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WO2025175360A1 - Combinaison d'antigènes et procédés et utilisations associés - Google Patents

Combinaison d'antigènes et procédés et utilisations associés

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Publication number
WO2025175360A1
WO2025175360A1 PCT/AU2025/050155 AU2025050155W WO2025175360A1 WO 2025175360 A1 WO2025175360 A1 WO 2025175360A1 AU 2025050155 W AU2025050155 W AU 2025050155W WO 2025175360 A1 WO2025175360 A1 WO 2025175360A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
amino acid
antigen combination
seq
antigens
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/AU2025/050155
Other languages
English (en)
Inventor
Drummer Heidi
Williams ELSIE
Zheng SHUNING
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Macfarlane Burnet Institute for Medical Research and Public Health Ltd
Original Assignee
Macfarlane Burnet Institute for Medical Research and Public Health Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2024900445A external-priority patent/AU2024900445A0/en
Application filed by Macfarlane Burnet Institute for Medical Research and Public Health Ltd filed Critical Macfarlane Burnet Institute for Medical Research and Public Health Ltd
Publication of WO2025175360A1 publication Critical patent/WO2025175360A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/20—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
    • G01N2470/04—Sandwich assay format
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/0078—Testing material properties on manufactured objects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the present invention relates to an antigen combination comprising Treponema pallidum (Tp) antigens.
  • the present invention also relates to vectors, polynucleotides, host cells and methods for producing such antigen combinations.
  • the present invention also relates to method and uses of such antigen combinations.
  • the present invention also relates to compositions, assays and kits and the methods and uses thereof.
  • Treponema pallidum causes the sexually transmitted infection known as syphilis in humans with approximately 7 million new infections reported in 2020 (WHO report). Syphilis is transmitted through sexual contact with infectious lesions, via blood transfusion, or from mother to foetus. Mother-to-foetal transmission is the most devastating form of infection causing adverse birth outcomes in 50-80% of cases which include still birth, neonatal death, prematurity, low birth weight and congenitally infected infants. Between 2012 and 2016, the prevalence of active syphilis increased in 78 countries (Korenromp et al., 2018).
  • Treponema pallidum (Tp) antigen combination suitable for detecting the presence of Tp antibodies in a sample.
  • the present inventors have developed an antigen combination comprising two or more or a combination of Tp antigens selected from: Tpl5 or an antigenic fragment thereof; Tpl7 or an antigenic fragment thereof: Tp47 or an antigenic fragment thereof; Tp0453 or an antigenic fragment thereof and/or TmpA or an antigenic fragment thereof.
  • the present application provides an antigen combination comprising three, four, or five Tp antigens selected from the group consisting of: Tpl5 or an antigenic fragment thereof; Tpl7 or an antigenic fragment thereof; Tp47 or an antigenic fragment thereof; Tp0453 or an antigenic fragment thereof; and/or TmpA or an antigenic fragment thereof.
  • the present invention provides an antigen combination comprising the Treponema pallidum antigens: Tp47 or an antigenic fragment thereof; Tp0453 or an antigenic fragment thereof; and TmpA an antigenic fragment thereof.
  • the antigen combination further comprises one or more binding molecule/s.
  • the binding molecule is selected from: a binding protein, a binding polynucleotide and a small molecule.
  • the binding molecule is selected from a: reproductive health binding molecule, a sexual health binding molecule, a women’s health binding molecule and a communicable disease binding molecule.
  • the binding protein is selected from an antibody and a non- Treponema pallidum antigen. In an embodiment, the binding protein is a HIV antigen or antibody.
  • the antigen combination consists of: i) Tp47 or an antigenic fragment thereof; ii) Tp0453 or an antigenic fragment thereof; iii) TmpA or an antigenic fragment thereof; and iv) one or more binding molecules.
  • the antigen combination consists of: i) Tp47 or an antigenic fragment thereof; ii) Tp0453 or an antigenic fragment thereof; and iii) TmpA or an antigenic fragment thereof.
  • the antigen combination consists of: Tp47, Tp0453 and TmpA.
  • the present invention provides a vector or polynucleotide encoding one or more antigen(s) in the antigen combination as described herein.
  • the present invention provides a host cell comprising the vector or polynucleotide as described herein.
  • the present invention provides a method of producing the antigen combination as described herein, the method comprising expressing the vector or polynucleotide as described herein in a host cell or cell-free expression system.
  • the present invention provides a lysate or extract from a cell as described herein, wherein the extract comprises one or more of the antigens as described herein.
  • the present invention provides a composition comprising the antigen combination as described herein.
  • the present invention provides a method of detecting the presence or absence of an active syphilis infection in a sample from a subject, the method comprising exposing the sample from the subject to the antigen combination as described herein under conditions which enable an antibody-antigen complex to form and detecting the presence or absence of an antibody-antigen complex.
  • the antibody-antigen complex is an IgA-specific antibodyantigen complex.
  • the present invention provides a kit for detecting the presence or absence of Treponema pallidum antibodies in a sample from a subject, wherein the assay or kit comprises the antigen combination as described herein.
  • the present invention provides a kit for detecting an active syphilis infection in a sample from a subject, wherein the kit comprises the antigen combination as described herein.
  • the present invention provides an assay for detecting the presence or absence of Treponema pallidum antibodies in a sample from a subject comprising the antigen combination as described herein.
  • Tp0453 or the antigenic fragment thereof is a recombinant antigen.
  • TmpA refers to a T. pallidum an inner-membrane lipoprotein also known as Tp44.5.
  • TmpA comprises the amino acid sequence as set forth in SEQ ID NO: 3, or an amino acid sequence at least 95% identical thereto, or amino acid sequence at least 96% identical thereto, or amino acid sequence at least 97% identical thereto, or amino acid sequence at least 98% identical thereto, or amino acid sequence at least 99% identical thereto.
  • Tp 47 comprises an amino acid about 95 to 100%, about 96 to 100%, about 97 to 100%, about 98 to 100%, or about 99 to 100% identical to SEQ ID NO: 3, or an antigenic fragment thereof.
  • TmpA comprises the amino acid sequence set forth in SEQ ID NO: 18.
  • a TmpA antigenic fragment is a fragment of SEQ ID NO: 3 comprising immunogenic activity.
  • a TmpA antigen fragment comprises the amino acid sequence set forth in any one or more of SEQ ID NOs: 115 to 160.
  • a TmpA antigen fragment comprises the amino acid sequence set forth in any one or more of SEQ ID NOs: 115 to 124.
  • a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 115.
  • a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 116 In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 117.
  • a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 130. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 131 In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 132. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 133. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 134 In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 135. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 136.
  • a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 137. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 138. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 139. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 140 In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 141. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 142.
  • a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 143 In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 144. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 145. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 146 In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 147. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 148.
  • a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 155 In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 156. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 157. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 158 In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 159. In an embodiment, a TmpA antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 160.
  • TmpA or antigenic fragment thereof is a recombinant antigen.
  • Tpl 5 refers to a T. pallidum the inner-membrane lipoprotein.
  • Tpl 5 comprises the amino acid sequence as set forth in SEQ ID NO: 4, or an amino acid sequence at least 95% identical thereto, or amino acid sequence at least 96% identical thereto, or amino acid sequence at least 97% identical thereto, or amino acid sequence at least 98% identical thereto, or amino acid sequence at least 99% identical thereto.
  • Tp 47 comprises an amino acid about 95 to 100%, about 96 to 100%, about 97 to 100%, about 98 to 100%, or about 99 to 100% identical to SEQ ID NO: 4, or an antigenic fragment thereof.
  • Tpl5 comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • a Tpl5 antigenic fragment is a fragment of SEQ ID NO: 4 comprising immunogenic activity.
  • a Tpl5 antigen fragment comprises the amino acid sequence set forth in any one or more of SEQ ID NOs: 161 to 198.
  • a Tpl5 antigen fragment comprises the amino acid sequence set forth in any one or more of SEQ ID NOs: 161 to 170.
  • a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 161.
  • a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 162.
  • a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 163.
  • a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 164. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 165. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 166 In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 167. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 168. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 169.
  • a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 170 In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 171. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 172. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 173. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 174 In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 175.
  • a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 176. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 177. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 178 In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 179. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 180. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 181.
  • a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 182 In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 183. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 184. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 185. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 186 In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 187.
  • a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 188. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 189. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 190 In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 191. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 192. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 193.
  • a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 194 In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 195. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 196. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 197. In an embodiment, a Tpl5 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 198
  • Tpl5 or an antigenic fragment is a recombinant antigen.
  • Tpl7 refers to a T. pallidum inner-membrane lipoprotein.
  • Tpl7 comprises the amino acid sequence as set forth in SEQ ID NO: 5, or an amino acid sequence at least 95% identical thereto, or amino acid sequence at least 96% identical thereto, or amino acid sequence at least 97% identical thereto, or amino acid sequence at least 98% identical thereto, or amino acid sequence at least 99% identical thereto.
  • Tp 47 comprises an amino acid about 95 to 100%, about 96 to 100%, about 97 to 100%, about 98 to 100%, or about 99 to 100% identical to SEQ ID NO: 5, or an antigenic fragment thereof.
  • Tpl5 comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • a Tpl7 antigenic fragment is a fragment of SEQ ID NO: 5 comprising immunogenic activity.
  • a Tpl7 antigen fragment comprises the amino acid sequence set forth in any one or more of SEQ ID NOs: 199 to 233.
  • a Tpl7 antigen fragment comprises the amino acid sequence set forth in any one or more of SEQ ID NOs: 199 to 202.
  • a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 199.
  • a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 200
  • a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 201.
  • a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 202. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 203. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 204 In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 205. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 206. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 207.
  • a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 208 In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 209. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 210. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 211. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 212 In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 213.
  • a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 214. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 215. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 216 In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 217. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 218. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 219.
  • a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 220 In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 221. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 222. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 223. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 224 In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 225.
  • a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 226. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 227. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 228 In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 229. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 230. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 231. In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 232 In an embodiment, a Tpl7 antigen fragment comprises the amino acid sequence set forth in SEQ ID NO: 233.
  • the antigen combination as described herein further comprises one or more one or more binding molecule/s.
  • the binding molecule is a non-Treponemal binding molecule (does not bind an antigen, antibody, or biomarker indicative of Treponema pallidum).
  • binding refers to the interaction of a binding molecule with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen.
  • a binding molecule recognises and binds to a specific protein structure rather than to proteins generally. If a binding molecule binds to epitope “A”, the presence of a molecule containing epitope “A” (or free, unlabelled “A”), in a reaction containing labelled “A” and the binding molecule, will reduce the amount of labelled “A” bound to the binding molecule.
  • the term “specifically binds” or “specific for X” shall be taken to mean a binding molecule of the disclosure reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen or antigens or cell expressing same than it does with alternative antigens or cells.
  • a molecule that specifically binds to an antigen binds that antigen with greater affinity (e.g., 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold greater affinity), avidity, more readily, and/or with greater duration than it binds to other antigens. It is also understood by reading this definition that, for example, a molecule that specifically binds to a first antigen may or may not specifically bind to a second antigen.
  • the binding molecule binds an antibody, antigen or biomarker indicative of a disease or condition as described herein.
  • biomarker refers to indicators of typical biological processes, pathogenic processes, or pharmacological reactions to therapy. Numerous biomarkers exist, and may be descriptive, prognostic or predictive. Biomarkers include enzymes, antigens and lipids, and can be found in any biological sample taken from a subject. Biomarkers include complex biomarkers or combinations of markers. The skilled person will be aware of suitable biomarkers for monitoring the development of disease, see, for example, Bodaghi et al., (2023); Yang et al., (2022); Califf (2016), Masson et al., (2019).
  • the biomarker is an inflammatory marker.
  • the biomarker is an interferon.
  • the biomarker is a chemokine.
  • the biomarker is a cytokine.
  • the biomarker is a CD antigen.
  • the biomarker is any biochemical biomarker that can be detected by an antibody.
  • the biomarker is alanine transaminase (ALT).
  • the biomarker is blood sugar level.
  • the biomarker is complete blood count.
  • the biomarker is creatinine.
  • the biomarker is C-reactive protein.
  • the biomarker is glial fibrillary acidic protein (GFAP).
  • the biomarker is hematocrit (HCT).
  • HCT hematocrit
  • the biomarker is Haemoglobin (Hgb).
  • the biomarker is red blood cell count (RBC).
  • RBC red blood cell count
  • the biomarker is thyroid- stimulating hormone (TSH).
  • TSH thyroid- stimulating hormone
  • the biomarker is triglyceride.
  • the biomarker is troponin.
  • the biomarker is ubiquitin carb oxy -terminal hydrolase LI (UCH-L1).
  • the disease or condition is selected from one of the following groups: reproductive health, sexual health, women’s health, and communicable diseases.
  • reproductive health refers to a condition or disease relating to the reproductive system and to its functions and processes.
  • reproductive health refers to female reproductive health.
  • reproductive health refers to male reproductive health.
  • a condition or disease relevant to “sexual health” refers to a condition or disease relating to a sexually transmitted infection, disease, or condition.
  • sexual health refers to women’s and men’s sexual health.
  • sexual health refers to women’s sexual health.
  • sexual health refers to men’s sexual health.
  • a condition or disease relevant to “women’ s health” refers broadly to a condition or disease related to physical and mental health problems that are of exclusive concern for women.
  • a condition or disease relevant to “communicable diseases” refers to a disease that can spread from subject to subject, for example by sharing of bodily fluids, skin contact and close proximity between subjects.
  • Antigens and antibodies directed to testing for conditions and diseases related to reproductive health, sexual health, women’s health, and communicable diseases can be tested for using known antigens and antibodies, as utilised in commercially available kits and assays, for example provided by health providers, of which the skilled person will be aware.
  • HIV antigens and antibodies directed to HIV proteins can be used to detect a disease or condition relevant to any one or more of reproductive health, sexual health, women’s health, or a communicable disease.
  • the binding molecule is selected from a: reproductive health binding molecule, a sexual health binding molecule, a women’s health binding molecule and a communicable disease binding molecule.
  • the binding molecule is a binding protein.
  • the binding protein is an antibody as described herein.
  • the binding protein is a non-Tp antigen as described herein.
  • a polypeptide may be defined by the extent of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by having a greater % identity to one reference amino acid sequence than to another.
  • the query sequence is at least 50 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 50 amino acids.
  • the GAP analysis aligns two sequences over their entire length.
  • the polypeptide has an antigenic activity of at least 10% of the activity of the reference polypeptide.
  • the polypeptide has an antigenic activity of about 10 to 100% of the activity of the reference polypeptide.
  • the polypeptide/protein comprises an amino acid sequence which is at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
  • the polypeptide/protein comprises an amino acid sequence which is about 90 to 100%, more preferably about 91 to 100%, more preferably about 92 to 100%, more preferably about 93 to 100%, more preferably about 94 to 100%, more preferably about 95 to 100%, more preferably about 96 to 100%, more preferably about 97 to 100%, more preferably about 98 to 100%, more preferably about 99 to 100%, more preferably about 99.1 to 100%, more preferably about 99.2 to 100%, more preferably about 99.3 to 100%, more preferably about 99.4 to 100%, more preferably about 99.5 to 100%, more preferably about 99.6 to 100%, more preferably about 99.7 to 100%, more preferably about 99.8 to 100 %, and even more preferably about 99.9% identical to the relevant nominated SEQ ID NO.
  • amino acid sequence mutants of the polypeptides defined herein can be prepared by introducing appropriate nucleotide changes into a polynucleotide defined herein, or by in vitro synthesis of the desired polypeptide.
  • Such mutants include for example, deletions, insertions, or substitutions of residues within the amino acid sequence.
  • a combination of deletions, insertions and substitutions can be made to arrive at the final protein, provided that the final polypeptide product possesses the desired characteristics.
  • Mutant (altered) polypeptides can be prepared using any technique known in the art, for example, using directed evolution or rational design strategies (see below).
  • the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified.
  • the sites for mutation can be modified individually or in series for example, by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
  • Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
  • Substitution mutants have at least one amino acid residue in the polypeptide removed and a different residue inserted in its place. Such conservative substitutions are shown in Table 1 under the heading of “exemplary substitutions”.
  • a mutant/variant polypeptide has only, or not more than, one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in Table 1. Mutants with desired activity may be engineered using standard procedures in the art such as by performing random mutagenesis, targeted mutagenesis, or saturation mutagenesis on known genes of interest, or by subjecting different genes to DNA shuffling.
  • the isolated and/or recombinant protein or antigenic fragment thereof comprises a signal sequence to aid secretion of the protein form a cell or a tag to aid purification of the protein.
  • the tag is a protein tag.
  • the protein tag is a FLAG-tag or HIS-tag.
  • FLAG-tagged or “FLAG-tag” refers to a polypeptide tag that can be added to a protein using recombinant DNA technology i.e. modification of the nucleotide sequence encoding the polypeptide.
  • non-Tp antigens are antigens of a pathogen.
  • non-Tp antigens are antigens that are a biomarker of a disease or condition as described herein.
  • suitable biomarkers that can be detected by antibodies and aptamers, including but not limited to cytokines, inflammatory markers, interferons, chemokines, lipids and CD antigens.
  • the biomarker is a lipid.
  • the biomarker is cardiolipin (detected with an anti-cardiolipin antibody).
  • the biomarker is not cardiolipin.
  • suitable biomarkers are any other biochemical biomarker detectable by an antibody.
  • biomarker antigens that are detectable by specific antibody binding.
  • biomarker antigens that are detectable by aptamer are a protein.
  • an enzyme, an enzyme fragment, a substrate for enzyme activity, or product of enzymatic activity are suitable biomarkers.
  • suitable methods for detection and/or quantification of proteins and enzymatic reactions including but not limited to western blot, ELISA, chemiluminescence and mass spectrometry.
  • biomarkers are detectable by enzyme assay.
  • biomarkers are lipids, for example, cardiolipin.
  • the antigen combination as described herein comprises one or more non-Tp antigens from a non-Tp pathogen or a biomarker relevant to a condition or disease selected from one or more of: reproductive health, sexual health, women’s health or a communicable disease.
  • the non-Tp antigen is a reproductive health antigen. In an embodiment, the non-Tp antigen is a sexual health antigen. In an embodiment, the non-Tp antigen is a women’s health antigen. In an embodiment, the non-Tp antigen is a communicable disease antigen. In an embodiment, the non-Tp antigen is HIV. In an embodiment, the non-Tp antigen is cardiolipin.
  • the antigen combination as described herein comprises one or more antibodies to a non-Tp pathogen (non-Tp antibodies) or a biomarker, for example, antibodies directed to proteins relevant to a condition or disease selected from one or more of: reproductive health, sexual health, women’s health or a communicable disease.
  • the non-Tp antibody is a reproductive health antibody.
  • the non-Tp antibody is a sexual health antibody.
  • the non-Tp antibody is a women’s health antibody.
  • the non-Tp antibody is a communicable disease antibody.
  • the non-Tp antibody is HIV.
  • the non-Tp antibody is cardiolipin.
  • the non-Tp antibody is an anti-lipid antibody.
  • an anti-cardiolipin antibody is an anti-lipid antibody.
  • the binding molecule is a binding polynucleotide.
  • the polynucleotide is DNA or RNA.
  • the polynucleotide is a mixture of DNA and RNA and/or can contain one or more modified bases or base analogues.
  • a binding polynucleotide is complementary to a single-stranded oligonucleotide or a single ‘sense’ strand, for example, mRNA, precursorRNA, siRNA, miRNA, pri-miRNA, single stranded DNA overhangs, aptamer, and short sequences of artificial DNA or RNA designed to bind a specific target (probe).
  • aptamer is a single stranded nucleic acid that has a three-dimensional conformation capable of recognising and binding an antigen as described herein.
  • the binding molecule is a small molecule which binds an antigen as described herein.
  • a “small molecule” refers to a chemical compound or molecule having a molecular weight below 2000 Daltons, preferably below 1500 Daltons, more preferably below 1000 Daltons, still more preferably below 750 daltons, yet more preferably below 500 Daltons.
  • the small molecule is not a polypeptide.
  • the antigen combination of the present invention comprises individual antigens and/or fragments thereof that are mixed together, or pooled. In an embodiment, the antigen combination of the present invention is mixed or pooled with one or more binding proteins as described herein. In some embodiments, the antigen combination of the present invention comprises individual antigens and/or fragments thereof that are collocated or specifically arranged on the same substrate material. For example, the antigen combination is striped onto a membrane, for example, a nitrocellulose membrane. In some embodiments, the antigen combination is pooled and then applied to a substrate, for example a membrane. In some embodiments, the antigen combination is applied sequentially to a substrate, for example a membrane.
  • antigens of the antigen combination are arranged on different areas of the same substrate, for example a membrane. It will be apparent to the skilled person that any combination of these approaches is contemplated by the present application. For example, a pool of two antigens is applied to a substrate and an individual antigen is applied to a substrate to produce an antigen combination of three antigens. Similarly, the antigens may be combined in any order, and over any suitable duration.
  • an anti-lipid antibody line is striped onto a solid substrate. In some embodiments, an anti-cardiolipin antibody line is striped onto a solid substrate.
  • fusion protein is a protein consisting of at least two domains that are encoded by separate genes that have been joined so that they are transcribed and translated as a single unit, producing a single polypeptide.
  • the fusion protein comprises at least two antigens or fragments thereof as described herein.
  • the fusion protein comprises at least three antigens or fragments thereof as described herein.
  • the fusion protein comprises at least four antigens or fragments thereof as described herein.
  • the fusion protein comprises at least five antigens or fragments thereof as described herein.
  • the fusion protein comprises at least two Tp antigens or fragments thereof as described herein. In an embodiment, the fusion protein comprises at least three Tp antigens or fragments thereof as described herein. In an embodiment, the fusion protein comprises at least four Tp antigens or fragments thereof as described herein. In an embodiment, the fusion protein comprises two to five antigens or fragments thereof as described herein. In an embodiment, the fusion protein comprises three to five antigens or fragments thereof as described herein. In an embodiment, the fusion protein comprises four or five antigens or fragments thereof as described herein. In an embodiment, the fusion protein comprises five antigens or fragments thereof as described herein.
  • the present invention provides an antigen combination, or fusion protein comprising two Tp antigens selected from the group consisting of Tpl5, Tpl7, Tp47, Tp0453 and TmpA, wherein at least one antigen is TmpA and/or Tp0453, and wherein the antigen combination or fusion protein does not comprise more than two Tp antigens.
  • the present invention provides an antigen combination, or fusion comprising three or four Tp antigens selected from the group consisting of Tpl5, Tpl7, Tp47, Tp0453 and TmpA, wherein at least one antigen is Tp0453, and wherein the antigen combination, or fusion protein does not comprise more than four Tp antigens.
  • the present invention provides an antigen combination comprising the Tp antigens: Tp47 or an antigenic fragment thereof; Tp0453 or an antigenic fragment thereof; and TmpA or an antigenic fragment thereof.
  • the antigen combination further comprises one or more binding molecules as described herein.
  • the antigen combination consists of: Tp47 or an antigenic fragment thereof; Tp0453 or an antigenic fragment thereof; TmpA or an antigenic fragment thereof; and one or more binding molecules as described herein.
  • the antigen combination consists of: Tp47 or an antigenic fragment thereof; Tp0453 or an antigenic fragment thereof; and TmpA or an antigenic fragment thereof.
  • the antigen combination consists of: Tp47, Tp0453 and TmpA.
  • the antigen combination for detection of active syphilis does not include Tpl7 or an antigenic fragment thereof. In some embodiments, the antigen combination for detection of active syphilis consists of: Tp47 or an antigenic fragment thereof; Tp0453 or an antigenic fragment thereof; and TmpA or an antigenic fragment thereof.
  • the antigen combination for detection of active syphilis consists of: Tp47, Tp0453 and TmpA or an antigenic fragment thereof.
  • At least two of Tp47, Tp0453 and TmpA are present in a fusion protein, optionally comprising a linker.
  • Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 2: 1 : 1.
  • Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 2:2: 1. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 2: 1 :2. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 1 :2: 1. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 1 : 1 :2. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 1 :2:2.
  • Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 3:2:3. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 3: 1 :2. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 2:3: 1. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 2: 1 :3. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 2:2:3.
  • Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 2:3:2. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 1 :2:3. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 1 : 1:3. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 3:2:3. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 3:3:2.
  • Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 3:3: 1. In some embodiments, Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 1 :3: 1.
  • Tp antigens Tp47:TpO453:TmpA are present in a ratio of about 1 : 1 : 1, wherein the ratio includes variations up to 20% of each ratio.
  • Tp47:TpO453:TmpA are present in a ratio of about 50:25:25.
  • Tp47:TpO453:TmpA are present in a ratio of about 40:20:40.
  • Tp47:TpO453:TmpA are present in a ratio of about 45:23:33.
  • the total antigen concentration is between about 0.1 mg/mL to about 2 mg/mL. In some embodiments, the total antigen concentration is between about 0.5 mg/mL to about 1 mg/mL. In some embodiment, the total antigen concentration is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0 mg/mL. In some embodiments, the total antigen concentration is about 0.1 mg/mL. In some embodiments, the total antigen concentration is about 0.2 mg/mL.
  • the total antigen concentration is about 0.3 mg/mL. In some embodiments, the total antigen concentration is about 0.4 mg/mL. In some embodiments, the total antigen concentration is about 0.5 mg/mL. In some embodiments, the total antigen concentration is about 0.6 mg/mL. In some embodiments, the total antigen concentration is about 0.7 mg/mL. In some embodiments, the total antigen concentration is about 0.8 mg/mL. In some embodiments, the total antigen concentration is about 0.9 mg/mL. In some embodiments, the total antigen concentration is about 1 mg/mL. In some embodiments, the total antigen concentration is about 1.1 mg/mL. In some embodiments, the total antigen concentration is about 1.2 mg/mL.
  • the total antigen concentration is about 1.3 mg/mL. In some embodiments, the total antigen concentration is about 1.4 mg/mL. In some embodiments, the total antigen concentration is about 1.5 mg/mL. In some embodiments, the total antigen concentration is about 1.6 mg/mL. In some embodiments, the total antigen concentration is about 1.7 mg/mL. In some embodiments, the total antigen concentration is about 1.8 mg/mL. In some embodiments, the total antigen concentration is about 1.9 mg/mL. In some embodiments, the total antigen concentration is about 2 mg/mL.
  • the condition is HIV.
  • the antigen combination further comprises one or more of the antigens in Table 2 to assess for HIV.
  • the antigen combination further comprises HIV antigens gp41, gpl20, and p24.
  • Table 2 Proteins of the Human Immunodeficiency virus and their function.
  • the antigen combination, composition, assay, kit, methods or uses as described herein further comprise an excipient.
  • a functional excipient that improves the sensitivity and/or specificity of the antigen combination for detecting Tp in a sample.
  • the excipient is any sugar.
  • the excipient is selected from the group comprising sucrose, galactose, maltose, glucose, lactose, and trehalose.
  • the excipient is sucrose.
  • the excipient is galactose.
  • the excipient is maltose.
  • the excipient is glucose.
  • the excipient is lactose.
  • the excipient is trehalose. In some embodiments, the excipient is present in an amount of 0.1 to 1% (w/v). In some embodiments, the excipient is present in an amount of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0% (w/v). In some embodiments, the excipient is present in an amount of 0.5% of the total antigen concentration. For example, the excipient is sucrose and is present in an amount of 0.5% (w/v) of the total antigen concentration.
  • the excipient is present in an amount of about 1.0 to 5% (w/v). In some embodiments, the excipient is present in an amount of about 1.2, 1.5, 1.7, 2.0, 2.2, 2.5, 2.7, 3.0, 3.2, 3.5, 2.7, 4.0, 4.2, 4.5, 4.7, or 5.0% (w/v).
  • the present invention provides a composition comprising the antigen combination as described herein. In an aspect, the present invention provides a composition comprising one or antigens or antigenic fragments of the antigen combination as described herein.
  • the composition comprises a single Tp antigen or an antigenic fragment thereof. In an embodiment, the composition comprises two Tp antigens or antigenic fragments thereof. In an embodiment, the composition comprises three Tp antigens or antigenic fragments thereof. In an embodiment, the composition comprises four Tp antigens or antigenic fragments thereof. In an embodiment, the composition comprises five Tp antigens or antigenic fragments thereof. In an embodiment, the composition comprises one or more non-Tp antigens and one or more binding molecules as described herein. In an embodiment, the composition comprises one or more non-Tp antigens or non-Tp antibodies as described herein. In an embodiment, the composition in a form selected from a: solution, powder, liquid, emulsion, lyophilised, dried, frozen, or freeze-dried.
  • the composition is a liquid. In an embodiment, the composition is a solution. In an embodiment, the composition is a powder. In an embodiment, the composition is an emulsion. In an embodiment, the composition is lyophilised. In an embodiment, the composition is dried. In an embodiment, the composition is frozen. In an embodiment, the composition is freeze-dried. In an embodiment, the composition is stored in one form and used in another. For example, the composition is stored in solution and is used dried (immobilised), for example on a test strip. For example, the composition is dried, lyophilised, or freeze-dried when stored and used in solution.
  • sensitivity refers to a methods ability to correctly identify those with the disease (true positive rate, i.e. without false negatives).
  • the specificity of the antigen combination as described herein for detecting active syphilis infection in subjects is about 95%.
  • the antigen combination as described herein are able to correctly classify between about 51% to about 79% of past-treated syphilis subjects. In some embodiments, the antigen combination as described herein are able to correctly classify about 58% of past-treated syphilis subjects. In some embodiments, the antigen combination as described herein are able to correctly classify about 71% of past-treated syphilis subjects. In some embodiments, the antigen combination as described herein are able to correctly classify about 80% of past-treated syphilis subjects. In some embodiments, the antigen combination as described herein are able to correctly classify between about 76% and about 100% of subjects.
  • the antigen combination as described herein are able to correctly classify subjects with a sensitivity of about 95%. In some embodiments, the antigen combination as described herein are able to correctly classify subjects with a sensitivity of about 97%.
  • the antigen combination as described herein are able to correctly classify subjects with a specificity of about 83%. In some embodiments, the antigen combination as described herein are able to correctly classify subjects with a specificity of about 84%. In some embodiments, the antigen combination as described herein are able to correctly classify subjects with a specificity of about 86%. In some embodiments, the antigen combination as described herein are able to correctly classify subjects with a specificity of about 92%.
  • the antigen combination as described herein have a sensitivity of up to and including about 100% for syphilis infection. In some embodiments, the antigen combination as described herein have a sensitivity of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% for active syphilis infection.
  • the antigen combination as described herein have a sensitivity of up to and including 100% for active syphilis infection. In some embodiments, the antigen combination, as described herein have a sensitivity of between about 80% to 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a sensitivity of between about 86% to 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a sensitivity of between about 86% to about 92% for active syphilis infection. In some embodiments, the antigen combination as described herein have a sensitivity of between about 90% to 100% for active syphilis infection.
  • the antigen combination as described herein have a sensitivity of between about 93% to 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a sensitivity of between about 95% to 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a sensitivity of between about 97% to 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a sensitivity of between 98% to 100% for active syphilis infection.
  • the antigen combination as described herein have a sensitivity of 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% for active syphilis infection.
  • the antigen combination as described herein have a sensitivity of about 97% for active syphilis infection. In some embodiments, the antigen combination as described herein have a sensitivity of about 98% for active syphilis infection. In some embodiments, the antigen combination as described herein have a sensitivity of about 100% for active syphilis infection.
  • the antigen combination as described herein have a specificity of up to and including 100% for syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% for active syphilis infection.
  • the antigen combination as described herein have a specificity of up to and including 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of at least 95% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of at least 94% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of at least 90 % for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of at least 80 % for active syphilis infection.
  • the antigen combination as described herein have a specificity of between 51% and 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of between 51% and 79% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of between 55% and 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of between 58% and 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of between 63% and 100% for active syphilis infection.
  • the antigen combination as described herein have a specificity of between 76% and 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of between 78% and 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of between 80% and 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of between 83% and 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of between 86% and 100% for active syphilis infection.
  • the antigen combination as described herein have a specificity of between 92% and 100% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of between 94% and 100% for active syphilis infection.
  • the antigen combination as described herein have a specificity of about 55% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of about 58% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of about 63% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of about 78% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of about 80% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of about 83% for active syphilis infection.
  • the antigen combination as described herein have a specificity of about 84% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of about 86% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of about 92% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of about 94% for active syphilis infection. In some embodiments, the antigen combination as described herein have a specificity of about 100% for active syphilis infection.
  • the antigen combination as described herein have a specificity of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98, 99, or 100% for active syphilis infection.
  • the antigen combination described herein is between 50- 100% more effective at detecting active syphilis than other reported methods.
  • the antigen combination described herein is about 50%, 60%, 70%, 80%, 90% and up to 100% more effective at detecting active syphilis than other reported methods.
  • the antigen combination as described herein reduces false positive detection of active syphilis by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and up to 100%.
  • the antigen combination as described herein reduces false positive detection of active syphilis by up to 80%.
  • the combination of two or more of the antigens described herein reduce the misidentification of past-treated syphilis as active syphilis. In some embodiments, combinations of two or more antibodies described herein reduce false positive active syphilis diagnoses in patients with past-treated syphilis.
  • the antigen combination does not detect a past-treated syphilis infection.
  • the sensitivity and/or specificity of the antigen combination as described herein for detecting syphilis infection is in subjects never diagnosed with syphilis. In one embodiment, the specificity for detecting syphilis infection in never infected subjects is 100%. In one embodiment, the specificity for detecting syphilis infection in never diagnosed subjects is about 94%.
  • the sensitivity and/or specificity of the antigen combination as described herein for detecting syphilis infection is in subjects with past- treated syphilis. In one embodiment, the specificity for detecting syphilis infection in past-treated subjects is about 63%. In one embodiment, the specificity for detecting syphilis infection in past-treated subjects is about 78%. In one embodiment, the sensitivity for detecting syphilis infection in past-treated subjects is about 90%. In one embodiment, the sensitivity for detecting syphilis infection in past-treated subjects is about 84.7%. In some embodiments, the sensitivity and/or specificity of the antigen combination as described herein for detecting syphilis infection is in subjects with active syphilis.
  • the sensitivity of the antigen combination as described herein for detecting syphilis infection is in subjects with active syphilis is about 90%. In some embodiments, the sensitivity of the antigen combination as described herein for detecting syphilis infection is in subjects with active syphilis is about 93%. In some embodiments, the sensitivity of the antigen combination as described herein for detecting syphilis infection is in subjects with active syphilis is about 95%. In some embodiments, the sensitivity of the antigen combination as described herein for detecting syphilis infection is in subjects with active syphilis is 100%.
  • the specificity of the antigen combination as described herein for detecting syphilis infection is in subjects with active syphilis is about 55%. In some embodiments, the specificity of the antigen combination as described herein for detecting syphilis infection is in subjects with active syphilis is about 58%. In some embodiments, the specificity of the antigen combination as described herein for detecting syphilis infection is in subjects with active syphilis is about 63%. In some embodiments, the specificity of the antigen combination as described herein for detecting syphilis infection is in subjects with active syphilis is about 78%.
  • the sensitivity and/or specificity of the antigen combination as described herein for detecting an active syphilis infection is in subjects never diagnosed with syphilis.
  • the sensitivity and/or specificity of the antigen combination as described herein for detecting an active syphilis infection is in subjects with past-treated syphilis.
  • the sensitivity and/or specificity of the antigen combination as described herein for detecting an active syphilis infection is in subjects with active syphilis.
  • the antigen combination as described herein have a sensitivity of about 95%. In some embodiments, the antigen combination as described herein have a sensitivity of about 95% when subjects who are ⁇ 6 months post-last infection are excluded. In some embodiments, the antigen combination as described herein have a sensitivity of about 94% when subjects who have had more than one past infection are excluded. In some embodiments, the antigen combination as described herein have a sensitivity of about 94% when subjects who have had more than two past infections are excluded. In some embodiments, the antigen combination as described herein have a sensitivity of about 95% when subjects who are ⁇ 6 months post-last infection and have had more than two past infections are excluded.
  • the antigen combination as described herein have a specificity of about 86%. In some embodiments, the antigen combination as described herein have a specificity of about 90% when subjects who are ⁇ 6 months post-last infection are excluded. In some embodiments, the antigen combination as described herein have a specificity of about 91% when subjects who have had more than one past infection are excluded. In some embodiments, the antigen combination as described herein have a specificity of about 90% when subjects who have had more than two past infections are excluded. In some embodiments, the antigen combination as described herein have a specificity of about 92% when subjects who are ⁇ 6 months post-last infection and have had more than two past infections are excluded.
  • Antibodies include modifications in a variety of forms including, for example, but not limited to, domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH, as described for a camelid), a dimer of the light chain variable region (VLL), Fv fragments containing only the light (VL) and heavy chain (VH) variable regions which may be joined directly or through a linker, or Fd fragments containing the heavy chain variable region and the CHI domain.
  • domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH, as described for a camelid), a dimer of the light chain variable region (VLL), Fv fragments containing only the light (VL) and heavy chain (VH) variable regions which may be joined directly or through a linker, or Fd fragments containing the heavy chain variable region and the CHI domain.
  • Antibodies can consist of VHH regions, in isolation or multiple VHH domains joined directly with linkers, and then connected to the Fc region comprising CH2 and CH3 domains of an immunoglobulin.
  • a scFv consisting of the variable regions of the heavy and light chains linked together to form a single-chain antibody (Bird et al., 1988; Huston et al., 1988) and oligomers of scFvs such as diabodies and triabodies are also encompassed by the term "antibody”.
  • fragments of antibodies such as Fab, (Fab')2 and FabFc2 fragments which contain the variable regions and parts of the constant regions.
  • the Chothia definition (which is based on the location of the structural loop regions; See, e.g., Chothia et al., (1987), the AbM definition (which is a compromise between the Kabat and Chothia definitions and is based on Oxford Molecular's AbM antibody modelling software), the IMGT definition (see, e.g., https://www.imgt.org/IMGTindex/CDR.php) and the method described by Kontermann and Diibel (2010).
  • the antibody is a Tp antibody.
  • Anti-Tp antibodies may be produced by the subject as a result of Tp infection, or may be developed synthetically, by means known in the art, for example, by antigen injection into a host organism.
  • Commercially available anti-Tp antibodies include Creative Biolabs VAnt-Wyb212, VAnt-Wyb210, and CABT-NS1525 (directed to Tp47, Tpl7, and TmpA respectively.
  • the antibody is a non-Tp antibody as described herein.
  • the antigen combination has lower/improved false positive rate for detecting active versus past-treated syphilis infections. In some embodiments, the antigen combination has lower/improved false positive rate for detecting active versus past-treated syphilis infections than an antigen combination comprising Tpl7 or a fragment thereof. In some embodiments, the antigen combination has lower/improved false positive rate for detecting active versus past-treated syphilis infections than an antigen combination comprising Tpl7.
  • a “detectable label” is a molecular or atomic tag or marker that generates or can be induced to generate an optical or other signal or product that can be detected visually or by using a suitable detector.
  • Detectable labels are well-known in the art and include, for example, metal labels, magnetic labels, beads, fluorescent labels, chemical labels, radionucleotides, coloured particles, quantum dots, fluorescent latex particles, carbon nanoparticles, luminescent label, chemiluminescence based label, liposome based probes, Raman-active tags, a prosthetic group, a contrast agent, an ultrasound agent, and protein labels (Song et al., 2008; Nuntawong et al., 2022; Muyldermans, 2013).
  • the detectable label is detectable via a smartphone (Zangheri et al., 2015).
  • the detectable label is directly detectable, for example, by visual inspection. In an embodiment, the detectable label is indirectly detected, for example, by enzymatic reaction.
  • the detectable label is gold.
  • an IgA-specific gold label is gold.
  • the detectable label is a nanoparticle label.
  • Suitable nanoparticle labels are known in the art, and include labels which have colloidal stability in solution under various conditions and temperatures, susceptibility for detection over a large (and useful) dynamic range, efficiency and reproducibility of conjugation (without the loss of chemical and biological integrity and activity), lack of or very low nonspecific binding characteristics (ensuring a high signal-to-noise ratio), commercial availability at low cost, and utilise an easy and scalable conjugation procedure.
  • Tp antigen binding proteins and/or binding proteins of a non-Tp condition comprising an antibody variable region are conjugated to coloured particles for visual analysis, for example, colloidal gold and/or latex microspheres.
  • the detectable label is colloidal gold.
  • the detectable label is latex.
  • the latex label is tagged with a detector reagent, for example coloured or fluorescent dyes or magnetic or paramagnetic components.
  • the detectable label is a carbon label, for example, carbon nanotubes. Detectable labels may be enzymatically modified to improve sensitivity of the assay. Polymer encapsulation and/or surface blocking may also be used to improve sensitivity, for example by reducing background noise of fluorescent nanoparticle labels such as quantum dots.
  • a detectable label is an enzyme.
  • enzymes useful in the disclosure include, without limitation, alkaline phosphatase and horseradish peroxidase.
  • the enzyme can be, for example, luciferase.
  • the enzyme can be linked to the antibody by conventional chemical methods, or it can be expressed together with the antibody as a fusion protein.
  • the enzyme is horseradish peroxidase.
  • Radioisotopes useful as detectable labels in the disclosure are well known in the art and can include 3H, 11C, 18F, 35S, 64Cu, 67Ga, 68Ga, 99mTc, U lin, 1231, 1241, 1251, and 1311.
  • the detectable label is selected from one or more of: red intense microspheres (Merck e.g. catalogue numbers FR180380637 and FR180380638), cellulose nanobeads, latex beads, alkaline phosphatase, horseradish peroxidase, colloidal gold, gold nanoshells, europium, fluorescent label and a luminescent label.
  • the detectable label is red intense microspheres.
  • the detectable label is cellulose nanobeads.
  • the detectable label is latex beads.
  • the detectable label is alkaline phosphatase.
  • the detectable label is horseradish peroxidase.
  • the detectable label is colloidal gold.
  • the detectable label is gold nanoshells.
  • the detectable label is europium.
  • the detectable label is red fluorescent protein.
  • the detectable label is green fluorescent protein.
  • the fluorescent label is selected from, but not limited to, Green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), fluorescein (FITC), alexa fluor, 5,6-carboxymethyl fluorescein, texas red, nitrobenz-2-oxa-l,3- diazol-4-yl (NBD), coumarin, dansyl chloride, rhodamine, 4'-6-diamidino-2- phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), and rhodamine (5,6- tetramethyl rhodamine), Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, mNeonGreen, mUKG, AcGFP, ZsGreen, Cloverm Sapphire, T-Sapphire,
  • the fluorescent protein is RFP. In an embodiment, the fluorescent protein is GFP.
  • the absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm).
  • the luminescent label is selected from: aequorin, firefly luciferase, renilla luciferase, gaussia luciferase, bacterial luciferase and nanoluc.
  • the magnetic label is a magnetic or paramagnetic compound, such as, iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferrous- chromium-cobalt, nickel-ferrous, cobalt- platinum, or strontium ferrite.
  • one or more of the antigens comprise a detectable label.
  • the detectable label is selected from a: metal label, magnetic label, bead, colorimetric label, radioactive label, enzymatic label, luminescent label, fluorescent label, antibody, quantum dot, fluorescent latex particle, chemiluminescence based label, liposome-based probe and a Raman-active tag.
  • the detectable label is specific to IgA antibodies.
  • the antibody is an anti-IgA gold.
  • the antigens and/or antigen combination of the disclosure can be produced intracellularly, in the periplasmic space, or directly secreted into the medium.
  • the particulate debris either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration.
  • supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. Supernatants can also be used directly for purification.
  • the present disclosure further provides a polynucleotide encoding an antigen or fusion protein, or a plurality of antigens of the disclosure.
  • the polynucleotide is a recombinant DNA sequence.
  • a recombinant DNA sequence is a plasmid.
  • an antigen or antigen combination of the disclosure can be modified to include a tag to facilitate purification or detection (examples of such are described above).
  • the resulting protein is then purified using methods known in the art, such as affinity purification.
  • a protein comprising a hexa-his tag is purified by contacting a sample comprising the protein with nickelnitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-His-tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein.
  • Ni-NTA nickelnitrilotriacetic acid
  • a ligand or antibody that binds to a tag is used in an affinity purification method.
  • a host cell comprising the vector or polynucleotide of the disclosure.
  • the host cell is a eukaryotic cell.
  • the host cell is a bacterial cell.
  • provided herein is a method of producing an antigen combination of the disclosure which comprises expressing the vector or polynucleotide disclosed herein in a host cell or cell-free expression system.
  • a lysate or extract from a cell disclosed herein wherein the extract comprises one or more of the antigens of the present disclosure.
  • the present disclosure further provides a polypeptide encoding an antigen or fusion protein, or a plurality of antigens of the disclosure.
  • a polynucleotide encoding one or more of the antigens described herein, or the antigen combination as described herein is placed into one or more expression construct/s, e.g., expression vector(s), which is/are then transfected into a host cell, such as a bacterial cell, a yeast cell, an insect cell, or a mammalian cell, for example bacterial cell, for example an E. coli cell.
  • the antigen combination may be produced using one vector or polynucleotide encoding all of the antigens in the combination, or several vectors or polynucleotides encoding one or more of the antigens in the combination.
  • nucleotides encoding the antigens of the antigen combination are combined in a single polynucleotide.
  • nucleotides encoding the antigens of the antigen combination are encoded by two or more polynucleotides, for example for expression and subsequent combination and thereby production of the antigen combination disclosed herein.
  • Exemplary bacterial cells include E. coli.
  • Exemplary mammalian cells include simian COS cells, Human Embryonic Kidney (HEK) cells and their derivatives, Chinese Hamster Ovary (CHO) cells, Hela, Human embryonic kidney 293 cells (HEK293), human osteosarcoma U2OS, A549, HT1080, Cath. -a-differentiated cells (CAD), Pl 9, NIH 3T3, L929, N2a, Hep G2 or myeloma cells that do not otherwise produce immunoglobulin protein.
  • CAD Cath. -a-differentiated cells
  • Pl 9 NIH 3T3, L929, N2a, Hep G2 or myeloma cells that do not otherwise produce immunoglobulin protein.
  • Molecular cloning techniques to achieve these ends are known in the art and described, for example in Ausubel (ed, 1988 including all updates until present) or Sambrook et al., (1989).
  • a wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids, as would be suitable for generating the antigens disclosed herein, and thereby the antigen combination disclosed herein.
  • Methods of producing recombinant antigens are also known in the art, as are methods of producing recombinant antibodies. See, for example, Fox and Klass (1989); US4816567; US7923221and US7022500.
  • the nucleic acid is operably linked to a promoter.
  • promoter is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and/or external stimulus, or in a tissue specific manner.
  • the nucleic acid is codon optimised for bacterial cell culture.
  • Methods of codon optimisation will be apparent to the skilled person.
  • tools for codon optimisation include, for example, GeneArt GeneOptimizer (Thermofisher®) or GenSmart® (GeneScript®).
  • the nucleic acid will comprise an N-terminal sequence to aid expression in a host cell.
  • the nucleic acid comprises a nucleotide sequence selected from: SEQ ID NOs: 11 to 15 or a codon optimised version thereof, or a sequence at least 70%, at least 80%, at least 85%, at least 90, at least 95%, or at least 98% identical thereto.
  • the sequence is between about 70 to 100% identical thereto.
  • the sequence is between about 80 to 100% identical thereto.
  • the sequence is between about 90 to 100% identical thereto.
  • the sequence is between about 95 to 100% identical thereto.
  • the sequence is between about 98 to 100% identical thereto.
  • the nucleic acid comprises the nucleotide sequence SEQ ID NO: 7 or a codon optimised version thereof, or a sequence at least 70%, at least 80%, at least 85%, at least 90, at least 95%, or at least 98% identical thereto.
  • the sequence is between about 70 to 100% identical thereto.
  • the sequence is between about 80 to 100% identical thereto.
  • the sequence is between about 90 to 100% identical thereto.
  • the sequence is between about 95 to 100% identical thereto.
  • the sequence is between about 98 to 100% identical thereto.
  • the nucleic acid comprises the nucleotide sequence SEQ ID NO: 8 or a codon optimised version thereof, or a sequence at least 70%, at least 80%, at least 85%, at least 90, at least 95%, or at least 98% identical thereto.
  • the sequence is between about 70 to 100% identical thereto.
  • the sequence is between about 80 to 100% identical thereto.
  • the sequence is between about 90 to 100% identical thereto.
  • the sequence is between about 95 to 100% identical thereto.
  • the sequence is between about 98 to 100% identical thereto.
  • the nucleic acid comprises the nucleotide sequence SEQ ID NO: 15 or a codon optimised version thereof, or a sequence at least 70%, at least 80%, at least 85%, at least 90, at least 95%, or at least 98% identical thereto.
  • the sequence is between about 70 to 100% identical thereto.
  • the sequence is between about 80 to 100% identical thereto.
  • the sequence is between about 90 to 100% identical thereto.
  • the sequence is between about 95 to 100% identical thereto.
  • the sequence is between about 98 to 100% identical thereto.
  • the present invention provides a vector or polynucleotide encoding three or more antigen(s) in the antigen combination as described herein.
  • the vector or polynucleotide as described herein wherein the vector or polynucleotide encodes at least two of the following: i) Tp47; ii) Tp47 antigenic fragment; iii) Tp0453; iv) Tp0453 antigenic fragment; v) TmpA; vi) TmpA antigenic fragment; and vii) one or more binding proteins.
  • the vector or polynucleotide encodes i), iii) and viii); or i), iii), viii) and ivii).
  • the vector or polynucleotide encodes Tp47 or an antigenic fragment thereof; Tp0453 or an antigenic fragment thereof; and TmpA or an antigenic fragment thereof.
  • the vector or polynucleotide is codon optimised.
  • Methods and resources for codon-optimisation are known to those skilled in the art and include algorithmic models for sequence selection and codon design.
  • the described invention contemplates all motivations for codon optimisation, including but not limited to increasing host suitability, increasing yield, increasing purity, and increasing yield compared to using a non-codon optimised vector or polynucleotide.
  • the vector(s) or polynucleotide(s) disclosed herein are a DNA construct.
  • different vectors or polynucleotides encode each of the antigens of the present disclosure.
  • different vectors or polynucleotides encode each of Tp47 or an antigenic fragment thereof; Tp0453 or an antigenic fragment thereof; and TmpA or an antigenic fragment thereof.
  • different vectors or polynucleotides encode each of Tp47 or an antigenic fragment thereof; Tp0453 or an antigenic fragment thereof; TmpA or an antigenic fragment thereof; and one or more binding proteins as described herein.
  • the antigens of the present application are encoded on two or more vectors.
  • the antigens of the present application are encoded on a single vector.
  • different vectors or polynucleotides encoding the antigens of the disclosure are expressed in the same cell.
  • different vectors or polynucleotides encoding the antigens of the disclosure are expressed in the different cells.
  • the vectors or polynucleotides of the disclosure encode a fusion protein comprising one or more of the antigens disclosed herein.
  • the antigens of the antigen combination are encoded by a fusion protein.
  • the vectors or polynucleotides of the disclosure encode a fusion protein comprising one or more of Tp47 or an antigenic fragment thereof; Tp0453 or an antigenic fragment thereof; and TmpA or an antigenic fragment thereof.
  • antigen combinations and compositions described herein are suitable for in vitro use, for example in an assay format known to a person skilled in the art, including as an immunoassay, chromatographic assay or a homogenous assay.
  • the term “assay” is a procedure or method for measuring the biochemical and/or immunological activity of a sample. For example, an assay for detecting the presence or absence of Treponema pallidum antibodies in a sample from a subject.
  • immunoassay refers to assays using immunoglobulins or parts thereof that are capable of detecting and quantifying a desired biomarker.
  • the immunoassay may be one of a range of immune assay formats known to the skilled addressee. A wide range of immunoassay techniques are available, such as those described in Wild D. “The Immunoassay Handbook” Nature Publishing Group, 4th Edition, 2013 and subsequent innovations.
  • the immunoassay is selected from: electrochemiluminescence (ELICA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent ELISA, fluorescent immunosorbent assay (FIA), a bead-type immunoassay and a particle-based immunoassay (e.g. mesoscale delivery platform (MSD)).
  • detectable-groups include, for example and without limitation: fluorochromes, enzymes, epitopes for binding a second binding reagent (for example, when the second binding reagent/antibody is a mouse antibody, which is detected by a fluorescently-labelled antimouse antibody), for example an antigen or a member of a binding pair, such as biotin.
  • patient sample anti-TP antigen IgA antibodies are detected using a lateral flow format.
  • patient sample anti-TP antigen IgA antibodies are detected and/or measured using ELISA.
  • the immunoassay is a wet-system immunoassay, for example, a wet-system lateral flow test.
  • wet-system refers to an immunoassay, such as but not limited to a lateral flow test, in which the test reagents are in solution, and specifically, wherein the visualisation reagent is in solution.
  • a “dry-system” refers to an immunoassay, such as but not limited to a lateral flow test, in which at least one of the visualisation reagents , for example, a gold conjugate is immobilised. For example, immobilised on a solid support.
  • a system is still a dry system if some of the reagents, for example a running buffer are a liquid, as long as a visualisation agent, such as a gold conjugate, is in dried/solid/immobilised format.
  • a bead-type immunoassay is selected from a Luminex LabMAP assay, Bio-Plex Multiplex immunoassay (Bio-Rad).
  • the Luminex LabMAP system can be utilized.
  • the LabMAP system incorporates polystyrene microspheres that are dyed internally with two spectrally distinct fluorochromes. Using precise ratios of these fluorochromes, an array is created consisting of different microsphere sets with specific spectral addresses. Each microsphere set can possess a different reactant on its surface. Because microsphere sets can be distinguished by their spectral addresses, they can be combined, allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel.
  • the assay is a homogenous assay, meaning an assay format allowing an assay-measurement by a simple mix and read procedure without the necessity to process samples by separating or washing. Such assays do not include an immunosorbent solid phase step.
  • the homogenous assay is time- resolved Forster resonance energy transfer (FRET).
  • the assay is a flow cytometry-, bead array-, lateral flow-, cartridge-, microfluidic- or immunochromatographic-based method or the like.
  • the assay is a point-of-care assay.
  • the point-of-care assay reader is an Axxin AX-2X-type reader, Leelu (Lumos diagnostics), or equivalent or modified device.
  • the device may be modified to include LEDs and filters of the appropriate wavelength for the subject assays.
  • the assay detects T. pallidum. In an embodiment, the assay further detects a non- T. pallidum condition as described herein. In some embodiments, the assay detects active syphilis infection. In some embodiments, the assay detects past- treated or latent syphilis infection. In some embodiments, the assay detects active syphilis and another non-L. pallidum condition. In some embodiments, the assay detects active syphilis and HIV.
  • the assay is “one-step” assay.
  • a one-step assay refers to an assay, for example a lateral flow assay, which requires only one buffer introduction post sample application.
  • a one-step test can be read at 15 minutes after the reagents are added.
  • a one-step test may include simultaneous sample and running buffer addition, or may include a time interval between the addition of the sample and the addition of the running buffer.
  • a one-step assay for detecting the presence or absence of Treponema pallidum antibodies in a sample from a subject is performed when using an AtomoRapidTM Pascal cassette.
  • the assay may comprise the steps of pricking a subject’s finger and then reading the displayed result, because the detection, measuring, and/or analysis steps are integrated into the AtomoRapidTM Pascal cassette.
  • a one-step assay achieves a sensitivity of 95% and/or a specificity of 86%. In some embodiments, a one-step assay achieves a 95% sensitivity and/or a 92% specificity. In some embodiments, a one-step assay achieves a positive predictive value (PPV) of 83%. In some embodiments, a one-step assay achieves a PPV of 90%. In some embodiments, a one-step assay achieves a negative predictive value (NPV) of 96%.
  • PPV positive predictive value
  • NPV negative predictive value
  • assays use fresh samples. In some embodiments, assays use defrosted plasma. In some embodiments, assays use a combination of fresh and defrosted sample. For example, defrosted plasma mixed in about a 50:50 ratio with freshly collected blood cells. For example, defrosted plasma mixed in about a 50:50 ratio with finger prick blood. For example, defrosted plasma mixed in about a 50:50 ratio with serum.
  • the present invention provides an assay comprising an antigen combination as described herein.
  • the present invention provides an assay comprising an antigen combination as described herein immobilised on a solid support.
  • the present invention provides an assay comprising an antigen combination as described herein, and a binding protein for a non-'/; pallidum condition as described herein.
  • the assay as described herein comprises an antigen combination for detecting T. pallidum antibodies (detection protein), for example by forming an antigen-antibody complex, and a binding protein for capturing the T. pallidum antigenantibody complex (a capture protein).
  • detection protein can be used to directly or indirectly measure the level of T. pallidum in a sample.
  • the detection protein comprises a detectable label.
  • a detectable label is added to the detection protein.
  • a “capture protein” is used to capture T. pallidum antigen-antibody complex in the sample.
  • the capture protein is an antibody.
  • the capture agent is bound/immobilised to a solid support.
  • immobilised is to be understood to involve various methods and techniques to fix proteins onto specific matrices, e.g. as described in WO99/56126 or WO02/26292.
  • immobilisation can serve to stabilise the proteins so that its activity is not reduced or adversely modified by biological, chemical or physical exposure, especially during storage or in single-batch use.
  • the capture agent is bound to the solid support in the presence of one or more excipients as described herein.
  • the solid support is a membrane made from a polymer such as nitrocellulose, polyvinylidene fluoride, nylon, polyethersulfone, cellulose, glass fibres, polyester, polypropylene, polytetrafluoroethylene or other similar material.
  • the membrane is made from nitrocellulose.
  • the lateral flow device is a microfluidic lateral flow device.
  • the solid support is a polycarbonate and silicon based support, for example but not limited to polydimethylsiloxane (PDMS).
  • the present disclosure provides a lateral flow assay for detecting T. pallidum in a sample.
  • a lateral flow assay requires a liquid sample, which moves, generally by capillary action, through various zones of polymeric strips, on which molecules for interaction with the analytes are immobilised.
  • Lateral flow immunoassays also known as ‘immunochromatographic strip tests’ operate on the same principles as enzyme-linked immunosorbent assays (ELISA). In essence, these tests run a liquid sample along the surface of a membrane or filter paper with reactive molecules that show a visual positive or negative result depending on the presence of a particular analyte.
  • Lateral flow assays comprise a chromatographic matrix for separation of components of a sample, enabling analysis and/or detection and/or quantification of a sample analyte of interest.
  • Lateral flow assays may comprise several membranes for detection and or quantification of several different analytes from the same or different samples.
  • a lateral flow assay device is a device configured to receive a sample or samples at a sample region or sample pad(s), and to provide for the sample to move laterally, via, e.g. wicking, by capillary action from the sample region to a detection region.
  • the lateral flow assay device further comprises one or more conjugation region(s) or conjugation pad(s), wherein the lateral flow assay device is configured to provide for lateral flow of a sample from a sample region/pad to one or more conjugation region(s)/pad(s) prior to reaching a detection region.
  • a sample region is in contact with a conjugation region and the conjugation region is in contact with one end of a detection region such that the lateral flow assay device is configured to allow a sample to flow from the sample region, to a conjugation region and finally to a detection region.
  • the device further comprises an absorbent region in contact with a detection region such that the device is configured to allow the flow of a sample from a sample region to a detection region and finally to the absorbent region.
  • a lateral assay device typically has a backing card onto which an optional sample region, an optional conjugation region, the detection region, and an optional absorbent region are mounted.
  • the backing card provides support for the pads and membranes of the actual assay but are otherwise is not involved in the reaction or flow of the sample and analyte.
  • Backing cards are for example made of polyvinylchloride (PVC).
  • PVC polyvinylchloride
  • the assembly of pads and solid supports such as membranes on the backing card will typically be in a plastic housing although this is not required.
  • the housing may have at least one opening (“sample port”) over the sample pad for application of the sample. For example, one to five openings, for example one to four openings, for example one to three openings, for example one to two openings, for example one opening.
  • a one-step assay may have one sample port.
  • a two-step assay may have two or more sample port/s.
  • the control and test zones are visible (e.g. via an opening or window) to detect or measure the bound label.
  • the housing prevents the user from applying the sample anywhere except the sample pad.
  • the housing also serves to protect the strip from inadvertent splash onto the membrane.
  • External labelling on the housing can also be used to indicate the position of test and control lines and provide other information.
  • Housings can be obtained as off-the-shelf cassettes or custom-designed to fit around the strip. Internal pins and bars can be used to hold the strip in place relative to the sample port and viewing window. They hold the materials in fluid communication with one another while the test strip is running.
  • the “sample region”, if present, receives the sample upon application and promotes the even distribution of the sample onto the detection region or conjugation region, if present.
  • sample region or pad may also comprise additional components such as proteins, detergents, viscosity enhancers and buffer salts in order to process the sample (e.g. separation of sample components, removal of interferences, adjustment of pH, increasing the viscosity, solubilising components and/or preventing non-specific binding between conjugate and analyte or other components or to the reaction membrane).
  • additional components such as proteins, detergents, viscosity enhancers and buffer salts in order to process the sample (e.g. separation of sample components, removal of interferences, adjustment of pH, increasing the viscosity, solubilising components and/or preventing non-specific binding between conjugate and analyte or other components or to the reaction membrane).
  • the “conjugate region” or “conjugation region”, if present, comprises a dried and mobilizable composition comprising, for example, the labelled antibody or labelled protein described herein.
  • the conjugation region When sample flows into the conjugation region, the labelled antibody or labelled protein lifts off the conjugate region material, and moves with the sample front into the detection region. If applicable, the conjugation region will also comprise the dried and mobilizable control conjugate.
  • the conjugate pad comprises a first Tp binding protein. In some embodiments, the Tp binding protein is detectably labelled.
  • the lateral flow assay device does not comprise a separate conjugate region or pad.
  • the sample is mixed with a composition comprising the labelled antibody or labelled protein disclosed herein in a separate container, prior to migration along the lateral flow assay device.
  • Such devices may be referred to as lateral flow assay dipsticks.
  • a sample from a subject may be contacted with the composition described herein in a separate container to create a mixed solution, and then a lateral flow assay device comprising a detection region may be dipped into the solution such that it migrates along the detection region to the test and control zones.
  • Patient sample antibody complex formation with Tp antigen combination is visualised by, for example, anti-IgA gold conjugate.
  • the running buffer is integrated into the cassette.
  • test results can be read and/or quantitated by an automated reader.
  • an automated reader For example, the Lumos LeeLu or Cerberus reader (Lumos, Carlsbad).
  • test results can be interpreted through visual assessment, for example of the assay, for example, the test strip, or, for example, by plotting the numerical readout of the test line intensities, for example in an automated manner.
  • the running buffer comprises one or more of the following: 1 X DPBS, about 2% Synperonic F108, about 1% Tween 20, about 1.8 mg/mL K2EDTA and a pH of about 7.4. In some embodiments, the running buffer comprises 1 X DPBS. In some embodiments, the running buffer comprises about Synperonic F108. In some embodiments, the running buffer comprises about 2% Synperonic F108. In some embodiments, the running buffer comprises about Tween 20. In some embodiments, the running buffer comprises about 1% Tween 20. In some embodiments, the running buffer comprises about K2EDTA.In some embodiments, the running buffer comprises about 1.8 mg/mL K2EDTA.
  • the methods, kits, and assays comprise a microfluidic lateral flow device.
  • a solid support for use in a microfluidic lateral flow device comprising the antigen combination described herein.
  • the solid support is a polycarbonate based support.
  • the solid support is a silicon based support, for example, polydimethylsiloxane (PDMS).
  • a lateral flow assay device of the present application comprises the lateral flow test strips described herein in the AtomoRapidTM Pascal cassette.
  • AtomoRapidTM Pascal cassette comprises one or more of the following features: the antigen combination striped onto nitrocellulose membranes; anti-IgA (DCNDx) gold conjugate pads(s); membrane laminated together with an absorbent pad and/or a blood retention pad; a sample pad; a conjugate pad; and a gold rehydration pad.
  • components are laminated together on an adhesive backing card from which test strips for integration into the cassette are formed. For example, the test strips are 4 mm.
  • the present invention provides a kit comprising a binding protein as described herein.
  • the kit comprises a strip, chip or cartridge for use on point-of care device.
  • the kit comprises at least one of: a lateral flow assay, ELISA and an Luminex® assay.
  • the kit is designed for use by a health care practitioner.
  • the kit is a self-test kit designed for home use.
  • the present invention provides a kit for detection of Tp antibodies in a sample, the kit comprising using the antigen combination, assay, or device disclosed herein.
  • kits for performing any of the methods disclosed herein comprising at least one of the antigen combination, assays, or devices disclosed herein.
  • the kit comprises positive and/or negative controls.
  • the kit comprises calibration samples.
  • the kit comprises one or more of enzyme conjugate, substrate for enzyme conjugate, buffer solution, detectable label, excipient and washing solution.
  • the kit comprises an excipient.
  • the excipient is any sugar.
  • the kit comprises sucrose.
  • the kit comprises sucrose, wherein the sucrose is used in an amount of 0.1 to 5% (w/v). In some embodiments, the kit comprises sucrose, wherein the sucrose is used in an amount of 01 to l%(w/v). In some embodiments, the kit comprises sucrose, wherein the sucrose is used in an amount of 0.5% (w/v).
  • the kit comprises a detectable label and the label is an antibody.
  • the kit comprises a detectable label and the label is gold.
  • the gold is an anti-IgA gold.
  • the present disclosure provides various methods for detecting Treponema pallidum. It will be apparent from the description herein that the present disclosure provides various methods/ uses for diagnosing/prognosing and/or monitoring conditions/treatments associated with Tp, and optionally a combination of Tp and a non-Tp disease or condition as described herein.
  • the present invention provides a method of detecting the presence or absence of Treponema pallidum antibodies in a sample from a subject, the method comprising exposing the sample from the subject to the antigen combination as described herein under conditions which enable an antibody-antigen complex to form, and detecting the presence or absence of an antibody-antigen complex.
  • the present invention provides a method of detecting the presence or absence of an active syphilis infection in a sample from a subject, the method comprising exposing the sample from the subject to the antigen combination as described herein under conditions which enable an antibody-antigen complex to form, and detecting the presence or absence of an antibody-antigen complex.
  • an antigen/antibody complex is detected.
  • a skilled person will be aware of suitable ways to detect an antibody/antigen complex, as known in the art or described herein.
  • an IgA-specific antibody/antigen complex is detected.
  • an antibody/antigen complex wherein at least one bound antibody specifically binds IgA.
  • the present invention provides use of the antigen combination as described herein, the kit as described herein, or the lateral flow device as described herein for detecting the presence or absence of Tp antibodies in a sample.
  • the present invention provides use of the antigen combination as described herein, the kit as described herein, the assay as described herein or the lateral flow device as described herein for detecting an active syphilis infection in a subject.
  • the present invention provides a method of treating an active syphilis infection in a subject, the method comprising: i) contacting a biological sample from the subject with the antigen combination as described herein; and ii) directly or indirectly detecting the presence of an antibody-antigen complex, wherein the presence of the antibody-antigen complex indicates that a subject has an active syphilis infection; and iii) administering a treatment for an active syphilis infection.
  • the treatment is administered if an antibody/antigen complex is detected.
  • the treatment comprises administering an antibiotic.
  • the treatment comprises administering penicillin.
  • Tpl5, Tpl7, Tp47 and TmpA were expressed in E. coli BL21 Star(DE3) cells (Invitrogen) in LB supplemented with 100 pg/mL ampicillin (Tpl5, Tp47 and TmpA) or 50 pg/mL Kanamycin (Tpl7) at 37 °C. Expression was induced by addition of 0.1 mM IPTG at 18 °C overnight.
  • Cells were harvested, resuspended in 50 mM Sodium Phosphate, 200 mM Sodium Chloride, 10 mM Imidazole, pH 7.6, with 1 mM phenylmethyl sulfonyl (PMSF) for protease inhibition added, and lysed by sonication (Branson). Proteins were purified from clarified cell lysate by affinity chromatography using HisTrap FF (Cytiva) with 50 mM Sodium Phosphate, 200 mM NaCl, 10 mM imidazole, pH 7.6 and elution with 20-500 mM imidazole gradient.
  • PMSF phenylmethyl sulfonyl
  • Recombinant protein was further purified by gel-filtration chromatography using a Superose 12 10/30 (Amersham) or Hiload 16/600 Superdex 200pg (Cytiva) pre-equilibrated with 50 mM Sodium Phosphate, 500 mM Sodium Chloride, pH 7.6.
  • Tp0453 was purchased from Genscript (custom order).
  • the first test line contains T. pallidum antigens, either individually or combined at different concentrations, in the presence or absence of 0.5% (w/v) sucrose.
  • the second test line contains 0.2 mg/mL Protein L (Sigma), which forms a complex with kappa chain IgA antibodies present in the sample and/or a highly specific IgA gold conjugate.
  • Test lines were striped using an IsoFlow dispenser (Imagene Technology) at a dispense rate of 0.100 pL/mm and dried for 2h at 37°C.
  • test lines are visualised and quantitated using an automated reader, such as the Axxin AX-2X reader (Axxin Ltd, Melbourne).
  • nitrocellulose membranes (Vivid90, Cytiva or Hi-flow90, Millipore) were striped as outlined above, but at higher antigen concentrations (1.3-1.4 mg/mL total protein) than those used in the two-step test (0.65-0.7 mg/mL).
  • Anti-IgA (DCNDx) gold conjugate pads were made as previously described.
  • the nitrocellulose membrane (NCM) was then laminated together with an absorbent pad (CF6, GE Healthcare) at the top of the NCM and a blood retention pad (FR-l(0.35), mdi) at the bottom.
  • defrosted plasma was mixed in a 50:50 ratio with freshly collected blood cells from venous blood draws of never infected human volunteers. Cells were collected by spinning freshly collected blood at 1000 x g for 5 mins and removing the top plasma layer. From this mix, 10 pL of reconstituted blood sample was pipetted into the blood collection unit of the AtomoRapidTM Pascal cassette and applied to the sample pad. Running buffer was then released to the test strip by pushing down on the test to burst an integrated blister prefilled with running buffer.
  • Running buffer consisted of 1 X Dulbecco’s phosphate buffered saline (DPBS), 2% Synperonic F108, 1% Tween20, 1.8 mg/mL K2EDTA, pH 7.4. The test was allowed to sit for 15 minutes, then visualised and the test and control lines quantitated by an automated reader such as the Lumos LeeLu or Cerberus reader (Lumos, Carlsbad) modified to fit the Pascal cassette. The test results were interpreted through visual assessment of the photographed test strips, as well as by plotting the numerical readout of the test line intensities.
  • the integrated lancet unit of the AtomoRapidTM Pascal cassette was used to prick the patient or volunteer’s finger, and the blood collection channel was placed into the fingerpick blood drop to collect 10 pL of blood via capillary action. This was then applied to the test strip and the test conducted as previously described.
  • Example 2 Selection of antigens for evaluation as candidate antigens in diagnostic test development
  • the syphilis spirochete has an outer membrane, peptidoglycan space and inner membrane.
  • the five proteins examined here are shown in their putative locations and are located on the inner surface of the outer membrane, and the inner membrane ( Figure 1).
  • the spirochete is unusual in that it does not possess lipids in its membrane and presents relatively few antigens to the immune system. These features are believed to contribute to its ability to evade immunity.
  • the putative size of each antigen in T. pallidum varies between 15 and 47 kDa.
  • Tpl5 is a lipoprotein
  • Tp47 is a lipoprotein and carboxypeptidase
  • Tpl7 is a periplasmic lipoprotein and has been described as a target for the generation of antibodies.
  • the five proteins were expressed in E. coli. Reducing SDS-PAGE performed on purified T. pallidum antigens shows they are highly purified, and their observed molecular mass corresponds to the expected mass. Each antigen was expressed in E. coli and purified using nickel affinity chromatography followed by gel filtration ( Figure 3).
  • a total antigen concentration of 0.7 mg/mL comprised of Tp47:TpO453:TmpA in a ratio of 2: 1 : 1 + 0.5% (w/v) sucrose provides the best sensitivity and specificity for this patient sample set.
  • the response of the test line can be tuned with varying the total concentration of antigen (Figure 17).
  • test line signal intensity e.g. OD of gold-conjugate, membrane identity, volume of sample applied
  • the exact ideal total antigen concentration may vary substantially if other parameters are varied. This is demonstrated in Figure 18, wherein decreasing the OD of the gold in the conjugate pad from the standard OD 7.2 to an OD of 5.2 decreases the test line strength.
  • suitable antigen concentrations range from between 0.1 mg/mL to 2 mg/mL total antigen.
  • slight variations in antigen ratio 50:25:25 vs 40:20:40 vs 45:23:33, Figure 19
  • the exact percentages of patients identified as active or past- treated varied, but the overall strength of the signal was similar, as an increase in the ability to accurately determine positive patients was always accompanied by an increase in past-treated patients being incorrectly identified as active Figure 19D).
  • the patient test panel decreases from 40 patients with active syphilis, 40 past-treated and 17 never infected to 39 active, 32 past-treated and 17 never infected patients.
  • the sensitivity of the test is unchanged at 95%, but the specificity increases from 86% to 92%.
  • the individual patient test line data following this exclusion can be seen in Figure 23.
  • TPHA assay is a standard serological test for total patient antibodies towards T. pallidum antigens, and patients typically remain TPHA positive for life post any infection with syphilis, even after successful treatment.
  • results have been used to calculate positive and negative predictive values for each test, with 24B showing results from the full patient panel and 24C the results once excluding patients >2 prior infections and ⁇ 6 months post last infection.
  • test comprises fragments of the T. pallidum antigens instead of one or more of the full-length proteins.
  • Emini Surface Accessibility Prediction (Emini et al., 1978) (2) Parker Hydrophilicity Prediction (Parker et al., 1986) (3) Kolaskar & Tongaonkar Antigenicity (Kolaskar and Tongaonkar 1990) (4) Chou & Fasman Beta-Turn Prediction (Chou and Fasman 1978) (5) Karplus & Schulz Flexibility Prediction (Karplus and Schulz 1985) (6) Bepipred Linear Epitope Prediction 2.0 (Jespersen et al., 1986). Outputs were ranked by highest scoring regions, and the sequences of each protein that encompass the top 20 highest scoring regions (excluding the recombinant protein tags) are shown underlined in Table 4.
  • the unique combination of treponemal antigens Tp0453, Tp47 and TmpA that differentiates active syphilis infection from past-treated and never infected cases developed here can be adapted to alternative lateral flow test formats and simplified to enhance usability.
  • the antigens and the components of the lateral flow device were adapted to fit a commercially available integrated device, the AtomoRapid Pascal cassette.
  • the lancet, a blister containing running buffer and a blood collection unit are contained with the lateral flow cassette.
  • Adaptation of the test antigens to this device allowed for reduction of the number of steps, the need for additional timed intervals, and shortened the time to result to 15 minutes.
  • the sensitivity and specificity against the same panel of active, past-treated and never infected patients was identical to that in the traditional cassette format. This demonstrates how the Tp0453, Tp47 and TmpA test antigens can be used in alternative cassette types.
  • Example 15 The effect of removing short (40-42 amino acid) regions of Tp antigens Tp47 and TmpA
  • Table 5 Table of key sequences.

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Abstract

La présente invention concerne une combinaison d'antigènes comprenant des antigènes Treponema pallidum (Tp). La présente invention concerne également des vecteurs, des polynucléotides, des cellules hôtes et des procédés pour produire de telles combinaisons d'antigènes. La présente invention concerne également un procédé et des utilisations de telles combinaisons d'antigènes. La présente invention concerne également des compositions, des dosages et des kits et leurs procédés et utilisations.
PCT/AU2025/050155 2024-02-23 2025-02-24 Combinaison d'antigènes et procédés et utilisations associés Pending WO2025175360A1 (fr)

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AU2024900445A AU2024900445A0 (en) 2024-02-23 Antigen combination and methods and uses thereof
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