means for detecting said reporter molecule, a suitable solid surface support means such as a microplate and/or additional reagents. The components of the kits may be packaged either in aqueous media or in lyophilized form. When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided. Additionally, a microplate or other suitable solid surface support means may be provided pre-bound to one or more T. pallidum antigens.

[0167] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional containers into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the reagent vials and other kit components in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[0168] Irrespective of the number or type of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the immunogenic composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.

VIII. EXAMPLES

[0169] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Chancre immunity in rabbits post-infection with T. pallidum

[0170] Adult male New Zealand White rabbits were obtained from

Myrtle's Rabbitry (Thompson Station, Tennessee). Rabbits were housed individually at 18 to 20 °C and given antibiotic-free food and water. Rabbits were tested for evidence of Treponema paraluiscuniculi infection using a Macro- Vue RPR card test (Becton- Dickinson, Cockeysville, Maryland). Only seronegative animals were included in this study. Three rabbits were each infected with 4 x 10⁸ Treponema pallidum Nichols strain organisms by intratesticular injection. To test for resistance to reinfection at 94 days post infection, the three previously infected rabbits and two control rabbits were inoculated intradermally on their shaved backs at 6 sites with 10⁴ freshly extracted T. pallidum. The animals were shaved and examined daily for the occurrence of erythema and induration (which together constituted lesion development). Syringe aspirates of selected lesions were used to verify the presence of T. pallidum by darkfield microscopy. The animals that had been previously infected exhibited chancre immunity (Turner and Hollander, 1957), i.e. they did not develop lesions following intradermal challenge with T. pallidum; in contrast, the control rabbits developed lesions at an average of 18 days post intradermal inoculation. Thus, as previously reported, the rabbits infected for ~3 months exhibited protective immunity against reinfection.

Example 2

Identification of antigenic proteins by NEPHGE two-dimensional electrophoresis

[0171] To identify proteins eliciting a humoral immune response during experimental infection of rabbits, sera were collected at time points up to 84 days post intratesticular inoculation with virulent T. pallidum. Three rabbits were each infected with 4 x 10⁸ Treponema pallidum Nichols strain organisms by intratesticular injection. Sera were collected 2 days before infection as well as 7, 14, 28, 56 and 84 days post infection. To provide a preliminary analysis of immunoreactivity, sera collected from these rabbits at 28 days and 84 days post infection were tested for reactivity with T. pallidum proteins separated by NEPHGE two-dimensional electrophoresis.

[0172] 2DGE was performed using 5x10⁷ Percoll purified (Hanff et al, 1984) Treponema pallidum subsp. pallidum Nichols per gel. Briefly, T. pallidum were subjected to nonequilibrium pH gradient electrophoresis (NEPHGE) with ampholines ranging from (pH 3.5 to 10) (Amersham, Piscataway, New Jersey) as described by O'Farrell et al. The second dimension consisted of SDS-PAGE with an 8% to 20% polyacrylamide gradient. Gels were subsequently stained using silver (Guevara et al., 1982) or transferred electrophoretically to PVDF membranes (Millipore, Billerica, Massachusetts) for two hours at 1240 mA (Towbin et ah, 1979). Membranes were preincubated overnight with agitation at 4° C in phosphate-buffered saline with 0.1% Tween 20 (PBST) containing 5% milk. Reactivity with pooled prebleed sera, 28-day and 84-day post-infection sera, and 28-day and 84-day E. co//-absorbed sera was examined. Membranes were incubated with serum diluted 1:5000 in PBST for one hour at room temperature with agitation. Membranes were then washed four times with PBST and incubated with donkey anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000 dilution; Pierce, Rockford, Illinois) for one hour. Membranes were washed four times with PBST, and developed using the Supersignal West Pico Chemiluminescent substrate (Pierce, Rockford, Illinois).

[0173] At both the 28 and 84 day time points, roughly 40 to 50 polypeptide spots were visible (Fig. 1). While some of the prominent antigens had been described before (e.g. GroEL, the 47 kDa lipoprotein, treponemal membrane protein A (TmpA) and the flagellar proteins FIaA, FIaBl, and FlaB2), many of the spots could not be identified by their location in the gel pattern. Some differences were evident when comparing the 28-day (Fig. IA) and 84-day (Fig. IB) serum reactivity, including heightened reactivity of the latter to a series of three large, basic 17 kDa spots which may represent different charge variants of the T. pallidum 17 kDa lipoprotein Tp 17 (Norris, 1993). Absorption of the immune rabbit serum with E. coli lysates was performed to reduce the background reactivity with residual E. coli antigens present in the immunoassay wells, and resulted in greatly reduced immunoblot reactivity to GroEL, the FIaB polypeptides, and other unidentified antigens.

Example 3

An immunoassay to identify antigenic proteins in the T. pallidum proteome

[0174] An immunoassay that utilizes a protein array was employed to identify antigenic proteins present in the T. pallidum proteome (McKevitt et ah, 2003; Sehr et ah, 2001)(see figure 2 for a schematic of this assay). Of the 1039 predicted genes in the T. pallidum genome, 908 have been fused to glutathione-S-transferase (GST) using the Univector Cτe-loxP recombination system (Liu et ah, 1998). Construction of the GST Clone Set as has been described previously by McKevitt et ah [0175] Protein arrays were generated using the GST-fusion protein set deposited into 96-Well ELISA plates coated with glutathione. In brief, E. coli BL21*DE3 hosting the plasmid constructs containing individual T. pallidum ORFs were inoculated into 1 mL LB media containing 25 μg/mL kanamycin, 100 μg/mL ampicillin and 2% glucose in a 96 well plate. Following incubation with shaking overnight at 37 ⁰C, 100 μL of the culture was added to 1.5 mL 2YT containing 25 μg/mL kanamycin and 100 μg/mL ampicillin. The cultures were incubated in 2 mL wells containing micro-stir bars in a 96 well format at 30 ⁰C for 5 hours followed by addition of IPTG (0.1 niM final), and incubation for an additional 5 hours. Cells were then pelleted and stored at - 80 ⁰C. Each pellet was subjected to three rounds of freeze-thawing prior to addition of 220 μL of Bacterial Protein Extraction Reagent (B-PER) (Pierce, Rockford, Illinois) containing 0.375 mg/mL lysozyme and 420 ng/mL DNasel, used to lyse the pellet. The resuspended pellets were stirred vigorously with a micro stir bar at room temperature for 10 minutes. Reacti-Bind Glutathione Coated White 96-Well Plates (Pierce, Rockford, Illinois) were washed three times with 210 μL of PBS (pH 7.4), 0.05% Tween 20 (buffer A) using a Elx50 Auto Strip Washer (Bio-Tek, Winooski, Vermont). 110 μL of each cell lysate was added to the glutathione-coated plates followed by incubation at room temperature for two hours. The plates were washed with buffer A five times and the wells were then blocked with 150 μL PBS (pH 7.4) containing 5% dry milk at room temperature for one hour.

[0176] To prepare absorbed rabbit serum, E. coli BL21*D£J was grown overnight at 37 ⁰C in 150 ml of LB liquid media. Cultures were centrifuged and the resulting cell pellets were freeze-thawed two times at -80 ⁰C. The cell pellet was re- suspended in 5 mL TE (pH 8.0) and subjected to cell lysis with a French Press. The lysate was centrifuged for ten minutes at 13,000 rpm and 4°C, and the supernatant was used in subsequent absorptions. A mixture of 45 μL rabbit serum, 405 μL PBS (pH 7.4) and 250 μL BL21*DE3 cell lysate supernatant was mixed on a rocking platform for two hours at room temperature. Absorbed serum was stored at -20° C until use.

[0177] In order to systematically identify antigenic proteins, sera pooled from the three infected rabbits were incubated in each well of the protein arrays, and proteins reactive with antibodies in the sera were identified by chemiluminescence with an anti-rabbit IgG antibody conjugated to horseradish peroxidase (Fig. 2). In brief, absorbed rabbit serum (0.70 mL) was diluted in 12 niL buffer A for a final serum dilution of 1 :282. 110 μL of absorbed serum solution was added to each well followed by incubation for two hours at room temperature. The plates were then washed five times with buffer A. A 1:12,000 dilution of donkey anti-rabbit IgG horseradish peroxidase conjugate (110 μL, Heavy + Light chain specific and affinity purified [Pierce, Rockford, Illinois]) was added to each plate well and incubated for one hour at room temperature. The plates were washed five times before 150 μL of SuperSignal ELISA Pico Chemiluminescent Substrate (Pierce, Rockford, Illinois) was added. Light emission from each plate well was monitored at 5 and 15 -minute intervals with a Genios plate reader (Tecan, Durham, North Carolina) for 200 milliseconds; the results at these two time points were comparable and averaged. Each experiment was repeated two times from the start, i.e. from the point of growing the E. coli cultures. The results of these experiments were combined and used for statistical analysis.

[0178] Each plate contained immobilized GST without a fusion protein as a negative control in order to identify statistically reactive proteins arrayed on the same plate. That is, the relative light units detected from the negative control were compared to the relative light units detected from immobilized GST-fusion proteins using a Students t-Test; p values were reported for each comparison. In order to increase the stringency upon which rabbit specific antigens were selected, a GST-fusion protein was required to exhibit ap value less than 0.0005 and display an average signal of 1.5 times greater than the average signal from wells containing the negative control.

[0179] The ability of a protein to elicit an immune response was inferred when the reactivity of the fusion protein was significantly greater than the background reactivity of the immunoassay. When this experiment was conducted, 882 GST-fusion proteins were available for over-expression in E. coli. Analysis of the immunoassay data collected from these 882 arrayed proteins indicated that 106 proteins exhibited reactivity with antibodies at higher than background levels (Table 1). Examination of T. pallidum literature generated a list of 29 proteins known to elicit an immune response in humans or rabbits. Of these 29 proteins, 27 were included in the array and 22 of these were detected as antigens (Table 1). Reactivity of 81% of the known immunogenic proteins indicates that the assay provides a representative view of the immunoproteome. The identified proteins, however, are likely to be an incomplete set of all the antigens that react with serum antibodies from infected rabbits for several reasons. First, only 882 of the 1039 proteins in the T. pallidum proteome were assayed. Second, some of the T. pallidum proteins may be poorly expressed in the system. GST was fused to the amino terminus of the recombinantly expressed T. pallidum proteins and antibodies against GST was used to ascertain the expression of this portion of the fusion protein, as described previously in McKevitt et al. This previous study indicated that the expression levels as measured by anti-GST antibodies did not correlate with high reactivity with antibodies from T. pallidum infected rabbits. However, this approach does not preclude the possibility that the T. pallidum region of the fusion protein is truncated or improperly folded.

[0180] Five previously identified antigens including proteins TP0868 (FIaBl), TP0792 (FlaB2), TP0870 (FlaB3), TP0897 (TprK) and TP0349 (PpiB) did not react with antibody in the sera at levels above background. DNA sequencing of the relevant clones indicated that these genes did not have mutations within their coding regions, suggesting that the false negative results are not due to errors introduced during cloning. The flagellar FIaBl, FlaB2 and FlaB3 exhibit high sequence similarity in the N- and C-terminal regions with the corresponding E. coli proteins (Norris, 1993), and reactive antibodies were removed in the pre-absorption steps as described above. It was surprising that the binding of rabbit antibodies from 84 days post-infection to the TprK wells was not detected, in that TprK is known to elicit a strong humoral and cell mediated immune response (Centurion-Lara et al., 2004; Morgan et ah, I^IΆ; Morgan et ah, 2002b). Examination of expression of the GST-TprK fusion protein by immunoblotting with anti-GST antibody indicates that the fusion is very poorly expressed in E, coli suggesting the lack of reactivity with rabbit sera is due to the absence of the protein in the lysate.

[0181] Functional classifications have been assigned to 56 of the 106 antigens identified (Table 1). These antigens fall among ten different functional classes including cell envelope, cellular processes, energy metabolism and translation. Proteins associated with the cell envelope are the most represented class (35%). Included in this class are four flagellar proteins involved in T. pallidum motility, numerous membrane proteins and lipoproteins believed to elicit a host inflammatory response during infection (Salazar et at, 2002). Indeed, the five proteins yielding the highest chemiluminescent signal were all experimentally demonstrated (Akins et ah, 1993; Chamberlain et at, 1989; Shevchenko et at, 1989; Swancutt et at, 1990) or predicted (Falquet et at, 2002) to be lipoproteins. Proteins predicted to be secreted beyond the cytoplasm, as indicated by the presence of a putative signal sequence (Fraser et at, 1998; McKevitt et at, 2003), are heavily represented among the antigenic proteins. The bias towards proteins with predicted signal sequences is very strong among the antigens exhibiting the highest signal in the immunoassay. For example, 74% of the 35 proteins exhibiting the highest reactivity have putative signal sequences while 46% of the next reactive set encodes signal sequences and only 25% of the final 36 proteins have predicted signal sequences (Table 1).

[0182] Adherence of treponemes to host tissues may be a critical step for T. pallidum to disseminate and establish infection in multiple tissue types. Host extracellular matrix components such as fibronectin, collagen I, collagen IV and laminin are considered adhesion targets of T. pallidum (Cameron, 2003). Recently, Cameron identified TP0751 as an antigenic laminin binding protein, based on genome-wide prediction of outer membrane proteins and analysis of its adherence and antigenic properties (Cameron, 2003). TP0751 was also identified as an antigen in the immunoassay utilized in this study (Table 1). Based on the results of PSORT (Nakai, 1991) prediction for outer membrane localization, five other members of the immunoproteome are potentially surface-localized hypothetical proteins and thus may also be involved in host-pathogen interactions including TP0453, TP0693, TP0856, TP0956 and TP1002.

[0183] Several other antigenic proteins are of interest based on results from other bacterial pathogens. For example, the HtrA protease encoded by TP0841 is recognized by rabbit antibodies (Table 1) and has also been identified as an antigen in Haemophilis influenzae (Loosmore, 1998), Chylamydia pneumoniae (Montigiani , 2002) and Chylamydia trachomatis (Sanchez-Campillo , 1999). In addition, the HtrA protease from H. influenzae has been shown to induce protective immunity in an animal model (Loosmore, 1998). Another antigen of interest is enolase, which is encoded by TP0817 (Table 1). Although enolase is a known glycolytic enzyme, it is expressed on the surface of both C. pneumoniae and group A Streptococcus (Montigiani et at, 2002; Pancholi and Fischetti, 1998). In group A Streptococcus, the enzyme has also been shown to be an adhesin that binds to host plasmin (Pancholi and Fischetti, 1998). Finally, the FKBP- type peptidyl-prolyl cis/trans isomerase encoded by TP0862 is recognized by rabbit antibodies and has also been shown to be surface localized in C. pneumoniae (Montigiani et ah, 2002) and Legionella pneumophila (Fischer et al, 1992) where it is thought to be involved in the initiation of infections.

Table 1: A list consisting of 106 antigens that react with serum antibodies from T. pallidum infected rabbits.

rPQ660: flagellar hook-associated protein 1 (flgK) 1491 ₅8 Cell envelope; Surface structures rP0823: desulfoferrodoxin (rbo) 1476 83 Energy metabolism; Electron transport

TP0171: lipoprotein, 15 kDa (tρpl5) 1473 123 Cell envelope; Membranes, lipoproteins, and porins rP0470: conserved hypothetical protein 1421 99 + TP0327: cationic outer membrane protein (ompH) 1412 47 + TP0841: periplasmio serine protease DO (htrA-2) 1355 72 Translation; Degradation of proteins, peptides, and glycopeptides

TP0751: hypothetical protein (laminin binding protein) 1348 42 +

TP0750: hypothetical protein 1294 54 + rPQ₅Q9: alkyl hydroperoxide reductase (ahpC) 1267 51 Cellular processes; Detoxification

TP0583: hypothetical protein 1233 56

IPO 163: ABC transporter, periplasmic binding protein (troA) 1054 81 Transport and binding proteins;

TP0942: hypothetical protein 1052 37

TP0298: exported protein (tpn38b) 1023 37 Transport and binding; rP0395: integrase / recombinase (xprB) 1012 78 Replication; DNA replication, restriction, recombination, and repair

TPQ456: hypothetical protein 996 32

TP0072: conserved hypothetical protein 983 34

TP0122: phosphoenolpyruvate carboxykinase (pckA) 967 60 Energy metabolism; Glycolysis rPQQS6: oxaloacetate decarboxylase, subunit alpha (oadA) 965 20 Amino acid biosynthesis;

Arginine, Proline Energy metabolism rP0₅07: ATP-dependent CIp protease proteolytic component (clpP-1) 944 44 Translation; Degradation of proteins, peptides, and glycopeptides rP0S71: Tp7Q protein 943 39 +

TP0247: N-acetylmuramoyl-L-alanine amidase (amiA) 927 57 + Cell envelope; Murein sacculus and peptidoglycan

FP0216: heat shock protein 70 (dnaK) 925 63 Cellular processes; Chaperones rP0817: enolase (eno) 920 61 Energy metabolism; Glycolysis rP036₅: chemotaxis protein (cheX) 918 47

TP0329: serine hydroxymethyltransferase (glyA) 889 37 Amino acid biosynthesis; Serine family

TP0127: hypothetical protein 871 129

TP0474: conserved hypothetical protein 852 24

TP0220: anti-sigma F factor antagonist (spoIIAA-1) 814 40 Regulatory functions rP0622: hypothetical protein 793 51

TP0604: ribosome recycling factor 788 28 Translation; Translation factors

TP0222: hypothetical protein 783 85

TP0037: D-specifϊc D-2-hydroxyacid dehydrogenase 736 61 Energy metabolism;

Fermentation rP0₆63: outer membrane protein, putative 712 58 Cell envelope; Surface structures

TP0767: translation elongation factor O (fiisA-2) 692 29 Translation; Translation factors

TP0369: hypothetical protein 684 35 +

TP06₅₅: spermidine/putrescine ABC transporter (potD) 668 24 + Transport and binding proteins;

Amino acids, peptides and amines

ΓP0₅46: periplasmic serine protease, putative 650 77 Translation; Degradation of proteins, peptides, and glycopeptides rP08₅6: hypothetical protein 631 37 + rP08₅8: hypothetical protein 628 23 +

TP0240: ribosomal protein L7/L12 (rplL) 601 35 Translation; Ribosomal proteins rP0030: heat shock protein (groEL) 601 28 Cellular processes; Chaperones

TPQ₆₀₆: ribosomal protein S2 (rpsB) 599 32 Translation; Ribosomal proteins

TP0465-. hypothetical protein 564 40

TPlOlO: nucleoside-diphosphate kinase (ndk) 559 39 Purines, pyrimidines, nucleosides, and nucleotides; Nucleotide and nucleoside interconversions

IP0437: hypothetical protein 549 10

TP0085: PTS system, nitrogen regulatory HA component (ptsN-1) 545 36 Transport and binding proteins;

IP0494: conserved hypothetical protein 524 30

TP0490: conserved hypothetical protein 521 24

TP0412: conserved hypothetical protein 510 23

TP0399: flagellar basal-body M ring protein (fliF) 503 25 Cell envelope; Surface structures rP0₅37: triosephosphate isomerase (tpi) 494 29 Energy metabolism; Glycolysis

TP0047: conserved hypothetical protein 481 11 rPQQ29: UDP-N-acetylglucosamine 1-carboxyvinyltransferase 465 36 Cell envelope; Murein sacculus (murA) and peptidoglycan

TP1038: bacterioferrin (TpFl) 4₅7 25 Cell envelope; Surface polysaccharides and antigens rP0368: hypothetical protein 4₅₆ 20 +

TP1017: alanyl-tRNA synthetase (alaS) 38S 52 Translation; Amino acyl tRNA synthetases rP031₅: hypothetical protein 379 18 rP1040: lysyl-tRNA synthetase (lysS-2) 373 26 Translation; Amino acyl tRNA synthetases

TP1039: adenine phosphoribosyltransferase (apt) 364 32 Salvage of nucleosides and nucleotides

Example 4

Humoral immune response in rabbits over the course of infection [0184] The GST fusion proteins were next used to monitor the development of the humoral immune response in rabbits against T. pallidum infection (Fig. 4, Table 2). Serum was collected and pooled from three rabbits prior to inoculation of T. pallidum and again at 7, 14, 28, 56, and 84 days after intratesticular inoculation. For this analysis, 74 of the 106 reactive proteins identified in the global screen were selected (Table 2).

[0185] Overall, little reactivity was observed between serum antibodies from uninfected rabbits and T. pallidum proteins (Fig. 4A). At 7 days post infection, however, significant levels of antibodies against the following proteins were detected in rabbit sera: TP0971- membrane antigen TpD₅ TP0574- 47 kDa carboxypeptidase and TP0684- methylgalactoside ABC transporter, periplasmic galactose-binding protein (MglB-2) (Fig. 4B). Antibodies against two other proteins, TP0727-flagellar hook protein (FIgE) and TP0319-membrane lipoprotein (TmpC), were also present in concentrations slightly greater than that observed prior to infection.

[0186] Antibodies present in rabbit sera 14 days post infection bound to 17 of the 74 proteins arrayed (Fig. 4C). The most reactive proteins at this time point corresponded with those seen at 7 days post infection, including MglB-2, the 47 kD carboxypeptidase and TmpC. An increase in the diversity of antibodies detected in infected rabbit serum can be visualized from 7 to 28 days post infection. Interestingly, there was little difference in reactivity between 28 and 56 days post infection.

[0187] The level of antibodies against some antigens developed early and then declined by 84 days. For example, the assay signal for the five proteins, TP0037 (D-lactate dehydrogenase), TP0329 (serine hydroxymethyl transferase), TP0841 (periplasmic serine protease DO), TP0618 (hypothetical protein) and TP0971 (membrane lipoprotein, TpD)₅ was greater at 14 days post infection than at 84 days post infection. The same can be said for 31 proteins at 28 days post infection, and 33 proteins at 56 days post infection (Table 2).

[0188] In contrast, the development of antibodies against 11 T. pallidum antigens was relatively low until 84 days post infection. This group of proteins included TP0327, TP0453, TP0693, TP0954, TP0292, TP0225, TP0625, TP0772, TP0257, TP0956 and TP0993. The antibody concentrations against these 11 proteins at 7, 14, 28 and 56 days post infection remained low (equal to or less than half of the signal for those same 11 proteins at 84 days post infection). A significant increase in antibody levels against these 11 proteins was observed at 84 days post infection. The importance of this finding is that rabbits typically exhibit only partial immunity to re-infection one to two months after intratesticular inoculation of T. pallidum. However, by three months post inoculation rabbits exhibit chancre immunity (Turner and Hollander, 1957). The increase in antibodies against one or all 11 of these proteins may be responsible for preventing T. pallidum re-infection in rabbits and therefore represent potentially protective antigens.

Table 2 Rabbit humoral immune response over the course of a T. pallidum infection.

Sample ORF Priorto STDV³ 7 Days STDV³ 14 Days STDV³ 28 Days STDV³ 56 Days STDV³ 84 Days STDV³

Number' Infection² P.I.²'⁴ P.I.² P.I.² P.I.² P.I.²'

1 gst 293 47 354 65 521 48 757 229 1140 341 380 115

2 gst 285 69 343 SO 517 41 737 228 1102 213 424 215

3 TP444 241 61 283 18 601 88 964 220 1110 315 735 218

4 TP222 264 79 310 40 487 77 815 177 1241 81 757 212

5 TP474 262 57 311 37 508 61 619 80 936 164 795 132

6 TP434 267 52 342 40 571 102 796 78 1004 277 799 284

7 TP663 272 60 345 50 620 55 1131 105 1361 325 826 160

8 TP583 289 69 349 34 575 94 3637 253 3602 280 843 98

9 TP30 400 57 454 60 669 102 1062 204 1282 205 864 158

10 TP72 253 52 328 46 563 84 1512 136 1646 107 888 98

11 TP37 287 54 390 33 1031 150 1752 124 1894 271 892 228

12 TP456 232 69 320 64 508 141 744 200 899 244 948 145

13 TPlOOl 221 76 282 54 451 91 1411 336 1491 344 965 225

14 TP56 235 45 319 38 528 99 672 171 922 285 970 32

15 TP88 249 49 304 45 486 74 1607 537 1681 374 1001 262

16 TP247 230 61 278 41 556 107 1478 552 1540 408 1021 433

17 TP877 220 38 287 37 513 81 643 138 965 193 1025 121

18 TP216 227 41 255 44 640 45 1586 137 1749 84 1053 106

19 TP509 282 66 310 41 545 70 781 70 1001 166 1063 193

20 TP163 255 66 307 44 531 122 1075 290 1425 444 1085 486

. 21 TP942 264 49 290 34 537 60 620 157 862 209 1211 262

22 TP369 356 69 347 35 572 102 853 194 1019 122 1233 38

23 TP571 234 64 290 39 507 108 835 248 1107 212 1284 89

24 TP750 266 77 310 48 806 174 3625 495 3477 271 1374 46

25 TP470 224 51 284 41 444 86 686 108 823 111 1374 339

26 TP365 287 66 322 44 523 48 2924 168 2671 181 1453 30

27 TPS23 247 54 298 32 547 90 672 178 869 181 1479 232

28 TP171 222 49 302 57 502 141 1066 87 1164 169 1652 219

29 TP841 274 81 349 60 2396 472 5065 774 4803 453 1669 212

30 TP433 239 46 265 26 461 72 805 191 924 152 1713 163

31 TP298 275 45 304 39 512 57 1091 225 1307 107 1767 363

32 TP751 328 108 350 50 593 117 2309 151 2385 247 1767 189

33 TP957 248 63 293 38 553 82 1070 189 1205 99 1778 336

34 TP769 520 139 553 35 1309 15 4710 259 4785 541 1902 28

35 TP327 273 57 306 33 454 80 749 120 851 168 1969 220

36 TP329 1682 234 1692 265 3307 363 2835 520 2925 563 2041 281

37 TP660 320 32 356 35 587 80 1242 691 1418 739 2119 1494

38 TP3S9 252 55 303 44 2375 269 6961 1130 6289 541 2392 338

39 TP468 230 42 279 34 492 53 2227 355 2094 175 2466 153

40 TP862 240 50 351 27 724 81 4467 159 3914 217 2561 236

41 TP453 258 65 301 59 499 76 967 170 1238 119 2758 626

42 TP895 266 78 323 47 601 133 1469 324 1731 212 2792 436

43 TP307 252 49 285 33 656 57 1911 90 2002 225 3044 290

44 TP633 246 46 301 37 525 69 1927 65 2015 192 3127 101

45 TP136 261 52 312 39 567 97 2477 235 2497 62 3295 436

46 TP469 241 51 298 55 718 328 1972 783 2101 688 3296 1449

47 TP965 261 55 403 39 956 84 3063 77 2919 305 3370 162

48 TP133 210 46 284 54 584 126 3545 348 3334 147 3572 613

49 TP821 356 52 440 65 788 123 3304 377 3058 409 3593 872

50 TP486 260 59 344 56 823 176 2922 239 2803 21 3596 163

51 TP618 248 56 288 39 6257 188 7433 481 7219 184 3790 68

52 TP789 338 81 388 37 822 59 4024 221 3989 861 3950 508

53 TP693 257 64 291 55 683 220 1604 307 1682 149 4402 702

54 TP954 285 64 339 60 688 93 1532 522 1813 440 4857 640

55 TP271 252 52 297 55 642 69 5331 684 5334 272 5090 602

56 TPl83 228 50 392 70 1638 500 13936 430 13334 485 5407 683

57 TP727 284 64 552 78 2433 180 9667 851 8897 152 6056 324

58 TP292 249 56 303 48 618 106 2842 231 2802 116 6629 384

59 TP1002 528 106 649 96 1189 190 6043 575 5296 264 6765 285

60 TP319 246 52 752 151 7025 1152 15747 798 14380 1359 7856 939

61 TP567 283 56 343 47 1752 122 10393 446 10039 392 8306 528

62 TP225 257 39 340 43 746 97 4762 880 4340 1030 8803 2641

63 TP1016 310 73 341 35 1540 133 9532 794 8699 346 8853 234

64 TP463 248 49 292 32 3314 377 10944 458 10368 51 9185 942

65 TP971 228 53 1776 531 12402 3488 15250 2307 14321 3165 9559 1523

66 TP625 248 61 313 48 675 196 1544 587 1515 349 9581 1071

67 TP772 242 59 317 62 842 256 4158 1044 3733 577 9941 1703

68 TP257 254 62 338 62 988 99 5264 794 4587 516 10126 2185 69 TP326 243 60 381 123 3368 700 11469 2571 10629 820 11839 1008

70 TP956 364 102 393 64 607 78 1056 35 1170 210 12414 2153

71 TPlOO 271 61 320 59 1124 155 11227 396 10593 365 13226 582

72 TP574 252 61 1948 434 12997 2460 18774 1888 17302 2641 17085 1915

73 TP768 247 58 422 55 6488 704 21036 877 22500 4723 19462 1298

74 TP993 279 70 469 166 2719 569 9866 974 9151 750 21028 1294

75 TP684 254 71 3169 657 15129 2170 20163 1406 19609 2606 22047 1851

76 TP435 378 72 458 51 3351 141 23067 473 21898 364 26513 813

1 Sample number corresponds to the sample number on Figure 4

2 The average chemiluminescence data from three experiments is presented in light units and is graphed in Figure 4

3 Three experimental data points were used to calculate the standard deviation

4 P.I. is post infection

Example 4

Immunoassay of human humoral immune response [0189] To identify T. pallidum proteins that elicit a humoral immune response in syphilis patients, the immunoassay was again utilized. Human serum samples were previously collected in Harris County, Texas from normal human subjects and from patients diagnosed with primary, secondary and early latent syphilis. Arrays using human serum differ from rabbit serum arrays in that there are many variables that cannot be avoided when working with sera collected from humans diagnosed with syphilis. For example, the rabbits are housed in temperature-controlled environments, the infections are synchronized and monitored daily, and the number of organisms used to infect each rabbit is consistent between animals. With human sera, patients vary considerably in terms of the extent of the infection or host immune status. In an attempt to minimize the effect of patient-specific anomalies, the patients' sera from various stages of disease were pooled for these experiments. The sera were pooled prior to the ELISA experiments as follows: six sera pooled for normal human sera, two patients' sera pooled for primary, nine patients' sera pooled for secondary, and five patients' sera was pooled for early latent samples.

[0190] To prepare absorbed human serum, E. coli BL21* DE3 was grown overnight at 37 °C in 150 niL of LB liquid media. Cultures were centrifuged and the resulting cell pellets were re-suspended in 10 mL Bacterial Protein Extraction Reagent (B-PER) (Pierce, Rockford, Illinois) containing 0.375 mg/niL lysozyme and 420 ng/niL DNasel, then shaken for 10 minutes at room temperature. The lysate was centrifuged for ten minutes at 10,000 rpm and 4⁰C. The supernatant was used in subsequent absorptions. For a 96 well plate, a mixture of 10 μL human serum, 11 niL PBS (pH 7.4) containing 1% dry milk and 1 mL BL21 *DE3 cell lysate supernatant was mixed on a rocking platform for two hours at room temperature, just prior to being used. Prepared absorbed human serum had a final serum dilution of 1 : 1200.

[0191] 110 μL of absorbed serum solution was added to each well followed by incubation for two hours at room temperature. The plates were then washed eight times with buffer A. A 1 : 12,000 dilution of goat anti-human IgG horseradish peroxidase conjugate (110 μL, Heavy + Light chain specific and affinity purified [Pierce, Rockford, Illinois]) was added to each plate well and incubated for one hour at room temperature. The plates were washed eight times before 150 μL of SuperSignal ELISA Pico Chemiluminescent Substrate (Pierce, Rockford, Illinois) was added. Light emission from each plate well was monitored at 5 and 15-minute intervals with a Genios plate reader (Tecan, Durham, North Carolina) for 200 milliseconds; the results at these two time points were comparable and averaged. Chemiluminescence was monitored once at 10 minutes after peroxidase substrate addition, and the experiments were repeated three times. Due to the increased background associated with the use of human serum when compared to rabbit serum, it was necessary to analyze the data from the human serum arrays differently than the rabbit serum arrays. To this end, the human serum data is displayed as a ratio of the chemiluminescence detected from a sample well containing a T. pallidum protein fused to GST to the chemiluminescence detected from a sample well containing only immobilized GST.

[0192] For the initial screening of reactivity, the pool of sera from five patients with early latent syphilis was used. Of the 908 proteins examined for reactivity with pooled early latent human serum, 26 proteins exhibited a ratio of 2.0 or greater while 34 displayed a ratio of 1.5 or greater. The 26 proteins are all present among the 106 proteins found to react with rabbit sera (Table 1). A panel of 90 antigenic proteins was selected for an analysis of reactivity with human sera from patients with primary, secondary, or early latent syphilis. Of the 90 proteins screened, 34 exhibited reactivity to antibodies at one or more of the syphilitic stages (Table 3). Sixteen were reactive with the pooled sera from each stage and thus may represent good candidates for immunodiagnostic antigens (Table 3). The characterization of antigens such as MglB-2, TpN47 carboxypeptidase and TmpC may be useful in immunodiagnosis in that these antigens give rise to strong, rapid antibody responses that may increase the sensitivity of diagnosis during the early stages of infection.

[0193] The 34 proteins reactive with human syphilitic sera were also members of the rabbit antigen collection from Table 1, whereas one protein, TP0974 (hypothetical protein), did not produce a detectable reaction with the rabbit sera tested. Of the 34 proteins, 16 were previously reported in the T, pallidum literature as antigens (Table 3). As seen in Table 3, no significant interactions were detected when normal human serum was incubated with the arrayed proteins. The most reactive stage was the early latent stage. Only two of the 34 proteins, TPOl 33 (hypothetical protein) and TPO 136 (hypothetical protein) did not exhibit reactivity during this stage, while twelve proteins exhibit reactivity only during early latent syphilis. Of the 34 proteins that exhibit reactivity to one or more of the human sera types, 22 fall among the top 26 reactive antigens that bind to sera antibodies from T. pallidum infected rabbits indicating a correlation between high reactivity with rabbit sera and reactivity with human sera.

[0194] In the assay, 12 proteins were reactive (ratio > 2.0) with the early latent pool, but were not reactive with sera from primary or secondary syphilis patients. If the development of the humoral immune response between secondary and early latent syphilis in humans coincides with protective immunity, then the 12 proteins that exhibited reactivity only during early latency are of great interest. Four of the 12 proteins, including TPO 163 (ABC transporter, periplasmic binding protein), TP0216 (heat shock protein 70), TP0292 (outer membrane protein) and TP 1038 (bacterioferrin) have been previously identified as antigens. Seven of the remaining 8 proteins, including TP0277 (carboxyl-terminal protease), TP0327 (cationic outer membrane protein), TP0470 (conserved hypothetical protein), TP0750 (hypothetical protein), TP0789 (hypothetical protein), TP0954 (conserved hypothetical protein) and TP0956 (conserved hypothetical protein) were identified as novel antigens using sera collected from rabbits (Table 1). Included in the group of 12 early-latent specific antigens are 4 proteins that exhibited elevated reactivity at 84 days post infection in rabbits infected with T. pallidum. The significance of this finding is that by 84 days post infection, rabbits exhibit chancre immunity to re-infection but not before, a stage in the rabbit infection that parallels early latency. The one antigen that appears to be unique to the human humoral immune response, TP0974 (hypothetical protein), is present in the group of 12 antigens.

[0195] Table 3 Human humoral immune response at the primary, secondary, and early latent stages of T. pallidum infection

Example 5

Identification of adhesions using phage display [0196] Since it is known that T. pallidum binds to human extracellular matrix protein fibronection, (Cameron et al., 2004; Fitzgerald et al., 1984), the next step was to use the clone set identified in the above examples to identify a fibronectin binding protein. For this purpose, phage display was used to express the T. pallidum proteins. The genome sequence of T, pallidum strain Nichols was analyzed for genes encoding putative secreted and extra cellular proteins. A total of 165 candidate genes were identified and the corresponding univectors were converted into phage display vectors by Cre-lox recombination phage display. Each of the 165 phage display constructs was used to produce phage particles and each of these phage preparations was used to screen for clones that interact with fibronectin using a phage ELISA protocol modified from Deshayes et al. (Deshayes et al., 2002).

[0197] Briefly, E. coli cells harboring the phage display vector encoding the candidate genes were grown individually in 96 deep well plates for phage production. After induction, the supernatants containing phage particles were applied to 96 well plates coated with fibronectin. Following an extensive wash, phages binding to fibronectin were detected using antibodies to an M 13 phage coat protein.

[0198] From the ELISA data in Fig. 5, it was apparent that the background levels of binding of the phage were relatively high. Examination of the clones providing the highest binding signals may be a useful means of screening for fibronectin binding proteins (Table 4). For example, the clone exhibiting the highest signal in the ELISA assay was TPOl 55, which had previously been identified as a fibronectin binding protein (Cameron et al., 2004). The clone exhibiting the next highest signal was TPO 136 (Fig. 5). This is a hypothetical protein of unknown function. TPOl 36 was positively identified in the antigen screens with both rabbit and human sera (Brinkman et al., 2006; McKevitt et al., 2005). Also of interest were TP0020, which was annotated as 76K protein but whose function was unknown, and TP0326, which has previously been shown to be a protective antigen present on the surface of T. pallidum (Cameron et al., 2000). Table 4. Putative fibronectin proteins.

Gene Function Signal

TP0155 Fibronectin binding protein 0.100 +/- 0.035

TP0136 predicted coding region 0.091 +/- 0.032

TP0020 76K protein 0.081 +/- 0.029

TP0326 outer membrane protein 0.080 +/- 0.028

TP0034 ABC transporter, periplasmic binding protein 0.079 +/- 0.028

TP0410 protein-export membrane protein (secD) 0.075 +/- 0.027

TP0216 heat shock protein 70 (dnaK) 0.073 +/- 0.026

TP0144 ABC transporter, periplasmic binding protein 0.067 +/- 0.024

TP0013 predicted coding region 0.067 +/- 0.024

TP0346 predicted coding region 0.065 +/- 0.023

Example 6

Purification and characterization of TP0136 protein [0199] In order to confirm the suggestion that TP0136 binds fibronectin as indicated in the initial ELISA results in Fig. 5 , the TPO 136 protein was expressed in E. coli with a 6x Histidine-tag sequence for purification. High level expression of TPOl 36 resulted in aggregation and inclusion body formation of the protein. Extensive optimization of protein expression and purification conditions allowed the purification of the insoluble TPOl 36 in the presence of 8M urea, followed by dialysis into phosphate buffered saline (PBS) solution. The refolded TPO 136 protein was soluble and the antigenicity of the purified protein was verified by ELISA using rabbit immune sera. The purified TPO 136 protein was also immobilized and detected with sera from patients with primary, secondary, and early latent syphilis, as well as with rabbit sera collected from sequential time points during an experimental rabbit infection. TPO 136 was significantly antigenic during primary, secondary, and early latent human disease stages as well as at days 14 and 90 in the rabbit model of infection, as determined by a p value < .005 when compared with the negative controls normal human sera and prechallenge rabbit sera by the Student two-tailed t test.

[0200] The purified TPOl 36 protein was also tested for fibronectin binding by ELISA (Fig. 6). For these experiments fibronectin as well as superfibronectin and the extracellular matrix protein (ECM) laminin were immobilized and probed with purified TPOl 36 protein. It was found that TP0136 bound significantly to the ECM proteins fibronectin and laminin as indicated a p value < .005 when compared with attachment to the negative control BSA by the Student two-tailed t test (Fig. 6 ). The binding appeared specific in that significant binding to BSA, fetuin or collagen was not detected.

[0201] Once TPOl 36 was verified as a fibronectin binding protein, the region responsible for the binding was examined. For this purpose, a peptide array consisting of overlapping peptides encompassing the TPOl 36 protein was synthesized using standard FMOC chemistry and probed with fibronectin (Reineke et al.₅ 2001). The amino acid sequence of TPOl 36 was arrayed on a cellulose membrane using a sliding 12 amino acid peptide window that moved 5 amino acids for each sequential peptide along the sequence of TPO 136. This resulted in a peptide array such that the first spot on the peptide array contained amino acids 1-12 of the sequence, the second spot contained amino acids 5-17 of the sequence, and so on until the end of the amino acid sequence. The specific peptides that bound fibronectin were SEQ ID NO. 35 17- TPSIPGAIYGIV- 28, SEQ ID NO. 36 70-ATDGNTFVLACV-81, SEQ ID NO. 37 75 -TFVL AC VPGTGV- 86. These candidate peptides are synthesized as soluble peptides and tested for the ability to bind fibronectin and to disrupt the interaction between TPOl 36 and fibronectin.

[0202] Next, the cellular location of endogenous TPO 136 was determined. The location of the protein was examined by immunofluorescence with rabbit-anti- TPOl 36 antibodies (Cox et al., 1995). In this assay, antibodies to known periplasmic protein FIaA were used in double label fluorescence as a control for outer membrane integrity. Using a secondary anti-rabbit conjugated to alexafluor488 identified fluorescent treponemes in the absence of detergent suggesting an outer membrane location, but fluorescent spirochetes were also observed, albeit at lower intensity, in the secondary antibody alone control. Using directly conjugated rabbit-anti-TP0136 produced markedly dimmer fluorescence with detergent, and have failed to produce fluorescence at all in the absence of detergent. This may indicate that TPOl 36 is either periplasmic, or that the directly conjugated antibodies were unable to produce fluorescence within the detectable range of the microscope. Similar studies using immunoTEM with anti-136 antibodies and a gold conjugated secondary antibody were done in the absence of detergent (Haapasalo et al., 1992). Gold particles were observed bound to the spirochete in the absence of detergent suggesting an outer membrane location of TP0136. Example 7

Antigengic Challenge of TP0136

[0203] The ability of TPO 1.36 to act as a protective antigen to subsequent challenge with infectious T. pallidum was assessed with the rabbit model of infection to perform a challenge study. Three New Zealand white rabbits were bled and immunized with TP0136/adjuvant. Bleeding and immunization was repeated at 21 day intervals for a total of 4 immunizations. On day 84, 3 immunized rabbits as well as 3 nonimmunized control rabbits were challenged intradermally on their shaved backs with 100 μL of live T. pallidum suspension at eight sites per rabbit (10⁴ organisms/site). The animals were observed daily for lesion appearance and development, and one representative site from each animal was biopsied at day 20 post challenge for verification of the presence of treponemes by dark field microscopy. The animals were further observed until all lesions healed. As shown in Table 5, immunization with TPO 136 caused a statistically significant delay in ulceration when compared to control rabbits, but did not provide clear protection from infection with T. pallidum in the rabbit model system. The fact that the immunized rabbits had a statistically significant delay in time to chancre formation suggested immunization had some effect on the infective process.

[0204] Table 5. Immunization test of TP0136

Syphilitic lesion development following intradermal challenge of immunized rabbits

St Dev

Number of Mean Date Mean Date

Immunization Lesions/Number of of

Antigen ID of Sites Ulceration Ulceration

TPO 136 154 8/8 28.1 4.3

TPOl 36 165 8/8 30 7.1

TPOl 36 170 8/8 26.8 1

None 279 8/8 19.6 3.1

None 280 8/8 20.5 2.5

None 281 6/8 23.7 1

Example 8

Hetergencity among T. pallidum isolates

[0205] The sequence heterogeneity of the TPO 136 gene was studied among different isolates. PCR amplification followed by DNA sequencing of the TP0136 gene from the Treponeama pallidum ssp pallidum street strain 14, as well as two yaws strains Treponema pallidum ssp pertenue Gauthier strain, and Treponema pallidum ssp pertenue Samoan F strain was performed. The data revealed multiple nucleotide changes and small insertions and deletions among strains, indicating TPO 136 may be under selective pressure from the host immune response.

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[0208] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Claims

CLAIMSWhat is claimed is:

1. An immunogenic composition, comprising at least one polypeptide selected from the group consisting of TPO 133, TPO 136, TP0277, TP0327, TP0463, TP0470, TP0486, TP0625, TP0639, TP0727, TP0750, TP0769, TP0772, TP0789, TP0954, TP0956, TP0974, and TP0993 and a pharmaceutically acceptable carrier.

2. The immunogenic composition of claim 1 wherein one or more polypeptides has at least 70% homology to a polypeptide selected from the group consisting of TPOl 33, TP0136, TP0277, TP0327, TP0463, TP0470, TP0486, TP0625, TP0639, TP0727, TP0750, TP0769, TP0772, TP0789, TP0954, TP0956, TP0974, and TP0993.

3. The immunogenic composition of claim 1 wherein at least one polypeptide is selected from the group consisting of TP0277, TP0327, TP0470, TP0750, TP0789, TP0954, TP0956 and TP0974.

4. The immunogenic composition of claim 1, further comprising one or more polypeptides selected from the group consisting of TPOlOO, TPO 163, TP0216, TP0225, TP0257, TP0292, TP0319, TP0326, TP0435, TP0574, TP0684, TP0767, TP0768, TP0971, TP1016 and TP1038.

5. The immunogenic composition of claim 4 wherein one or more polypeptides has at least 70% homology to a polypeptide selected from the group consisting of TPO 133, TPOl 36, TP0277, TP0327, TP0463, TP0470, TP0486, TP0625, TP0639, TP0727, TP0750, TP0769, TP0772, TP0789, TP0954, TP0956, TP0974, TP099, TPOlOO, TPO 163, TP0216, TP0225, TP0257, TP0292, TP0319, TP0326, TP0435, TP0574, TP0684, TP0767, TP0768, TP0971, TP1016 and TP1038.

6. The immunogenic composition of claim 4 wherein at least one polypeptide is selected from the group consisting of TP0277, TP0327, TP0470, TP0750, TP0789, TP0954, TP0956, TP0974, TP0163, TP0216, TP0292 and TP1038.

7. A method of inducing an immune response in a subject comprising administering to the subject the immunogenic composition of claim 1.

8. A method of treating T. pallidum infection in a subject comprising administering to the subject the immunogenic composition of claim 1.

9. A method of inducing an immune response in a subject comprising the step of administering to the subject an immunogenic composition comprising one or more polypeptides selected from the group consisting of TP0133, TP0136, TP0277, TP0327, TP0463, TP0470, TP0486, TP0625, TP0639, TP0727, TP0750, TP0769, TP0772, TP0789, TP0954, TP0956, TP0974, and TP0993.

10. The method of claim 9 wherein one or more polypeptides has at least 70% homology to a polypeptide selected from the group consisting of TPO 133, TPOl 36, TP0277, TP0327, TP0463, TP0470, TP0486, TP0625, TP0639, TP0727, TP0750, TP0769, TP0772, TP0789, TP0954, TP0956, TP0974, and TP0993.

11. The method of claim 9 wherein at least one polypeptide is selected from the group consisting of TP0277, TP0327, TP0470, TP0750, TP0789, TP0954, TP0956 and TP0974.

12. The method of claim 9 wherein the immunogenic composition further comprises one or more polypeptides selected from the group consisting of TPOlOO, TPO 163, TP0216, TP0225, TP0257, TP0292, TP0319, TP0326, TP0435, TP0574, TP0684, TP0767, TP0768, TP0971, TP1016 and TP1038.

13. The method of claim 12 wherein one or more polypeptides has at least 70% homology to a polypeptide selected from the group consisting of TPOl 33, TPOl 36, TP0277, TP0327, TP0463, TP0470, TP0486, TP0625, TP0639, TP0727, TP0750, TP0769, TP0772, TP0789, TP0954, TP0956, TP0974, TP099, TPOlOO, TP0163, TP0216, TP0225, TP0257, TP0292, TP0319, TP0326, TP0435, TP0574, TP0684, TP0767, TP0768, TP0971, TP1016 and TP1038.

14. The method of claim 12 wherein at least one polypeptide is selected from the group consisting of TP0277, TP0327, TP0470, TP0750, TP0789, TP0954, TP0956, TP0971, TP0163, TP0216, TP0292, and TP1038.

15. A method of detecting T. pallidum antibodies in a sample comprising the steps of: a. obtaining a sample from a subject;

b. mixing said sample with one or more polypeptide antigens; and

c. detecting the presence of an immune complex.

16. A method of staging a syphilis infection in a subject comprising the step of detecting T. pallidum antibodies as defined in claim 15.

17. The method of claim 16, wherein the T. pallidum antigen is selected from the group consisting of SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34.