B-cell epitopes of treponema pallidum antigens for use in a syphilis vaccine

Abstract

B-cell epitopes of Treponema pallidum and vaccines against syphilis based on the same are described herein. The epitopes described herein can be concatenated or presented on a delivery scaffold such as a scaffold protein, virus-like particle, or nanoparticle for delivery as a vaccine. The present disclosure also provides methods of stimulating an anti-syphilis immune response in a subject using the epitopes and / or vaccine described herein.

Description

B-CELL EPITOPES OF TREPONEMA PALLIDUM ANTIGENS FOR USE IN A SYPHILIS VACCINE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 499,106, filed on April 28, 2023, which is incorporated herein by reference in its entirety as if fully set forth herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant Nos. R01AI042143, R01AI063940, R01AI123196, and U19AI44133 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING

[0003] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 31G4061.xml. The file is 69,075 bytes, was created on April 8, 2024, and is being submitted electronically via Patent Center. FIELD OF THE DISCLOSURE

[0004] The current disclosure describes B-cell epitopes of Treponema pallidum and vaccines against syphilis based on the same. The present disclosure also describes epitopes in a concatenated form or expressed on a delivery scaffold such as a scaffold protein, a virus-like particle, or a nanoparticle for use in vaccines. The vaccines can be used to selectively elicit antibodies capable of neutralizing T. pallidum. BACKGROUND OF THE DISCLOSURE

[0005] After years of steady decline during the 1990's, syphilis has become resurgent in many developed countries including the United States (US), Canada, and China, as well as in many European nations. Furthermore, it continues to be endemic in developing countries, where more than 90% of cases occur. The World Health Organization (WHO) estimates that the global syphilis burden might be as high as 22.3 million cases worldwide in adults, with an incidence of 7.1 million newly infected adults in 2020. Congenital syphilis (CS), caused by vertical transmission of the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum) is the most common infection associated with fetal loss or stillbirth in resource-poor settings. Every year, 1.4 million pregnant women become infected globally.305,000 of these women will experience fetal loss or stillbirth, while 215,000 infants will be born prematurely and with clinical signs of infection. The WHO estimated that in 2016, there were 661,000 infants born with congenital syphilis including 143,000 early fetal deaths and stillbirths, 61,000 neonatal deaths, 41,000 preterm or low-birth weight births, and 109,000 infants with a clinical diagnosis of congenital syphilis. Although the majority of congenital syphilis cases occur in countries of sub-Saharan Africa and South America, CS resurgence has also been affecting developed countries with high maternal  care standards. In the US alone, CS incidence nearly tripled from 2017 to 2021, from 941 to 2855 cases. Altogether, these data indicate a need for new syphilis preventive measures. SUMMARY OF THE DISCLOSURE

[0006] The current disclosure provides Treponema pallidum vaccine epitopes specifically designed to be recognized by B cells to fight infection. In particular implementations, T. pallidum epitopes include sequences as set forth in SEQ ID NOs: 1-12. In particular implementations, an epitope described herein can be expressed on a delivery scaffold such as a scaffold protein, a virus-like particle, or a nanoparticle. In other implementations, an epitope described herein can be expressed as a concatemer including a chimeric concatemer.

[0007] Various implementations of the present disclosure relate to syphilis epitopes and vaccines that can be used to elicit or increase an immune response to syphilis. The syphilis epitopes described herein can be used in a vaccine to prevent or decrease the severity of a syphilis infection. In particular implementations, the syphilis vaccine includes an epitope described in SEQ ID NOs: 1-12. In particular implementations, the syphilis vaccine includes an epitope described in SEQ ID NOs: 1-12 expressed on a delivery scaffold. In particular implementations, the syphilis vaccine includes an epitope described in SEQ ID NOs: 1-12 expressed as a concatemer.

[0008] In particular implementations, a vaccine as described herein includes one or more of the syphilis epitopes described herein and an adjuvant. BRIEF DESCRIPTION OF THE FIGURES

[0009] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0010] FIGs.1A, 1B. (1A) 3D model of Nichols strain TprC (TP0117) generated using I-TASSER. Loops are predicted to be surface exposed. Variability among strains is found in loops without white arrow (loops 5, and 9-11), while loops indicated with white arrow are conserved among strains. ES: extracellular space, OM: outer membrane, P: periplasm (1B) Circular dichroism (CD) analysis of re-folded full-length recombinant Nichols TprC. CD spectrum analysis yielded 4% a-helix, 48% β-sheet, 48% random coil. Analysis of non-refolded TprC was 100% random coil (not shown).

[0011] FIG.2. Multiple sequence alignment of the COOH-terminal region (amino acids (aa) 405-600) of five TprC variants found in T. pallidum subsp. pallidum isolates, where the most antigenic variability is located. External loop (ExL) 5 (not shown) is also diverse. Discrete variable regions (DVRs) are highlighted in black. ExL sequences are labeled in the top sequence.

[0012] FIG.3. Schematic of T. pallidum tprK gene (TP0897) and mechanism of antigenic variation of the variable (V) regions. The tprK gene (top) represents the expression site for the TprK protein. Seven discrete V regions (termed V1-V7) are located within the tprK open reading frame (ORF) (Centurion-Lara et al., Molecular Microbiology. 2004;52(6):1579-96 (“Centurion-Lara 2”)). Dark grey and white (C) regions are conserved among all tprK genes  sequenced to date. Cassettes that represent diverse V regions donor cassettes are found at the 5' and 3' side of the tprD gene (bottom, in light gray). Portions of these donor cassettes can be copied and inserted into the tprK locus, creating variant V region sequences. There are 52-54 donor sites, and a subset are shown in this schematic. The four NH2-terminal conserved loops of the protein (C1-C4) targeted by the TprK component of our vaccine design are represented in white.

[0013] FIGs. 4A, 4B. (4A) Transcription of IFNy by PBMC from control and immunized rabbits incubated with mitogen (ConA) or T. pallidum vaccine candidate. No IFN-y was produced by PBMC from unimmunized controls incubated with vaccine candidate. Among tested adjuvants, the Ribi-like adjuvant induced (4A) high levels of IFNγ transcription and (4B) significant protection against chancre progression (*p<0.01). This shows the efficacy of the Ribi adjuvant although antigens other than TprC and TprK were used.

[0014] FIG.5. Phagocytosis of T. pallidum in the presence of antisera against recombinant peptides, compared to normal (NRS) and infected (IRS, positive control) rabbit serum. TprK antiserum is directed against the protein conserved NH2-terminus. TprC / D antiserum is to the Tpr Subfamily I group of gene products. FlaA is a periplasmic protein (also negative control). *p<0.05 vs. NRS.

[0015] FIGs.6A, 6B. Primary chancre development in rabbits immunized with TprK conserved NH2-termial region (AA 37-273 of, for example, SEQ ID NO: 40) (6A) and unimmunized rabbits (6B). Pictures were taken 23 days post- challenge. Morgan et al., Infect Immun.2002;70(12):6811-6. This work was performed with the previously commercially available Ribi adjuvant.

[0016] FIG. 7. Phagocytosis of T. pallidum in the presence of polyclonal antisera against recombinant peptides compared to normal (NRS, negative control) and infected (IRS, positive control) rabbit serum. Tpr antisera are directed against TprC COOH-terminus and TprC central regions. *p<0.05 vs. NRS.

[0017] FIG.8. Schematic representation of the experiments. Sera collected from immunized animals will be used to evaluate B-cell epitopes (procedure #1, ELISA) and ability to opsonize (procedure #2, opsonophagocytosis assay) or neutralize (procedure #3, neutralization assay) T. pallidum. PBMCs were used to evaluate INF-y production upon stimulation (procedure #4, ELISA after stimulation) to evaluate induction of a Th1-type response. ELISA, opsonophagocytosis and neutralization assays were repeated with monoclonal antibodies (procedure #5).

[0018] FIG.9. B-cell epitope mapping using sera from rabbits (n=3) immunized with full-length refolded TprC (from the Nichols strain) and Titermax adjuvant. Sequences of recognized peptides are identified by black stars.

[0019] FIG.10. B-cell epitope mapping using synthetic peptides based on variants of the TprC sequence and sera from rabbits infected long-term (90 days) with different T. pallidum strains. Letters underneath peptides indicate strain specific variants: M=MexicoA, N=Nichols, 8=Sea81-4, U=UW249, and B=Bal3. TprC sequences of Chicago and Bal73- 1 strains are identical to Nichols. Together, the MexicoA, Sea81-4, and Nichols sequences encompass the known spectrum antigenic variability in these proteins. Peptides 1, 14, 18, 26-30, 46, 47, 51, 54-56 are all predicted to be found in TprC external loops. (Molini et al., Front Immunol.2022, 13:862491). From left to right, the x-axis reads: 1, 2,  3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, M 21, 22, M 22, 23, M23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, N 45, 46, N 46, 47, N 47, 48, N 48, 49, 50, 51, N 51, 52, N52, 53, 8 B 54, M N U 54, 8 B 55, N U 55, M U 55, 8 B N 56, M U 56, and No Ag.

[0020] FIGs.11A-11C. Effect of immunization on lesion development in rabbits infected with the syphilis agent. (11A) Rabbits immunized with the TprC antigen have attenuated lesion development when the lesion volume of immunized rabbits (black lines) is compared to the lesion volume of control animals, supporting vaccine efficacy. The “dome” shaped line indicates that over time lesions increase in volume, and then decrease due to healing. (11B) Rabbits immunized with the TprK antigen have attenuated lesion development when the lesion volume of immunized rabbits (black lines) is compared to the lesion volume of control animals, supporting vaccine efficacy. (11C) Picture of lesions in infected rabbits. Left: control rabbit not vaccinated Right: Test rabbit vaccinated with the TprC antigen, showing a decrease in lesion volume 21 days post-infection, supporting vaccine efficacy.

[0021] FIGs.12A-12C.Effect of immunization on treponemal burden in rabbits infected with the syphilis agent. (12A) Treponemal cell burden was estimated by counting bacterial cells under the microscope in lesion aspirates from rabbits immunized with TprK and TprC, compared to control animals that were not immunized but were challenged with either the Nichols or SS14 strain. Rabbits immunized with TprK and TprC have significantly fewer treponemes in their lesions compared to controls infected with the Nichols strain, supporting vaccine efficacy. (12B) Treponemal cell burden was estimated by real-time quantitative PCR in lesion aspirates from rabbits immunized with TprK and TprC, compared to control animals that were not immunized but challenged with either the Nichols or SS14 strain. Rabbits immunized with TprK and TprC have significantly fewer treponemes in their lesions compared to controls infected with the Nichols strain, supporting vaccine efficacy. (12C) Percentage of lesion ulceration in rabbits immunized with TprK, and TprC showing that fewer lesion ulcerate in rabbits immunized with The TprK and TprC antigens, supporting the efficacy of TprC-and TprK-based vaccines.

[0022] FIG.13. Visual assessment of delayed-type hypersensitivity (DTH) at 48 hours after challenge of immunized rabbits. Control rabbits do not exhibit DTH at injection sites, but rabbits immunized with TprK and TprC do. This result supports that immunization induced a T-cell response in animals.

[0023] FIGs.14A, 14B. Time to orchitis development is delayed upon receiving lymph nodes from immunized rabbits after challenge. Another way to assess vaccine efficacy is to harvest lymph nodes from vaccinated / challenge animals and inject them into one testis of a naive rabbit. If the naïve rabbit develops syphilis more slowly compared to the control rabbits, it means that the immunized animals were protected. (14A and 14B) Animals that received lymph nodes from rabbits vaccinated with TprC (14A) or TprK (14B) developed syphilis more slowly than controls, supporting vaccine efficacy.

[0024] FIG. 15. Example of epitope mapping using sera from TprC-immunized animals. To define the peptides included in this disclosure, sera from rabbits immunized with the TprC antigen were evaluated using an enzyme-linked immunosorbent assay (ELISA) to define which epitopes were predominantly recognized on regions of the protein  mapping at the host-pathogen interface. In this figure, sera were tested against a pool of peptides corresponding to external loops (ExL) of the vaccine candidate. In this figure, one can appreciate how certain ExLs (e.g., ExL1, 3, and 6) are more strongly recognized. Subsequently, the same experiment was repeated using single peptides that span each ExL, to identify the specific epitopes that elicited the protective response. Bar graph is stacked such that 214 is the bottom block and 207 is the top block. FlrC: Full-length recombinant TprC antigen.

[0025] FIGs. 16A, 16B. Example of epitope mapping using sera from TprK-immunized animals. To define the peptides included in this disclosure, sera from rabbits immunized with the TprK antigen were evaluated using an enzyme-linked immunosorbent assay (ELISA) to define which epitopes were predominantly recognized on regions of the protein mapping at the host-pathogen interface. Sera were tested against a pool of peptides corresponding to conserved external loops (CExL) of the vaccine candidate. In the panels of this figure, one can appreciate how CExL1 (16A) and CEXL2 (16B), are more strongly recognized. Subsequently, the same experiment was repeated using single peptides that span each ExL, to identify the specific epitopes that elicited the protective response. From left to right, the x-axis is labeled as integers from 1 – 40.

[0026] FIG.17. Alignment of amino acid sequences of the predicted protein sequences encoded at the tprD, tprC, tprF, and tprI loci to show where the peptides identified here map on the protein sequence and the identity level across Tpr antigens. The TprF truncated proteins in syphilis strains are also included in the alignment for clarity purposes. DVR: Discrete variable regions. EL: External loops predicted by 3D models. The last letter on the left column (strain name) indicates the locus where the predicted protein sequence is encoded.

[0027] FIG.18. Sequences for T. pallidum strains aligned in FIG.17.

[0028] FIG.19. TprK and TprC sequences. DETAILED DESCRIPTION

[0029] The current disclosure provides T. pallidum vaccine epitopes specifically designed to be recognized by B cells to fight infection. An epitope includes specific amino acids that contact the binding portions of an immunoglobulin or antibody. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, or any combination thereof, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0030] An “epitope” includes any determinant capable of being bound by an antigen-binding protein, such as a B cell receptor (BCR). An epitope is a region of molecule that is bound by a binding protein that targets that region of a molecule, and when that region of a molecule is a protein, includes specific residues that directly contact the binding protein. In particular implementations, an “epitope” denotes the binding site on a protein target bound by a corresponding binding domain. The binding domain binds to a linear epitope, (e.g., an epitope including a stretch of 5 to 12 consecutive amino acids), to a three-dimensional structure formed by the spatial arrangement of several short stretches of the protein target, or a combination thereof. Three-dimensional epitopes recognized by a binding domain,  e.g., by the epitope recognition site of a BCR or BCR fragment, can be thought of as three-dimensional surface features of an epitope molecule. These features fit precisely with the corresponding binding site of the binding domain. Accordingly, binding between the binding domain and its target protein is facilitated. In particular implementations, an epitope can be considered to have two levels: (i) the “covered patch” which can be thought of as the shadow a BCR or a binding domain would cast; and (ii) the individual participating side chains and backbone residues. Binding is then due to the aggregate of ionic interactions, hydrogen bonds, and hydrophobic interactions.

[0031] B cells mediate the humoral immune system through antibody secretion that neutralizes antigens. B cells are stimulated when the antigen receptor, which is part of the paratope, recognizes antigenic epitopes. Epitopes recognized by B cells can be classified into two types: continuous and discontinuous epitopes. Continuous epitopes (also referred to as linear or sequential epitopes) are short peptide fragments (15 amino acids in size) of an antigen protein that are specifically identified by certain antibodies. Discontinuous epitopes include amino acid residues that are not sequential in their primary structure but involve a folding mechanism that forms into a region that is close together.

[0032] In particular implementations, there are multiple strains of T. pallidum. In particular implementations, the T. pallidum strain includes a Nichols strain, a Bal3 strain, a Sea81-4 strain, UW249 strain, a MexicoA strain, an Iraq strain, a SamoaD strain, a Gauthier strain, and a TP0316 strain. In particular implementations, the T. pallidum strain includes a Nichols strain, a Sea81-4 strain, and a MexicoA strain.

[0033] Sequences for multiple T. pallidum strains are in FIG.18. These sequences are aligned in FIG.17.

[0034] In particular implementations, a T. pallidum epitope includes a sequence set forth in Table 1. Table 1. TprK- and TprC-specific Epitopes. Variants of the same epitope are listed with the same External Loop (ExL) number. Epitope Specificity and ExL Sequence SEQ ID NO

[0035] As indicated, in particular implementations, the syphilis vaccine includes a subunit vaccine. A subunit vaccine can refer to a vaccine that includes only a subunit (e.g., a single protein or protein fragment) of the pathogen that stimulates an immune response against the pathogen. For instance, a subunit vaccine omits a whole live or killed pathogen.

[0036] The term “concatenate” is broadly used to describe linking together into a chain or series. It is used to describe the linking together of nucleotide or amino acid sequences into a single nucleotide or amino acid sequence, respectively. The term “concatemerize” should be interpreted to recite: “concatenate.” Herein, a “concatemer” is a molecule containing multiple copies of a sequence linked in series.

[0037] In particular implementations, a concatemer includes 2, 3, 4, 5, 6, 7, 8, 9, or more copies of any of SEQ ID NOs: 1-12. In particular implementations, a concatemer includes 3 copies of SEQ ID NO: 1. In particular implementations, a concatemer includes 2 copies of SEQ ID NO: 2. In particular implementations, a concatemer includes 4 copies of SEQ ID NO: 3. In particular implementations, a concatemer includes 3 copies of SEQ ID NO: 4. In particular implementations, a concatemer includes 5 copies of SEQ ID NO: 5. In particular implementations, a concatemer includes 6 copies of SEQ ID NO: 6. In particular implementations, a concatemer includes 7 copies of SEQ ID NO: 7. In particular implementations, a concatemer includes 9 copies of SEQ ID NO: 8. In particular implementations, a concatemer includes 8 copies of SEQ ID NO: 9. In particular implementations, a concatemer includes 2 copies of SEQ ID NO: 10. In particular implementations, a concatemer includes 3 copies of SEQ ID NO: 11. In particular implementations, a concatemer includes 3 copies of SEQ ID NO: 12.

[0038] In particular implementations, a concatemer includes 2, 3, 4, 5, 6, 7, 8, 9, or more copies of any of cExL1, cExL2, or cExL3. In particular implementations, a concatemer includes cExL1-cExL1-cExL1, cExL1-cExL1-cExL1, cExL1-cExL1-cExL1-cExL1-cExL1, cExL1-cExL1-cExL1-cExL1-cExL1- cExL1- cExL1- cExL1- cExL1, cExL2-cExL2, cExL2-cExL2-cExL2, cExL2-cExL2-cExL2-cExL2, cExL3-cExL3, cExL3-cExL3-cExL3, cExL3-cExL3-cExL3-cExL3, or cExL3-cExL3-cExL3-cExL3-cExL3-cExL3.

[0039] In particular implementations, a concatemer includes 2, 3, 4, 5, 6, 7, 8, 9, or more copies of any of ExL1, ExL3, ExL4, ExL6, ExL9, or ExL10. In particular implementations, a concatemer includes ExL1- ExL1, ExL1- ExL1- ExL1, ExL1- ExL1- ExL1- ExL1, ExL1- ExL1- ExL1- ExL1- ExL1- ExL1, ExL1- ExL1- ExL1- ExL1- ExL1- ExL1- ExL1- ExL1- ExL1, ExL3- ExL3, ExL3- ExL3- ExL3, ExL3- ExL3- ExL3- ExL3, ExL3- ExL3- ExL3- ExL3- ExL3, ExL3- ExL3- ExL3- ExL3- ExL3- ExL3- ExL3- ExL3, ExL4-ExL4, ExL4- ExL4- ExL4, ExL4- ExL4- ExL4- ExL4- ExL4- ExL4- ExL4, ExL6- ExL6, ExL6- ExL6- ExL6, ExL6- ExL6- ExL6- ExL6- ExL6, ExL9- ExL9, ExL9- ExL9- ExL9, ExL9- ExL9- ExL9- ExL9- ExL9- ExL9- ExL9- ExL9, ExL10- ExL10, or ExL10- ExL10- ExL10.

[0040] In particular implementations, a concatemer includes a chimeric concatemer. A chimeric concatemer includes at least 2 of the sequences selected from SEQ ID NOs: 1-12. In particular implementations, a chimeric concatemer includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a first sequence selected from any one of SEQ ID NOs: 1-12 and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a second sequence selected from any one of SEQ ID NOs: 1-12, wherein the first  and second sequence are not the same. In particular implementations, the chimeric concatemer can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of a third, fourth, fifth, or more sequences.

[0041] In particular implementations, a chimeric concatemer includes SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 operatively linked together.

[0042] In particular implementations, a chimeric concatemer includes SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 operatively linked together. In particular implementations, a chimeric concatemer includes SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and / or SEQ ID NO: 12 operatively linked together. In particular implementations, a chimeric concatemer includes SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 operatively linked together. In particular implementations, a chimeric concatemer includes SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 12 operatively linked together. In particular implementations, a chimeric concatemer includes SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 12 operatively linked together.

[0043] In particular implementations, a chimeric concatemer includes cExL1-cExL2-cExL1, cExL1-cExL2- cExL3, cExL3-cExL2-cExL3, cExL2-cExL1-cExL2, cExL1-cExL3-cExL3, cExL1-cExL2-cExL2-cExL1, cExL1-cExL1-cExL2- cExL3, cExL1-cExL3-cExL1-cExL3, cExL1-cExL1-cExL1-cExL2, cExL2-cExL3-cExL3-cExL3, cExL2-cExL2-cExL1- cExL1, cExL2-cExL1-cExL1-cExL2, cExL3-cExL1-cExL3-cExL1, cExL3-cExL1-cExL2-cExL1, cExL3-cExL1-cExL1- cExL3, or cExL1-cExL2-cExL3-cExL1-cExL2-cExL3.

[0044] In particular implementations, a chimeric concatemer includes ExL1-ExL9-ExL10, ExL3-ExL6-ExL3, ExL4- ExL1-ExL1, ExL6-ExL1-ExL6, ExL9-ExL9-ExL9, ExL10-ExL3-ExL4, ExL1-ExL1-ExL4-ExL4, ExL3-ExL4-ExL3-ExL4, ExL4-ExL1-ExL9-ExL1, ExL6-ExL3-ExL10-ExL1, ExL9-ExL4-ExL3-ExL1, ExL10-ExL6-ExL6-ExL10, ExL1-ExL3- ExL4-ExL6-ExL9- ExL10, or ExL9-ExL4-ExL3-ExL1-ExL6-ExL10.

[0045] In particular implementations, a chimeric concatemer includes cExL1-ExL1-cExL1, cExL2-ExL3-cExL2, cExL3-ExL4-ExL6, ExL1-ExL6-cExL2, ExL3-ExL9-ExL1, ExL4-ExL10-ExL4, ExL6-cExL1-cExL3, ExL9-cExL2-cExL1, ExL10-cExL3-ExL10, cExL1-cExL1-ExL1-ExL1,cExL2-ExL3-ExL4-cExL3, cExL3-ExL6-ExL10-ExL9, ExL1-cExL1- ExL6-cExL3, ExL3-cExL1-ExL9-cExL1, ExL4-ExL4-ExL10-ExL4, ExL6-cExL3-cExL3-cExL1, ExL9-ExL3-ExL3-cExL2, ExL10-cExL3-ExL3-cExL2, cExL1-ExL6-cExL2-cExL3-ExL9-ExL1-ExL3-ExL4-ExL10, or cExL1-cExL2- cExL3-ExL1- ExL3-ExL4-ExL6-ExL9-ExL10.

[0046] Various implementations of the present disclosure relate to syphilis epitopes and vaccines that can be used to elicit or increase an immune response to syphilis. The syphilis epitopes described herein can be used in a vaccine to prevent or decrease the severity of a syphilis infection. In particular implementations, the syphilis vaccine includes an epitope described in SEQ ID NOs: 1-12. In particular implementations, the syphilis vaccine includes an epitope described in SEQ ID NOs: 1-12 expressed on a delivery scaffold. In particular implementations, the syphilis vaccine includes an epitope described in SEQ ID NOs: 1-12 expressed as a concatemer.

[0047] In particular implementations, the syphilis epitope is presented on a delivery scaffold. In particular implementations, the delivery scaffold includes a scaffold protein, a virus-like particle, or a nanoparticle.

[0048] In particular implementations, a vaccine as described herein includes one or more of the syphilis epitopes described herein and an adjuvant. Adjuvants are described in more detail elsewhere herein.

[0049] Aspects of the current disclosure are now described in more supporting detail as follows: (I) Delivery Scaffolds; (II) Recombinant Production; (III) Composition for Administration; (IV) Methods of Use; (V) Kits; (VI) Example Clauses; (VII) Experimental Examples; and (VIII) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0050] (I) Delivery Scaffolds. The epitopes disclosed herein can be expressed on a delivery scaffold. A delivery scaffold can confer desired properties to the epitope, for example, a delivery scaffold may improve the immunogenicity of an antigen, e.g., by altering the structure of the antigen, by altering uptake and processing of the antigen, and / or by allowing the antigen to bind to a binding partner. In particular implementations, a delivery scaffold includes an epitope- scaffold protein, a virus-like particle, or a nanoparticle. An epitope expressed on a delivery scaffold forms an epitope- scaffold molecule. In particular implementations, an epitope-scaffold molecule includes an epitope expressed on an epitope-scaffold protein, an epitope expressed on a virus-like particle, or an epitope expressed on a nanoparticle.

[0051] An epitope-scaffold protein is a chimeric protein that includes an epitope sequence fused to a heterologous "acceptor" scaffold protein. Design of the epitope-scaffold is performed, for example, computationally in a manner that preserves the native structure and conformation of the epitope when it is fused onto the heterologous scaffold protein. The use of such scaffold proteins is well known in the art and such methods and techniques are described in WO 2011 / 050168 and US 2010 / 0068217 and the skilled person can follow methods described therein and apply them to the present invention.

[0052] Scaffold proteins are useful for creating immunogens of the present invention in that they hold contact residues in the immunogen in the proper spatial orientation to facilitate interaction between such residues and contact residues of the antigen.

[0053] “Superposition” epitope-scaffolds are based on scaffold proteins having an exposed segment with similar conformation as the target epitope—the backbone atoms in this “superposition-region” can be structurally superposed onto the target epitope with minimal root mean square deviation (RMSD) of their coordinates. Suitable scaffolds are identified by computationally searching through a library of protein crystal structures; epitope-scaffolds are designed by putting the epitope residues in the superposition region and making additional mutations on the surrounding surface of the scaffold to prevent clash or other interactions with the antibody.

[0054] “Grafting” epitope-scaffolds utilize scaffold proteins that can accommodate replacement of an exposed segment with the crystallized conformation of the target epitope. For each suitable scaffold identified by computationally searching through all protein crystal structures, an exposed segment is replaced by the target epitope and the surrounding sidechains are redesigned (mutated) to accommodate and stabilize the inserted epitope. Finally,  as with superposition epitope-scaffolds, mutations are made on the surface of the scaffold and outside the epitope, to prevent clash or other interactions with the antibody. Grafting scaffolds require that the replaced segment and inserted epitope have similar translation and rotation transformations between their N- and C-termini, and that the surrounding peptide backbone does not clash with the inserted epitope. One difference between grafting and superposition is that grafting attempts to mimic the epitope conformation exactly, whereas superposition allows for small structural deviations.

[0055] “De novo” epitope-scaffolds are computationally designed from scratch to optimally present the crystallized conformation of the epitope. This method is based on computational design of a novel fold (Kuhlman, B. et al.2003 Science 302:1364-1368). The de novo allows design of immunogens that are both minimal in size, so they do not present unwanted epitopes, and also highly stable against thermal or chemical denaturation.

[0056] The epitope-scaffold protein can include an appropriate protein from nature or synthetic. The epitope-scaffold protein can be derived from bacterial proteins, viral proteins, or eukaryotic proteins. In particular implementations, the epitope-scaffold protein includes a Treponema pallidum protein (Tp0751), small-synthetic β-barrel protein, Protein Z from S. aureus, Cag-Z from H. pylori, p26 capsid protein from equine infectious anemia virus, or transmembrane domain of diphtheria toxin (DTT).

[0057] Treponema pallidum protein (Tp0751), also referred to as pallilysin, is a protein from Treponema pallidum that displays a propensity to interact with the extracellular matrix. Structural analysis revealed an eight-stranded beta-barrel with a profile of short, conserved regions consistent with a non-canonical lipocalin fold. In particular implementations, Tp0751 [Treponema pallidum] includes the sequence: GSMASHGNAPPAPVGGAAQTHTQPPVQTAMRIALWNRATHGEQGALQHLLAGLWIQTEISPNSGDIHPLLFFDREHA EITFSRASVQEIFLVDSAHTHRKTVSFLTRNTAISSIRRRLEVTFESHAVIHVRAVEDVARLKIGSTSMWDGQYTRYHA GPASAPSPAAA (SEQ ID NO: 42).

[0058] A small beta barrel is a protein structural domain, highly conserved throughout evolution and hence exhibits a broad diversity of functions. Small beta barrels function as a single domain protein, or as part of a multi-domain protein. They also function as quaternary structures through toroid rings assembled from individual small beta barrels. Small beta barrels interact with RNA, DNA and protein; sometimes with two partners simultaneously. Small barrels are evolutionary ancient, being found in viruses, bacteria, archaea and eukaryotes and act as fundamental components in diverse biological processes. In particular implementations, the epitope-scaffold protein includes a small-synthetic β- barrel protein. In particular embodiments, the small-synthetic β-barrel includes the sequence: MEQKPGTLMVYVVVGYNTDNTVDVVGGAQYAVSPYLFLDVGYGWNNSSLNFLEVGGGVSYKVSPDLEPYVKAGFE YNTDNTIKPTAGAGALYRVSPNLALMVEYGWNNSSLQKVAIGIAYKVKD (SEQ ID NO: 60).

[0059] Staphylococcal protein A (SpA) is a cell-wall-bound pathogenicity factor from the bacterium Staphylococcus aureus. The protein A specific binding protein, or Z-domain contains three well-defined alpha-helices corresponding to polypeptide segments Lys7 to Leu17 (helix 1), Glu24 to Asp36 (helix 2), and Ser41 to Ala54 (helix 3). In particular  implementations, protein A [Staphylococcus aureus] includes the sequence: MKKKKIYSIRKLGVGIASVTLGTLLISGGVTPAANAAQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANV LGEAQKLNDSQAPKADAQQNKFNKDQQSAFYEILNMPNLNEEQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPKA DNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLP NLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPKEEDNNKPGKEDGNKPGKEDGNKPGKEDNKKPGKEDGNK PGKEDNKKPGKEDGNKPGKEDGNKPGKEDGNKPGKEDGNKPGKEDGNGVHVVKPGDTVNDIAKANGTTADKIAAD NKLADKNMIKPGQELVVDKKQPANHADANKAQALPETGEENPFIGTTVFGGLSLALGAALLAGRPSPNYKNKQYTTID IILSKPILTYIR (SEQ ID NO: 43).

[0060] In particular implementations, the Z-domain of protein A includes the sequence: ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 44).

[0061] Cag-Z from Heliobacter pylori, regulates Cagβ-mediated CagA transport. The protein includes a single compact L-shaped domain, composed of seven alpha-helices including 70% of the total residues. The presence of a disordered C-terminal tail and the nature of the molecular surface suggest that CagZ may participate in the interaction of effector proteins with one or more components of the H. pylori type IV secretion system on the cytoplasmic side of the inner membrane. In particular implementations, CagZ [Heliobacter pylori] includes the sequence: MELGFNETERQKILDSNKSLMGNANEVRDKFIQNYAASLKDSNDPQDFLRRVQELRINMQKNFISFDAYYNYLNNLVL ASYNRCKQEKTFAESTIKNELTLGEFVAEISDNFNNFMCDEVARISDLVASYLPREYLPPFIDGNMMGVAFQILGIDDF GKKLNEIVQDIGTKYIILSKNKTYLTSLERAKLITQLKLNLE (SEQ ID NO: 45).

[0062] Equine infectious anemia virus (EIAV) is a lentivirus having a single-stranded RNA genome of 8.2 kb and contains three major genes, gag (structural), pol (enzymes), and env (surface glycoproteins), which are flanked by long terminal repeats (LTRs). The protein p26, encoded by the gag gene, is a capsid protein with high antigenicity. In particular implementations, p26 (gag protein) [equine infectious anemia virus] includes the sequence: MGDPLTWSKALKKLEKVTVSGSQKLTTGNCNWALSLVDLFHDTNFTKEKDWQLRDVIPLLEDVSQTLSGLEREAFEK TWWAISAVKMGLQINNAGDGKASFQLLKVKYEKKATGKRQPEPPEEYPIMIDGAGNRNFRPLTPRGYTTWVNTIQQN NLLNEASVNLFGILSVDCTSEEMNAFLDVVPGQAGQKQVLLDLLDKIAEDWDNRHPLPNPPLVAPAQGPIPMTARFIR GLGVPRERQMEPAFDQFRQTYRQWIIEAMTEGIKIMIGKPKAQNIRQGPKEPYPEFVDRLLSQIKSEGHPTEITKFLTD TLTIQNANEECKNAMRHLRPEDTLEEKLYACRDIGTTKQKMMLFAKALQAGLAGSMKGGIIKGGPPRAKQTCYNCGK PGHLSSQCRAPKVCFKCKEPGHFSKQCKALPKNGKQGAQGRPQKQTFPVQQSHPAKSNQGERVQTQNLYPDLTQ MKQDYQIGKEAPQDPEDLNLSSLWE (SEQ ID NO: 46).

[0063] The transmembrane (T) domain of diphtheria toxin (DTT) is known to participate in the pH-dependent translocation of the catalytic C domain of the toxin across the endosomal membrane. The transmembrane domain includes nine alpha-helices, two pairs of which are unusually apolar. In particular implementations, the T domain of diptheria toxin [Corynebacterium diphtheriae] includes the sequence: SCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANY  AAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAY NFVESIINLFQVVHNSYNRPA (SEQ ID NO: 47).

[0064] In various implementations, the epitope can be placed anywhere in the epitope-scaffold protein, as long as the resulting epitope-scaffold protein can be specifically bound by molecules of the immune system (e.g., antibodies) or properly processed for presentation on antigen presenting cells. Methods for determining if a particular epitope- scaffold protein is specifically bound by its cognate binding molecule are disclosed herein and known to the person of ordinary skill in the art (see, for example, International Application Pub. Nos. WO 2006 / 091455 and WO 2005 / 111621). In addition, the formation of an antibody-antigen complex or MHC-epitope complex can be assayed using a number of well-defined diagnostic assays including conventional immunoassay formats to detect and / or quantitate antigen- specific antibodies. Such assays include, for example, enzyme immunoassays, e.g., ELISA, cell-based assays, flow cytometry, radioimmunoassays, and immunohistochemical staining. Numerous competitive and non-competitive protein binding assays are known in the art and many are commercially available.

[0065] In particular implementations, the epitope can be expressed on a virus-like particle (VLP). A VLP is a non- replicating, viral shell, derived from any of several viruses. VLPs are generally composed of one or more viral proteins, such as, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs lack the viral components that are required for virus replication and thus represent a highly attenuated form of a virus. The VLP can display a polypeptide (e.g., a Treponema pallidum epitope) that is capable of eliciting an immune response when administered to a subject. VLPs and methods of their production are known and familiar to the person of ordinary skill in the art, and viral proteins from several viruses are known to form VLPs. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. The formation of such VLPs can be detected by any suitable technique. Examples of suitable techniques known in the art for detection of VLPs in a medium include, e.g., electron microscopy techniques, dynamic light scattering (DLS), selective chromatographic separation (e.g., ion exchange, hydrophobic interaction, and / or size exclusion chromatographic separation of the VLPs) and density gradient centrifugation. Further, VLPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristic density banding. See, for example, Baker et al. (1991) Biophys. J.60:1445-1456; and Hagensee et al. (1994) J. Virol.68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider-Ohrum and Ross, Curr. Top. Microbiol. Immunol., 354: 53073, 2012).

[0066] In particular implementations, the virus-like particle is derived from a virus selected from adeno-associated virus (AAV), Budgerigar fledgling disease virus (BFDV), Bluetongue virus (BTV), Ebola, Enterovirus 71, Goose hemorrhagic polyoma virus (GHPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis δ virus (HDV), Hepatitis E virus (HEV), human immunodeficiency virus (HIV), Human papillomavirus (HPV), Infectious bursal disease virus (IBDV), influenza (e.g., Influenza A, Influenza A H1N1, Influenza A H3N2, avian influenza (H5N3)), Marburg virus (MVD), bacteriophage MS2, Indian peanut clump virus (IPCV), Newcastle disease virus (NDV), Norovirus (No), Norwalk virus (Nv), Physalis mottle virus (PhMV), Polyomavirus (e.g., human polyomavirus or JC polyomavirus),  parvovirus (e.g., Porcine parvovirus (PPV), canine parvovirus), Rabbit hemorrhagic disease virus (RHDV), Rotavirus, Semliki-Forest virus, Sever acute respiratory syndrome (SARS), Simian immunodeficiency virus (SIV), Simian virus 40 (SV40), Swine vesicular disease virus (SVDV), and so on. In particular implementations, the virus-like particle is derived from HEV, HIV, HPV, NDV, polyomavirus, or parvovirus. In particular implementations, the virus-like particle is an L1 capsid protein of HPV type 16. In particular implementations, the epitope is expressed on the DE loop of the L1 capsid protein of HPV type 16.

[0067] A VLP can be any size but viruses typically range in size from 20 nm – 200 nm. In particular implementations, a VLP can be 0 – 500 nm in size, 20-200 nm in size, 30-100 nm in size, or 50-60 nm in size. In particular implementations, a VLP is 50-60 nm in size. In particular implementations, a VLP is 55 nm in size. A VLP that is 55 nm in size has optimal drainage to the lymph nodes.

[0068] In particular implementations, an epitope can be expressed on a nanoparticle. The term nanoparticle includes particles of spherical, elliptical, elongated shape or irregular structure. Nanoparticles can include one or more materials. Nanoparticles also include nanocapsules and polyplex particles. Nanocapsules are nanoparticles containing a reservoir, for example an oil reservoir, which is surrounded by a polymer wall (Maria J. Alonso, Microparticulate systems for the delivery of proteins and vaccines, S. Cohen And Howard Bernstein, Marcel Dekker, New York 1996, p206). A nanoparticle has dimensions controlled on the nanometer scale. For example, a nanoparticle typically has at least one dimension less than 1000 nm or 1 to 1000 nm. Still a nanoparticle can refer to particles with larger dimensions. A nanoparticle can have a diameter of 5000 nm, 2000 nm, 2500 nm, 1000 nm, 900 nm, 700 nm, 500 nm, 250 nm, 100 nm, 50 nm, 30 nm, 10 nm, 1 nm, or any size therebetween. In particular implementations, a nanoparticle is 0 – 500 nm in size, 20-200 nm in size, 30-100 nm in size, or 50-60 nm in size. In particular implementations, a nanoparticle is 50- 60 nm in size. In particular implementations, a nanoparticle is 55 nm in size.

[0069] A nanoparticle can include one or more materials, for instance. In particular implementations, the nanoparticle is made of self-assembling proteins including ferritin, lumazine, and encapsulin. To construct nanoparticles including the disclosed epitope, the epitope is linked to a subunit of the nanoparticle (such as ferritin, lumazine, or encapsulin). The fusion protein self-assembles into a nanoparticle under appropriate conditions. In other implementations, the epitope can be linked to the nanoparticle using linker chemistry or a peptide linker.

[0070] In particular implementations, a self-assembling protein can be used in a nanoparticle. Self-assembling proteins include ferritin, lumazine, and encapsulin. Ferritin is a protein whose primary function is intracellular iron storage. Ferritin is a protein whose main function is intracellular iron storage. Ferritin is composed of 24 subunits, each composed of four alpha-helical bundles that self-assemble into quaternary structures with octahedral symmetry (Cho KJ et al. J Mol Biol.2009; 390: 83). -98). Several high-resolution structures of ferritin were determined by confirming that Helicobacter pylori ferritin was composed of 24 identical protomers, whereas in animals it had ferritin light and heavy chains that could assemble alone or bind at different rates into 24 subunit particles. (Granier T. et al. J Biol Inorg  Chem.2003, 8:105-111; Lawson DM et al. Nature.1991, 349:541-544). Ferritin self-assembles into nanoparticles with strong thermal and chemical stability. Thus, ferritin nanoparticles are well suited for antigen delivery and exposure.

[0071] Lumazine synthetase (LS) is also well suited as a nanoparticle platform for antigen presentation. Responsible for the penultimate catalytic step in the biosynthesis of riboflavin, LS is an enzyme present in a variety of organisms including archaea, bacteria, fungi, and plants (Weber S.E. Flavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology.2014). The LS monomer is 150 amino acids long and includes a beta-sheet with tandem alpha-helical flanking on its sides. A number of different quaternary structures have been reported for LS, showing morphological diversity ranging from homopentamers to symmetrical assembly of 12 pentamers forming 150 Å diameter capsids. LS cages of more than 100 subunits have also been described (Zhang X. et al. J Mol Biol.2006, 362:753-770).

[0072] Encapsulins, novel protein cage nanoparticles isolated from the thermophilic bacterium Thermotoga maritima, can also be used as a platform to present antigens on the surface of self-assembling nanoparticles. The encapsulin is assembled from 60 copies of the same 31 kDa monomer with a thin, icosahedral T = 1 symmetrical cage structure with inner and outer diameters of 20 and 24 nm, respectively (Sutter M. et al. Nat Struct Mol Biol.2008, 15: 939-947). Although the exact function of the encapsulin in T. maritima is not yet clearly understood, its crystal structure has recently been resolved, and its function is related to the oxidative stress response of DyP (destaining peroxidase) and Flp (ferritin-like) proteins.

[0073] The term “nanoparticle” can also be used to describe other types and materials of particles including nanoshells, nanobeads, or nanodots. Nanoparticles can be made from organic matter and / or inorganic matter. They can be made of any suitable materials that allow for the conjugation of epitopes disclosed herein to their surface. Examples of suitable materials include: ceramics, gold, carbon, glass (silica), polymers, and magnetic materials. Suitable polymers include polystyrene, poly-(methyl methacrylate), poly-(lactic acid), (poly-(lactic-co -glycolic acid)), polyesters, polyethers, polyolefϊns, polyalkylene oxides, polyamides, polyurethanes, polysaccharides, celluloses, polyisoprenes, methylstyrene, acrylic polymers, thoria sol, latex, nylon, Teflon cross- linked dextrans (e.g., Sepharose), chitosan, agarose, and cross-linked micelles. Additional examples include carbon graphited, titanium dioxide, and paramagnetic materials. See, e.g., "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. In particular implementations, microparticles can be made of one or more materials. In particular implementations, a nanoparticle includes a gold nanoparticle, a carbon nanoparticle or a silica nanoparticle.

[0074] (II) Recombinant Production. In particular implementations, the proteins and vaccines disclosed herein are produced from a gene using a protein expression system. Protein expression systems can utilize DNA constructs (e.g., chimeric genes, expression cassettes, expression vectors, recombination vectors) including a nucleic acid sequence encoding the protein or proteins of interest operatively linked to appropriate regulatory sequences. In particular implementations, such DNA constructs are not naturally-occurring DNA molecules and are useful for introducing DNA into host-cells to express selected proteins of interest. In particular implementations, a DNA construct that encodes a  vaccine protein can be inserted into cells (e.g., bacterial, mammalian, insect, etc.), which can produce the vaccine protein encoded by the DNA construct.

[0075] Operatively linked refers to the linking of DNA sequences (including the order of the sequences, the orientation of the sequences, and the relative spacing of the various sequences) in such a manner that the encoded protein is expressed. Methods of operatively linking expression control sequences to coding sequences are well known in the art. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1982; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1989.

[0076] Expression control sequences are DNA sequences involved in any way in the control of transcription or translation. Suitable expression control sequences and methods of making and using them are well known in the art. Expression control sequences generally include a promoter. The promoter may be inducible or constitutive. It may be naturally occurring, may be composed of portions of various naturally occurring promoters, or may be partially or totally synthetic. Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley and Reynolds, Nucleic Acids Res., 15, 2343-2361, 1987. Also, the location of the promoter relative to the transcription start may be optimized. See, e.g., Roberts et al., Proc. Natl. Acad. Sci. USA, 76:760-764, 1979.

[0077] The promoter may include, or be modified to include, one or more enhancer elements. In particular implementations, the promoter includes a plurality of enhancer elements. Promoters including enhancer elements can provide for higher levels of transcription as compared to promoters that do not include them.

[0078] For efficient expression, the coding sequences can be operatively linked to a 3’ untranslated sequence. In particular implementations, the 3’ untranslated sequence can include a transcription termination sequence and a polyadenylation sequence. The 3’ untranslated region can be obtained, for example, from the flanking regions of genes.

[0079] In particular implementations, a 5’ untranslated leader sequence can also be employed. The 5’ untranslated leader sequence is the portion of an mRNA that extends from the 5’ CAP site to the translation initiation codon.

[0080] In particular implementations, a “hisavi” tag can be added to the N-terminus or C-terminus of a gene by the addition of nucleotides coding for the Avitag amino acid sequence, “GLNDIFEAQKIEWHE” (SEQ ID NO: 48), as well as the 6xhistidine tag “HHHHHH” (SEQ ID NO: 49). The Avitag avidity tag can be biotinylated by a biotin ligase to allow for biotin-avidin or biotin-streptavidin based interactions for protein purification, as well as for immunobiology (such as immunoblotting or immunofluorescence) using anti-biotin antibodies. The 6xhistidine tag allows for protein purification using Ni-2+affinity chromatography. Other tags include: Flag tag (DYKDDDDK; SEQ ID NO: 50), Xpress tag (DLYDDDDK; SEQ ID NO: 51), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 52), Polyglutamate tag, HA tag (YPYDVPDYA; SEQ ID NO: 53), Myc tag (EQKLISEEDL; SEQ ID NO: 54), Strep tag (which refers the original STREP®tag (WRHPQFGG; SEQ ID NO: 55), STREP®tag II (WSHPQFEK SEQ ID NO: 56 (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 57), Softag 3 (TQDPSRVG; SEQ ID NO: 58), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 59).

[0081] In particular implementations, syphilis vaccines can be produced using, for example, human suspension cells and / or the Daedalus expression system as described in Pechman et al., Am J Physiol 294: R1234-R1239, 2008. The Daedalus system utilizes inclusion of minimized ubiquitous chromatin opening elements in transduction vectors to reduce or prevent genomic silencing and to help maintain the stability of decigram levels of expression. This system can bypass tedious and time-consuming steps of other protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg / l of correctly folded and post-translationally modified, endotoxin-free protein of up to 70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications. Bandaranayake et al., Nucleic Acids Res., 2011 (Nov); 39(21). In some instances, purification by chromatography may not be needed due to the purity of manufacture according the methods described herein.

[0082] In particular implementations, an epitope concatemer is produced. In particular implementations, the concatemer-encoding sequence is obtained by joining pre-synthesized DNA fragments by overlapping PCR, which are then cloned into an expression vector. In particular implementations, the expression vector includes the E. coli expression vector pET-32. In particular implementations, the concatemer-encoding sequence includes a tag for purification. In particular implementations, the tag is a 5xHis-tag.

[0083] In particular implementations, an epitope-VLP (epitope expressed on a VLP) is produced by the epitope(s) sequence being inserted into a p16L1L2 vector using primer extension to replace a portion of the delivery scaffold followed by Infusion cloning. Following plasmid synthesis, VLPs will be produced according to the protocol developed by Buck and colleagues (“Buck”; Buck et al., Journal of virology.2004, 78(2):751-7; and Cardone et al., MBio.2014, 5(4):e01104-14). Briefly, plasmids are used to transfect 7x106cells. In particular implementations, the cells are human embryonic kidney (HEK) cell line 293T, previously modified to express high levels of SV40 LT antigen to drive L1 expression from the p16L1L2 vector. Next, cells are collected and lysed using detergent. VLPs will be given time to mature by incubation of the cell lysates at an incubation temperature. In particular implementations, the incubation temperature is 30°C-50°C. In particular implementations, the incubation temperature is 37°C. VLPs can be harvested by centrifugation. In particular implementations, centrifugation includes for a select time at a select temperature with a select speed. In particular implementations, the select time is 0-12 hours, 1-5 hours, or 2-4 hours. In particular implementations, the select time is 3.5 hours. In particular implementations, the select temperature is 0-50°C, 5-30°C, 10-20°C, or 15-18°C. In particular implementations, the select temperature s 16°C. In particular implementations, the select speed is 10,000xg to 900,000xg, 100,000xg to 500,000xg, or 200,000xg to 300,000xg. In particular implementations, the select speed is 234,000xg. In particular implementations, centrifugation includes ultracentrifugation through an Optiprep (60% iodixanol) step gradient for 3.5 hours at 16°C at 234,000xg.

[0084] In particular implementations, the DNA constructs can be introduced by transfection, a technique that involves introduction of foreign DNA into the nucleus of eukaryotic cells. In particular implementations, the proteins can be  synthesized by transient transfection (e.g., in which the DNA does not integrate with the genome of the eukaryotic cells, but the genes are expressed for 24-96 hours). Various methods can be used to introduce the foreign DNA into the host-cells, and transfection can be achieved by chemical-based means including by the calcium phosphate, by dendrimers, by liposomes, and by the use of cationic polymers. Non-chemical methods of transfection include electroporation, sono-poration, optical transfection, protoplast fusion, impalefection, and hydrodynamic delivery. In particular implementations, transfection can be achieved by particle-based methods including gene gun techniques, wherein the DNA construct is coupled to a nanoparticle of an inert solid which is then "shot" directly into the target- cell's nucleus. Other particle-based transfection methods include magnet assisted transfection and impalefection.

[0085] Nucleic acid sequences encoding proteins disclosed herein can be derived by those of ordinary skill in the art. Nucleic acid sequences can also include one or more of various sequence polymorphisms, mutations, and / or sequence variants (e.g., splice variants or codon optimized variants). In particular implementations, the sequence polymorphisms, mutations, and / or sequence variants do not affect the function of the encoded protein.

[0086] Sequence information provided by public databases can be used to identify additional gene and protein sequences that can be used with the systems and methods disclosed.

[0087] (III) Composition for Administration. Syphilis therapeutics (epitopes, concatenated epitopes, epitope- scaffold molecules, vaccines, vectors, or cells modified to express a vaccine disclosed herein) can be formulated alone or in combination into compositions for administration to subjects. In particular implementations, the syphilis therapeutics include immunogenic compositions. An immunogenic composition refers to an agent that stimulates an innate and / or an adaptive immune response in a subject.

[0088] Salts and / or pro-drugs of syphilis therapeutics can also be used.

[0089] A pharmaceutically acceptable salt includes any salt that retains the activity of the syphilis therapeutic and is acceptable for pharmaceutical use. A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.

[0090] Suitable pharmaceutically acceptable acid addition salts can be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.

[0091] Suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, arginine and procaine.

[0092] A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage of a syphilis therapeutic or by hydrolysis of a biologically labile group.

[0093] In particular implementations, compositions disclosed herein include a syphilis therapeutic of at least 0.1% w / v or w / w of the composition; at least 1% w / v or w / w of composition; at least 10% w / v or w / w of composition; at least  20% w / v or w / w of composition; at least 30% w / v or w / w of composition; at least 40% w / v or w / w of composition; at least 50% w / v or w / w of composition; at least 60% w / v or w / w of composition; at least 70% w / v or w / w of composition; at least 80% w / v or w / w of composition; at least 90% w / v or w / w of composition; at least 95% w / v or w / w of composition; or at least 99% w / v or w / w of composition.

[0094] Exemplary generally used pharmaceutically acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and / or delivery vehicles.

[0095] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0096] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0097] An exemplary chelating agent is EDTA.

[0098] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0099] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0100] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the syphilis therapeutic or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on therapeutic weight.

[0101] The compositions disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intramuscular, intravesicular, oral and / or subcutaneous administration and more particularly by  intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intramuscular, intravesicular, and / or subcutaneous injection.

[0102] For injection, compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the formulation can be in lyophilized and / or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0103] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For oral solid formulations such as powders, capsules and tablets, suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g., lactose, sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and / or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents can be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. can also be used.

[0104] Compositions can be formulated as an aerosol. In particular implementations, the aerosol is provided as part of an anhydrous, liquid or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers can also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may also be formulated including a powder mix of syphilis therapeutic composition and a suitable powder base such as lactose or starch.

[0105] Compositions can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salts.

[0106] Additionally, compositions can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers including at least one syphilis therapeutic. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release one or more syphilis therapeutics following administration for a few weeks up to over 100 days. Depot preparations can be administered by injection; parenteral injection; instillation; or implantation into soft tissues, a body cavity, or occasionally into a blood vessel with injection through fine needles.

[0107] Depot formulations can include a variety of bioerodible polymers including poly(lactide), poly(glycolide), poly(caprolactone) and poly(lactide)-co(glycolide) (PLG) of desirable lactide:glycolide ratios, average molecular weights, polydispersities, and terminal group chemistries. Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers.

[0108] The use of different solvents (for example, dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof) can alter microparticle size and structure in order to modulate release characteristics. Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl- 2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.

[0109] Exemplary release modifiers can include surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68 / F127), polysorbates, Span® (Croda Americas, Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), sucrose acetate isobutyrate (SAIB), salts, and buffers.

[0110] Excipients that partition into the external phase boundary of microparticles such as surfactants including polysorbates, dioctylsulfosuccinates, poloxamers, PVA, can also alter properties including particle stability and erosion rates, hydration and channel structure, interfacial transport, and kinetics in a favorable manner.

[0111] Additional processing of the disclosed sustained release depot formulations can utilize stabilizing excipients including mannitol, sucrose, trehalose, and glycine with other components such as polysorbates, PVAs, and dioctylsulfosuccinates in buffers such as Tris, citrate, or histidine. A freeze-dry cycle can also be used to produce very low moisture powders that reconstitute to similar size and performance characteristics of the original suspension.

[0112] In certain examples, compositions include a vaccine adjuvant and / or a secondary syphilis treatment. Examples of secondary syphilis treatments are described elsewhere herein.

[0113] In certain implementations, the compositions disclosed herein may be combined with one or more adjuvants to enhance the immune response. In other implementations, the compositions are prepared without adjuvants, and thus are available for administration as non-adjuvant compositions.

[0114] In particular implementations a squalene-based adjuvant can be used. Squalene is part of the group of molecules known as triterpenes, which are all hydrocarbons with 30 carbon molecules. Squalene can be derived from certain plant sources, such as rice bran, wheat germ, amaranth seeds, and olives, as well as from animal sources, such as shark liver oil. In particular implementations, the squalene-based adjuvant is MF59® (Novartis, Basel, Switzerland). An example of a squalene-based adjuvant that is similar to MF59® but is designed for preclinical research use is Addavax™ (InvivoGen, San Diego, CA). MF59 has been FDA approved for use in an influenza vaccine, and studies indicate that it is safe for use during pregnancy (Tsai T, et al. Vaccine.2010.17:28(7):1877-80; Heikkinen T, et al. Am J Obstet Gynecol.2012.207(3):177). In particular implementations, squalene-based adjuvants can include 0.1%-20% (v / v) squalene oil. In particular implementations, squalene-based adjuvants can include 5%(v / v) squalene  oil. In particular implementations, a squalene-based adjuvant includes the RIBI Adjuvant System ® (RAS®) (Corxia Corporation, Seattle, WA). RAS® (Corxia Corporation) is a commercial adjuvant system that uses a small quantity of metabolizable squalene oil and Tween 80 surfactant in which the antigen is incorporated before emulsion in water.

[0115] In some implementations, the adjuvant may be alum. Alum refers to a family of salts that contain a monovalent cation, and a trivalent metal, such as aluminum or chromium. Aluminum hydroxide, aluminum phosphate, and potassium aluminum sulphate (alum) can each be used as an adjuvant in a vaccine. In particular implementations, vaccines can include alum in the amounts of 1-1000µg / dose or 0.1mg-10mg / dose.

[0116] Saponin-containing adjuvants can also be combined with the immunogens disclosed herein. Saponins are glycosides derived from the bark of the Quillaja saponaria Molina tree. Usually, saponins are obtained using a multi- step purification process resulting in the formation of several fractions. As used herein, the term "saponin fraction from Quillaja saponaria Molina" is used generically to describe a semi-purified or specified saponin fraction of Quillaja saponaria, or a substantially purified fraction thereof.

[0117] Liposome-based adjuvants may act as both delivery systems for subunit antigens and as immunopotentiators, and they are highly versatile adjuvants, as they can be tailored through (i) the choice of lipid composition, (ii) the inclusion of immunostimulating compounds, (iii) the choice of formulation method and (iv) the mode of antigen and immunostimulator association. In particular implementations, the adjuvant is a liposome-based adjuvant.

[0118] In particular implementations, a vaccine adjuvant can include any kind of Toll-like receptor ligand (TLR-based) or combinations thereof (e.g. CpG, Cpg-28 (a TLR9 agonist), polyriboinosinic polyribocytidylic acid (Poly(I:C)), α- galactoceramide, MPLA, Motolimod (VTX-2337, a novel TLR8 agonist developed by VentiRx), IMO-2055 (EMD1201081), TMX-101 (imiquimod), MGN1703 (a TLR9 agonist), G100 (a stabilized emulsion of the TLR4 agonist glucopyranosyl lipid A), Entolimod (a derivative of Salmonella flagellin also known as CBLB502), Hiltonol (a TLR3 agonist), and Imiquimod), and / or inhibitors of heat-shock protein 90 (Hsp90), such as 17-DMAG (17- dimethylaminoethylamino-17-demethoxygeldanamycin). In particular implementations, an adjuvant can include a TLR4 agonist glucopyranosyl lipid A (GLA) formulated in stable emulsion (GLA SE; Odegard et al. (2016) Vaccine 34(1): 101-109). In particular implementations, an adjuvant includes resiquimod (TLR 7 / 8 agonist), muramyl dipeptide (MDP, a NOD2 agonist), synthetic diacylated lipoprotein FSL-1 (TLR2 / TLR6 agonist), or GLA.

[0119] In particular implementations, one or more STING agonists are used as a vaccine adjuvant. "STING" is an abbreviation of "stimulator of interferon genes", which is also known as "endoplasmic reticulum interferon stimulator (ERIS)", "mediator of IRF3 activation (MITA)", "MPYS" or "transmembrane protein 173 (TM173)".

[0120] In particular implementations, STING agonists include cyclic molecules with one or two phosphodiester linkages, and / or one or two phosphorothioate diester linkages, between two nucleotides. This includes (3',5')-(3',5') nucleotide linkages (abbreviated as (3',3')); (3',5')-(2',5') nucleotide linkages (abbreviated as (3',2')); (2',5')-(3',5') nucleotide linkages (abbreviated as (2',3')); and (2',5')-(2',5') nucleotide linkages (abbreviated as (2',2')). "Nucleotide" refers to any nucleoside linked to a phosphate group at the 5', 3' or 2' position of the sugar moiety.

[0121] In particular implementations, STING agonists include c-AIMP; (3’,2’)c-AIMP; (2’,2’)c-AIMP; (2’,3’)c-AIMP; c- AIMP(S); c-(dAMP-dIMP); c-(dAMP-2’FdIMP); c-(2’FdAMP-2’FdIMP); (2’,3’)c-(AMP-2’FdIMP); c-[2’FdAMP(S)- 2’FdIMP(S)]; c-[2’FdAMP(S)-2’FdIMP(S)](POM)2; and DMXAA. Additional examples of STING agonists are described in WO2016 / 145102.

[0122] In another implementation, the adjuvant used in a composition of the invention herein is an attenuated lipid A derivative (ALD). ALDs are lipid A-like molecules that have been altered or constructed to lower pyrogenicity, local Shwarzman reactivity and toxicity. Useful ALDs include monophosphoryl lipid A (MLA) and 3-deacylated monophosphoryl lipid A (3D-MLA). See for example U.S. Pat. No.4,436,727 issued Mar.13, 1984, assigned to Ribi ImmunoChem Research, Inc., which discloses monophosphoryl lipid A and its manufacture. U.S. Pat. No.4,912,094 and reexamination certificate B IU.S. Pat. No.4,912,094 to Myers, et al., also assigned to Ribi ImmunoChem Research, Inc., embodies 3-deacylated monophosphoryl lipid A and a method for its manufacture.

[0123] In particular implementations, an adjuvant includes a Ribi-like adjuvant. In particular implementations, a Ribi- like adjuvant includes mono-phosphoryl lipid A (MPLA, a TLR4 agonist) from S. minnesota, synthetic trehalose dimycolate (TDM), and cell wall skeleton (CWS, a TLR2 and TLR4 agonist) from M. bovis. In particular implementations, a Ribi-like adjuvant includes mono-phosphoryl lipid A (MPLA, a TLR4 agonist) from S. minnesota, synthetic trehalose dimycolate (TDM), and muramyl dipeptide (MDP).

[0124] In particular implementations, an adjuvant can include a carbomer-lecithin-based adjuvant (e.g., Adjuplex™, Millipore Sigma, Burlington, MA; Wegmann et al. (2015) Clin Vaccine Immunol. CVI-00736).

[0125] Other immune stimulants can also be used as vaccine adjuvants. Additional exemplary small molecule immune stimulants include TGF-β inhibitors, SHP-inhibitors, STAT-3 inhibitors, and / or STAT-5 inhibitors. Exemplary siRNA capable of down-regulating immune-suppressive signals or oncogenic pathways (such as kras) can be used whereas any plasmid DNA (such as minicircle DNA) encoding immune-stimulatory proteins can also be used.

[0126] In particular implementations, the immune stimulant may be a cytokine and or a combination of cytokines, such as IL-2, IL-12 or IL-15 in combination with IFN-α, IFN-β or IFN-γ, or GM-CSF, or any effective combination thereof, or any other effective combination of cytokines. The above-identified cytokines stimulate TH1 responses, but cytokines that stimulate TH2 responses may also be used, such as IL-4, IL-10, IL-11, or any effective combination thereof. Also, combinations of cytokines that stimulate TH1 responses along with cytokines that stimulate TH2 responses may be used.

[0127] In particular implementations, the adjuvant induces a strong Th1-type response. Molecules that induce a strong Th1-type response include TLR2, TLR4, and NOD2 agonists. Molecules that induce a strong Th1-type response include MPLA, TDM, synthetic diacylated lipoprotein FSL-1 (TLR2 / TLR6 agonist), and muramyl dipeptide (MDP, a NOD2 agonist). In particular implementations, the adjuvant includes GLA, risiquimod, MDP, FSL-1, and / or Qui-A. In particular implementations, the adjuvant includes TiterMax® Gold (Titermax USA, Inc., Norcross, GA).

[0128] Any composition disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0129] (IV) Methods of Use. Methods disclosed herein include treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens) and research animals (e.g., monkeys, rats, mice, fish) with compositions disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0130] An “effective amount” is the amount of a composition necessary to result in a desired physiological change in the subject. For example, an effective amount can provide an immunogenic effect. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an in vitro assay, an animal model or clinical study relevant to the assessment of an infection’s development, progression, and / or resolution, as well as the effects of the infection. An immunogenic composition can be provided in an effective amount, wherein the effective amount stimulates an immune response.

[0131] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of an infection or displays only early signs or symptoms of an infection such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the infection. Thus, a prophylactic treatment functions as a preventative treatment against an infection and / or the potential effects of an infection.

[0132] Particular uses of the compositions include use as prophylactic vaccines. Vaccines increase the immunity of a subject against a particular infection. Therefore, "syphilis vaccine" can refer to a treatment that increases the immunity of a subject against syphilis. In certain examples, a vaccine may be administered prophylactically, for example to a subject that is immunologically naive (e.g., no prior exposure or experience with syphilis). In particular implementations, a vaccine may be administered therapeutically to a subject who has been exposed to Treponema pallidum or syphilis. Thus, a vaccine can be used to ameliorate a symptom and / or complication associated with syphilis, examples of each of which are described elsewhere herein.

[0133] In particular implementations, a syphilis vaccine is a therapeutically effective composition including a Treponema pallidum epitope that induces an immune response in a subject against syphilis. The skilled artisan will appreciate that the immune system generally is capable of producing an innate immune response and an adaptive immune response. An innate immune response generally can be characterized as not being substantially antigen specific and / or not generating immune memory. An adaptive immune response can be characterized as being substantially antigen specific, maturing over time (e.g., increasing affinity and / or avidity for antigen), and in general can  produce immunologic memory. Even though these and other functional distinctions between innate and adaptive immunity can be discerned, the skilled artisan will appreciate that the innate and adaptive immune systems can be integrated and therefore can act in concert.

[0134] In particular implementations, administration of a syphilis vaccine can further include administration of one or more adjuvants. The term “adjuvant” refers to material that enhances the immune response to a vaccine antigen and is used herein in the customary use of the term. The precise mode of action is not understood for all adjuvants, but such lack of understanding does not prevent their clinical use for a wide variety of vaccines.

[0135] "Immune response" refers to a response of the immune system to produce Treponema pallidum neutralizing antibodies in response to B cells binding T. pallidum epitopes. In particular implementations, the immune response causes production of antibodies to neutralize T. pallidum. In particular implementations, an immune response to a syphilis vaccine can be an innate and / or adaptive response. In particular implementations, an adaptive immune response can be a "primary immune response" which refers to an immune response occurring on the first exposure of a "naive" subject to T. pallidum or vaccine. For example, in the case of a primary antibody response, after a lag or latent period of from 3 to 14 days depending on, for example, the composition, dose, and subject, antibodies to T. pallidum can be produced. Generally, IgM production lasts for several days followed by IgG production and the IgM response can decrease. Antibody production can terminate after several weeks but memory cells can be produced. In particular implementations, an adaptive immune response can be a "secondary immune response", "anamnestic response," or "booster response" which refer to the immune response occurring on a second and subsequent exposure of a subject to T. pallidum or vaccine. Generally, in a secondary immune response, memory cells respond to the T. pallidum or vaccine and therefore the secondary immune response can differ from a primary immune response qualitatively and / or quantitatively. For example, in comparison to a primary antibody response, the lag period of a secondary antibody response can be shorter, the peak antibody titer can be higher, higher affinity antibody can be produced, and / or antibody can persist for a greater period of time.

[0136] In particular implementations, an immune response against T. pallidum will include antibody production against T. pallidum.

[0137] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of an infection and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the infection or effects of the infection. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the infection and / or reduce, control or eliminate side effects of the infection.

[0138] In particular implementations a therapeutic treatment can reduce, control, or eliminate a primary infection with T. pallidum. In particular implementations a therapeutic treatment can reduce or eliminate the symptoms of syphilis. In particular implementations, a therapeutically effective amount reduces or prevents transmission of T. pallidum and / or syphilis.

[0139] In particular implementations, a therapeutically effective amount alleviates or reduces the severity or occurrence of symptoms and / or complications associated with T. pallidum infection. Exemplary symptoms include skin rash (e.g., on palms of the hands and soles of the fee), sores (e.g., mucous patches in mouth, vagina, or penis), warty patches, fever, general ill feeling, loss of appetite, muscle and joint pain, swollen lymph nodes, vision changes, hair loss, heart damage, tumors, and central nervous system disorders (e.g., neurosyphilis).

[0140] In particular implementations, a therapeutically effective amount reduces the duration of infection with syphilis for a subject as compared to a subject that has not received a syphilis vaccine disclosed herein.

[0141] In particular implementations, a therapeutically effective amount reduces the time to sustained non-detectable syphilis in the sera, blood, or urine in a patient infected with the bacteria as compared to a subject that has not received a syphilis vaccine disclosed herein.

[0142] In particular implementations, a therapeutically effective amount reduces the spread and symptoms as compared to a subject that has not received a syphilis vaccine disclosed herein.

[0143] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular implementations, administered dosages may accomplish more than one treatment type.

[0144] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of infection, stage of infection, effects of infection, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0145] Useful doses can range from 0.1 to 5 µg / kg or from 0.5 to 1 µg / kg. In other examples, a dose can include 1 µg / kg, 15 µg / kg, 30 µg / kg, 50 µg / kg, 55 µg / kg, 70 µg / kg, 90 µg / kg, 150 µg / kg, 350 µg / kg, 500 µg / kg, 750 µg / kg, 1000 µg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0146] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly).

[0147] In some implementations, the syphilis vaccine described herein can be administered in combination or alternation with a secondary syphilis treatment. In particular implementations, the secondary syphilis treatment includes antibiotics such as penicillin (e.g., Benzathine penicillin G), amoxicillin, ampicillin, doxycycline, tetracycline, or ceftriaxone, or combinations thereof.

[0148] In some implementations, the syphilis vaccine disclosed herein can be used as a booster vaccine, to increase or modify or alter immune responses induced by a prior syphilis vaccine. In some implementations, the syphilis vaccine disclosed herein can be used as a booster vaccine following infection and recovery from syphilis.

[0149] The syphilis vaccine described herein can be administered on top of the current standard of care for syphilis patients, or in combination or alternation with any other compound or therapy that the healthcare provider deems beneficial for the patient. The combination and / or alternation therapy can be therapeutic, adjunctive, or palliative.

[0150] The pharmaceutical compositions described herein can be administered by, without limitation, injection, inhalation, infusion, perfusion, lavage or ingestion. Routes of administration can include intravenous, intradermal, intramucosal, intraarterial, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intramuscular, intravesicular, oral, subcutaneous, and / or sublingual administration and more particularly intramuscular, subcutaneous, and / or intradermal injection.

[0151] (V) Kits. Also disclosed herein are kits including one or more containers including one or more of the Treponema pallidum epitope sequences described herein, nucleic acids encoding Treponema pallidum epitope sequences, vectors, Treponema pallidum epitope sequences on a delivery scaffold, modified cells (e.g., cells modified to express a vaccine disclosed herein), and / or compositions and / or adjuvants, anti-infective agents, or secondary vaccines described herein. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.

[0152] The Example Clauses and Example below are included to demonstrate particular implementations of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific implementations disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0153] (VI) Example Clauses. 1. A An epitope-scaffold molecule including an epitope having a sequence as set forth in at least one of SEQ ID NOs: 1-12 or a sequence having at least 98% sequence identity to the sequence as set forth in at least one of SEQ ID NOs.1-12; and a delivery scaffold. 2. The epitope-scaffold molecule of clause 1, wherein the epitope includes the sequence as set forth in SEQ ID NO: 1 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 1. 3. The epitope-scaffold molecule of clause 1 or 2, wherein the epitope includes the sequence as set forth in SEQ ID NO: 2 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 2. 4. The epitope-scaffold molecule of any one of clauses 1-3, wherein the epitope includes the sequence as set forth in SEQ ID NO: 3 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 3.5. The epitope-scaffold molecule of any one of clauses 1-4, wherein the epitope includes the sequence as set forth in SEQ ID NO: 4 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 4. 6. The epitope-scaffold molecule of any one of clauses 1-5, wherein the epitope includes the sequence as set forth in SEQ ID NO: 5 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 5. 7. The epitope-scaffold molecule of any one of clauses 1-6, wherein the epitope includes the sequence as set forth in SEQ ID NO: 6 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 6. 8. The epitope-scaffold molecule of any one of clauses 1-7, wherein the epitope includes the sequence as set forth in SEQ ID NO: 7 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 7. 9. The epitope-scaffold molecule of any one of clauses 1-8, wherein the epitope includes the sequence as set forth in SEQ ID NO: 8 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 8. 10. The epitope-scaffold molecule of any one of clauses 1-9, wherein the epitope includes the sequence as set forth in SEQ ID NO: 9 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 9. 11. The epitope-scaffold molecule of any one of clauses 1-10, wherein the epitope includes the sequence as set forth in SEQ ID NO: 10 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 10. 12. The epitope-scaffold molecule of any one of clauses 1-11, wherein the epitope includes the sequence as set forth in SEQ ID NO: 11 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 11. 13. The epitope-scaffold molecule of any one of clauses 1-12, wherein the epitope includes the sequence as set forth in SEQ ID NO: 12 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 12. 14. The epitope-scaffold molecule of any one of clauses 1-13, wherein the delivery scaffold includes a scaffold protein, a virus-like particle, or a nanoparticle. 15. The epitope-scaffold molecule of clause 14, wherein the scaffold protein includes a protein from T. pallidum, S. aureus, H. pylori, equine infectious anemia virus, or diphtheria toxin. 16. The epitope-scaffold molecule of clauses 14 or 15, wherein the scaffold protein includes a T. pallidum protein (Tp0751) from T. pallidum, a Protein A from S. aureus, a Cag-Z from H. pylori, a p26 capsid protein from equine infectious anemia virus, or a transmembrane domain of diphtheria toxin (DTT).17. The epitope-scaffold molecule of clause 14, wherein the scaffold protein includes a synthetic protein. 18. The epitope-scaffold molecule of clause 17, wherein the synthetic protein includes a small-synthetic β-barrel protein. 19. The epitope-scaffold molecule of clause 14, wherein the virus-like particle is derived from adeno-associated virus (AAV), Budgerigar fledgling disease virus (BFDV), Bluetongue virus (BTV), Ebola, Enterovirus 71, Goose hemorrhagic polyoma virus (GHPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis δ virus (HDV), Hepatitis E virus (HEV), human immunodeficiency virus (HIV), Human papillomavirus (HPV), Infectious bursal disease virus (IBDV), influenza (e.g., Influenza A, Influenza A H1N1, Influenza A H3N2, avian influenza (H5N3)), Marburg virus (MVD), bacteriophage MS2, Indian peanut clump virus (IPCV), Newcastle disease virus (NDV), Norovirus (No), Norwalk virus (Nv), Physalis mottle virus (PhMV), Polyomavirus (e.g., human polyomavirus or JC polyomavirus), parvovirus (e.g., Porcine parvovirus (PPV), canine parvovirus), Rabbit hemorrhagic disease virus (RHDV), Rotavirus, Semliki-Forest virus, Sever acute respiratory syndrome (SARS), Simian immunodeficiency virus (SIV), Simian virus 40 (SV40), or Swine vesicular disease virus (SVDV). 20. The epitope-scaffold molecule of clauses 14 or 19, wherein the virus-like particle is derived from HEV, HIV, HPV, NDV, polyomavirus, or parvovirus. 21. The epitope-scaffold molecule of clause 20, wherein the HPV includes HPV type 16. 22. The epitope-scaffold molecule of any one of clauses 14-21, wherein the virus-like particle includes an L1 capsid protein of HPV type 16. 23. The epitope-scaffold molecule of any one of clauses 1-22, wherein the epitope is expressed on a DE loop of the L1 capsid protein of HPV type 16. 24. The epitope-scaffold molecule of any one of clauses 1-23, wherein the delivery scaffold is 50-60 nm in diameter. 25. The epitope-scaffold molecule of any one of clauses 1-24, wherein the delivery scaffold is 55 nm in diameter. 26. The epitope-scaffold molecule of clause 14, wherein the nanoparticle includes self-assembling proteins. 27. The epitope-scaffold molecule of clause 26, wherein the self-assembling proteins include ferritin, lumazine, or encapsulin. 28. The epitope-scaffold molecule of clause 14, wherein the nanoparticle includes a gold nanoparticle, a carbon nanoparticle, or a silica nanoparticle. 29. The epitope-scaffold molecule of any one of clauses 14-28, wherein the nanoparticle is 10-1000 nm in diameter. 30. The epitope-scaffold molecule of any one of clauses 14-29, wherein the nanoparticle is 50-60 nm in diameter. 31. The epitope-scaffold molecule of any one of clauses 14-30, wherein the nanoparticle is 55 nm in diameter.32. A concatenated epitope including at least two copies of a sequence selected from any of SEQ ID NOs: 1-12 or a sequence having at least 98% sequence identity to any of SEQ ID NOs: 1-12. 33. The concatenated epitope of any one of clauses 32, wherein the concatenated epitope includes: 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 1 or a sequence having at least 98% sequence identity to SEQ ID NO: 1; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 2 or a sequence having at least 98% sequence identity to SEQ ID NO: 2; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 3 or a sequence having at least 98% sequence identity to SEQ ID NO: 3; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 4 or a sequence having at least 98% sequence identity to SEQ ID NO: 4; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 5 or a sequence having at least 98% sequence identity to SEQ ID NO: 5; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 6 or a sequence having at least 98% sequence identity to SEQ ID NO: 6; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 7 or a sequence having at least 98% sequence identity to SEQ ID NO: 7; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 8 or a sequence having at least 98% sequence identity to SEQ ID NO: 8; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 9 or a sequence having at least 98% sequence identity to SEQ ID NO: 9; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 10 or a sequence having at least 98% sequence identity to SEQ ID NO: 10; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 11 or a sequence having at least 98% sequence identity to SEQ ID NO: 11; and / or 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 12 or a sequence having at least 98% sequence identity to SEQ ID NO: 12. 34. The concatenated epitope of clauses 32 or 33, wherein the concatenated epitope includes SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 operatively linked together. 35. The concatenated epitope of any one of clauses 32-34, wherein the concatenated epitope includes SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 operatively linked together. 36. A composition including:(i) an epitope including the sequence as set forth in at least one of SEQ ID NOs: 1-12 or a sequence having at least 98% sequence identity to the sequence as set forth in at least one of SEQ ID NOs: 1-12, an epitope-scaffold molecule including the sequence as set forth in at least one of SEQ ID NOs: 1-12 or a sequence having at least 98% sequence identity to the sequence as set forth in at least one of SEQ ID NOs: 1-12 and a delivery scaffold, or a concatenated epitope including the sequence as set forth in at least two of SEQ ID NOs: 1-12 or a sequence having at least 98% sequence identity to the sequence as set forth in of SEQ ID NOs: 1-12; and (ii) a pharmaceutically acceptable carrier. 37. The composition of clause 36, further including an adjuvant. 38. The composition of clause 37, wherein the adjuvant includes a squalene-based adjuvant, an aluminum- based adjuvant, a saponin-based adjuvant, a liposome-based adjuvant, a Toll-like receptor (TLR)-based adjuvant, a STING agonist, an attenuated lipid A derivative (ALD), or a carbomer-lecithin-based adjuvant. 39. The composition of clause 38, wherein the aluminum-based adjuvant includes aluminum hydroxide, aluminum phosphate, or potassium aluminum sulphate (alum). 40. The composition of clause 38, wherein the saponin-based adjuvant includes Quillaja saponaria. 41. The composition of clause 38, wherein the TLR-based adjuvant includes resiquimod, muramyl dipeptide, synthetic diacylated lipoprotein FSL-1, glucopyranosyl lipid A (GLA). 42. The composition of clauses 38 or 41, wherein the TLR-based adjuvant includes GLA formulated in stable emulsion (GLA SE). 43. The composition of clause 38, wherein the ALD includes monophosphoryl lipid A (MLA). 44. The composition of any one of clauses 37-43, wherein the adjuvant includes MLA-Alum. 45. The composition of any one of clauses 37-44, wherein the adjuvant includes a monophosphoryl lipid A, synthetic trehalose dimycolate, and cell wall skeleton. 46. The composition of any one of clauses 37-45, wherein the adjuvant includes a monophosphoryl lipid A, synthetic trehalose dimycolate, and muramyl dipeptide. 47. The composition of any one of clauses 37-46, wherein the adjuvant induces a Th1-type response. 48. A method of stimulating an anti-syphilis immune response in a subject, the method including administering to the subject a therapeutically effective amount of the composition of any one of clauses 36-47, thereby stimulating the anti-syphilis immune response in the subject. 49. The method of clause 48, wherein the subject is syphilis seropositive. 50. The method of clause 48, wherein the subject is a syphilis seronegative subject, and wherein the therapeutically effective amount reduces the likelihood of the syphilis seronegative subject becoming syphilis seropositive.  51. The method of any one of clauses 48-50, wherein the administering to the subject includes injection, inhalation, infusion, perfusion, lavage, or ingestion. 52. The method of any one of clauses 48-51, wherein the administering includes intramuscular, subcutaneous, or intradermal administration. 53. A syphilis vaccine including an epitope including a sequence as set forth in at least one of SEQ ID NOs: 1- 12 or a sequence having at least 98% sequence identity to at least one of SEQ ID NOs: 1-12.

[0154] (VII) Experimental Examples.

[0155] Example 1. Diverse and Conserved Sequences of TprC and TprK Variants. Two antigens have been identified among T. pallidum surface-exposed proteins belonging to the Tpr family of virulence factors that confer substantial protection to syphilis in the rabbit model. Immunity to these antigens promoted pathogen clearance by opsonophagocytosis and attenuated chancre development at challenge sites compared to controls. These two vaccine candidates were the conserved i) NH2-terminal region shared by the TprC protein, and ii) the conserved NH2-terminus of the TprK protein (Giacani et al., J Bacteriol.2012, 194(16):4208-25 ("Giacani 1”); Giacani et al., J Immunol.2010, 184(7):3822-9 (“Giacani 2”); Centurion-Lara et al., J Exp Med.1999, 189(4):647-56 (“Centurion-Lara 1”); Anand et al., J Bacteriol.2012, 194(9):2321-33; Giacani et al., Infect Immun.2007, 75(1):104-12 ("Giacani 3”); Giacani et al., Infect Immun.2005, 73(6):3817-22; and Lukehart, et al., Vaccine, 2022, 40(52):7676-7692).

[0156] T. pallidum outer membrane hosts very few and a very low density of surface-exposed integral membrane proteins (Radolf et al., Proc Natl Acad Sci USA.1989, 86(6):2051-5). Finding these rare outer membrane proteins (OMPs) is compared to "finding the holy grail" in the field, as OMP identification would allow a deeper understanding of the host-pathogen interaction dynamics. Progress toward OMP identification has been hindered by limitations such as the inability to steadily culture this pathogen, even though this might change in the future. Although T. pallidum can be propagated in rabbits by intratesticular passage every 10-12 days, low treponemal yields from infected animals (107- 1010cells per animal, depending on the strain) and the presence of contaminating rabbit material make it difficult to evaluate the bacterial surface. Furthermore, the uncommon fragility of T. pallidum outer membrane makes direct identification of OMPs very difficult using approaches such as surface immunofluorescence (Penn et al., J Gen Microbiol.1985, 131(Pt 9):2349-57; and Blanco et al., Emerg Infect Dis.1997, 3(1):11-20). Lack of genetic tools for this pathogen further limits the array of techniques applicable for OMP identification. Mining of the T. pallidum genome using computational approaches to predict OMPs, coupled with functional and biophysical assays to confirm OMP predictions (Cox et al., Infect Immun.2010, 78(12):5178-94; and Centurion-Lara A, et al., PLoS Negl Trop Dis.2013, 16(7):e2222 ("Centurion-Lara 3”)) has recently populated the list of the bona fide and putative T. pallidum OMPs with 30 candidates. Several of them, however, such as BamA (Tp0326) (Cameron et al. J Infect Dis.2000, 181(4):1401- 13), OmpW (Tp0126) (Giacani et al., unpublished), and surface adhesins (Tp0136) (Brinkman et al., Infect Immun. 2008, 76(5):1848-57) have failed to induce protection in immunization / challenge experiments. The T. pallidum repeat (Tpr) antigens that are the focus of this example are showing significant protection to the infection.

[0157] Recognition of the Tpr family of paralogous proteins occurred with the sequencing of the T. pallidum Nichols strain genome (Fraser et al., Science.1998, 281(5375):375-88). These proteins were immediately identified as putative virulence factors based on homology with the surface-exposed Msp porin / adhesin of the oral pathogen Treponema denticola (Fenno et al., J Bacteriol.1997, 179(4):1082-9; and Fenno et al., Infect Immun.1998, 66(5):1869-77). This group of genes alone accounts for 2% of the very small (1.13 Mb) T. pallidum genome. Throughout its evolution to become an obligate human pathogen, T. pallidum has eliminated genes encoding important metabolic activities (such as oxidative phosphorylation, synthesis of fatty acids, nucleotides, and most amino acids) (Norris and Sell, J Immunol. 1984, 133(5):2686-92), but has retained the tpr-encoding genes likely because they play a key role in T. pallidum biology and disease pathogenesis. The members of the Tpr family are divided into three subfamilies according to sequence homology. Subfamily I member TprC and Subfamily III member TprK are the focus of this example.Additionally, as T. pallidum has a very low-density of integral proteins in its outer membrane, evidencethat the tprK,and tprC, genes are highlyexpressed in this pathogen (“Giacani 3”; Smajs et al., J Bacteriol. 2005, 187(5):1866-74)further supports their selection for vaccine design. A three-dimensional model of both TprC generated with homology- based modeling programs such as I-TASSER (Yang et al., Nature methods.2015, 12(1):7-8), predict these proteins to be typical β-barrel outer membrane proteins (OMPs) with 11 external loops (ExL, FIG.1A). Relying on 3D models is still utilized because crystallization has not been accomplished for any Tpr protein to date. However, experimental evidence for this structure was gained in the laboratory by performing circular dichroism (CD) on recombinant full-length TprC from the Nichols strain of T. pallidum after protein purification and refolding (FIG.1B). CD spectrum analysis using Dichroweb (Whitmore and Wallace, Nucleic Acids Res.2004, 32(Web Server issue):W668-73) supported that the relative proportions of beta-sheet (48%), alpha-helix (4%), and random coil (48%) in the refolded protein are consistent with the TprC 3D structure shown in FIG.1A, suggesting that 3D predictions are a reasonable representation of the antigen's native structure. Slightly different antigenic variants of TprC protein (encoded by the tprC gene, tp0117) were described ("Centurion-Lara 3”; and Pinto et al., Nat Microbiol. 2016, 2:16190). If the TprC regions harboring sequence diversity are highlighted in the 3D model (FIG.1A, white arrows), they fall within predicted surface- exposed loops.

[0158] FIG.2 shows a multiple sequence alignment of the COOH-terminal region (AA 405-600) of five variants of TprC. Noteworthy is that although several TprC variants were described thus far in syphilis isolates, the whole spectrum of known variability within the predicted external loops of these variants is limited and TprC variants are the result of combinations of a small number of antigenically different "external loop" sequences. Three TprC protein variants (from the Nichols, MexicoA, and Sea81-4 strains) cover the whole spectrum of known loop variants in this protein ("Centurion- Lara 3”).

[0159] TprK is also a bona fide OMP of T. pallidum (“Giacani 1”; “Giacani 2”; and “Centurion-Lara 1”) During infection,TprK is the most highly expressed (“Giacani 3”) and most immunogenic of the Tprs, containing B- cell epitopes (“Giacani 3”; and Leader et al., Infect Immun. 2003, 71(10):6054-7). This protein is the T. pallidum to undergo intra-strain antigenic variation during the course of infection.

[0160] Generation of diversity in TprK occurs through segmental gene conversion in seven discrete variable (V)regions named V1-V7 (FIG.3), that are separated from each other by conserved sequences (“Giacani 1”). TprK modelssupport that this protein has a β-barrel structure with 11 surface-exposed loops, seven of which are predicted to harborthe variable regions (“Giacani 2”) (“V regions” in FIG. 3), while the remaining four, all located in the protein NH2-terminus, are conserved (FIG. 3). The TprK conserved region is already known to contain targets for opsonizing antibodies and to be protective in immunization / challenge experiments. The role of the TprK V region in allowing immune evasion during infection was repeatedly shown (“Giacani 2”; LaFond et al., Infect Immun.2006, 74(3):1896- 906; LaFond et al., J Bacteriol. 2003, 185(21):6262-8; and Reid et al., Infect Immun. 2014, 82(12):4959-67), and although vaccines based on TprK fragments containing V regions were tried (Morgan et al., Infect Immun. 2002, 70(12):6811-6 (“Morgan 1”)), the TprK NH2-terminal fragment (that harbors the four predicted conserved loops) will be focused on for the vaccine.

[0161] Example 2. Adjuvants. The Ribi-like adjuvant used in early immunization experiments with the conserved TprK (“Centurion-Lara 1”; and Sun et al., Microbes Infect.2004, 6(8):725-37 (“Sun”)) and TprC (“Morgan 1”) contained mono-phosphoryl lipid A (MPLA, a TLR4 agonist) from S. minnesota, synthetic trehalose dimycolate (TDM, a MINCLE receptor agonist) and cell wall skeleton (CWS, a TLR2 and TLR4 agonist) from M. bovis. This adjuvant has not been manufactured for over a decade. Thus, other adjuvants were tested in combination with the antigens representing the small conserved regions of TprK and TprC. This list also included adjuvants for human use, such as alum, in addition to several custom-made emulsion-based adjuvants containing various combinations of immunomodulatory components such as glucopyranosyl lipid (GL), risiquimod, muramyl dipeptide (MDP, a NOD2 agonist), FSL-1, and Qui- A (a saponin adjuvant), as well as the commercially available Titermax Gold. Results showed that although all adjuvants triggered high antibody titers to the immunogens (not shown), significant protection was induced with adjuvants inducing a Th1-type response such as those containing strong TLR2, TLR4, and NOD2 agonists (MPLA, TDM, FSL-1, and MDP). FIGs.4A, 4B shows that i) antigen-stimulated PBMCs from protected rabbits produced significantly higher IFNγ levels compared to PBMCs from rabbits that received a "non-protective" adjuvant, and ii) that immunization with antigen+Ribi-like significantly reduced the number of ulcerating lesions at challenge sites (which is a correlate of protection). The Ribi-like formulation, was produced by the Seattle- based Infectious Disease Research Institute (IDRI) to be virtually identical to the Ribi, but with MDP instead of CWS. The results of this example suggest that for antigen optimization in the rabbit model, the use of the original Ribi adjuvant formula has favorable antigen optimization.

[0162] Example 3. Evaluate protective ability of recombinant full-length TprC and TprK antigens toinformvaccine design. Antisera raised against the conserved TprK NH2-terminal region (AA 37-273 of, for example, SEQ ID NO: 40, predicted to contain four conserved surface-exposed loops) (“Giacani 2”), are opsonic for T. pallidum (FIG.5), and rabbits immunized with the same antigen were significantly protected against infectious challenge (“Morgan 1”;  and Morgan et al., Infect Immun. 2003, 71(10):5605-12 ("Morgan 2”)). In TprK-immunized animals, in fact, lesion development and progression to ulceration was significantly attenuated compared to controls (FIG.6), and treponemal burden at challenge sites reduced. In TprK-immunized animals, 20% of lesions from these animals contained T. pallidum compared to 94% of the lesions in unimmunized controls (“Morgan 1”; and "Morgan 2”). These results show that the TprK conserved fragment contains targets of opsonic antibodies, and immunity to these targets promotes early treponemal clearance. Similar results were obtained in opsonophagocytosis assays performed with immune sera to the NH2-terminal conserved region shared by TprC variants (AA 21-284 of, for example, SEQ ID NO: 41, containing fourpredicted surface loops) (FIG. 5).Furthermore, immunization with this TprC conserved peptideprovided significantprotection against infectious challenge. Upon challenge with the Nichols strain, for example, ulcerative chancres developed in 8% of the inoculation sites in immunized rabbits compared to 83% of the inoculation sites in controls(p<0.01) (“Sun”).However, immunization with the TprK and TprC conservedfragments did not provide sterilizingimmunity in this example, in that all immunized animals do become infected, as evidenced by reactivity in the VDRL serologic test (“Centurion-Lara 1”; and “Sun”), which confirmed active infection. Rabbit infectivity tests (RITs) were also performed to assess presence of live T. pallidum in tissues from some rabbits immunized with TprK and then challenged. In these experiments, transfer of lymph nodes and testicular tissue from immunized / challenged rabbits to naive rabbits always caused seroconversion of the recipients ("Centurion-Lara 1”), showing lack of complete immunity to infection in vaccinated animals.

[0163] Treponemal clearance from early syphilis lesions is mediated by opsonophagocytosis of the pathogen by activated macrophages (Baker-Zander and Lukehart, J Infect Dis.1992, 165(1):69-74; and Baker-Zander et al., FEMS Immunol Med Microbiol. 1993, 6(4):273-9). T. pallidum opsonophagocytosis experiments using polyclonal antisera directed against the TprC central region (AA 285-437 of, for example, SEQ ID NO: 41, containing three predicted surface loops), and COOH- region (AA 438-598 of, for example, SEQ ID NO: 41, also containing three predicted loops), respectively showed that these sera enhanced treponemal opsonophagocytosis compared to controls (FIG.7).

[0164] Example 4. Evaluate the protective ability of chimeric concatemers based on protective B-cell epitopesof TprC and TprK.Epitopes for concatemer construction were selected and include those recognized by mAbs withopsonic and neutralizing ability. Additionally, the TprC and TprK B-cell epitopes that fall within the predicted proteins loops and are seen by immune sera in animals exhibiting significant protection compared to controls were cloned. The concatemer- encoding sequence was obtained by joining pre-synthesized DNA fragments by overlapping PCR, which was then cloned into the E. coli expression vector pET-32 to add a 5xHis-tag for purification. Concatemers were produced as codon-optimized genes for E. coli to maximize likelihood of expression. Targets were amplified using primers with 5' overhangs to allow ligase-independent cloning (LIC) into the pET-32 vector, as previously done for the OspC-based vaccine for B. burgdorferi. Antigens were purified using affinity chromatography and dialyzed against PBS containing arginine and glutamic acid (50 mM each), to improve solubility (Golovanov et al., Journal of the American Chemical Society.2004, 126(29):8933-9).

[0165] Tpr antigens are relatively large hydrophobic proteins (60 kDa) that require extensive work to be purified and refolded into an antigen that is similar to its native counterpart. Antigen solubility does not generally improve even when smaller portions of the whole proteins are expressed in E. coli, as hydrophobic regions alternate with hydrophilic ones throughout the primary sequence of these antigens. In addition, as the result of immunizing with sequences representing both hydrophilic surface loops and the hydrophobic β-barrel scaffolding, some antibodies are inevitably elicited by portions of the proteins that are not surface exposed and therefore will not opsonize T. pallidum. Early B-cell epitope mapping experiments performed in the lab using overlapping peptides and sera from rabbits immunized (but not challenged) with the Nichols TprC protein with a "non- protective" adjuvant (Titermax Gold) showed indeed that much of the humoral reactivity elicited by immunization was directed to the protein scaffolding, rather than the surface loops (FIG.9). It is believed that directing the humoral response to surface-exposed sequences might be an effective strategy to achieve higher protection. To this end, based on the results of the experiments carried on above, two synthetic genes encoding for chimeric concatemers of protective B-cell epitopes of the TprC variants are considered promising candidates, and TprK conserved fragment. These chimeric genes can be used to produce recombinant antigens for immunization.

[0166] Example 5. Evaluate the protective ability of VLPs carrying protective B-cell epitopesof TprC and TprK.VLPs are supra-molecular protein structures that carry many of the characteristics of viruses, and that can be exploited in vaccine development strategies. As VLP-based vaccines lack a viral genome and have lost the ability to replicate, they constitute a very safe template for vaccine development. VLPs, including those derived from the HPV16 L1 protein, form a unique repetitive surface structure making them highly immunogenic vaccine templates. The rigid and repetitive surface structure facilitates cross-linking of B-cell receptors and binding to multimeric components of the innate immune system with high avidity (Wang and Roden, Expert review of vaccines. 2013, 12(2):129-41; Chen et al., Cellular immunology.2011, 269(1):5-9; and Stanley et al., Vaccine.2012, 30(3):F83-F7). VLP-based vaccines mainly target B- cells and induce potent antibody responses following activation of T helper cells and presentation by antigen presenting cells (APCs), making them an ideal vector for T. pallidum antigens. Additional advantages include a size (55 nm) that is optimal for drainage to the lymph nodes. The size of the sequences that can be cloned into the L1 DE loop of chimeric HPV16 L1-based VLPs is limited to 15-20 amino acids in length. B-cell epitope mapping using synthetic peptides representing the Nichols TprC COOH-region with antisera from long-term infected rabbits suggests that the sequences to be cloned do not exceed 20 amino acids (FIG.10).

[0167] Experimental procedures to obtain chimeric VLPs were performed using data acquired on B-cell epitope mapping for TprC and TprK. Epitopes of max 20 amino acids were expressed into the DE loop of the L1 protein by engineering a p16L1L2 vector (Buck and Thompson, Current protocols in cell biology.2007, Chapter 26:Unit 261 ("Buck”)) using primer extension to replace the L1 DE loop with the desired sequence followed by InFusion cloning (Takara Bio, USA) after the modified L1 gene is sequenced to ensure lack of undesired mutations. Following plasmid synthesis, VLPs were produced according to the protocol developed by Buck and colleagues at the NIH (“Buck”; Buck  et al., Journal of virology.2004, 78(2):751-7; and Cardone et al., MBio.2014, 5(4):e01104-14). Briefly, plasmids were used to transfect 7x106human embryonic kidney (HEK) cell line 293TT, previously modified to express high levels of SV40 LT antigen to drive L1 expression from the p16L1L2 vector. After 48 hours, cells were collected and lysed using detergent. VLPs were given time to mature by overnight incubation of the cell lysates at 37°C. VLPs were harvested by ultracentrifugation through an Optiprep (60% iodixanol) step gradient for 3.5 hours at 16°C at 234,000xg. L1 antibodies that bind to conformational epitopes were used to recognize properly folded L1. Presence of the T. pallidum- specific epitope were assessed using western blotting with Tpr-specific antisera and mass spectrometry.

[0168] Example 6. Effect of immunization on lesion development in rabbits infected with the syphilis agent. For some of these experiments, a total of 60µg of each antigen was given over the course of 5 immunizations (20µg in the initial immunization and 10µg in the boost injections). During each immunization, antigens were administered intramuscularly (400 µl), subcutaneously (400 µl distributed in four sites), and intradermally (100 µl in two sites). Three weeks after the final boost, however, rabbits were challenged with the Nichols strain in ten sites on their shaved backs, using 105T. pallidum cells per injection site to evaluate protection to infection. One day prior to infectious challenge, immune serum was collected from all rabbits to define i) antibody titer to antigens to correlate to protection, and perform ii) B-cell epitope mapping also using synthetic peptides, and iii) opsonization / neutralization assays to assess production of functional antibodies. Additionally, iv) PBMCs from these animals were isolated and frozen for measurement of IFNγ production, and antigen-specific B-cell isolation to develop mAbs from the rabbit that showed the highest level of protection in each of the three groups. The epitope-specificity of the Tpr monoclonal antibodies were determined using synthetic peptides, and the mAbs were tested for opsonic and neutralizing ability. As negative controls for these experiments, normal rabbit serum was used. Opsonization assays were conducted as previously described (Shaffer et al., Infect Immun.1993, 61(2):781-4). Briefly, after incubation of treponemes with control or test sera plus rabbit peritoneal macrophages, the un-ingested treponemes were washed off, and macrophages were fixed and stained for T. pallidum. An observer blinded to the test conditions deterined the percentage of macrophages containing ingested T. pallidum on triplicate slides per condition. Macrophages incubated with treponemes + normal rabbit serum, and macrophages incubated without treponemes were the negative controls. Pooled sera from long term- infected rabbits serve as positive controls. Each antiserum was tested in triplicate per macrophage donor, with at least 3 macrophage donors. Neutralization was performed. Treponemes were incubated with heated control or test sera, with and without added active rabbit complement, under microaerophilic conditions for 16 hours, followed by intradermal inoculation onto the backs of normal rabbits. Animals were monitored for 1 month. Neutralization of treponemes is indicated by lack of lesion appearance at an inoculation site. Negative controls include NRS and all sera incubated in the absence of active complement. Positive controls include infection-immune serum relevant to the target strain being used in the assay, incubated with active complement. Each condition is injected into two sites per rabbit, 3 rabbits per assay; 2 assays per test serum. The proportion of lesions appearing at injection sites for each condition was compared to the values for positive and negative controls. Induction of Th1-type immunity was assessed prior to challenge by  measurement of IFNγ release in PBMC incubated with the antigens. PBMC was purified by Ficoll-Hypaque and cultured for 8 and 24 hours with the antigens or ConA (positive control). IFNγ release was measured using the LSBio Rabbit IFNγ ELISA kit.

[0169] Following sera and PBMC collection, rabbits were challenged with the Nichols strain of T. pallidum to assess the level of protection induced by these immunizations. The rabbit backs were kept clipped and observed daily for 1 month for i) development of chancres (expected at day 5 at 105 / site challenge dose), ii) measurement of lesion diameter, ii) evidence and time of ulceration, and iii) time of healing. Biopsies were harvested from 2 challenge sites per rabbit at 24 hours post-challenge for analysis of IFNγ mRNA levels (to confirm DTH and a Th1-type response) as described (Godornes et al., Cytokine.2007, 38:1-7). Biopsies were harvested from 2 challenge sites per rabbit at the time when lesions in the unimmunized control rabbits were on the brink of ulceration; these were processed for qPCR and reverse transcription (RT)-qPCR to determine total (DNA) and live (mRNA) T. pallidum burden, normalized to the message for the rabbit housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) (for RNA quantification) or rabbit cystic fibrosis transmembrane conductance regulator (CFTR) gene (for DNA quantification). At this same time, aspirates taken from each lesion (those not affected by previous biopsy, 6 per rabbit) were examined by dark-field (DF) microscopy to quantify number of DF+ lesions, and number of treponemes seen in positive lesions in 100 observation fields. Fisher's exact test and Wilcoxon rank-sum test will be used to compare outcomes.

[0170] Rabbits immunized with the TprC or TprK antigen were observed to have attenuated lesion development when the lesion volume of immunized rabbits (black lines) is compared to the lesion volume of control animals, supporting vaccine efficacy (FIGs.11A, 11B). Over time lesions increased in volume, and then decrease due to healing. The Treponemal cell burden was estimated by counting bacterial cells under the microscope in lesion aspirates from rabbits immunized with TprK and TprC compared to control animals that were not immunized but were challenged with either the Nichols or SS14 strain. Treponemal cell burden was also estimated by real-time quantitative PCR in lesion aspirates from rabbits (FIG.12B) Rabbits immunized with TprK and TprC were observed to have significantly fewer treponemes in their lesions compared to controls infected with the Nichols strain (FIG.12A). Lesion ulceration was lower in rabbits immunized with The TprK and TprC antigens. Immunization induced a T-cell response in animals as shown by the delayed-type hypersensitivity (DTH) at 48 hours after challenge for immunized rabbits (FIG.13). Another way to assess vaccine efficacy is to harvest lymph nodes from vaccinated / challenge animals and inject them into one testis of a naive rabbit. If the naïve rabbit develops syphilis more slowly compared to the control rabbits, it means that the immunized animals were protected. Animals that received lymph nodes from rabbits vaccinated with TprC (FIG. 14A) or TprK (FIG.14B) developed syphilis more slowly than controls.

[0171] Example 7. Epitope Mapping. To define the peptides included in this disclosure, sera from rabbits immunized with the TprC antigen (FIG. 15) or the TprK antigen (FIG. 16A, 16B) were evaluated using an enzyme-linked immunosorbent assay (ELISA) to define which epitopes were predominantly recognized on regions of the protein mapping at the host-pathogen interface. In this figure, sera were tested against a pool of peptides corresponding to  external loops (ExL) of the vaccine candidate. For TprC, one can appreciate how certain ExLs (e.g., ExL1, 3, and 6) are more strongly recognized. Subsequently, the same experiment was repeated using single peptides that span each ExL, to identify the specific epitopes that elicited the protective response. For TprK, one can appreciate how CExL1 and CEXL2 are more strongly recognized.

[0172] (VIII) Closing Paragraphs.

[0173] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be used for realizing implementations of the disclosure in diverse forms thereof.

[0174] The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1, 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in implementations where it would be appropriate.

[0175] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0176] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (non-polar): Proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0177] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol.157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser (−0.8); Trp (−0.9); Tyr (−1.3); Pro (−1.6); His (−3.2); Glutamate (−3.5); Gln (−3.5); aspartate (−3.5); Asn (−3.5); Lys (−3.9); and Arg (−4.5).

[0178] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0179] As detailed in US 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (−0.4); Pro (−0.5±1); Ala (−0.5); His (−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); Ile (−1.8); Tyr (−2.3); Phe (−2.5); Trp (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0180] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side- chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistically-significant degree.

[0181] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0182] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987);  and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0183] Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 µg / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 µg / ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g.5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0184] "Specifically binds" refers to an association of a binding domain (of, for example, a CAR binding domain) to its cognate binding molecule with an affinity or Ka(i.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M-1, while not significantly associating with any other molecules or components in a relevant environment sample. Binding domains may be classified as "high affinity" or "low affinity".  In particular implementations, "high affinity" binding domains refer to those binding domains with a Kaof at least 107M-1, at least 108M-1, at least 109M-1, at least 1010M-1, at least 1011M-1, at least 1012M-1, or at least 1013M-1. In particular implementations, "low affinity" binding domains refer to those binding domains with a Kaof up to 107M-1, up to 106M-1, up to 105M-1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) ofbinding interaction with units of M (e.g., 10-5M to 10-13M). In certain implementations, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka(equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd(dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off-rate (Koff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE®analysis (see also, e.g., Scatchard, et al., 1949, Ann. N.Y. Acad. Sci.51:660; and U.S. Patent Nos.5,283,173, 5,468,614, or the equivalent).

[0185] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and R. I. Freshney, ed. Animal Cell Culture (1987).

[0186] As will be understood by one of ordinary skill in the art, each implementation disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the implementation to the specified elements, steps, ingredients or components and to those that do not materially affect the implementation. As used herein, the term “based on” is equivalent to “based at least partly on,” unless otherwise specified.

[0187] Unless otherwise indicated, all numbers expressing quantities, properties, conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary  rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0188] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0189] The terms “a,” “an,” “the” and similar referents used in the context of describing implementations (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate implementations of the disclosure and does not pose a limitation on the scope of the disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of implementations of the disclosure.

[0190] Groupings of alternative elements or implementations disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0191] Certain implementations are described herein, including the best mode known to the inventors for carrying out implementations of the disclosure. Of course, variations on these described implementations will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for implementations to be practiced otherwise than specifically described herein. Accordingly, the scope of this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the  above-described elements in all possible variations thereof is encompassed by implementations of the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0192] The present disclosure refers to various articles, books, patent documents, and other references. Each one of these references is hereby incorporated by reference in its entirety.

Claims

CLAIMS What is claimed is:

1. An epitope-scaffold molecule comprising an epitope having a sequence as set forth in at least one of SEQ ID NOs: 1-12 or a sequence having at least 98% sequence identity to the sequence as set forth in at least one of SEQ ID NOs.1-12; and a delivery scaffold.

2. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 1 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

1.

3. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 2 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

2.

4. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 3 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

3.

5. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 4 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

4.

6. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 5 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

5.

7. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 6 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

6.

8. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 7 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

7.

9. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 8 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

8.

10. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 9 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

9.

11. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 10 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

10.

12. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 11 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

11.

13. The epitope-scaffold molecule of claim 1, wherein the epitope comprises the sequence as set forth in SEQ ID NO: 12 or a sequence having at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

12.

14. The epitope-scaffold molecule of claim 1, wherein the delivery scaffold comprises a scaffold protein, a virus- like particle, or a nanoparticle.

15. The epitope-scaffold molecule of claim 14, wherein the scaffold protein comprises a protein from T. pallidum, S. aureus, H. pylori, equine infectious anemia virus, or diphtheria toxin.

16. The epitope-scaffold molecule of claim 14, wherein the scaffold protein comprises a T. pallidum protein (Tp0751) from T. pallidum, a Protein A from S. aureus, a Cag-Z from H. pylori, a p26 capsid protein from equine infectious anemia virus, or a transmembrane domain of diphtheria toxin (DTT).

17. The epitope-scaffold molecule of claim 14, wherein the scaffold protein comprises a synthetic protein.

18. The epitope-scaffold molecule of claim 17, wherein the synthetic protein comprises a small-synthetic β- barrel protein.

19. The epitope-scaffold molecule of claim 14, wherein the virus-like particle is derived from adeno-associated virus (AAV), Budgerigar fledgling disease virus (BFDV), Bluetongue virus (BTV), Ebola, Enterovirus 71, Goose hemorrhagic polyoma virus (GHPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis δ virus (HDV), Hepatitis E virus (HEV), human immunodeficiency virus (HIV), Human papillomavirus (HPV), Infectious bursal disease virus (IBDV), influenza (e.g., Influenza A, Influenza A H1N1, Influenza A H3N2, avian influenza (H5N3)), Marburg virus (MVD), bacteriophage MS2, Indian peanut clump virus (IPCV), Newcastle disease virus (NDV), Norovirus (No), Norwalk virus (Nv), Physalis mottle virus (PhMV), Polyomavirus (e.g., human polyomavirus or JC polyomavirus), parvovirus (e.g., Porcine parvovirus (PPV), canine parvovirus), Rabbit hemorrhagic disease virus (RHDV), Rotavirus, Semliki-Forest virus, Sever acute respiratory syndrome (SARS), Simian immunodeficiency virus (SIV), Simian virus 40 (SV40), or Swine vesicular disease virus (SVDV).

20. The epitope-scaffold molecule of claim 14, wherein the virus-like particle is derived from HEV, HIV, HPV, NDV, polyomavirus, or parvovirus.

21. The epitope-scaffold molecule of claim 20, wherein the HPV comprises HPV type 16.

22. The epitope-scaffold molecule of claim 14, wherein the virus-like particle comprises an L1 capsid protein of HPV type 16.

23. The epitope-scaffold molecule of claim 1, wherein the epitope is expressed on a DE loop of the L1 capsid protein of HPV type 16.

24. The epitope-scaffold molecule of claim 1, wherein the delivery scaffold is 50-60 nm in diameter.

25. The epitope-scaffold molecule of claim 1, wherein the delivery scaffold is 55 nm in diameter.

26. The epitope-scaffold molecule of claim 14, wherein the nanoparticle comprises self-assembling proteins.

27. The epitope-scaffold molecule of claim 26, wherein the self-assembling proteins comprise ferritin, lumazine, or encapsulin.

28. The epitope-scaffold molecule of claim 14, wherein the nanoparticle comprises a gold nanoparticle, a carbon nanoparticle, or a silica nanoparticle.

29. The epitope-scaffold molecule of claim 14, wherein the nanoparticle is 10-1000 nm in diameter.

30. The epitope-scaffold molecule of claim 14, wherein the nanoparticle is 50-60 nm in diameter.

31. The epitope-scaffold molecule of claim 14, wherein the nanoparticle is 55 nm in diameter.

32. A concatenated epitope comprising at least two copies of a sequence selected from any of SEQ ID NOs: 1- 12 or a sequence having at least 98% sequence identity to any of SEQ ID NOs: 1-12.

33. The concatenated epitope of claim 32, wherein the concatenated epitope comprises: 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 1 or a sequence having at least 98% sequence identity to SEQ ID NO: 1; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 2 or a sequence having at least 98% sequence identity to SEQ ID NO: 2; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 3 or a sequence having at least 98% sequence identity to SEQ ID NO: 3; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 4 or a sequence having at least 98% sequence identity to SEQ ID NO: 4; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 5 or a sequence having at least 98% sequence identity to SEQ ID NO: 5; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 6 or a sequence having at least 98% sequence identity to SEQ ID NO: 6; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 7 or a sequence having at least 98% sequence identity to SEQ ID NO: 7; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 8 or a sequence having at least 98% sequence identity to SEQ ID NO: 8; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 9 or a sequence having at least 98% sequence identity to SEQ ID NO: 9; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 10 or a sequence having at least 98% sequence identity to SEQ ID NO: 10; 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 11 or a sequence having at least 98% sequence identity to SEQ ID NO: 11; and / or 2, 3, 4, 5, 6, 7, 8, 9, or more copies of SEQ ID NO: 12 or a sequence having at least 98% sequence identity to SEQ ID NO:

12.

34. The concatenated epitope of claim 32, wherein the concatenated epitope comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 operatively linked together.

35. The concatenated epitope of claim 32, wherein the concatenated epitope comprises SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 operatively linked together.

36. A composition comprising:(i) an epitope comprising the sequence as set forth in at least one of SEQ ID NOs: 1-12 or a sequence having at least 98% sequence identity to the sequence as set forth in at least one of SEQ ID NOs: 1-12, an epitope-scaffold molecule comprising the sequence as set forth in at least one of SEQ ID NOs: 1-12 or a sequence having at least 98% sequence identity to the sequence as set forth in at least one of SEQ ID NOs: 1-12 and a delivery scaffold, or a concatenated epitope comprising the sequence as set forth in at least two of SEQ ID NOs: 1-12 or a sequence having at least 98% sequence identity to the sequence as set forth in of SEQ ID NOs: 1-12; and (ii) a pharmaceutically acceptable carrier.

37. The composition of claim 36, further comprising an adjuvant.

38. The composition of claim 37, wherein the adjuvant comprises a squalene-based adjuvant, an aluminum- based adjuvant, a saponin-based adjuvant, a liposome-based adjuvant, a Toll-like receptor (TLR)-based adjuvant, a STING agonist, an attenuated lipid A derivative (ALD), or a carbomer-lecithin-based adjuvant.

39. The composition of claim 38, wherein the aluminum-based adjuvant comprises aluminum hydroxide, aluminum phosphate, or potassium aluminum sulphate (alum).

40. The composition of claim 38, wherein the saponin-based adjuvant comprises Quillaja saponaria.

41. The composition of claim 38, wherein the TLR-based adjuvant comprises resiquimod, muramyl dipeptide, synthetic diacylated lipoprotein FSL-1, glucopyranosyl lipid A (GLA).

42. The composition of claim 38, wherein the TLR-based adjuvant comprises GLA formulated in stable emulsion (GLA SE).

43. The composition of claim 38, wherein the ALD comprises monophosphoryl lipid A (MLA).

44. The composition of claim 37, wherein the adjuvant comprises MLA-Alum.

45. The composition of claim 37, wherein the adjuvant comprises a monophosphoryl lipid A, synthetic trehalose dimycolate, and cell wall skeleton.

46. The composition of claim 37, wherein the adjuvant comprises a monophosphoryl lipid A, synthetic trehalose dimycolate, and muramyl dipeptide.

47. The composition of claim 37, wherein the adjuvant induces a Th1-type response.

48. A method of stimulating an anti-syphilis immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition of claim 36, thereby stimulating the anti- syphilis immune response in the subject.

49. The method of claim 48, wherein the subject is syphilis seropositive.

50. The method of claim 48, wherein the subject is a syphilis seronegative subject, and wherein the therapeutically effective amount reduces the likelihood of the syphilis seronegative subject becoming syphilis seropositive.

51. The method of claim 48, wherein the administering to the subject comprises injection, inhalation, infusion, perfusion, lavage, or ingestion.

52. The method of claim 48, wherein the administering comprises intramuscular, subcutaneous, or intradermal administration.

53. A syphilis vaccine comprising an epitope comprising a sequence as set forth in at least one of SEQ ID NOs: 1-12 or a sequence having at least 98% sequence identity to at least one of SEQ ID NOs: 1-12.

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