WO2014183166A1 - Antigenic polypeptides of dientamoeba fragilis and antibodies thereto - Google Patents

Antigenic polypeptides of dientamoeba fragilis and antibodies thereto Download PDF

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Publication number
WO2014183166A1
WO2014183166A1 PCT/AU2014/050035 AU2014050035W WO2014183166A1 WO 2014183166 A1 WO2014183166 A1 WO 2014183166A1 AU 2014050035 W AU2014050035 W AU 2014050035W WO 2014183166 A1 WO2014183166 A1 WO 2014183166A1
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Prior art keywords
seq
amino acids
polypeptide
antibody
sequence
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PCT/AU2014/050035
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French (fr)
Inventor
John Timothy Ellis
Joel Leonard Nicholas BARRATT
Damien John STARK
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University Of Technology, Sydney
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Priority claimed from AU2013901678A external-priority patent/AU2013901678A0/en
Application filed by University Of Technology, Sydney filed Critical University Of Technology, Sydney
Publication of WO2014183166A1 publication Critical patent/WO2014183166A1/en

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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
    • G01N33/56905—Protozoa
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • the present invention relates generally to antigenic polypeptides of Dienl amoeba fragilis and antibodies thereto.
  • the present invention also relates generally to methods and kits for the generation of antibodies to D. fragilis and methods and kits for the detection of D. fragilis.
  • Dientamoeba fragilis is a protozoan parasite that inhabits the human bowel, causing a gastrointestinal disorder characterized by abdominal pain, nausea, anorexia, vomiting and diarrhea. Of these symptoms, abdominal pain and diarrhea are most common. Although originally described as a rare and harmless commensal, the pathogenic potential of D. fragilis is becoming increasingly apparent. With the improvement of techniques for the recovery and identification of D. fragilis. this organism is now among the most frequently encountered intestinal protozoa in humans. In Australia, D. fragilis prevalence in patients presenting with gastrointestinal complaints has been reported to be between 0.4% and 16.8%.
  • the present invention is directed to the D. fragilis enoJase polypeptide and fragments thereof, isolated polypeptides containing antigenic determinants of the D. fragilis enolase polypeptide, and isolated antibodies and antigen-binding fragments thereof that bind to the antigenic determinants within the D. fragilis enolase polypeptide, and thus bind to the D. fragilis enolase polypeptide and D. fragilis parasites.
  • the invention is also directed to methods and kits associated with (he polypeptides and antibodies of the invention.
  • the invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to an antigenic determinant within the D. fragilis enolase polypeptide set forth in SEQ ID NO:l or a variant thereof having at least about 85% sequence identity with the sequence set forth in SEQ ID NO:l.
  • the variant has at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98% or 99% sequence identity with the sequence set forth in SEQ ID NO: 1.
  • the antibodies and antigen-binding fragments of the invention may be monoclonal or polyclonal.
  • the antibodies or antigen-binding fragments are linked to a detectable label, such as, for example, a fluorescent label, a chcmiluminescent label, an enzymatic label, gold particle, biotin or streptavidin.
  • the antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 453-467 of SEQ ID NO: I or corresponding amino acids in the variant.
  • the antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 264-272 of SEQ ID NO:l or corresponding amino acids in the variant.
  • the antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 177-189 of SEQ ID NO:l or coircsponding amino acids in die varianl.
  • Ihe antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 212-222 of SEQ TD NO:l or corresponding amino acids in the variant.
  • the antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 294-302 of SEQ ID NO:l or corresponding amino acids in the variant.
  • the antibodies and antigen- binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 319-328 of SEQ ID NO:l or corresponding amino acids in the variant.
  • the antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 409-419 of SEQ ID NQ:1 or corresponding amino acids in the variant.
  • the antibodies and antigen-binding fragments of the invention can be used to detect the presence of a D. fragilis cnolase polypeptide and or a D. fr gilis parasite in a sample.
  • the antibodies and antigen-binding fragments are used in diagnosing a D. fragilis infection in a subject.
  • the invention provides a method for the detection of a D. fragilis enolase polypeptide in a sample, comprising contacting the sample with an antibody or antigen-binding fragment of the invention and detecting binding of the antibody or antigen-binding fragment to the enolase polypeptide.
  • the invention also provides a method for the detection of a D. fragilis parasite in a sample, comprising contacting the sample with an antibody or antigen binding fragment of the invention and detecting binding of the antibody or antigen-binding fragment to the D. fragilis parasite.
  • Also provided arc methods for the diagnosis of D. fragilis infection in a subject, comprising contacting a sample from the subject with an antibody or antigen-binding fragment of the invention and detecting binding of the antibody or antigen binding fragment to a D. fragilis parasite in the sample.
  • the sample comprises blood, serum, stool, gastric lavage or urine.
  • the sample is a stool sample.
  • the detection can be performed in any suitable manner. In one example, detection is performed by direct immunofluorcscence. In another example, detection is performed by indirect immunofluorescence.
  • kits comprising any one or more of the antibodies or antigen-binding fragments described herein.
  • the kits are used for detecting the presence of a D. fragilis enolase polypeptide and/or a D. fragilis parasite in a sample.
  • the kits of the invention can also be used for diagnosing D. fragilis infection in a subject.
  • the kit also contains a labelled secondary antibody,
  • the present invention provides an isolated polypeptide comprising the sequence of amino acids set forth in SEQ TD NO:1 or 2, or a sequence of amino acids having at least about 85% sequence identity with (he sequence set forth in SEQ ID NO: I or 2,
  • the isolated polypeptide comprises a sequence of amino acids having at least about 86%, 87%, 88%. 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence set forth in SEQ ID NO: 1 or 2.
  • the present invention provides an isolated polypeptide, comprising the sequence of amino acids set forth in SEQ ID NO:3 or 10.
  • the present invention provides an isolated polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:3 or the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:3, wherein the variant has at least 75%, 80%, 85% or 90% sequence identity with the sequence set forth in SEQ ID NO:3, and wherein (he isolated polypeptide is less than 300 amino acids in length.
  • the polypeptide comprises the sequence of amino acids set forth in SEQ ID NO:3 or 10.
  • the polypeptide consists of the sequence set forth in SEQ ID NO:3 or 10.
  • the present invention provides an isolated polypeptide, comprising the sequence of amino acids set forlh in SEQ ID NO:4 or 11.
  • the invention provides an isolated polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:4 or the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:4. wherein the variant has at least 75%, 80% or 85% sequence identity with the sequence of amino acids set forth in SEQ ID NO:4, and wherein (he isolated polypeptide is less lhan 300 amino acids in length.
  • (he polypeptide comprises the sequence of amino acids set forth in SEQ ID NO:4 or 11.
  • Ihe polypeptide consists of the sequence set forth in SEQ ID NO:4 or 11.
  • the polypeptides of the invention are linked to at least one moiety.
  • the polypeptides can be linked to another antigenic determinants, a T helper cell epitope or polypeptide that comprises a T helper cell epitope, an immunostiinulatory molecule, a targeting agent, a polymer, a multimerisation domain, a detectable label or an affinity tag.
  • the polypeptides arc linked to a T cell epitope or polypeptide that comprises a T cell epitope.
  • the polypeptides of the invention are used to elicit antibodies that specifically bind to a D. fragilis cnolase polypeptide.
  • methods for eliciting antibodies that specifically bind to a D. fragilis enolase polypeptide comprising administering a polypeptide of the invention to a subject, thereby eliciting antibodies that specifically bind to a D. fragilis enolase polypeptide.
  • the polypeptides of the invention are used to elicit antibodies that specifically bind to a D. fragilis parasite. Accordingly, also provided are methods for eliciting antibodies that specifically bind to a D. fragilis parasite, comprising administering a polypeptide of the invention to a subject, thereby eliciting antibodies that specifically bind to a D. fragilis parasite.
  • the present invention also provides nucleic acid molecules encoding any one or more of the polypeptides described herein.
  • Figure 1 is a three dimensional modeling (in space fill formal) of the D. fragilis enolase polypeptide showing the predicted protein structure. The locations of the antigenic determinants arc shown in dark grey: Panel A: antigenic determinant A; Panel B: antigenic determinant B; Panel C: antigenic determinant C; Panel D: antigenic determinant D; Panel E: antigenic determinant E; Panel F: antigenic determinant F (indicated with an arrow): Panel G: antigenic determinant G (indicated with an arrow): Panel H: antigenic determinants A-G; Panel 1: antigenic determinants A-G with amino acids that compose the intracellular loop of the protein and the transmembrane helices obscured, i.e. showing only the extracellular portion of the protein.
  • Figure 2 represents an indirect immunofluorescence assay of D. fragilis trophozoites stained with polyclonal antibody preparation A.
  • Row A D. fragilis subjected to mild fixation and stained with polyclonal antibody preparation A at a dilution of 1:10.
  • Row B Negative control using only the secondary antibody at. a 1:80 dilution.
  • Row C Close-up of D. fragilis stained with polyclonal antibody preparation A at a dilution of 1 : 10.
  • Row D D. fragilis fixed in SAF and stained with polyclonal antibody preparation A at a dilution of 1:10.
  • Column 1 Bright field micrographs.
  • Column 2 Corresponding fluorescent micrographs of the same cells shown in column 1.
  • Column 3 Overlay of fluorescent micrographs on the bright field micrographs.
  • Figure 3 represents an indirect immunofluorescence assay of D. fragilis trophozoites using polyclonal antibody preparation B.
  • Rows A and B D. fragilis subjected to mild fixation and stained with polyclonal antibody preparation B at a dilution of 1:10.
  • Row C Negative control using only the secondary antibody at a 1:80 dilution.
  • Row D D. fragilis fixed in SAF and stained with polyclonal antibody preparation B at a dilution of 1:10.
  • Column 1 Bright field micrographs.
  • Column 2 Corresponding fluorescent micrographs of the same cells shown in column I.
  • Column 3 Overlay of fluorescent micrographs on the bright field micrographs.
  • Figure 4 represents an indirect immunofluorescence assay of Entamoeba moshkovskii cells using polyclonal antibody preparation A.
  • Row A E. moshkovskii cells stained with polyclonal antibody preparation A at a dilution of 1 :10.
  • Row B Negative control using only the secondary antibody at a 1 :80 dilution.
  • Column 1 Bright field micrographs.
  • Column 2 Corresponding fluorescent micrographs of the same cells shown in column I.
  • Column 3 Overlay of fluorescent micrographs on the bright Held micrographs.
  • Figure 5 represents an indirect immunofluorescence assay of Entamoeba moshkovskii cells using polyclonal antibody preparation B.
  • Row A £. moshkovskii cells stained with polyclonal antibody preparation A as a positive control.
  • Row B Negative control using only the secondary antibody at a 1:80 dilution.
  • Row C E. moshkovskii cells stained with polyclonal antibody preparation B at a dilution of 1:10.
  • Column 1 Bright Held micrographs.
  • Column 2 Corresponding fluorescent micrographs of the same cells shown in column 1.
  • Column 3 Overlay of fluorescent micrographs on the bright field micrographs.
  • Figure 6 represents an indirect immunofluorescence assay of Entamoeba histolytica cells using polyclonal antibody preparation A.
  • Row A D. fragilis trophozoites stained with polyclonal antibody preparation A at a dilution of 1:10 as a positive control.
  • Row B Negative control of D. fragilis trophozoites using only the secondary antibody at a 1 :80 dilution.
  • Row C E. histolytica cells stained with polyclonal antibody preparation A at a dilution of 1:10.
  • Row D Negative control of £. histolytica cells using only the secondary antibody at a 1:80 dilution.
  • Column 1 Bright field micrographs.
  • Column 2 Corresponding fluorescent micrographs of the same cells shown in column 1.
  • Column 3 Overlay of fluorescent micrographs on the bright field micrographs.
  • Figure 7 represents an indirect immunofluorescence assay of E. histolytica cells using polyclonal antibody preparation B.
  • Row A £>. fragilis trophozoites stained widi polyclonal antibody preparation B at a dilution of 1:10 as a positive control.
  • Row B Negative control of D. fragilis trophozoites using only the secondary antibody at a 1 :80 dilution.
  • Rows C and D E. histolytica cells stained with polyclonal antibody preparation B at a dilution of 1:10.
  • Column 1 Bright field micrographs.
  • Column 2 Corresponding fluorescent micrographs of the same cells shown in column 1.
  • Column 3 Overlay of fluorescent micrographs on the bright field micrographs.
  • Figure 8 represents an indirect immunofluorescence assay of Blastocysts sp. cells using polyclonal antibody preparation A.
  • Row A D. fragilis trophozoites stained with polyclonal antibody preparation A at a dilution of 1:10 as a positive control.
  • Row B Negative control of Blastocysts cells using only the secondary antibody at a 1:80 dilution.
  • Row C Blastttcystis cells stained with polyclonal antibody preparation A at a dilution of 1:10.
  • Column I Bright field micrographs.
  • Column 2 Corresponding fluorescent micrographs of the same cells shown in column I.
  • Column 3 Overlay of fluorescent micrographs on the bright field micrographs.
  • Figure 9 represents an indirect immunofluorescence assay of Blastocysts sp. cells using polyclonal antibody preparation B.
  • Row A D. fragilis trophozoites stained with polyclonal antibody preparation B at a dilution of 1:4 as a positive control.
  • Row B Negative control of Blastocysts cells using only the secondary antibody at a 1:80 dilution.
  • Row C Blasiocysiis cells stained with polyclonal antibody preparation B at a dilution of 1:10.
  • Column 1 Bright field micrographs.
  • Column 2 Corresponding fluorescent micrographs of the same cells shown in column I .
  • Column 3 Overlay of fluorescent micrographs on the bright field micrographs.
  • Figure 10 represents an indirect immunofluorescence assay of Giardia trophozoites using polyclonal antibody preparation A.
  • Row A D. fragilis trophozoites stained with polyclonal antibody preparation A at a dilution of 1:10 as a positive control.
  • Row B Negative control of D. fragilis trophozoites using only the secondary antibody at a 1:80 dilution.
  • Row C Giardia trophozoites stained with polyclonal antibody preparation A at a dilution of 1:10.
  • Row D Negative control of Giardia trophozoites using only the secondary antibody at a 1:80 dilution.
  • Column 1 Bright field micrographs.
  • Column 2 Corresponding fluorescent micrographs of the same cells shown in column 1.
  • Column 3 Overlay of fluorescent micrographs on the bright field micrographs.
  • Figure 11 represents an indirect immunofluorescence assay of Giardia trophozoites using polyclonal antibody preparation B.
  • Row A D. fragilis trophozoites stained with polyclonal antibody preparation B at a dilution of 1 :10 as a positive control.
  • Row B Negative control of Z . fragilis trophozoites using only the secondary antibody at a 1:80 dilution.
  • Row C Giardia trophozoites stained with polyclonal antibody preparation B at a dilution of 1:10.
  • Column 1 Bright field micrographs.
  • Column 2 Corresponding fluorescent micrographs of the same cells shown in column I.
  • Column 3 Overlay of fluorescent micrographs on the bright field micrographs. BRIEF DESCRIPTION OF THE SEQUENCES
  • an antimicrobial agent means one antimicrobial agent or more than one antimicrobial agent.
  • antigenic determinant' * refers to a region of an antigen or protein ⁇ e.g. an enolase protein) that is specifically recognized by antibodies and B cells. i.e. the portion to which the antibody paratope binds.
  • Antigenic determinant and epipe are used interchangeably herein.
  • isolated with reference to a nucleic acid molecule, peptide, or polypeptide means that the nucleic acid molecule, peptide, or polypeptide is substantially free of cellular material or other contaminating proteins from the cells from which the nucleic acid molecule, peptide, or polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily delectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC).
  • TLC thin layer chromatography
  • HPLC high performance liquid chromatography
  • an isolated peptide preparation produced by recombinant methods generally has less than about 30%, more typically less than about 20%. 10% or 5% (w/w), of cellular material, and an isolated peptide preparation produced by chemical synthesis generally has less than about 30%, more typically less than about 20%. 10% or 5% (w/w), of chemical precursors or other chemicals.
  • corresponding amino acid residues (or positions) refer to residues (or positions) that occur at aligned loci within the primary amino acid sequence of a protein..
  • polypeptides are aligned by any method known to those of skill in the art. Such methods typically maximize matches, and include methods such as using manual alignments and by using the numerous alignment programs available (for example. BLASTP) and others known to those of skill in the art.
  • BLASTP the numerous alignment programs available
  • aligning the sequences of polypeptides one skilled in the art can identify corresponding residues, using conserved and identical amino acid residues as guides.
  • aligning the sequences of the cnolase polypeptide set forth in SEQ ID NO: 1 with another enolase polypeptide one of skill in the art can identify corresponding residues using conserved and identical amino acid residues as guides.
  • antibody refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically, such as recombinantiy, produced, including any fragment thereof containing at least a portion of the variable region of the immunoglobulin molecule that retains the binding specificity ability of the full-length immunoglobulin.
  • an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen- binding domain (antibody combining site).
  • Antibodies include antibody fragments.
  • antibody includes synthetic antibodies, recombinantiy produced antibodies, multispccifk antibodies (e.g., bispecific antibodies), human antibodies, non- human antibodies, humanized antibodies, chimeric antibodies, intrabodies. and antibody fragments, such as, but not limited to. Fab fragments, Fabl fragments. F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv). Fd fragments. Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFab), diabodics, anti-idiot ypic (anti-Id) antibodies, or antigen- binding fragments of any of the above.
  • Fab fragments fragments, Fabl fragments.
  • Fd fragments Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFab
  • Antibodies provided herein include members of any immunoglobulin type (e.g.. IgG, IgM. IgD, IgE, IgA and IgY), any class (e.g. IgGl. IgG2, lgG3, lgG4, IgAl and IgA2) or subclass (e.g., IgG2a and lgG2b).
  • immunoglobulin type e.g.. IgG, IgM. IgD, IgE, IgA and IgY
  • any class e.g. IgGl. IgG2, lgG3, lgG4, IgAl and IgA2
  • subclass e.g., IgG2a and lgG2b.
  • an antibody or antigen-binding fragment thereof refers to the ability of the antibody or antigen-binding fragment to form one or more noncovalent bonds with a cognate antigen, by noncovalent interactions between the antibody combining site(s) of (lie antibody and the antigen.
  • the antigen can be an isolated antigen such as an isolated protein (e.g. an enolase polypeptide) or presented on thc surface of a cell, such as D. fragiUs trophozoite.
  • an antibody lhat specifically binds to a polypeptide or cell herein is one that binds with an affinity constant t.
  • Affinity constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays (e.g. ELISA). or surface plasmon resonance (SP ). Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (e.g.. Biacore 2000. Biacore AB. Upsala, Sweden and GE Healthcare Life Sciences).
  • an "antigenic variant” refers to a variant of a reference polypeptide tha has that same antigenicity as the reference polypeptide.
  • an antigenic variant of a polypeptide set forth in SEQ ID NO:3 is a variant that is recognized by the same antibody or antibodies that recognize the polypeptide set forth in SEQ ID NO:3.
  • an antigenic variant of a polypeptide set forth in SEQ ID NO:3 can be used to elicit antibodies that bind to both the variant and to the polypeptide set forth in SEQ ID NO:3.
  • the antigenic variant may contain amino acid substitutions, deletions and/or additions compared to the sequence of the reference polypeptide.
  • antigenic variants include fragments of the reference polypeptide lhat are recognized by the same antibody or antibodies lhat recognize the reference sequence or polypeptide.
  • Enolases are present in a wide variety of pathogenic and non-pathogenic organisms, including Archac, bacteria and eukaryotic organisms. Enolases are responsible for the reversible conversion of D-2- phosphogiyceraie (2POA) and phosphoenolpyruvalc (PEP) in glycolysis and gluconeogencsis. In some organisms, enolases are involved in pathogenicity.
  • 2POA D-2- phosphogiyceraie
  • PEP phosphoenolpyruvalc
  • the enolase of pathogenic Streptococcus pyogenes and the insect parasite Aphidius ervi is excreted to the extracellular environment where it mediates degradation of host tissues and immune evasion.
  • the surface-associated enolase of Trichomonas vaginalis can bind human plasminogen and release plasmin, which may facilitate penetration of 7 * . vaginalis to the cell basement membrane to enable association with fibroncctin and laminin. which in turn could ensure the parasite access to growth factors and nutrients.
  • the D. fragilis enolasc protein is a surface-associated protein.
  • An exemplary D. fragilis enolase protein comprises the sequence of amino acids set forth in SEQ ID NO:l . Sequence analysis and three dimensional modeling of this enolase polypeptide indicate that amino acids 1 to 141 of SEQ ID NO: 1 reside within the cell; amino acids 142 to 176 comprise the transmembrane helices; and amino acids 177 to 467 are extracellular. It is predicted that the polypeptide contains a further 10-12 amino acids at the N-terminus of the protein, which is intracellular.
  • polypeptides comprising a sequence of amino acids set forth in SEQ ID NO: 1, or a polypeptide having at least 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%. 95%. 96%, 97%. 98% or 99% sequence identity to the enolase polypeptide set forth in SEQ ID NO:l .
  • polypeptides comprising a sequence of amino acids set forth in SEQ ID NO:2, which corresponds to the extracellular portion (amino acids 177 to 467) of SEQ ID NO: l. or a polypeptide having at least 85%, 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%.
  • polypeptides described herein retain enolase activity, i.e. can catalyze the dehydration of 2- phospho-d-glycerate (PGA) to phosphoenolpyruvate (PEP) in glycolysis, and/or the hydration of PEP to PGA during gluconcogcncsis.
  • PGA 2- phospho-d-glycerate
  • PEP phosphoenolpyruvate
  • the extracellular portion of the D. fragilis enolase protein contains multiple antigenic determinants (or epitopes) to which antibodies can specifically bind.
  • Exemplary antigenic determinants comprise, or are located at or within, amino acids 264-272 of SEQ ID NO:l (antigenic determinant Bj, or amino acids 453-467 of SEQ ID NO: 1 (antigenic determinant A), or corresponding amino acids in another D. fragilis enolase protein.
  • antigenic determinants include those comprising, or located at or within, amino acids 177-189 (antigenic determinant C), 212-222 (antigenic determinant D), 294-302 (antigenic determinant F), 319-328 (antigenic determinant E) or 409-419 (antigenic determinant G) of SEQ ID NO:l or corresponding amino acids in another D. fragilis enolase protein.
  • Three dimensional modeling of the enolase polypeptide set forth in SEQ ID NO:l indicates the position of these antigenic determinants within the three dimcnsional structure ( Figure I). Accordingly, provided herein arc polypeptides that consist of. or comprise, one or more of antigenic determinants A, B, C. D, E, G and/or G.
  • polypeptides of the present invention that comprise the antigenic determinants described herein can be used to elicit antibodies that can bind to D.frngttis.
  • polypeptides comprising antigenic determinant A comprising, or located at or within, amino acids 453-467 of 5EQ ID NO: I can be used to elicit antibodies that bind to D. fr gilis.
  • polypeptides comprising antigenic determinant A comprising, or located at or within, amino acids 453-467 of SEQ ID NO:l also contain other surface-exposed residues within the enolase polypeptide, including bu not limited to. any one or more of residues at positions 430, 433, 434, 440, 441 , 442, 443, 446, 447, 450 or 4 1 of SEQ ID NO: 1.
  • Exemplary polypeptides of the present invention can thus consist of the sequence of amino acids LQIYEYLKEHDMLEE (SEQ ID NO:3; corresponding to amino acids 453-467 of SEQ ID NO:l), or consist of the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:3.
  • the antigenic variant can have at least 60%, 70%, 75%, 80%. 85%, 90% or 95% ⁇ sequence identity with the sequence of amino acids set forth in SEQ ID NO:3.
  • the polypeptides consist of a sequence of amino acids that has 1, 2. 3, 4 or 5 amino acid substitutions, additions and or deletions compared to the sequence of amino acids set forth in SEQ ID NO:3.
  • the polypeptide consists of the amino acid sequence CLQIY EYLKEH DMLEE (SEQ ID NO: 10).
  • the polypeptides of the present invention also include those polypeptides that comprise the sequence of amino acids set forth in SEQ ID NO: 3 or SEQ ID NO: 10, or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:3, such as an antigenic variant having at least 60%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity with the sequence of amino acids set forth in SEQ ID NO:3.
  • the polypeptides are less than 400. 300, 200. 100, 50.
  • Polypeptides comprising antigenic determinant B comprising, or located at or within, amino acids 264-272 of SEQ ID NO:l can also be used to elicit antibodies that bind to D. fragilis. This demonstrates that the region of the enolase polypeptide containing antigenic determinant B is accessible to antibodies when the enolase polypeptide is associated with the surface of the D. fragilis trophozoite.
  • the polypeptides comprising antigenic determinant B comprising, or located at or within, amino acids 264-272 of SEQ ID NO: I also contain other surface-exposed residues within the enolase polypeptide, including but not limited to, any one or more of residues at positions 230; 231, 232, 235, 238, 239, 240. 241, 242, 243. 244, 246. 247. 248, 259, 260, 273, 275, 276 or 277 of SEQ ID NO:l.
  • the polypeptides of the present invention consist of the sequence of amino acids A1SYIEQAM (SEQ ID NO:4; corresponding to amino acids 264- 272 of SEQ ID NO: I), or the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:4.
  • the antigenic variant has at least 75%, 80% or 85% sequence identity with the sequence of amino acids set forth in SEQ ID NO:4.
  • the polypeptides consist of a sequence of amino acids that has 1. 2 or 3 amino acid substitutions, additions and/or deletions compared to the sequence of amino acids set forth in SEQ ID NO:4.
  • the polypeptide can consist of the amino acid sequence CAISYIEQAM (SEQ ID NO:l 1).
  • the polypeptides of the present invention also include those polypeptides that comprise the sequence of amino acids set forth in SEQ ID NO:4 or SEQ ID NO: 1.1 , or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:4, such as an antigenic variant having at least 75%, 80% or 85% sequence identity to SEQ ID NO:4.
  • the polypeptides are less than 400, 300, 200, 100, 50. or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID NO:4 or SEQ ID NO:l 1, or a sequence having at least 75%, 80%. 85% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:4 or SEQ ID NO: 11.
  • Polypeptides of the present invention also include those that consist of the sequence of amino acids EQNMP Y LPQC (SEQ ID NO:5; corresponding to amino acids 177- 189 of SEQ ID NO:l), or consist of the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:5 having at least 75%, 84% or 90 sequence identity with the sequence of amino acids set forth in SEQ ID NO:5.
  • the polypeptides consist of a sequence of amino acids that has 1. 2 or 3 amino acid substitutions, additions and/or deletions compared to the sequence of amino acids set forth in SEQ ID NO:S.
  • polypeptides of the present invention also include (hose polypeptides that comprise the sequence of amino acids set forth in SEQ ID NO:5 or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:5 having at least 75%. 84% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:5.
  • the polypeptides are less than 400, 300, 200, 100, 50, or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID NO:5, or comprise a sequence having at least 75%, 84% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO: 5.
  • polypeptides of the present invention consist of the sequence of amino acids SPA GTPLPEQ (SEQ ID NO:6; corresponding to amino acids 212-222 of SEQ JD NO:l), or consist of the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:6 having at least 70%, 80%. or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:6.
  • the polypeptides consist of a sequence of amino acids that has 1, 2 or 3 amino acid substitutions, additions and/or deletions compared lo the sequence of amino acids set forth in SEQ ID NO:6.
  • the polypeptide consists of the amino acid sequence CSPAKGTPLPEQ (SEQ ID NO: 12).
  • polypeptides of the present invention also include those polypeptides that comprise the sequence of amino acids set forth in SEQ ID NO:6 or SEQ ID NO: 12, or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:6 having at least 70%. 80% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:6.
  • the polypeptides are less than 400, 300, 200, 100, 50, or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID NO:6 or SEQ ID NO: 12, or comprise a sequence having at least 70%, 80% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:4 or SEQ ID NO: 10.
  • Polypeptides of the present invention also can consist of the sequence of amino acids FYDAETKLY (SEQ ID NO:8; corresponding lo amino acids 294-302 of SEQ ID NO:1), or consist of the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:8 having at least 66%, 77%. or 88% sequence identity with the sequence of amino acids set forth in SEQ ID NO:8.
  • the polypeptides consist of a scqucncc ⁇ amino acids that has 1, 2 or 3 amino acid substitutions, additions and/or deletions compared to the sequence of amino acids set forth in SEQ ID NO:8.
  • the polypeptide consists of the amino acid sequence CFYDAETKLY (SEQ ID NO: 13).
  • the polypeptides of the present invention also include those polypeptides that comprise the sequence of amino acids set forth in SEQ ID NO:8 or SEQ ID NO: 13, or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO: 7 having at least 66%. 77%. or 88% sequence identity with the sequence of amino acids set forth in SEQ ID NO:8.
  • the polypeptides are less than 400, 300, 200, 100, SO. or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID N :8 or SEQ ID NO: 13, or comprise a sequence having at least 66%, 77%. or 88% sequence identity with the sequence of amino acids set forth in SEQ ID NO:8 or SEQ ID NO: 13.
  • Additional polypeptides of the present invention consist of the sequence of amino acids WADL1AKHPC (SEQ ID NO:7; corresponding to amino acids 177-189 of SEQ ID NO: 1), or a consist of the sequence of an antigenic variant of SEQ ID N :7 having at least 70%, 80% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:7.
  • the polypeptides consist of a sequence of amino acids that has I. 2 or 3 amino acid substitutions, additions and/or deletions compared to the sequence of amino acids set forth in SEQ ID NO: 7.
  • the polypeptides of the present invention also include polypeptides that are less than 400, 300, 200, 100. 50, or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID NO:7 or a sequence having at least 70%, 80% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:7.
  • polypeptides of the present invention consist of the sequence of amino acids VMVSHRSGETC (SEQ ID NO:9; corresponding to amino acids 409-419 of SEQ ID NO: 1 ), or the sequence of an antigenic variant of the polypeptide set froth in SEQ ID NO:9 having at least 70%. 80 or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:9.
  • the polypeptides consist of a sequence of amino acids that has 1, 2 or 3 amino acid substitutions, additions and/or deletions compared to the sequence of amino acids set forth in SEQ ID NO:9.
  • the polypeptides of the present invention also include polypeptides that are less than 400, 300, 200, 100, 50. or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID NO:9 or a scqucncc having at least 70%. 80% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:9.
  • polypeptides of the present invention containing the described antigenic determinants can be used as immunogens and administered to a subject to elicit antibodies specific for the antigenic determinant.
  • the resulting antibodies can specifically bind to the enolasc polypeptide of D. Jr ' agilis.
  • the polypeptides provided herein have a length of 7 amino acids to 400 amino acids, more typically 7 to 300, 7 to 200 or 7 to 100 amino acids, such as 9 amino acids to 100 amino acids, 9 amino acids to 75 amino acids, 9 amino acids to 50 amino acids, or 9 amino acids to 25 amino acids.
  • provided arc peptides that are 7, 8. 9, 10, 1 1. 12, 13, 14, 15, 16, 17, 18, 19, 20. 21, 22, 23, 24, 25, 26, 27. 28, 29, 30, 31, 32. 33, 34, 35, 36, 37. 38. 39, 40, 45, 50, 55, 60. 65. 70, 75, 80, 85, 90, 100, 200 or 300 amino acids in length.
  • the polypeptides of the present invention comprise two or more of the described antigenic determinants A, B, C. D, E, F or G.
  • Such polypeptides can be used, for example, to elicit antibodies specific for the two or more determinants present in the polypeptide.
  • included among the polypeptides of the present invention are those that comprise antigenic determinant B located at or within amino acids 264-272 and antigenic determinant A located at or within amino acids 453-467 of SEQ ID NO:1, or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:3 and the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:4.
  • polypeptides provided herein that contain at least one antigenic determinant can be linked to one or more other moieties, including, but not limited to, one or more other antigenic determinants, one or more T cell epitopes, one or more other immunostimulatory molecules, one or more targeting agents, one or more polymers, one or more proteins, one or more multimerisation domains, one or mote detectable labels, one or more affinity tags or any combination thereof.
  • the polypeptides can be linked to (he one or more other moieties by any method known in the art, including any chemical or recombinant method resulting in the formation of covalent and/or non-covalent bonds between the polypeptide and die one or more other moieties.
  • the resulting molecules can be used as immunogens that can be administered to a subject to elicit antibodies that bind to the enolasc polypeptide of D. fragilis.
  • the resulting molecules containing the polypeptides can be used in in vitro methods, such s screening methods, detection methods, diagnostic methods and purification methods.
  • the polypeptides can be linked to one or more T helper cell epitopes or a polypeptide comprising one or more T helper cell epitopes.
  • Any T helper cell epitope can be linked to the peptides provided the T helper epitope is recognized by T helper cells in the subject to which the peptide will be administered.
  • Promiscuous or universal T helper cell epitopes that are recognized in the context of different MHC backgrounds ⁇ i.e.
  • T helper cell epitopes can be identified using publicly accessible databases such as the immune Epitope Database and Analysis Resource (immuneepitope.org) and new T helper cell epitopes can be identified using methods well known in the art (sec e.g. Pira et al.. (2010) J Biomed Biotechnol). It is well within the skill of a skilled artisan to identify and select an appropriate T helper cell epitope for the desired purpose.
  • T helper ceil epitopes that can be linked to the polypeptides provided herein include, but are not limited to, those derived from microorganism proteins, such as viral proteins and bacterial proteins, as well as artificial or synthetic T helper cell epitopes (see e.g. U.S. Patent No. 6,713.301).
  • the T helper cell epitopes are from potent immunogens such as tetanus toxin, diptheria toxin, polio virus, pertussis toxin, the measles virus F protein, HIV gpl20 and HIV Gag proteins, and the hepatitis B virus surface antigen (HbsAg).
  • the T helper cell epitopes arc provided within the context of a larger protein.
  • the polypeptides of the present invention can be linked to a protein or polypeptide comprising a T helper cell epitope.
  • Exemplary proteins are carrier proteins, such as keyhole limpet haemocyanin or tetanus toxoid.
  • the polypeptides of the present invention may be linked to an affinity tag to, for example, facilitate purification.
  • affinity tags include, but are not limited to, chitin binding protein (CBP), maltose binding protein (MBP), glutathionc-S-transfcrase (GST), FLAG, His, c-myc and HA lags.
  • Detectable molecules including, but not limited to. fluorescent or chemi luminescent molecules, or biotin or strcptavidin. also can be Linked to the polypeptides.
  • the one or more other moieties linked to the provided polypeptides can be linked by any method known in the art, including chemical methods and recombinant methods.
  • polymers can he coupled to the polypeptides using standard chemical reactions.
  • Proteins e.g. carrier proteins such as ovalbumin, keyhole limpet hacmocyanin and bovine scrum albumin
  • MBS standard chemical coupling techniques
  • glutaraldchyde EDC
  • BDB coupling standard chemical coupling techniques
  • the polypeptides are linked to other peptides (such as those including a T cell epitope) or proteins by peptide synthesis methods or recombinant methods.
  • a polypeptide can be linked to a T cell epitope by sequentially synthesizing the polypeptide then the T cell epitope as a single polypeptide using standard methods (e.g. Fmoc solid phase synthesis).
  • DNA encoding the polypeptides can be operatively linked to DNA encoding the T cell epitope (or any other protein) and the entire DNA molecule expressed, such as using a bacterial expression system, to produce a single polypeptide containing the peptide and the T cell epitope.
  • linkage can be by covalent and/or non-covalent bonds, depending on the method of linkage employed.
  • a peptide linker or spacer is used to link the polypeptides and the one or more other moieties.
  • Peptide linkers typically are from 1 amino acid in length to 10 amino acids in length, although can be longer.
  • Non-limiting examples of peptide linkers that can be used herein include linkers having the sequence , K. K . GPGPG t.SEQ ID NO: 14), G, GG, GGG. GGGG (SEQ ID NO: 15), GGA, GA. GD, GSGGGG (SEQ ID NO: 16), GSGGGGS (SEQ ID NO: 17), GS. RS. RR, KKK. KKAA (SEQ ID NO:18), VE, and A AY.
  • polypeptides provided herein can be produced using any method known in the art, including peptide synthesis techniques and recombinant techniques.
  • the polypeptides arc synthesized using the Fmoc-polyamide mode of solid-phase peptide synthesis.
  • Other synthesis methods include solid phase t-Boc synthesis and liquid phase synthesis.
  • Other peptide moieties, such as T cell epitopes, can be sequentially synthesized and linked to the polypeptide.
  • Purification of the synthesized polypeptides can be performed by any one, or a combination of, techniques such as re-crystallization, size exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography and reverse-phase high performance liquid chromatography using, for example, Aconitril/water gradient separation.
  • the polypeptides are produced using recombinant methods well known in the art.
  • Nucleic acid encoding the polypeptides can be obtained by any suitable method, including, but not limited to, RT-PCR of Z). fragilis mRNA or synthesis of an oligonucleotide that encodes a polypeptide of the present invention.
  • nucleic acid molecules encoding a polypeptide of the present invention It is well within the skill of a skilled artisan to design a nucleic acid molecule thai encodes a polypeptide described herein, such as a polypeptide comprising one or more antigenic determinants located at or within amino acids 177-189. 212-222, 264-272. 294-302, 319- 328, 409-419 or 453-467 of SEQ ID NO:l.
  • Nucleic acid encoding the polypeptide can be cloned into an expression vector suitable for the expression system of choice, operably linked to regulatory sequences that facilitate expression of the heterologous nucleic acid molecule.
  • Many expression vectors are available and known to those of skill in the art for the expression of peptides. The choice of expression vector is influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan.
  • expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that arc used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells.
  • the polypeptides are expressed as fusions with another peptide or polypeptide.
  • the polypeptides can be produced as fusions by operably linking the nucleic acid encoding the peptide with nucleic acid encoding one or more T cell epitopes, ligands.
  • affinity tags e.g. a his6 tag or a myc tag, a GST fusion
  • Other examples of fusions include, but are not limited to, fusions of a signal sequence and a sequence for directing protein secretion.
  • the provided polypeptides can be purified using any method known to those of skill in the art including, but not limited to, SDS-PAGE, size fraction and size cxclusion chromatography, ammonium sulfate precipitation, chelate chromatography, ionic exchange chromatography and affinity chromatography. Affinity purification techniques can be used to improve the purity of the preparations.
  • expression constructs can be engineered to add an affinity tag to the polypeptide, such as a myc epitope, GST fusion or His6. which facilitates affinity purification using an anti-myc antibody, glutathione resin, or Ni-resin, respectively.
  • Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometry techniques.
  • analysis of the polypeptides is carried out using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis, fast atom bombardment (FAB) mass spectromelric analysis.
  • ALDI or ESI-Q-TOF mass spectromelric analysis is analyzed using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis, fast atom bombardment (FAB) mass spectromelric analysis.
  • the antigenic properties of the polypeptides can be assessed using a variety of methods known to those skilled in the art.
  • the ability of the polypeptides to induce an antibody response can be assessed by linking the polypeptides to one or more T helper cell epitopes or a protein carrier containing one or more T helper cell epitopes (e.g. keyhole limpet haemocyanin) and administering (such as by intravenous, intraperitoneal or intramuscular injection) the resulting immunogen to a subject one or more times.
  • the antibody response elicited can be assessed at various time points after immunization by sampling the blood of the subject and subjecting the sera to analysis using an appropriate assay, such as an ELISA.
  • the ELISA plate can be coated with the polypeptide or the D. fragilLs enolase set forth in SEQ ID NOs: 1 or 2.
  • Such methods can be used to determine the magnitude and specificity of an antibody response elicited by administration of the provided peptides when linked to one or more T helper cel.! epitopes.
  • the ability of the polypeptides to be recognized by antibodies, including polyclonal or monoclonal antibodies directed to D. fragilis * can be assessed by standard methods, including, but not limited to, ELISA, Western blot, dot blot, surface plasmon resonance and rapid flow tests (e.g. lateral or vertical flow test).
  • the polypeptides can be present in compositions, such as pharmaceutical compositions.
  • the pharmaceutical compositions can be formulated in any conventional manner by mixing a selected amount of polypeptide (including polypeptides linked to one or more other moieties), with one or more physiologically acceptable carriers or excipients. Selection of Ihc carrier or cxcipicnl is within the skill of die administering profession and can depend upon a number of parameters, such as the mode of administration (i.e., systemic, nasal, oral or any other mode). ⁇ some examples the pharmaceutical compositions are provided as a fluid. In other instances, the pharmaceutical compositions are provided in diicd form, such as desiccated or freeze-dried form.
  • compositions can be rehydratcd prior to administration to a subject by the addition of a suitable solution, such as water, buffer, saline or other suitable solution.
  • a suitable solution such as water, buffer, saline or other suitable solution.
  • the pharmaceutical compositions provided herein can be formulated for direct administration or can be formulated for dilution or other modification. Accordingly, the pharmaceutical compositions can be formulated in single (or unit) dosage forms or multiple dosage forms. Examples of single dose forms include ampoules and syringes. Examples of multiple dose forms include vials and bottles that contain multiple unit doses.
  • compositions containing the provided polypeptides can also include pharmaceutically active ingredients including, but not limited to, other antigens, adjuvants or immunomodulators, such as chemokincs. interlcukins and cytokines.
  • exemplary adjuvants include, but are not limited to, aluminium salts (e.g. aluminium hydroxide, aluminium phosphate and potassium aluminium sulphate; also referred to as Alum), iscoms. and saponin-based adjuvants (e.g. adjuvants containing Quil A or Quil A derivatives or fractions).
  • isolated antibodies such as isolated polyclonal and monoclonal antibodies (including antigen-binding fragments thereof, such as single-chain Fv (scFv).
  • Fab. Fab', F(ab')2, Fv, dsFv, diabody, Fd, and Fd' fragments that specifically bind to the antigenic determinants and polypeptides containing the antigenic determinants described herein.
  • provided arc isolated antibodies that recognize and bind to antigenic determinants comprising, or located at or within, amino acids 177-189, 212-222.
  • the antibodies of the present invention can bind to polypeptides comprising a sequence of amino acids set forth in any one of SEQ ID N0s:3-13, or a sequence of amino acids having at leasl 70%, 75%, 80%, 85%, 90% or 95% sequence identity to a sequence of amino acids set forth in any one of SEQ ID NOs:3-13.
  • the antibodies and anligenic-binding fragments thereof may be of any isotype (e.g. IgG (including FgGl, IgG2a, IgG2b, IgG3 and FgG4), IgM. IgA, IgD and IgE) and can be polyclonal or monoclonal, non-human (e.g. mouse, rat. rabbit, guinea pig) or human, or chimeric or humanized.
  • Exemplary of the antibodies described herein are those that recognize and bind to an antigenic determinant comprising, or located at or within, amino acids 264-272 of SEQ ID NO:1 (antigenic determinant B) or corresponding amino acids in a polypeptide having at least 85%. 86%, 87%, 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98% or 99% sequence identity with the sequence set forth in SEQ ID NO:l.
  • the antibodies can bind to polypeptides consisting of or comprising the sequence of amino acids AISYIEQA (SEQ ID NO:4) or CAISYIEQAM (SEQ ID NO: 1 1), or polypeptides consisting of or comprising a sequence of amino acids having at least 75%, 80% or 85% sequence identity with the sequence of amino acids set forth in SEQ ID NO:4 or SEQ ID NO: 10.
  • Exemplary antibodies also include those that recognize and bind to the antigenic determinant comprising,, or located at or within, amino acids 453-467 of SEQ ID NO:l (antigenic determinant B) or corresponding amino acids in variant having at least 85%, 86%, 87%. 88%. 89%, 90%, 91%, 92%. 93%. 94%. 95%. 96%, 97%, 98% or 99% sequence identity with the sequence set forth in SEQ ID NO:l.
  • Such antibodies can bind to polypeptides consisting of or comprising the sequence of amino acids LQIYEYLKEHDMLEE (SEQ ID NO:3) or CLQJYEYLKEHD LEE iSEQ ID NO: 10), or polypeptides consisting of or comprising a sequence of amino acids having at leasl 75%, 80%, 85%, 90% or 95% sequence identity with the sequence of amino acids set forth in SEQ ID NO:3 or SEQ ID NO:5.
  • the antibodies of the present invention specifically bind (o the polypeptides provided herein and thus may specifically bind to the D. fragilis enolase protein.
  • the antibodies and antigen binding fragments specifically bind to the cnolasc polypeptide when associated with the surface of a D. fragilis trophozoite.
  • antibodies of the present invention can specifically bind D. fragilis.
  • Techniques for preparing polyclonal and monoclonal antibodies against polypeptides are well known in the art. For example, polyclonal antibodies directed against an antigenic determinant described herein can be generated by administering to a subject (such as a non-human subject, e.g.
  • polypeptide described herein that contains that antigenic determinant and a T helper cell epitope.
  • the polyclonal antibodies produced following administration can then be isolated from the serum of the subject.
  • monoclonal antibodies specific for an antigenic determinant can be obtained by injecting a subject with a polypeptide that contains that antigenic determinant and a T helper cell epitope, then removing the spleen to obtain 6 lymphocytes.
  • B lymphocytes can be isolated from peripheral blood lymphocytes ⁇ PBL). The B lymphocytes from immunized subjects are then fused with myeloma cells to produce hybridomas, which are cloned.
  • Positive clones thai produce antibodies to the antigenic determinant are selected using standard techniques (e.g. ELISpot), culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques including, but not limited to, affinity chromatography with Protein-A SEPHAROSE®, size-exclusion chromatography, and ion- exchange chromatography. After the initial raising of antibodies to the antigenic determinant, the antibodies can be sequenced and subsequently prepared by recombinant techniques.
  • Monoclonal antibodies can also be produced from an antibody library.
  • total RNA can be extracted from peripheral blood B lymphocytes of a subject, such as a healthy subject or a subject that has been or is infected with D. fmgilis, and a cD A library constructed by amplifying ⁇ , ⁇ and chain antibody repertoires.
  • the cDNA library can then be used to make a display library, such as a phage display library in which antigen-binding fragments of antibodies, such as single chain Fv (scFv) fragments, are expressed on the surfaces of bacteriophages as fusion proteins with the bacteriophage coat protein.
  • scFv single chain Fv
  • Antibodies or fragments thereof that recognize and bind to antigenic determinants comprising- or located at or within, amino acids 177-189. 212-222. 264-272. 294-302. 319- 328. 409-419 or 453-467 of the D. fmgilis enolase protein set forth in SEQ ID NO:l or corresponding amino acids in another D. fmgilis enolase protein can then be screened and sclectcd using the polypeptides provided herein (hat contain such antigenic determinants. Typically, selection is performed using panning techniques, a process well known to those of skill in the art.
  • the antibodies and antigen-binding fragments of the present invention can be linked to one or more moieties, such as to facilitate detection.
  • the antibodies or antigen-binding fragments may be linked to a detectable label such a fluorescent, chcmilumincscent, enzyme, biotin/streplavidin or metabolic labels.
  • Non-limiting examples of labels that can be linked to the antibodies and antigen-binding fragments include biotin, streptavidin, alkaline phosphatase (AP), horseradish peroxidase (HRP), fluorescein isotmocyamUc (FITC), rhodaminc (tctramcthyl rhodaminc isothiocyanatc, T 1TC), green fluorescent protein (GFP). allophycocyanin, phycocyanin, phycoerythrin and phycoerytlirocyanin.
  • Antibodies and fragments thereof may be linked to one or more moieties using any method known in the art. For example, linkage may be through chemical conjugation using one of a variety of well known techniques, including but not limited to the use of NHS esters, hcterobifunctional reagents, carbodiirnides or sodium periodate.
  • binding properties of the antibodies of the present invention can be characterized by established methodologies, for example, EL1SA and Western blot. Binding affinity also can be determined. Any method known to one of skill in the art can be used to measure the binding properties of an antibody. In some examples, the binding properties of an antibody are assessed by performing a saturation binding assay, for example, a saturation ELTSA, whereby binding of the antibody to the polypeptide is assessed with increasing amounts of antibody. In such experiments, it is possible to assess whether the binding is dose- dependent and or saturable. In addition, the binding affinity can be extrapolated from the 50% binding signal.
  • a saturation binding assay for example, a saturation ELTSA
  • apparent binding affinity is measured in terms of its association constant (K a ) or dissociation constant (K ⁇ i) and determined using Scatchard analysis.
  • binding affinity to a target polypeptide can be assessed in a competition binding assay in where increasing concentrations of unlabeled protein is added, such as by radioimmunoassay (RIA) or ELISA.
  • RIA radioimmunoassay
  • ELISA ELISA
  • the ability of the antibodies to bind to D.fragilis also can be assessed using methods well known in the art. For example. thc binding of antibodies to D. fragilis trophozoites can be visualized by microscopy using direct or indirect fluorescence.
  • the polypeptides and antibodies provided herein can be utilized in a variety of methods.
  • the polypeptides can be used in methods for eliciting or inducing antibodies specific for an antigenic determinant described herein, and thus specific for D. fragilis methods for screening and selecting antibodies; methods for purifying antibodies; and methods for characterizing the binding properties of antibodies.
  • the antibodies described herein that bind to an antigenic determinant in the enolase polypeptide of D. fragilis (such as antigenic determinant A, B. C. D, E, F or G) can be used in methods for detecting the enolase polypeptide of D. fragilis in a sample, methods for detecting D. fragilis in a sample; methods for diagnosing D. fragilis infection in a subject; methods for characterizing the binding properties of other antibodies; and methods for isolating and purifying D. fragilis enolase polypeptides and fragments thereof
  • the polypeptides of the present invention may be administered to a subject to elicit antibodies that bind to the polypeptide.
  • administration of the polypeptides to a subject can elicit antibodies that bind to the D. fragilis enolase polypeptide, such as the enolase polypeptide associated with the surface of the D. fragilis trophozoite.
  • the polypeptides can be administered to a human or non-human subject, such as a rat, mouse, rabbit, goat, sheep, rabbit or guinea pig, and by any suitable route, including, but. not limited to.
  • polypeptides should be formulated in compositions suitable for the particular route of administration
  • the polypeptides provided herein can be administered to a subject one time or more than once, including 2, 3. 4. 5 or more times. If the polypeptides are administered more than one time, the time between dosage administration can be, for example, 1. 2. 3, 4. 5, 6, 7, 8. 9, 10, 1 1, 12 or more weeks, or 1, 2, 3, 4, 5, 6, 7, 8, , 10, 11, .12 or more months. Selecting an optimal protocol to elicit antibodies is well within the level of skill of the skilled artisan. In some examples, repeat doses of the polypeptide can be administered to a subjcct if Ihc antibody response elicited by the subject after the first dose is below desired level.
  • antibodies and/or antibody producing cells can be isolated from a biological sample, such as blood, (issue, cells or serum, from the subject.
  • a biological sample such as blood, (issue, cells or serum, from the subject.
  • polyclonal antibodies can be isolated from the scrum.
  • antibody producing cells can be isolated from the spleen or from peripheral blood lymphocytes (PBL) in the blood.
  • PBL peripheral blood lymphocytes
  • the polypeptides of the present invention can also be used to select, screen, isolate and/or purify antibodies that are specific for an antigenic determinant comprising, or located at or within, amino acids 177-189. 212-222, 264-272, 294-302, 319-328, 409-419 or 453-467 of the D. fragilis enolase protein set forth in SEQ ID NO: 1 or corresponding amino acids in another D. fragilis enolase protein.
  • capture techniques whereby the polypeptides are used to capture an antibody specific for the antigenic determinant present in the polypeptide.
  • the polypeptides are contacted with the antibodies, and those antibodies that arc bound to the polypeptides arc then selected and optionally isolated.
  • the polypeptides can be incubated with a samples containing the antibodies for a sufficient time to permit binding of antibodies to the polypeptides.
  • the polypeptides are immobilized, such as on a column or a plate, or are linked to beads, such as magnetic beads.
  • the polypcptidc-antibody complexes are washed to remove non-binding antibodies.
  • the stringency of the wash can be adjusted according to methods well known to the skilled artisan. After washing to remove unbound antibodies, bound antibodies can be eluted using one of several well known clution methods, typically by reduction of the pH of the solution. In some instances, selection methods are an iterative process with multiple rounds of capture, wash and clution.
  • the polypeptides can also be used to detect the presence of D. fragilis antibodies in a sample.
  • the sample may be a biological sample, such as blood, serum, stool, gastric lavage or urine.
  • the polypeptides of the present invention can be used to detect or diagnose D. fragilis infection in a subject, whereby the subject has raised antibodies specific to the enolase polypeptide.
  • Such methods can be performed using standard techniques such as ELISA, Western blot and dot blot.
  • the polypeptides can be contaclcd with the sample, and binding of antibodies in the sample to the polypeptide can be detected.
  • the polypeptides can be immobilized on a surface and contacted with the sample. Following washing, any antibody thai is bound to the polypeptide can be detected using a labeled secondary antibody.
  • the antibodies of the present invention can be used to detect the presence of the enolase polypeptide of D. fragilis in a sample by contacting the antibodies with a sample that may contain the enolase polypeptide under conditions that would facilitate binding of the antibodies to the polypeptide.
  • samples include, but arc not limited to, biological samples from a subject, such as blood, serum, stool, gastric lavage or urine, cultured parasites or cultured recombinant cells.
  • Detection of the presence of the enolase polypeptide can be performed by any method known in the art that detects the formation of a complex between the antibody and the enolase protein, including, but not limited to. rapid flow tests (e.g. lateral or vertical flow tests), ELISA, dot blot and Western blot.
  • the antibodies of the present invention can also be used to detect the presence of D. fragilis parasites in a sample. Jn some instances the sample is a biological sample, such as blood, serum, stool, gastric lavage or urine. The biological sample may have been processed further, such as by homogenization, fixation, concentration, dessication or some other form of processing. The antibodies of the present invention can therefore be used to diagnose D. fragilis infection in a subject by detecting the presence of D. fragilis in a sample from the subject. Typically, the sample is a stool sample.
  • Methods for detecting the presence of D. fragilis in a sample, and thus methods for diagnosing D. fragilis infection in a subject arc performed by contacting the sample with an antibody of the present invention under conditions that facilitate binding of the antibody to D. fragilis parasites in the sample.
  • the antibody binds to an antigenic determinant comprising, or located at or within, amino acids 264-272 of SEQ ID NO:l or corresponding amino acids in another D. fragilis enolase protein.
  • the antibody binds to the antigenic determinant comprising, or located at or within, amino acids 453-467 of SEQ ID NO:l or corresponding amino acids in another D. fragilis enolase protein.
  • the presence of D. fragilis in the sample is determined by delecting whether the antibody has bound to any D. fragilis parasites. Detection of the binding between the antibod and a D. fragilis parasite, such as a D. fragilis trophozoite, can be performed using any method known in the art. In some instances, the binding is detected directly by microscopy using antibodies of the present invention that contain a detectable label such as a fluorescent label, e.g.
  • Such methods typically involve contacting the antibodies with a sample containing the enolase polypeptides under conditions that facilitate binding of the antibodies to the enolase polypeptides.
  • the antibodies are immobilized, such as on a column or a plate, or are linked to beads, such as magnetic beads.
  • the antibody-enolase complexes are washed to remove non-bound proteins.
  • the enolase polypeptide can then be eluted using conditions well known to those skilled in the art. such as reduced pH of the elution solution.
  • the antibodies of the present invention also can be used in methods for characterizing the binding properties of other antibodies. For example, competition binding assays can be used to determine whether a given antibody binds to the same antigenic determinant, an overlapping antigenic determinant or a different antigenic determinant than any one or more of the antibodies described herein.
  • kits comprising one or more polypeptides and/or antibodies described herein. Such kits may be useful in performing any one or more methods described herein.
  • the kits provided herein optionally contain instructions for use, such as instructions for use in a method of the present invention. Additional reagents also may be included in the kits, including, but not limited to, diluents and one or more additional antibodies, such as a labeled secondary antibody.
  • Exemplary kits of the present invention include one or more antibodies of the present invention, such as one or more antibodies that bind to an antigenic determinant comprising, or located at or within, amino acids 264-272 of SEQ IT) NO:l or corresponding amino acids in another D.
  • kits may be used, for example, in methods for detecting the presence of enolase polypeptides and or D. fragilis parasites in a sample. Accordingly, such kits may be used for the diagnosis of D. fragilis infection in a subject, by detecting the presence of D. fragilis parasites in a sample from the subject.
  • kits may contain a detectable label, such as a fluorescent label or gold particles.
  • a detectable label such as a fluorescent label or gold particles.
  • the kits may be used in a direct flourescenl antibody test or rapid flow test (e.g. lateral or vertical flow lest).
  • the antibodies contained in the kit do not have a detectable label.
  • kits may be used, for example, in an indirect flourcsccnt antibody test
  • kits containing antibodies of the present invention that do not have a detectable label may also comprise a labeled secondary antibody, such as a fluorescently labeled secondary antibody, that binds to the Fc portion of the antibodies of the present invention.
  • D. fragilis trophozoites were cultured xenically at 37°C under microaerophilic conditions in a Loeffler's slope medium (modified from Barratt et al. (2010) Parasitology 137, 1867-1878). consisting of an inspissated horse scrum slope (5ml of horse autoclaved for 20 minutes at 89°C, dry) overlaid with 5ml of PBS and supplemented with 2-5mg of rice starch. RNA was extracted from D.
  • the top 44 of 100 hits from a blaslx search were cnolases from trichomonads or related parabasalian parasites (Tridwmonas vaginalis.
  • this putative Dientamoeba enolase possesses a conserved enolase metal binding site, an enolase substrate binding pocket, an enolase dimer interface and a triosephosphate isomerase (TIM) barrel domain.
  • the sequence also possesses a conserved enolase N-terminal domain. It was concluded that the closest homologue of T. vaginalis enolase AY895164 from Dientamoeba had been identified.
  • a 3D model of the enolase polypeptide set forth in SEQ ID NO: 1 was generated using the SWISS-MODEL workspace and using the automated mode.
  • the qmean score (a measure of model accuracy) of the model was 0.62, indicating that the enolase model is a reasonable depiction of the predicted structure.
  • amino acids in white represent those which are part of the extracellular loop of the and are exposed on the surface of the protein itself, though were not used for peptide design as described in Example 2. While these amino acids were not used for peptide design in this case, they arc likely to be accessible to antibodies and so arc also candidates for incorporation into any of peptides A to G (such as by making these peptides longer). Alternatively, these amino acids could be used to design completely new peptides based on the same principals used to design peptides A to G.
  • These surface- exposed amino acids include those at positions 198; 203; 205; 223; 224; 227; 228; 230; 231 ; 232; 235; 238; 239; 240; 241; 242; 243; 244; 246; 247; 248; 259; 260; 273; 275; 276; 277; 303: 305; 306; 307; 308: 310; 313; 314; 315; 317; 329; 330; 339; 342; 345: 346: 348; 349: 350; 353; 354; 355; 356; 367: 368; 370; 37.1 ; 372; 374: 375; 376; 377; 380: 389; 396; 400; 401 ; 403; 404; 405; 408; 420; 421 ; 422; 430; 433; 434; 440; 441 ; 442; 443; 446; 447;
  • the peptides were each conjugated to the immunogenic carrier protein keyhole limpet hemocyanin (KLH) via (he thiol group of the cysteine residue of each peptide, before being administrated to rabbits to produce antisera using standard techniques.
  • KLH keyhole limpet hemocyanin
  • the presence of anti-peplide antibodies in the resulting rabbit antisera was determined using a simple slot blotting technique. Briefly, peptide was blotted onto a nitrocellulose membrane using a PB 600 Slot Blot Filtration manifold system (Amersham Biosciences).
  • the peptide was then probed with the rabbit antisera (diluted 1:2 with PBS) and then with an anti-rabbit IgG antibody (raised in goats) conjugated to FITC (Sigma Aldrich, product number F0382) (diluted 1:80).
  • the blot was scanned using a Pharos FX Plus Molecular Imager (Bio-Rad) to determine the presence or absence of fluorescence.
  • the blot was accompanied by two negative controls; a pre-bleed sample from the rabbit (prior to immunization with the peptide) and a blot probed with the FITC conjugated antibody only (no rabbit antisera). It was confirmed thai antibodies specific for the relevant peptide were present in each antiserum composition.
  • the resulting suspension was then centrifuged at 7000g at 4°C for 15 minutes. The supernatant was discarded and the protein pellet was dissolved in 3 mL of PBS.
  • This antibody solution was dialysed overnight at 4°C against 1L of PBS with three buffer changes. The antibody solution was removed from the dialysis tubing and stored (temporary storage of several days at 4°C, long term storage -20°C to -80°C). Twenty-five mL of rabbit antisera yielded approximately 140mg of protein.
  • Tlie absorbance of each fraction was measured to identify the IgG containing fractions.
  • the IgG containing fractions were pooled and dialysed overnight at 4°C against 1L of PBS with three buffer changes.
  • the purity of the antibody preparation was evaluated by reducing a portion with dithiothrcitol (DTT) and subjecting it to polyacrylamidc gel electrophoresis.
  • the polyacrilimidc gel was then stained with Coomassie blue for observation of IgG heavy and light chains.
  • the polyclonal antibody preparations were stored at -20°C until use (long term storage -80°C).
  • the ability of the antibody preparations to bind to D. fragilis was assessed using an indirect immunofluorescence assay.
  • To prepare the D. fragilis trophozoites two day old cultures of D. fragilis were agitated slightly to suspend the culture sediments where the trophozoites reside. The cell suspension was collected and centrifuged at 700 g for 10 minutes to pellet cells. The supernatant was discarded and the cells were fixed by addition of PBS containing 3.7% formaldehyde and incubating for 10 minutes. Immediately after addition of the PBS-formaldehyde solution, the tube was gently shaken to thoroughly break apart the pellet. Cells were then centrifuged at 700g for 5 minutes and the supernatant was discarded.
  • This method was intended to be mild and non- jcrmcabilising, so as to assess the presence of a fluorescent signal on the surface of cells.
  • cells instead of fixing cells with 3.7% formaldehyde, cells were fixed by bathing for at least 24 hours in SAF fixative (Fronine Lab Supplies. Australia) in a 1:1 ratio of culture supcrnalant Co SAF. The cells were then immediately bathed in a large volume of PBS and centrifuged once more to remove any residual formaldehyde or SAF.
  • antibody preparations A and B exhibited a very strong reaction to the surface of Dientamoeka trophozoites ( Figures 2 and 3) after fixation with 3.7% formaldehyde in PBS. All other antibody preparations resulted in an extremely weak to completely absent fluorescent signal. Interestingly, each of antibody preparation A and B gave rise to slightly different fluorescent patterns.
  • Antibody preparation A produced a di fuse, even fluorescent signal across the surface of Dieniamaeba cells ( Figure 2). Staining with antibody preparation B produced a distinct ring pattern of fluorescence characterized by a densely stained fluorescent ring on the periphery of cells and a lighter fluorescent signal within the ring ( Figure 3).
  • Negative controls (cultured ceils treated with only the secondary antibody) did not fluoresce. Examination of fixed, unstained Dieniamaeba trophozoites demonstrated that they are not auto-fluorescent. Cells from control slides stained with antibody preparations A and B, though without the application of the FITC conjugated anti-rabbit IgG, also failed to fluoresce. The same staining profile was observed with the SAF fixation protocol.
  • Example 5 The same staining protocol described in Example 5 was used to determine whether the antibodies in the preparations A and B could bind to Entamoeba histolytica, E. tnoshkovskii and Giardia trophozoites and Blastocysts cells, thereby assessing specificity of the antibodies.

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Abstract

Provided herein are antigenic polypeptides of Dientamoeba fragilis and antibodies and antigen-binding fragments thereof that bind thereto. Embodiments of the invention relate to antibodies and antigen-binding fragments that specifically bind to antigenic determinants within D. fragilis enolase polypeptides, including the enolase polypeptide comprising the sequence set forth in SEQ ID NO:1, and to methods and kits for the detection of D. fragilis using such antibodies and antigen-binding fragments.

Description

ANTIGENIC POLYPEPTIDES OF DIENTAMOEBA FRAGILIS AND ANTIBODIES THERETO
FIELD OF THE INVENTION
[001] The present invention relates generally to antigenic polypeptides of Dienl amoeba fragilis and antibodies thereto. The present invention also relates generally to methods and kits for the generation of antibodies to D. fragilis and methods and kits for the detection of D. fragilis.
BACKGROUND OF THE INVENTION
[002] Dientamoeba fragilis is a protozoan parasite that inhabits the human bowel, causing a gastrointestinal disorder characterized by abdominal pain, nausea, anorexia, vomiting and diarrhea. Of these symptoms, abdominal pain and diarrhea are most common. Although originally described as a rare and harmless commensal, the pathogenic potential of D. fragilis is becoming increasingly apparent. With the improvement of techniques for the recovery and identification of D. fragilis. this organism is now among the most frequently encountered intestinal protozoa in humans. In Australia, D. fragilis prevalence in patients presenting with gastrointestinal complaints has been reported to be between 0.4% and 16.8%.
[003] Despite the increasing appreciation of its prevalence, infections by D. fragilis are frequently overlooked because of the lack of rapid and cost effective diagnostic tests for the diagnostic laboratory. This can be particular problematic as the clinical signs of infection associated with D. fragilis are the same as those seen for irritable bowel syndrome (IBS), which is a commonly diagnosed gut disorder, particularly in developed countries including the United States, Europe and Australia. Given the similarities in presentation, D. fragilis infection should be considered in the differential diagnosis of IBS and other diarrheal diseases.
[004] Although there has been a growing trend in developing new diagnostics for the detection of Dientamoeba in human stool specimens in recent years, these developments have focused on molecular tests based on the polymerase chain reaction (PCR). While these tests arc very sensitive and specific, they have not replaced standard microscopic cxamination of stained fecal preparations in most diagnostic Laboratories due to issues associated with cost and feasibility. Thus, there is a need for improved methods for detection otD. fragilis.
SUMMARY OF THE INVENTION
f005] The present invention is directed to the D. fragilis enoJase polypeptide and fragments thereof, isolated polypeptides containing antigenic determinants of the D. fragilis enolase polypeptide, and isolated antibodies and antigen-binding fragments thereof that bind to the antigenic determinants within the D. fragilis enolase polypeptide, and thus bind to the D. fragilis enolase polypeptide and D. fragilis parasites. The invention is also directed to methods and kits associated with (he polypeptides and antibodies of the invention.
[006] In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to an antigenic determinant within the D. fragilis enolase polypeptide set forth in SEQ ID NO:l or a variant thereof having at least about 85% sequence identity with the sequence set forth in SEQ ID NO:l. In some examples, the variant has at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98% or 99% sequence identity with the sequence set forth in SEQ ID NO: 1.
|0 7] The antibodies and antigen-binding fragments of the invention may be monoclonal or polyclonal. In some embodiments, the antibodies or antigen-binding fragments are linked to a detectable label, such as, for example, a fluorescent label, a chcmiluminescent label, an enzymatic label, gold particle, biotin or streptavidin.
[008] In particular embodiments, the antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 453-467 of SEQ ID NO: I or corresponding amino acids in the variant. In other embodiments, the antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 264-272 of SEQ ID NO:l or corresponding amino acids in the variant. In further embodiments, the antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 177-189 of SEQ ID NO:l or coircsponding amino acids in die varianl. In olhcr embodiments, Ihe antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 212-222 of SEQ TD NO:l or corresponding amino acids in the variant. In still further embodiments, the antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 294-302 of SEQ ID NO:l or corresponding amino acids in the variant. In other examples, the antibodies and antigen- binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 319-328 of SEQ ID NO:l or corresponding amino acids in the variant. In further examples, the antibodies and antigen-binding fragments of the invention bind to an antigenic determinant that comprises, or is located at or within, amino acids 409-419 of SEQ ID NQ:1 or corresponding amino acids in the variant.
[009] The antibodies and antigen-binding fragments of the invention can be used to detect the presence of a D. fragilis cnolase polypeptide and or a D. fr gilis parasite in a sample. In some embodiments, the antibodies and antigen-binding fragments are used in diagnosing a D. fragilis infection in a subject.
[010] Accordingly, in some aspects, the invention provides a method for the detection of a D. fragilis enolase polypeptide in a sample, comprising contacting the sample with an antibody or antigen-binding fragment of the invention and detecting binding of the antibody or antigen-binding fragment to the enolase polypeptide. The invention also provides a method for the detection of a D. fragilis parasite in a sample, comprising contacting the sample with an antibody or antigen binding fragment of the invention and detecting binding of the antibody or antigen-binding fragment to the D. fragilis parasite. Also provided arc methods for the diagnosis of D. fragilis infection in a subject, comprising contacting a sample from the subject with an antibody or antigen-binding fragment of the invention and detecting binding of the antibody or antigen binding fragment to a D. fragilis parasite in the sample.
[011] In some examples, the sample comprises blood, serum, stool, gastric lavage or urine. In particular embodiments, the sample is a stool sample. The detection can be performed in any suitable manner. In one example, detection is performed by direct immunofluorcscence. In another example, detection is performed by indirect immunofluorescence.
[012] The present invention also provides kits comprising any one or more of the antibodies or antigen-binding fragments described herein. In some examples, the kits are used for detecting the presence of a D. fragilis enolase polypeptide and/or a D. fragilis parasite in a sample. The kits of the invention can also be used for diagnosing D. fragilis infection in a subject. In particular embodiments, the kit also contains a labelled secondary antibody,
[013] In one aspect, the present invention provides an isolated polypeptide comprising the sequence of amino acids set forth in SEQ TD NO:1 or 2, or a sequence of amino acids having at least about 85% sequence identity with (he sequence set forth in SEQ ID NO: I or 2, In particular embodiments, the isolated polypeptide comprises a sequence of amino acids having at least about 86%, 87%, 88%. 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence set forth in SEQ ID NO: 1 or 2.
[014] In a further aspect, the present invention provides an isolated polypeptide, comprising the sequence of amino acids set forth in SEQ ID NO:3 or 10.
[015] In another aspect, the present invention provides an isolated polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:3 or the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:3, wherein the variant has at least 75%, 80%, 85% or 90% sequence identity with the sequence set forth in SEQ ID NO:3, and wherein (he isolated polypeptide is less than 300 amino acids in length. In some examples, the polypeptide comprises the sequence of amino acids set forth in SEQ ID NO:3 or 10. In further examples, the polypeptide consists of the sequence set forth in SEQ ID NO:3 or 10.
[016] In one aspect, the present invention provides an isolated polypeptide, comprising the sequence of amino acids set forlh in SEQ ID NO:4 or 11.
[017] In another aspect, the invention provides an isolated polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:4 or the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:4. wherein the variant has at least 75%, 80% or 85% sequence identity with the sequence of amino acids set forth in SEQ ID NO:4, and wherein (he isolated polypeptide is less lhan 300 amino acids in length. In one example, (he polypeptide comprises the sequence of amino acids set forth in SEQ ID NO:4 or 11. In another example, Ihe polypeptide consists of the sequence set forth in SEQ ID NO:4 or 11.
Γ0Ι8] In some embodiments, the polypeptides of the invention are linked to at least one moiety. For example, the polypeptides can be linked to another antigenic determinants, a T helper cell epitope or polypeptide that comprises a T helper cell epitope, an immunostiinulatory molecule, a targeting agent, a polymer, a multimerisation domain, a detectable label or an affinity tag. In particular examples, the polypeptides arc linked to a T cell epitope or polypeptide that comprises a T cell epitope.
[019] In some embodiments, the polypeptides of the invention are used to elicit antibodies that specifically bind to a D. fragilis cnolase polypeptide. Thus, also provided are methods for eliciting antibodies that specifically bind to a D. fragilis enolase polypeptide, comprising administering a polypeptide of the invention to a subject, thereby eliciting antibodies that specifically bind to a D. fragilis enolase polypeptide.
[020] In farther embodiments, the polypeptides of the invention are used to elicit antibodies that specifically bind to a D. fragilis parasite. Accordingly, also provided are methods for eliciting antibodies that specifically bind to a D. fragilis parasite, comprising administering a polypeptide of the invention to a subject, thereby eliciting antibodies that specifically bind to a D. fragilis parasite.
(021] The present invention also provides nucleic acid molecules encoding any one or more of the polypeptides described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[022] The present invention is further described herein, by way of non-limiting example only, with reference to the accompanying drawings.
[023] Figure 1 is a three dimensional modeling (in space fill formal) of the D. fragilis enolase polypeptide showing the predicted protein structure. The locations of the antigenic determinants arc shown in dark grey: Panel A: antigenic determinant A; Panel B: antigenic determinant B; Panel C: antigenic determinant C; Panel D: antigenic determinant D; Panel E: antigenic determinant E; Panel F: antigenic determinant F (indicated with an arrow): Panel G: antigenic determinant G (indicated with an arrow): Panel H: antigenic determinants A-G; Panel 1: antigenic determinants A-G with amino acids that compose the intracellular loop of the protein and the transmembrane helices obscured, i.e. showing only the extracellular portion of the protein.
[024] Figure 2 represents an indirect immunofluorescence assay of D. fragilis trophozoites stained with polyclonal antibody preparation A. Row A: D. fragilis subjected to mild fixation and stained with polyclonal antibody preparation A at a dilution of 1:10. Row B: Negative control using only the secondary antibody at. a 1:80 dilution. Row C: Close-up of D. fragilis stained with polyclonal antibody preparation A at a dilution of 1 : 10. Row D: D. fragilis fixed in SAF and stained with polyclonal antibody preparation A at a dilution of 1:10. Column 1: Bright field micrographs. Column 2: Corresponding fluorescent micrographs of the same cells shown in column 1. Column 3: Overlay of fluorescent micrographs on the bright field micrographs.
[025] Figure 3 represents an indirect immunofluorescence assay of D. fragilis trophozoites using polyclonal antibody preparation B. Rows A and B: D. fragilis subjected to mild fixation and stained with polyclonal antibody preparation B at a dilution of 1:10. Row C: Negative control using only the secondary antibody at a 1:80 dilution. Row D: D. fragilis fixed in SAF and stained with polyclonal antibody preparation B at a dilution of 1:10. Column 1: Bright field micrographs. Column 2: Corresponding fluorescent micrographs of the same cells shown in column I. Column 3: Overlay of fluorescent micrographs on the bright field micrographs.
[026] Figure 4 represents an indirect immunofluorescence assay of Entamoeba moshkovskii cells using polyclonal antibody preparation A. Row A: E. moshkovskii cells stained with polyclonal antibody preparation A at a dilution of 1 :10. Row B: Negative control using only the secondary antibody at a 1 :80 dilution. Column 1: Bright field micrographs. Column 2: Corresponding fluorescent micrographs of the same cells shown in column I. Column 3: Overlay of fluorescent micrographs on the bright Held micrographs.
[027] Figure 5 represents an indirect immunofluorescence assay of Entamoeba moshkovskii cells using polyclonal antibody preparation B. Row A: £. moshkovskii cells stained with polyclonal antibody preparation A as a positive control. Row B: Negative control using only the secondary antibody at a 1:80 dilution. Row C: E. moshkovskii cells stained with polyclonal antibody preparation B at a dilution of 1:10. Column 1: Bright Held micrographs. Column 2: Corresponding fluorescent micrographs of the same cells shown in column 1. Column 3: Overlay of fluorescent micrographs on the bright field micrographs.
[028] Figure 6 represents an indirect immunofluorescence assay of Entamoeba histolytica cells using polyclonal antibody preparation A. Row A: D. fragilis trophozoites stained with polyclonal antibody preparation A at a dilution of 1:10 as a positive control. Row B: Negative control of D. fragilis trophozoites using only the secondary antibody at a 1 :80 dilution. Row C: E. histolytica cells stained with polyclonal antibody preparation A at a dilution of 1:10. Row D: Negative control of £. histolytica cells using only the secondary antibody at a 1:80 dilution. Column 1: Bright field micrographs. Column 2: Corresponding fluorescent micrographs of the same cells shown in column 1. Column 3: Overlay of fluorescent micrographs on the bright field micrographs.
[029] Figure 7 represents an indirect immunofluorescence assay of E. histolytica cells using polyclonal antibody preparation B. Row A: £>. fragilis trophozoites stained widi polyclonal antibody preparation B at a dilution of 1:10 as a positive control. Row B: Negative control of D. fragilis trophozoites using only the secondary antibody at a 1 :80 dilution. Rows C and D: E. histolytica cells stained with polyclonal antibody preparation B at a dilution of 1:10. Column 1: Bright field micrographs. Column 2: Corresponding fluorescent micrographs of the same cells shown in column 1. Column 3: Overlay of fluorescent micrographs on the bright field micrographs.
[030] Figure 8 represents an indirect immunofluorescence assay of Blastocysts sp. cells using polyclonal antibody preparation A. Row A: D. fragilis trophozoites stained with polyclonal antibody preparation A at a dilution of 1:10 as a positive control. Row B: Negative control of Blastocysts cells using only the secondary antibody at a 1:80 dilution. Row C: Blastttcystis cells stained with polyclonal antibody preparation A at a dilution of 1:10. Column I: Bright field micrographs. Column 2: Corresponding fluorescent micrographs of the same cells shown in column I. Column 3: Overlay of fluorescent micrographs on the bright field micrographs. [031] Figure 9 represents an indirect immunofluorescence assay of Blastocysts sp. cells using polyclonal antibody preparation B. Row A: D. fragilis trophozoites stained with polyclonal antibody preparation B at a dilution of 1:4 as a positive control. Row B: Negative control of Blastocysts cells using only the secondary antibody at a 1:80 dilution. Row C: Blasiocysiis cells stained with polyclonal antibody preparation B at a dilution of 1:10. Column 1: Bright field micrographs. Column 2: Corresponding fluorescent micrographs of the same cells shown in column I . Column 3: Overlay of fluorescent micrographs on the bright field micrographs.
[032] Figure 10 represents an indirect immunofluorescence assay of Giardia trophozoites using polyclonal antibody preparation A. Row A: D. fragilis trophozoites stained with polyclonal antibody preparation A at a dilution of 1:10 as a positive control. Row B: Negative control of D. fragilis trophozoites using only the secondary antibody at a 1:80 dilution. Row C: Giardia trophozoites stained with polyclonal antibody preparation A at a dilution of 1:10. Row D: Negative control of Giardia trophozoites using only the secondary antibody at a 1:80 dilution. Column 1: Bright field micrographs. Column 2: Corresponding fluorescent micrographs of the same cells shown in column 1. Column 3: Overlay of fluorescent micrographs on the bright field micrographs.
[033] Figure 11 represents an indirect immunofluorescence assay of Giardia trophozoites using polyclonal antibody preparation B. Row A: D. fragilis trophozoites stained with polyclonal antibody preparation B at a dilution of 1 :10 as a positive control. Row B: Negative control of Z . fragilis trophozoites using only the secondary antibody at a 1:80 dilution. Row C: Giardia trophozoites stained with polyclonal antibody preparation B at a dilution of 1:10. Column 1: Bright field micrographs. Column 2: Corresponding fluorescent micrographs of the same cells shown in column I. Column 3: Overlay of fluorescent micrographs on the bright field micrographs. BRIEF DESCRIPTION OF THE SEQUENCES
Table 1:
SEQ ID NO: Description
1 D.fr gilis enolase polypeptide
2 D.fragilis enolase polypeptide - extracellular portion
3 Antigenic determinant A
LQIYEYLKEHDMLEE - amino acids 453-467 of SEQ ID NO:l
4 Antigenic determinant B
AISYIEQA - amino acids 264-272 of SEQ ID NO: 1
5 Antigenic determinant C / Peptide C
EQNMPKKYKLPQC - amino acids 177- 189 of SEQ ID NO: 1
6 Antigenic determinant D
SPAKGTPLPEQ - amino acids 212-222 of SEQ ID NO: 1
7 Antigenic determinant E / Peptide E
WADLIAKHPC- amino acids 319-328 of SEQ ID NO:l
8 Antigenic determinant F
FYDAET LY- amino acids 294-302 of SEQ ID NO: I
9 Antigenic determinant G / Peptide G
VMVSHRSGETC- amino acids 409-420 of SEQ ID NO:1
10 Peptide A - CLQIYEYL EHDMLEE
I I Peptide B - CAISYIEQA
12 Peptide D - CSPA GTPLPEQ
13 Peptide F - CFYDAET LY
14 Linker - GPGPG
15 Linker - GGGG
16 Linke - GSGGGG
17 Linker - GSGGGGS
19 Linker - KKAA
20 D.fragilis enolase transcriptome contig 41 1 1
21 D. fragilis enolase transcriptome contig 9 and enolase cDNA DETAILED DESCRIPTION
[034] The articles "a" and "an" are used herein to refer to one or to more than one (/.<?., to at least one) of the grammatical object of the article. By way of example, "an antimicrobial agent" means one antimicrobial agent or more than one antimicrobial agent.
[035] In the context of this specification, the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.
[036] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
[037) As used herein the term "antigenic determinant'* refers to a region of an antigen or protein {e.g. an enolase protein) that is specifically recognized by antibodies and B cells. i.e. the portion to which the antibody paratope binds. "Antigenic determinant" and "epitope" are used interchangeably herein.
[038] As used herein, "isolated" with reference to a nucleic acid molecule, peptide, or polypeptide means that the nucleic acid molecule, peptide, or polypeptide is substantially free of cellular material or other contaminating proteins from the cells from which the nucleic acid molecule, peptide, or polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily delectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC). used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not delectably alter the physical and chemical properties of the peptide. Accordingly, an isolated peptide preparation produced by recombinant methods generally has less than about 30%, more typically less than about 20%. 10% or 5% (w/w), of cellular material, and an isolated peptide preparation produced by chemical synthesis generally has less than about 30%, more typically less than about 20%. 10% or 5% (w/w), of chemical precursors or other chemicals. [039] As used herein, corresponding amino acid residues (or positions) refer to residues (or positions) that occur at aligned loci within the primary amino acid sequence of a protein.. Related or variant polypeptides are aligned by any method known to those of skill in the art. Such methods typically maximize matches, and include methods such as using manual alignments and by using the numerous alignment programs available (for example. BLASTP) and others known to those of skill in the art. By aligning the sequences of polypeptides, one skilled in the art can identify corresponding residues, using conserved and identical amino acid residues as guides. For example, by aligning the sequences of the cnolase polypeptide set forth in SEQ ID NO: 1 with another enolase polypeptide, one of skill in the art can identify corresponding residues using conserved and identical amino acid residues as guides.
[040] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically, such as recombinantiy, produced, including any fragment thereof containing at least a portion of the variable region of the immunoglobulin molecule that retains the binding specificity ability of the full-length immunoglobulin. Hence, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen- binding domain (antibody combining site). Antibodies include antibody fragments. As used herein, the term antibody includes synthetic antibodies, recombinantiy produced antibodies, multispccifk antibodies (e.g., bispecific antibodies), human antibodies, non- human antibodies, humanized antibodies, chimeric antibodies, intrabodies. and antibody fragments, such as, but not limited to. Fab fragments, Fabl fragments. F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv). Fd fragments. Fd' fragments, single-chain Fvs (scFv), single-chain Fabs (scFab), diabodics, anti-idiot ypic (anti-Id) antibodies, or antigen- binding fragments of any of the above. Antibodies provided herein include members of any immunoglobulin type (e.g.. IgG, IgM. IgD, IgE, IgA and IgY), any class (e.g. IgGl. IgG2, lgG3, lgG4, IgAl and IgA2) or subclass (e.g., IgG2a and lgG2b).
[041] As used herein, "specifically bind" with respect to an antibody or antigen-binding fragment thereof refers to the ability of the antibody or antigen-binding fragment to form one or more noncovalent bonds with a cognate antigen, by noncovalent interactions between the antibody combining site(s) of (lie antibody and the antigen. The antigen can be an isolated antigen such as an isolated protein (e.g. an enolase polypeptide) or presented on thc surface of a cell, such as D. fragiUs trophozoite. Typically, an antibody lhat specifically binds to a polypeptide or cell herein is one that binds with an affinity constant t. a) of about or at least 107- 108 M- (or a dissociation constant (K<j) of or about 10"7 M (100 nM) or 10"8 M (10 n ) or less). Affinity constants can be determined by standard kinetic methodology for antibody reactions, for example, immunoassays (e.g. ELISA). or surface plasmon resonance (SP ). Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (e.g.. Biacore 2000. Biacore AB. Upsala, Sweden and GE Healthcare Life Sciences).
[042] As used herein, an "antigenic variant" refers to a variant of a reference polypeptide tha has that same antigenicity as the reference polypeptide. For example, an antigenic variant of a polypeptide set forth in SEQ ID NO:3 is a variant that is recognized by the same antibody or antibodies that recognize the polypeptide set forth in SEQ ID NO:3. Accordingly, an antigenic variant of a polypeptide set forth in SEQ ID NO:3 can be used to elicit antibodies that bind to both the variant and to the polypeptide set forth in SEQ ID NO:3. The antigenic variant may contain amino acid substitutions, deletions and/or additions compared to the sequence of the reference polypeptide. Accordingly, antigenic variants include fragments of the reference polypeptide lhat are recognized by the same antibody or antibodies lhat recognize the reference sequence or polypeptide.
Antigenic polypeptides
[043] Provided arc polypeptides comprising antigenic determinants of D. fragilis. These antigenic determinants are within the enolasc protein of D. fragilis. Enolases are present in a wide variety of pathogenic and non-pathogenic organisms, including Archac, bacteria and eukaryotic organisms. Enolases are responsible for the reversible conversion of D-2- phosphogiyceraie (2POA) and phosphoenolpyruvalc (PEP) in glycolysis and gluconeogencsis. In some organisms, enolases are involved in pathogenicity. For example, the enolase of pathogenic Streptococcus pyogenes and the insect parasite Aphidius ervi is excreted to the extracellular environment where it mediates degradation of host tissues and immune evasion. The surface-associated enolase of Trichomonas vaginalis can bind human plasminogen and release plasmin, which may facilitate penetration of 7*. vaginalis to the cell basement membrane to enable association with fibroncctin and laminin. which in turn could ensure the parasite access to growth factors and nutrients.
[044] As demonstrated herein, the D. fragilis enolasc protein is a surface-associated protein. An exemplary D. fragilis enolase protein comprises the sequence of amino acids set forth in SEQ ID NO:l . Sequence analysis and three dimensional modeling of this enolase polypeptide indicate that amino acids 1 to 141 of SEQ ID NO: 1 reside within the cell; amino acids 142 to 176 comprise the transmembrane helices; and amino acids 177 to 467 are extracellular. It is predicted that the polypeptide contains a further 10-12 amino acids at the N-terminus of the protein, which is intracellular.
[045] Accordingly, provided are polypeptides comprising a sequence of amino acids set forth in SEQ ID NO: 1, or a polypeptide having at least 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%. 95%. 96%, 97%. 98% or 99% sequence identity to the enolase polypeptide set forth in SEQ ID NO:l . Also provided arc polypeptides comprising a sequence of amino acids set forth in SEQ ID NO:2, which corresponds to the extracellular portion (amino acids 177 to 467) of SEQ ID NO: l. or a polypeptide having at least 85%, 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%. 94%, 95%. 96%, 97%, 98% or 99% sequence identity to the polypeptide set forth in SEQ ID NO:2. In some aspects, the polypeptides described herein retain enolase activity, i.e. can catalyze the dehydration of 2- phospho-d-glycerate (PGA) to phosphoenolpyruvate (PEP) in glycolysis, and/or the hydration of PEP to PGA during gluconcogcncsis.
[046] As demonstrated herein, the extracellular portion of the D. fragilis enolase protein contains multiple antigenic determinants (or epitopes) to which antibodies can specifically bind. Exemplary antigenic determinants comprise, or are located at or within, amino acids 264-272 of SEQ ID NO:l (antigenic determinant Bj, or amino acids 453-467 of SEQ ID NO: 1 (antigenic determinant A), or corresponding amino acids in another D. fragilis enolase protein. Other antigenic determinants include those comprising, or located at or within, amino acids 177-189 (antigenic determinant C), 212-222 (antigenic determinant D), 294-302 (antigenic determinant F), 319-328 (antigenic determinant E) or 409-419 (antigenic determinant G) of SEQ ID NO:l or corresponding amino acids in another D. fragilis enolase protein. Three dimensional modeling of the enolase polypeptide set forth in SEQ ID NO:l indicates the position of these antigenic determinants within the three dimcnsional structure (Figure I). Accordingly, provided herein arc polypeptides that consist of. or comprise, one or more of antigenic determinants A, B, C. D, E, G and/or G.
(047] As described in the Examples, polypeptides of the present invention that comprise the antigenic determinants described herein can be used to elicit antibodies that can bind to D.frngttis. For example, polypeptides comprising antigenic determinant A comprising, or located at or within, amino acids 453-467 of 5EQ ID NO: I can be used to elicit antibodies that bind to D. fr gilis. This demonstrates that the region of the enolase polypeptide containing antigenic determinant A is accessible to antibodies when the enolase polypeptide is associated with the surface of the D. fragilis trophozoite. In some examples, the polypeptides comprising antigenic determinant A comprising, or located at or within, amino acids 453-467 of SEQ ID NO:l also contain other surface-exposed residues within the enolase polypeptide, including bu not limited to. any one or more of residues at positions 430, 433, 434, 440, 441 , 442, 443, 446, 447, 450 or 4 1 of SEQ ID NO: 1.
[048] Exemplary polypeptides of the present invention can thus consist of the sequence of amino acids LQIYEYLKEHDMLEE (SEQ ID NO:3; corresponding to amino acids 453-467 of SEQ ID NO:l), or consist of the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:3. The antigenic variant can have at least 60%, 70%, 75%, 80%. 85%, 90% or 95%· sequence identity with the sequence of amino acids set forth in SEQ ID NO:3. In some examples, the polypeptides consist of a sequence of amino acids that has 1, 2. 3, 4 or 5 amino acid substitutions, additions and or deletions compared to the sequence of amino acids set forth in SEQ ID NO:3. In one example, the polypeptide consists of the amino acid sequence CLQIY EYLKEH DMLEE (SEQ ID NO: 10). The polypeptides of the present invention also include those polypeptides that comprise the sequence of amino acids set forth in SEQ ID NO: 3 or SEQ ID NO: 10, or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:3, such as an antigenic variant having at least 60%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity with the sequence of amino acids set forth in SEQ ID NO:3. In some aspects, the polypeptides are less than 400. 300, 200. 100, 50. or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID NO:3 or SEQ ID NO: 1.0 or a sequence having at least 60%, 70%, 75%, 80%. 85% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:3 or SEQ ID NO: 10. [049] Polypeptides comprising antigenic determinant B comprising, or located at or within, amino acids 264-272 of SEQ ID NO:l can also be used to elicit antibodies that bind to D. fragilis. This demonstrates that the region of the enolase polypeptide containing antigenic determinant B is accessible to antibodies when the enolase polypeptide is associated with the surface of the D. fragilis trophozoite. In some examples, the polypeptides comprising antigenic determinant B comprising, or located at or within, amino acids 264-272 of SEQ ID NO: I also contain other surface-exposed residues within the enolase polypeptide, including but not limited to, any one or more of residues at positions 230; 231, 232, 235, 238, 239, 240. 241, 242, 243. 244, 246. 247. 248, 259, 260, 273, 275, 276 or 277 of SEQ ID NO:l. f050] In particular aspects, the polypeptides of the present invention consist of the sequence of amino acids A1SYIEQAM (SEQ ID NO:4; corresponding to amino acids 264- 272 of SEQ ID NO: I), or the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:4. In some instances, the antigenic variant has at least 75%, 80% or 85% sequence identity with the sequence of amino acids set forth in SEQ ID NO:4. In some examples, the polypeptides consist of a sequence of amino acids that has 1. 2 or 3 amino acid substitutions, additions and/or deletions compared to the sequence of amino acids set forth in SEQ ID NO:4. For example, the polypeptide can consist of the amino acid sequence CAISYIEQAM (SEQ ID NO:l 1). The polypeptides of the present invention also include those polypeptides that comprise the sequence of amino acids set forth in SEQ ID NO:4 or SEQ ID NO: 1.1 , or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:4, such as an antigenic variant having at least 75%, 80% or 85% sequence identity to SEQ ID NO:4. In some aspects, the polypeptides are less than 400, 300, 200, 100, 50. or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID NO:4 or SEQ ID NO:l 1, or a sequence having at least 75%, 80%. 85% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:4 or SEQ ID NO: 11.
[051] Polypeptides of the present invention also include those that consist of the sequence of amino acids EQNMP Y LPQC (SEQ ID NO:5; corresponding to amino acids 177- 189 of SEQ ID NO:l), or consist of the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:5 having at least 75%, 84% or 90 sequence identity with the sequence of amino acids set forth in SEQ ID NO:5. In some examples, the polypeptides consist of a sequence of amino acids that has 1. 2 or 3 amino acid substitutions, additions and/or deletions compared to the sequence of amino acids set forth in SEQ ID NO:S. The polypeptides of the present invention also include (hose polypeptides that comprise the sequence of amino acids set forth in SEQ ID NO:5 or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:5 having at least 75%. 84% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:5. Typically, the polypeptides are less than 400, 300, 200, 100, 50, or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID NO:5, or comprise a sequence having at least 75%, 84% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO: 5.
Γ052] Other polypeptides of the present invention consist of the sequence of amino acids SPA GTPLPEQ (SEQ ID NO:6; corresponding to amino acids 212-222 of SEQ JD NO:l), or consist of the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:6 having at least 70%, 80%. or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:6. In some examples, the polypeptides consist of a sequence of amino acids that has 1, 2 or 3 amino acid substitutions, additions and/or deletions compared lo the sequence of amino acids set forth in SEQ ID NO:6. In one example, the polypeptide consists of the amino acid sequence CSPAKGTPLPEQ (SEQ ID NO: 12). The polypeptides of the present invention also include those polypeptides that comprise the sequence of amino acids set forth in SEQ ID NO:6 or SEQ ID NO: 12, or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:6 having at least 70%. 80% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:6. In some aspects, the polypeptides are less than 400, 300, 200, 100, 50, or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID NO:6 or SEQ ID NO: 12, or comprise a sequence having at least 70%, 80% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:4 or SEQ ID NO: 10.
[0531 Polypeptides of the present invention also can consist of the sequence of amino acids FYDAETKLY (SEQ ID NO:8; corresponding lo amino acids 294-302 of SEQ ID NO:1), or consist of the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:8 having at least 66%, 77%. or 88% sequence identity with the sequence of amino acids set forth in SEQ ID NO:8. In some examples, the polypeptides consist of a scqucncc οΓ amino acids that has 1, 2 or 3 amino acid substitutions, additions and/or deletions compared to the sequence of amino acids set forth in SEQ ID NO:8. In one example, the polypeptide consists of the amino acid sequence CFYDAETKLY (SEQ ID NO: 13). The polypeptides of the present invention also include those polypeptides that comprise the sequence of amino acids set forth in SEQ ID NO:8 or SEQ ID NO: 13, or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO: 7 having at least 66%. 77%. or 88% sequence identity with the sequence of amino acids set forth in SEQ ID NO:8. In some aspects, the polypeptides are less than 400, 300, 200, 100, SO. or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID N :8 or SEQ ID NO: 13, or comprise a sequence having at least 66%, 77%. or 88% sequence identity with the sequence of amino acids set forth in SEQ ID NO:8 or SEQ ID NO: 13.
[054] Additional polypeptides of the present invention consist of the sequence of amino acids WADL1AKHPC (SEQ ID NO:7; corresponding to amino acids 177-189 of SEQ ID NO: 1), or a consist of the sequence of an antigenic variant of SEQ ID N :7 having at least 70%, 80% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:7. In some examples, the polypeptides consist of a sequence of amino acids that has I. 2 or 3 amino acid substitutions, additions and/or deletions compared to the sequence of amino acids set forth in SEQ ID NO: 7. The polypeptides of the present invention also include polypeptides that are less than 400, 300, 200, 100. 50, or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID NO:7 or a sequence having at least 70%, 80% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:7.
[055] Other polypeptides of the present invention consist of the sequence of amino acids VMVSHRSGETC (SEQ ID NO:9; corresponding to amino acids 409-419 of SEQ ID NO: 1 ), or the sequence of an antigenic variant of the polypeptide set froth in SEQ ID NO:9 having at least 70%. 80 or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:9. In some examples, the polypeptides consist of a sequence of amino acids that has 1, 2 or 3 amino acid substitutions, additions and/or deletions compared to the sequence of amino acids set forth in SEQ ID NO:9. The polypeptides of the present invention also include polypeptides that are less than 400, 300, 200, 100, 50. or 20 amino acids in length and comprise the sequence of amino acids set forth in SEQ ID NO:9 or a scqucncc having at least 70%. 80% or 90% sequence identity with the sequence of amino acids set forth in SEQ ID NO:9.
[056] As demonstrated in the Examples below, polypeptides of the present invention containing the described antigenic determinants can be used as immunogens and administered to a subject to elicit antibodies specific for the antigenic determinant. The resulting antibodies can specifically bind to the enolasc polypeptide of D. Jr'agilis. Generally, the polypeptides provided herein have a length of 7 amino acids to 400 amino acids, more typically 7 to 300, 7 to 200 or 7 to 100 amino acids, such as 9 amino acids to 100 amino acids, 9 amino acids to 75 amino acids, 9 amino acids to 50 amino acids, or 9 amino acids to 25 amino acids. For example, provided arc peptides that are 7, 8. 9, 10, 1 1. 12, 13, 14, 15, 16, 17, 18, 19, 20. 21, 22, 23, 24, 25, 26, 27. 28, 29, 30, 31, 32. 33, 34, 35, 36, 37. 38. 39, 40, 45, 50, 55, 60. 65. 70, 75, 80, 85, 90, 100, 200 or 300 amino acids in length.
[057] In some examples, the polypeptides of the present invention comprise two or more of the described antigenic determinants A, B, C. D, E, F or G. Such polypeptides can be used, for example, to elicit antibodies specific for the two or more determinants present in the polypeptide. For example, included among the polypeptides of the present invention are those that comprise antigenic determinant B located at or within amino acids 264-272 and antigenic determinant A located at or within amino acids 453-467 of SEQ ID NO:1, or comprise the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:3 and the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:4.
[058] The polypeptides provided herein that contain at least one antigenic determinant can be linked to one or more other moieties, including, but not limited to, one or more other antigenic determinants, one or more T cell epitopes, one or more other immunostimulatory molecules, one or more targeting agents, one or more polymers, one or more proteins, one or more multimerisation domains, one or mote detectable labels, one or more affinity tags or any combination thereof. The polypeptides can be linked to (he one or more other moieties by any method known in the art, including any chemical or recombinant method resulting in the formation of covalent and/or non-covalent bonds between the polypeptide and die one or more other moieties. In some instances, the resulting molecules can be used as immunogens that can be administered to a subject to elicit antibodies that bind to the enolasc polypeptide of D. fragilis. In other instances, the resulting molecules containing the polypeptides can be used in in vitro methods, such s screening methods, detection methods, diagnostic methods and purification methods.
1059} To assist in eliciting a humoral immune response to the antigenic dcterminant(s) when the polypeptides arc administrated to a subject, the polypeptides can be linked to one or more T helper cell epitopes or a polypeptide comprising one or more T helper cell epitopes. Any T helper cell epitope can be linked to the peptides provided the T helper epitope is recognized by T helper cells in the subject to which the peptide will be administered. Promiscuous or universal T helper cell epitopes that are recognized in the context of different MHC backgrounds {i.e. in a genetically diverse population) arc well known in the art and can be linked to the peptides provided herein (see e.g. Dicthelm-Okita et aL (2000) J. Inf. Dis. 181:1001-1009; Greenslein et al. (1992) J Immunol 148(12):3970- 3977). Known T helper cell epitopes can be identified using publicly accessible databases such as the immune Epitope Database and Analysis Resource (immuneepitope.org) and new T helper cell epitopes can be identified using methods well known in the art (sec e.g. Pira et al.. (2010) J Biomed Biotechnol). It is well within the skill of a skilled artisan to identify and select an appropriate T helper cell epitope for the desired purpose.
[060] T helper ceil epitopes that can be linked to the polypeptides provided herein include, but are not limited to, those derived from microorganism proteins, such as viral proteins and bacterial proteins, as well as artificial or synthetic T helper cell epitopes (see e.g. U.S. Patent No. 6,713.301). In some examples, the T helper cell epitopes are from potent immunogens such as tetanus toxin, diptheria toxin, polio virus, pertussis toxin, the measles virus F protein, HIV gpl20 and HIV Gag proteins, and the hepatitis B virus surface antigen (HbsAg). In some instances, the T helper cell epitopes arc provided within the context of a larger protein. Thus, the polypeptides of the present invention can be linked to a protein or polypeptide comprising a T helper cell epitope. Exemplary proteins are carrier proteins, such as keyhole limpet haemocyanin or tetanus toxoid.
[061 J The polypeptides of the present invention may be linked to an affinity tag to, for example, facilitate purification. Exemplary affinity tags include, but are not limited to, chitin binding protein (CBP), maltose binding protein (MBP), glutathionc-S-transfcrase (GST), FLAG, His, c-myc and HA lags. Detectable molecules, including, but not limited to. fluorescent or chemi luminescent molecules, or biotin or strcptavidin. also can be Linked to the polypeptides.
[062] The one or more other moieties linked to the provided polypeptides can be linked by any method known in the art, including chemical methods and recombinant methods. For example, polymers can he coupled to the polypeptides using standard chemical reactions. Proteins (e.g. carrier proteins such as ovalbumin, keyhole limpet hacmocyanin and bovine scrum albumin) can be conjugated to the polypeptides using standard chemical coupling techniques such as MBS, glutaraldchyde, EDC, or BDB coupling. In other examples, the polypeptides are linked to other peptides (such as those including a T cell epitope) or proteins by peptide synthesis methods or recombinant methods. For example, a polypeptide can be linked to a T cell epitope by sequentially synthesizing the polypeptide then the T cell epitope as a single polypeptide using standard methods (e.g. Fmoc solid phase synthesis). In other examples, DNA encoding the polypeptides can be operatively linked to DNA encoding the T cell epitope (or any other protein) and the entire DNA molecule expressed, such as using a bacterial expression system, to produce a single polypeptide containing the peptide and the T cell epitope. Accordingly, linkage can be by covalent and/or non-covalent bonds, depending on the method of linkage employed.
[063] In some examples, a peptide linker or spacer is used to link the polypeptides and the one or more other moieties. Peptide linkers typically are from 1 amino acid in length to 10 amino acids in length, although can be longer. Non-limiting examples of peptide linkers that can be used herein include linkers having the sequence , K. K . GPGPG t.SEQ ID NO: 14), G, GG, GGG. GGGG (SEQ ID NO: 15), GGA, GA. GD, GSGGGG (SEQ ID NO: 16), GSGGGGS (SEQ ID NO: 17), GS. RS. RR, KKK. KKAA (SEQ ID NO:18), VE, and A AY.
[064] The polypeptides provided herein can be produced using any method known in the art, including peptide synthesis techniques and recombinant techniques. In one example, the polypeptides arc synthesized using the Fmoc-polyamide mode of solid-phase peptide synthesis. Other synthesis methods include solid phase t-Boc synthesis and liquid phase synthesis. Other peptide moieties, such as T cell epitopes, can be sequentially synthesized and linked to the polypeptide. Purification of the synthesized polypeptides can be performed by any one, or a combination of, techniques such as re-crystallization, size exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography and reverse-phase high performance liquid chromatography using, for example, acelonitril/water gradient separation.
Γ065] In other examples, the polypeptides are produced using recombinant methods well known in the art. Nucleic acid encoding the polypeptides can be obtained by any suitable method, including, but not limited to, RT-PCR of Z). fragilis mRNA or synthesis of an oligonucleotide that encodes a polypeptide of the present invention. Accordingly, also provided are nucleic acid molecules encoding a polypeptide of the present invention. It is well within the skill of a skilled artisan to design a nucleic acid molecule thai encodes a polypeptide described herein, such as a polypeptide comprising one or more antigenic determinants located at or within amino acids 177-189. 212-222, 264-272. 294-302, 319- 328, 409-419 or 453-467 of SEQ ID NO:l.
[066] Nucleic acid encoding the polypeptide can be cloned into an expression vector suitable for the expression system of choice, operably linked to regulatory sequences that facilitate expression of the heterologous nucleic acid molecule. Many expression vectors are available and known to those of skill in the art for the expression of peptides. The choice of expression vector is influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that arc used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells.
[067] In some examples, the polypeptides are expressed as fusions with another peptide or polypeptide. For example, the polypeptides can be produced as fusions by operably linking the nucleic acid encoding the peptide with nucleic acid encoding one or more T cell epitopes, ligands. affinity tags (e.g. a his6 tag or a myc tag, a GST fusion) or other proteins. Other examples of fusions include, but are not limited to, fusions of a signal sequence and a sequence for directing protein secretion.
[068] Following expression, the provided polypeptides (including any fusions of the polypeptide with one or more other moieties) can be purified using any method known to those of skill in the art including, but not limited to, SDS-PAGE, size fraction and size cxclusion chromatography, ammonium sulfate precipitation, chelate chromatography, ionic exchange chromatography and affinity chromatography. Affinity purification techniques can be used to improve the purity of the preparations. As discussed above, expression constructs can be engineered to add an affinity tag to the polypeptide, such as a myc epitope, GST fusion or His6. which facilitates affinity purification using an anti-myc antibody, glutathione resin, or Ni-resin, respectively. Purity can be assessed by any method known in the art including gel electrophoresis and staining and spectrophotometry techniques. In some examples, analysis of the polypeptides is carried out using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis, fast atom bombardment (FAB) mass spectromelric analysis. ALDI or ESI-Q-TOF mass spectromelric analysis.
[069] The antigenic properties of the polypeptides can be assessed using a variety of methods known to those skilled in the art. For example, the ability of the polypeptides to induce an antibody response can be assessed by linking the polypeptides to one or more T helper cell epitopes or a protein carrier containing one or more T helper cell epitopes (e.g. keyhole limpet haemocyanin) and administering (such as by intravenous, intraperitoneal or intramuscular injection) the resulting immunogen to a subject one or more times. The antibody response elicited can be assessed at various time points after immunization by sampling the blood of the subject and subjecting the sera to analysis using an appropriate assay, such as an ELISA. For example, the ELISA plate can be coated with the polypeptide or the D. fragilLs enolase set forth in SEQ ID NOs: 1 or 2. Such methods can be used to determine the magnitude and specificity of an antibody response elicited by administration of the provided peptides when linked to one or more T helper cel.! epitopes. The ability of the polypeptides to be recognized by antibodies, including polyclonal or monoclonal antibodies directed to D. fragilis* can be assessed by standard methods, including, but not limited to, ELISA, Western blot, dot blot, surface plasmon resonance and rapid flow tests (e.g. lateral or vertical flow test).
[070] The polypeptides can be present in compositions, such as pharmaceutical compositions. The pharmaceutical compositions can be formulated in any conventional manner by mixing a selected amount of polypeptide (including polypeptides linked to one or more other moieties), with one or more physiologically acceptable carriers or excipients. Selection of Ihc carrier or cxcipicnl is within the skill of die administering profession and can depend upon a number of parameters, such as the mode of administration (i.e., systemic, nasal, oral or any other mode). ΙΛ some examples the pharmaceutical compositions are provided as a fluid. In other instances, the pharmaceutical compositions are provided in diicd form, such as desiccated or freeze-dried form. Such dried compositions can be rehydratcd prior to administration to a subject by the addition of a suitable solution, such as water, buffer, saline or other suitable solution. The pharmaceutical compositions provided herein can be formulated for direct administration or can be formulated for dilution or other modification. Accordingly, the pharmaceutical compositions can be formulated in single (or unit) dosage forms or multiple dosage forms. Examples of single dose forms include ampoules and syringes. Examples of multiple dose forms include vials and bottles that contain multiple unit doses.
[071] The compositions containing the provided polypeptides can also include pharmaceutically active ingredients including, but not limited to, other antigens, adjuvants or immunomodulators, such as chemokincs. interlcukins and cytokines. Exemplary adjuvants include, but are not limited to, aluminium salts (e.g. aluminium hydroxide, aluminium phosphate and potassium aluminium sulphate; also referred to as Alum), iscoms. and saponin-based adjuvants (e.g. adjuvants containing Quil A or Quil A derivatives or fractions).
Antibodies
[072] Provided herein are isolated antibodies, such as isolated polyclonal and monoclonal antibodies (including antigen-binding fragments thereof, such as single-chain Fv (scFv). Fab. Fab', F(ab')2, Fv, dsFv, diabody, Fd, and Fd' fragments) that specifically bind to the antigenic determinants and polypeptides containing the antigenic determinants described herein. Thus, in one aspect, provided arc isolated antibodies that recognize and bind to antigenic determinants comprising, or located at or within, amino acids 177-189, 212-222. 264-272, 294-302, 319-328, 409-419 or 453-467 of the D.fra lis enolase protein set forth in SEQ ID NO:l or corresponding amino acids in a variant enolase polypeptide having at least 85%, 86%, 87%, 88%, 89%. 90%, 91 %, 92%. 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence set forth in SEQ ID NO:l. Accordingly, the antibodies of the present invention can bind to polypeptides comprising a sequence of amino acids set forth in any one of SEQ ID N0s:3-13, or a sequence of amino acids having at leasl 70%, 75%, 80%, 85%, 90% or 95% sequence identity to a sequence of amino acids set forth in any one of SEQ ID NOs:3-13. The antibodies and anligenic-binding fragments thereof may be of any isotype (e.g. IgG (including FgGl, IgG2a, IgG2b, IgG3 and FgG4), IgM. IgA, IgD and IgE) and can be polyclonal or monoclonal, non-human (e.g. mouse, rat. rabbit, guinea pig) or human, or chimeric or humanized.
[073] Exemplary of the antibodies described herein are those that recognize and bind to an antigenic determinant comprising, or located at or within, amino acids 264-272 of SEQ ID NO:1 (antigenic determinant B) or corresponding amino acids in a polypeptide having at least 85%. 86%, 87%, 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98% or 99% sequence identity with the sequence set forth in SEQ ID NO:l. Thus, the antibodies can bind to polypeptides consisting of or comprising the sequence of amino acids AISYIEQA (SEQ ID NO:4) or CAISYIEQAM (SEQ ID NO: 1 1), or polypeptides consisting of or comprising a sequence of amino acids having at least 75%, 80% or 85% sequence identity with the sequence of amino acids set forth in SEQ ID NO:4 or SEQ ID NO: 10.
f074] Exemplary antibodies also include those that recognize and bind to the antigenic determinant comprising,, or located at or within, amino acids 453-467 of SEQ ID NO:l (antigenic determinant B) or corresponding amino acids in variant having at least 85%, 86%, 87%. 88%. 89%, 90%, 91%, 92%. 93%. 94%. 95%. 96%, 97%, 98% or 99% sequence identity with the sequence set forth in SEQ ID NO:l. Such antibodies can bind to polypeptides consisting of or comprising the sequence of amino acids LQIYEYLKEHDMLEE (SEQ ID NO:3) or CLQJYEYLKEHD LEE iSEQ ID NO: 10), or polypeptides consisting of or comprising a sequence of amino acids having at leasl 75%, 80%, 85%, 90% or 95% sequence identity with the sequence of amino acids set forth in SEQ ID NO:3 or SEQ ID NO:5.
(075] The antibodies of the present invention specifically bind (o the polypeptides provided herein and thus may specifically bind to the D. fragilis enolase protein. In particular embodiments, the antibodies and antigen binding fragments specifically bind to the cnolasc polypeptide when associated with the surface of a D. fragilis trophozoite. Thus, antibodies of the present invention can specifically bind D. fragilis. [076] Techniques for preparing polyclonal and monoclonal antibodies against polypeptides are well known in the art. For example, polyclonal antibodies directed against an antigenic determinant described herein can be generated by administering to a subject (such as a non-human subject, e.g. a mouse, rat or rabbit) a polypeptide described herein that contains that antigenic determinant and a T helper cell epitope. The polyclonal antibodies produced following administration can then be isolated from the serum of the subject. In other examples, monoclonal antibodies specific for an antigenic determinant can be obtained by injecting a subject with a polypeptide that contains that antigenic determinant and a T helper cell epitope, then removing the spleen to obtain 6 lymphocytes. Alternatively. B lymphocytes can be isolated from peripheral blood lymphocytes <PBL). The B lymphocytes from immunized subjects are then fused with myeloma cells to produce hybridomas, which are cloned. Positive clones thai produce antibodies to the antigenic determinant are selected using standard techniques (e.g. ELISpot), culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. f077] Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques including, but not limited to, affinity chromatography with Protein-A SEPHAROSE®, size-exclusion chromatography, and ion- exchange chromatography. After the initial raising of antibodies to the antigenic determinant, the antibodies can be sequenced and subsequently prepared by recombinant techniques.
[ 78] Monoclonal antibodies can also be produced from an antibody library. For example, total RNA can be extracted from peripheral blood B lymphocytes of a subject, such as a healthy subject or a subject that has been or is infected with D. fmgilis, and a cD A library constructed by amplifying μ, γ and chain antibody repertoires. The cDNA library can then be used to make a display library, such as a phage display library in which antigen-binding fragments of antibodies, such as single chain Fv (scFv) fragments, are expressed on the surfaces of bacteriophages as fusion proteins with the bacteriophage coat protein. Antibodies or fragments thereof that recognize and bind to antigenic determinants comprising- or located at or within, amino acids 177-189. 212-222. 264-272. 294-302. 319- 328. 409-419 or 453-467 of the D. fmgilis enolase protein set forth in SEQ ID NO:l or corresponding amino acids in another D. fmgilis enolase protein can then be screened and sclectcd using the polypeptides provided herein (hat contain such antigenic determinants. Typically, selection is performed using panning techniques, a process well known to those of skill in the art.
Γ079] The antibodies and antigen-binding fragments of the present invention can be linked to one or more moieties, such as to facilitate detection. For example, the antibodies or antigen-binding fragments may be linked to a detectable label such a fluorescent, chcmilumincscent, enzyme, biotin/streplavidin or metabolic labels. Non-limiting examples of labels that can be linked to the antibodies and antigen-binding fragments include biotin, streptavidin, alkaline phosphatase (AP), horseradish peroxidase (HRP), fluorescein isotmocyamUc (FITC), rhodaminc (tctramcthyl rhodaminc isothiocyanatc, T 1TC), green fluorescent protein (GFP). allophycocyanin, phycocyanin, phycoerythrin and phycoerytlirocyanin. Antibodies and fragments thereof may be linked to one or more moieties using any method known in the art. For example, linkage may be through chemical conjugation using one of a variety of well known techniques, including but not limited to the use of NHS esters, hcterobifunctional reagents, carbodiirnides or sodium periodate.
[080] The binding properties of the antibodies of the present invention, such as the ability to bind to the polypeptides provided herein, can be characterized by established methodologies, for example, EL1SA and Western blot. Binding affinity also can be determined. Any method known to one of skill in the art can be used to measure the binding properties of an antibody. In some examples, the binding properties of an antibody are assessed by performing a saturation binding assay, for example, a saturation ELTSA, whereby binding of the antibody to the polypeptide is assessed with increasing amounts of antibody. In such experiments, it is possible to assess whether the binding is dose- dependent and or saturable. In addition, the binding affinity can be extrapolated from the 50% binding signal. Typically, apparent binding affinity is measured in terms of its association constant (Ka) or dissociation constant (K<i) and determined using Scatchard analysis. For example, binding affinity to a target polypeptide can be assessed in a competition binding assay in where increasing concentrations of unlabeled protein is added, such as by radioimmunoassay (RIA) or ELISA. The ability of the antibodies to bind to D.fragilis also can be assessed using methods well known in the art. For example. thc binding of antibodies to D. fragilis trophozoites can be visualized by microscopy using direct or indirect fluorescence.
Methods
[081] The polypeptides and antibodies provided herein can be utilized in a variety of methods. For example, the polypeptides can be used in methods for eliciting or inducing antibodies specific for an antigenic determinant described herein, and thus specific for D. fragilis methods for screening and selecting antibodies; methods for purifying antibodies; and methods for characterizing the binding properties of antibodies. The antibodies described herein that bind to an antigenic determinant in the enolase polypeptide of D. fragilis (such as antigenic determinant A, B. C. D, E, F or G) can be used in methods for detecting the enolase polypeptide of D. fragilis in a sample, methods for detecting D. fragilis in a sample; methods for diagnosing D. fragilis infection in a subject; methods for characterizing the binding properties of other antibodies; and methods for isolating and purifying D. fragilis enolase polypeptides and fragments thereof
[082] The polypeptides of the present invention may be administered to a subject to elicit antibodies that bind to the polypeptide. Thus, administration of the polypeptides to a subject can elicit antibodies that bind to the D. fragilis enolase polypeptide, such as the enolase polypeptide associated with the surface of the D. fragilis trophozoite. The polypeptides can be administered to a human or non-human subject,, such as a rat, mouse, rabbit, goat, sheep, rabbit or guinea pig, and by any suitable route, including, but. not limited to. intramuscular, intradermal, transdermal, parenteral, intravenous, subcutaneous, intranasal, oral, intraperitoneal or topical administration, as well as by any combination of any two or more thereof. As would be understood by those in the art, the polypeptides should be formulated in compositions suitable for the particular route of administration
[083] The polypeptides provided herein can be administered to a subject one time or more than once, including 2, 3. 4. 5 or more times. If the polypeptides are administered more than one time, the time between dosage administration can be, for example, 1. 2. 3, 4. 5, 6, 7, 8. 9, 10, 1 1, 12 or more weeks, or 1, 2, 3, 4, 5, 6, 7, 8, , 10, 11, .12 or more months. Selecting an optimal protocol to elicit antibodies is well within the level of skill of the skilled artisan. In some examples, repeat doses of the polypeptide can be administered to a subjcct if Ihc antibody response elicited by the subject after the first dose is below desired level.
(084] Following administration of the polypeptides to the subject, antibodies and/or antibody producing cells, such as B cells, can be isolated from a biological sample, such as blood, (issue, cells or serum, from the subject. For example, polyclonal antibodies can be isolated from the scrum. In other instances, antibody producing cells can be isolated from the spleen or from peripheral blood lymphocytes (PBL) in the blood.
[085] The polypeptides of the present invention can also be used to select, screen, isolate and/or purify antibodies that are specific for an antigenic determinant comprising, or located at or within, amino acids 177-189. 212-222, 264-272, 294-302, 319-328, 409-419 or 453-467 of the D. fragilis enolase protein set forth in SEQ ID NO: 1 or corresponding amino acids in another D. fragilis enolase protein. These methods can be performed using capture techniques, whereby the polypeptides are used to capture an antibody specific for the antigenic determinant present in the polypeptide. The polypeptides are contacted with the antibodies, and those antibodies that arc bound to the polypeptides arc then selected and optionally isolated. For example, the polypeptides can be incubated with a samples containing the antibodies for a sufficient time to permit binding of antibodies to the polypeptides. In some examples, the polypeptides are immobilized, such as on a column or a plate, or are linked to beads, such as magnetic beads. Following incubation, the polypcptidc-antibody complexes are washed to remove non-binding antibodies. The stringency of the wash can be adjusted according to methods well known to the skilled artisan. After washing to remove unbound antibodies, bound antibodies can be eluted using one of several well known clution methods, typically by reduction of the pH of the solution. In some instances, selection methods are an iterative process with multiple rounds of capture, wash and clution.
[086] The polypeptides can also be used to detect the presence of D. fragilis antibodies in a sample. The sample may be a biological sample, such as blood, serum, stool, gastric lavage or urine. Thus, in some aspects, the polypeptides of the present invention can be used to detect or diagnose D. fragilis infection in a subject, whereby the subject has raised antibodies specific to the enolase polypeptide. Such methods can be performed using standard techniques such as ELISA, Western blot and dot blot. The polypeptides can be contaclcd with the sample, and binding of antibodies in the sample to the polypeptide can be detected. For example, the polypeptides can be immobilized on a surface and contacted with the sample. Following washing, any antibody thai is bound to the polypeptide can be detected using a labeled secondary antibody.
[087] The antibodies of the present invention can be used to detect the presence of the enolase polypeptide of D. fragilis in a sample by contacting the antibodies with a sample that may contain the enolase polypeptide under conditions that would facilitate binding of the antibodies to the polypeptide. Such samples include, but arc not limited to, biological samples from a subject, such as blood, serum, stool, gastric lavage or urine, cultured parasites or cultured recombinant cells. Detection of the presence of the enolase polypeptide can be performed by any method known in the art that detects the formation of a complex between the antibody and the enolase protein, including, but not limited to. rapid flow tests (e.g. lateral or vertical flow tests), ELISA, dot blot and Western blot.
[088] The antibodies of the present invention can also be used to detect the presence of D. fragilis parasites in a sample. Jn some instances the sample is a biological sample, such as blood, serum, stool, gastric lavage or urine. The biological sample may have been processed further, such as by homogenization, fixation, concentration, dessication or some other form of processing. The antibodies of the present invention can therefore be used to diagnose D. fragilis infection in a subject by detecting the presence of D. fragilis in a sample from the subject. Typically, the sample is a stool sample.
[089] Methods for detecting the presence of D. fragilis in a sample, and thus methods for diagnosing D. fragilis infection in a subject, arc performed by contacting the sample with an antibody of the present invention under conditions that facilitate binding of the antibody to D. fragilis parasites in the sample. In particular examples, the antibody binds to an antigenic determinant comprising, or located at or within, amino acids 264-272 of SEQ ID NO:l or corresponding amino acids in another D. fragilis enolase protein. In other examples, the antibody binds to the antigenic determinant comprising, or located at or within, amino acids 453-467 of SEQ ID NO:l or corresponding amino acids in another D. fragilis enolase protein.
[0901 The presence of D. fragilis in the sample is determined by delecting whether the antibody has bound to any D. fragilis parasites. Detection of the binding between the antibod and a D. fragilis parasite, such as a D. fragilis trophozoite, can be performed using any method known in the art. In some instances, the binding is detected directly by microscopy using antibodies of the present invention that contain a detectable label such as a fluorescent label, e.g. by direct immunofluorescence using a direct fluorescence antibody test, in other instances, the binding is detected indirectly by microscopy using a secondary antibody that binds to the Fc portion of an unlabellcd antibody of the present invention., wherein the secondary antibody contains a detectable label, such as such as a fluorescent label, e.g. by indirect immunofluorescence using an indirect fluorescent antibody lest <IFAT). Other methods to detect binding of the antibodies to D. fragilis include, but arc not limited to. whole parasite dot blot. f091] Methods of the present invention that utilize the antibodies described herein also include methods for purifying and isolating enolase polypeptides. Such methods typically involve contacting the antibodies with a sample containing the enolase polypeptides under conditions that facilitate binding of the antibodies to the enolase polypeptides. In some examples, the antibodies are immobilized, such as on a column or a plate, or are linked to beads, such as magnetic beads. Generally, the antibody-enolase complexes are washed to remove non-bound proteins. The enolase polypeptide can then be eluted using conditions well known to those skilled in the art. such as reduced pH of the elution solution.
[092] The antibodies of the present invention also can be used in methods for characterizing the binding properties of other antibodies. For example, competition binding assays can be used to determine whether a given antibody binds to the same antigenic determinant, an overlapping antigenic determinant or a different antigenic determinant than any one or more of the antibodies described herein.
Kits
[093] The present invention also provides kits comprising one or more polypeptides and/or antibodies described herein. Such kits may be useful in performing any one or more methods described herein. The kits provided herein optionally contain instructions for use, such as instructions for use in a method of the present invention. Additional reagents also may be included in the kits, including, but not limited to, diluents and one or more additional antibodies, such as a labeled secondary antibody. [094] Exemplary kits of the present invention include one or more antibodies of the present invention, such as one or more antibodies that bind to an antigenic determinant comprising, or located at or within, amino acids 264-272 of SEQ IT) NO:l or corresponding amino acids in another D. fragiUs enolase protein, and/or one or more antibodies that bind to the antigenic determinant comprising, or located at or within, amino acids 453-467 of SEQ ID NO:l or corresponding amino acids in another D. fragilis enolase protein. Such kits may be used, for example, in methods for detecting the presence of enolase polypeptides and or D. fragilis parasites in a sample. Accordingly, such kits may be used for the diagnosis of D. fragilis infection in a subject, by detecting the presence of D. fragilis parasites in a sample from the subject.
[095] The antibodies of the present invention that are included in such kits may contain a detectable label, such as a fluorescent label or gold particles. Thus, the kits may be used in a direct flourescenl antibody test or rapid flow test (e.g. lateral or vertical flow lest). In other examples, the antibodies contained in the kit do not have a detectable label. Such kits may be used, for example, in an indirect flourcsccnt antibody test In particular instances, kits containing antibodies of the present invention that do not have a detectable label may also comprise a labeled secondary antibody, such as a fluorescently labeled secondary antibody, that binds to the Fc portion of the antibodies of the present invention.
[096] Those skilled in the art will appreciate that the aspects and embodiments described herein arc susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes ail of (he steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
[097] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the present application. Further, the reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. [098] The present disclosure is further described by reference to the following non- limiting examples.
EXAMPLES
Example 1. Identification and sequencing of the D.fragilis enolase
[0991 D. fragilis trophozoites were cultured xenically at 37°C under microaerophilic conditions in a Loeffler's slope medium (modified from Barratt et al. (2010) Parasitology 137, 1867-1878). consisting of an inspissated horse scrum slope (5ml of horse autoclaved for 20 minutes at 89°C, dry) overlaid with 5ml of PBS and supplemented with 2-5mg of rice starch. RNA was extracted from D. fragilis culture sediments (from 2 day old cultures using TRIsure reagent (Bioline, catalogue number B1O-38032) and enriched for eukaryotic mR A's using oligo (dT)-ccllulose chromatography. Sequencing of the draft transcripiome was performed by the service provider AGRP. A single plate was sequenced on a Roche GS FLX 454 sequencer using the XL Plus chemistry followed by an additional half plate using the XLR70 chemistry. Assembly was carried out using the Roche GS Assembler program version 2.6.
[01001 Contigs obtained from the transcriptome assembly were used to construct a NCBJ blast database using the standalone makeblastdb program freely available from the NCBI website (version 2,2.25+). The enolase mRNA of Trichomonas vaginalis (Gcnbank Acc. No. AY89 164) was used as a query sequence using a standalone blastn search (version 2.2.25+) against this database to identify homologous cDNA sequences from D. fragilis. After blasting, four hits were obtained which were considered homologues of T. vaginalis enolase AY895164. based on their sequence similarity.
[0101] The top hit (SEQ ID NO:20) to sequence AY89 1 4 was then subjected to a blastn and blastx search on the NCBI web server to ensure that the sequence was trychomonad in origin rather than a bacterial hoinologuc originating from the prokaryotic support flora within the Dientamoeba cultures. The top 9 of 13 hits from a blastn search were enolase sequences from trichomonads (Histomonas meleagridis, Tritrichomonas foetus. Hypolrichomonas acosta and Trichomonas vaginalis). The closest hit was from an enolase sequence of Histomonas meleagridis (Genbank accession=FJ710l63 E-value=0.0 Idcntily=84%). The top 44 of 100 hits from a blaslx search were cnolases from trichomonads or related parabasalian parasites (Tridwmonas vaginalis. Spirotrichonympha leidyi, Pseudotrichonympha grassi Tridio ympha agilis, Holomastigotoides mirabile, Stephanonympha sp„ Histomonas meleagridis, Zo termopsis parabasalian symbionts, THtrichomonas foetus, Hypotrichomonas a osfa, Tetratrichomonas gall inar urn, Monocercomonas sp. ATCC 50210, rickomitus balracho um and Devescovinu sp. Nk9) plus on hit to Naegleria gruberi enolase(Genbank accession= EFC48029). The closest hit was from an enolase sequence of Trichomonas vaginalis (Genbank accession=EAY0652l E-valtie=0.0 Identify=77% Positives=84 ).
[01021 One of the four contig hits to sequence AY895164 (SEQ ID NO:19) was shown to encode a protein that was slightly longer than that encoded by SEQ ID NO:20, but with the same sequence over the overlapping region. This indicated that D. fragiUs likely has two or more copies of the putative enolase gene in its genome, giving rise to identical proteins when expressed.
[0103] The longer contig and thus predicted D. fragilis alpha enolase cDNA (SEQ ID NO: 19) was translated into its respective protein sequence in silico using the 'Translate* component of the freely available "Sequence manipulation suite' software (Stothard, (2000) Biotechniques 28, 1102. 1104), and is shown as SEQ ID NO:l. The 'Conserved Domains' feature of the NCB1 web server was used to examine the structural features of this putative Dientamoeha enolase. According to 'Conserved Domains', this putative Dientamoeba enolase possesses a conserved enolase metal binding site, an enolase substrate binding pocket, an enolase dimer interface and a triosephosphate isomerase (TIM) barrel domain. The sequence also possesses a conserved enolase N-terminal domain. It was concluded that the closest homologue of T. vaginalis enolase AY895164 from Dientamoeba had been identified.
Example 2. Peptide design and synthesis
[0104] Several online tools were used for prediction of suitable antigenic peptides derived from the enolase polypeptide of SEQ ID NO:l. These included the Kolaskar & Tongaonkar Antigenicity prediction tool (Kolaskar and Tongaonkar. (1990) FEBS Lett 276, 172-174). the Bepipred linear epitope prediction tool (Larsen et al., (2006) Immunoine Res 2, 2), and the BCEPRED server (Saha et al., (2004) Artificial Immune Systems. Springer Berlin/Heidelberg, pp. 197-204) which predicts B-ccll epitopes based on a range of different properties including hydrophilicity. flexibility, accessibility, turns, exposed surface residues, polarity and antigenic propensity. Consideration of peptides for synthesis was based on a consensus between the programs. When two or more programs predicted a similar peptide from the D. fragilis cnolase sequence, that peptide was considered a prime candidate for synthesis. If a cysteine residue was not present al the beginning or end of the native predicted peptide, a cysteine residue was added to one end of the peptide to allow conjugation of the peptide. Table 2 sets forth the peptides that were designed and subsequently synthesized, and shows the amino acids within the cnolase polypeptide set forth in SEQ TD NO:l to which the peptides correspond.
Table 2. Enolase Peptides
Peptide Start functional Amino add sequence End Position in SEQ group functional j j NO:l
group
A Acetyl (Ac) CLQIYEYLKEHD LEE Hydroxy (OH) 453-467
(SEQ ID NO:J0)
B Acetyl (Ac) CAISY1EQAM Amine (NH2) 264-272
(SEQ ID NO: U)
C Acetyl (Ac) EQNMPKK Y KLPQC Amine (NH2) 177-189
(SEQ ID O:5)
D Acetyl (Ac) CSPAKGTPLPEQ Amine (NH2) 212-222
(SEQ ID NO: 12)
E Acetyl (Ac) WADL1AKHPC Amine (NH2) 319-328
(SEQ ID O:7)
P Acetyl (Ac) CFYDAETKLY Amine (NH2> 294-302
(.SEQ ID NO: 13)
G Acetyl (Ac) VMVSHRSGETC Amine (NH2> 409-420
(SEQ ID NO:9) Example 3. Three dimensional modeling of D.fragitis enolase
Γ0Ι05] A 3D model of the enolase polypeptide set forth in SEQ ID NO: 1 was generated using the SWISS-MODEL workspace and using the automated mode. The qmean score (a measure of model accuracy) of the model was 0.62, indicating that the enolase model is a reasonable depiction of the predicted structure.
[01061 Structural models of the enolase protein indicating the location of each peptide on the structure as well as the locations of transmembrane helices, those parts of the protein which are predicted to reside within the cell and the parts of the protein which are predicted to reside outside the cell, were generated. Amino acids 1 to 141 of SEQ ID NO:l reside within the cell; amino acids 142 to 176 comprise the transmembrane helices; and amino acids 177 to 467 are extracellular. As shown in the 3D modeling using space fill format (Figure I). Peptides A-G (corresponding to antigenic determinants A-G) correspond to regions in the enolase protein that are in the extracellular portion of the polypeptide and appear to be on the external surface of the folded protein. Furthermore, these regions arc separated, distinct and non-overlapping, suggesting (hat antibodies against these epitopes would be unlikely to impede each other when binding to the enolase protein.
[0107] In panel I of Figure I, the amino acids in white represent those which are part of the extracellular loop of the and are exposed on the surface of the protein itself, though were not used for peptide design as described in Example 2. While these amino acids were not used for peptide design in this case, they arc likely to be accessible to antibodies and so arc also candidates for incorporation into any of peptides A to G (such as by making these peptides longer). Alternatively, these amino acids could be used to design completely new peptides based on the same principals used to design peptides A to G. These surface- exposed amino acids include those at positions 198; 203; 205; 223; 224; 227; 228; 230; 231 ; 232; 235; 238; 239; 240; 241; 242; 243; 244; 246; 247; 248; 259; 260; 273; 275; 276; 277; 303: 305; 306; 307; 308: 310; 313; 314; 315; 317; 329; 330; 339; 342; 345: 346: 348; 349: 350; 353; 354; 355; 356; 367: 368; 370; 37.1 ; 372; 374: 375; 376; 377; 380: 389; 396; 400; 401 ; 403; 404; 405; 408; 420; 421 ; 422; 430; 433; 434; 440; 441 ; 442; 443; 446; 447; 450 and 451 of SEQ JD NO: l. Example 4. Production and purification of polyclonal antibodies
[01081 The peptides were each conjugated to the immunogenic carrier protein keyhole limpet hemocyanin (KLH) via (he thiol group of the cysteine residue of each peptide, before being administrated to rabbits to produce antisera using standard techniques. The presence of anti-peplide antibodies in the resulting rabbit antisera was determined using a simple slot blotting technique. Briefly, peptide was blotted onto a nitrocellulose membrane using a PB 600 Slot Blot Filtration manifold system (Amersham Biosciences). The peptide was then probed with the rabbit antisera (diluted 1:2 with PBS) and then with an anti-rabbit IgG antibody (raised in goats) conjugated to FITC (Sigma Aldrich, product number F0382) (diluted 1:80). The blot was scanned using a Pharos FX Plus Molecular Imager (Bio-Rad) to determine the presence or absence of fluorescence. The blot was accompanied by two negative controls; a pre-bleed sample from the rabbit (prior to immunization with the peptide) and a blot probed with the FITC conjugated antibody only (no rabbit antisera). It was confirmed thai antibodies specific for the relevant peptide were present in each antiserum composition.
[01091 Approximately 25 mL of rabbit antisera was heated to 56°C for 30 minutes to inactivate complement. The serum was diluted 1 :2 in PBS (total volume 50 mL) and the IgG fraction was extracted using the ammonium sulfate precipitation technique. Briefly, one volume of saturated ammonium sulfate was added to the dilute sera (drop-wise with continuous mixing). The resulting antibody suspension was centrifuged (7000g at 4rtC for 15 minutes). The supernatant was discarded and the pellet was dissolved in 50mls of PBS followed by addition of 1 volume of saturated ammonium sulfate (drop-wise with frequent mixing). The resulting suspension was then centrifuged at 7000g at 4°C for 15 minutes. The supernatant was discarded and the protein pellet was dissolved in 3 mL of PBS. This antibody solution was dialysed overnight at 4°C against 1L of PBS with three buffer changes. The antibody solution was removed from the dialysis tubing and stored (temporary storage of several days at 4°C, long term storage -20°C to -80°C). Twenty-five mL of rabbit antisera yielded approximately 140mg of protein.
[01101 An affinity column was prepared for each peptide using a SulfoLink® Immobilization Kit for Peptides (Thermo Scientific, product number 44999) in accordance with manufacturer's instructions. Antisera were adjusted to contain an additional concentration of 300mM NaCl (resulting in binding buffer - normal PBS plus 300mM NaCl). The column was equilibrated wilh binding buffer (PBS plus 300mM NaCl) and the antibody solution was sterile filtered (0.2μηι filter) and applied to the column. Elution was performed using 0,2 M glycine (pH 2.5). Eluted fractions were collected into tubes containing 400μ) of neutralisation buffer (1M Tris pH9). Tlie absorbance of each fraction (at 280nm) was measured to identify the IgG containing fractions. The IgG containing fractions were pooled and dialysed overnight at 4°C against 1L of PBS with three buffer changes. The purity of the antibody preparation was evaluated by reducing a portion with dithiothrcitol (DTT) and subjecting it to polyacrylamidc gel electrophoresis. The polyacrilimidc gel was then stained with Coomassie blue for observation of IgG heavy and light chains. The polyclonal antibody preparations were stored at -20°C until use (long term storage -80°C).
[0111] This immunoaffinity chromatography recovered approximately 1.5-2 mg of anli- peptidc antibody for each peptide. Polyacrylamidc gel electrophoresis of the resulting antibody solutions revealed that the antibody preparations A-G (corresponding to the peptides A-G. respectively, that they were raised against) were extremely pure, containing only small traces of albumin and other serum proteins.
Example 5. Indirect fluorescent antibody staining of D. fragitis
Γ0112] The ability of the antibody preparations to bind to D. fragilis was assessed using an indirect immunofluorescence assay. To prepare the D. fragilis trophozoites, two day old cultures of D. fragilis were agitated slightly to suspend the culture sediments where the trophozoites reside. The cell suspension was collected and centrifuged at 700 g for 10 minutes to pellet cells. The supernatant was discarded and the cells were fixed by addition of PBS containing 3.7% formaldehyde and incubating for 10 minutes. Immediately after addition of the PBS-formaldehyde solution, the tube was gently shaken to thoroughly break apart the pellet. Cells were then centrifuged at 700g for 5 minutes and the supernatant was discarded. This method was intended to be mild and non- jcrmcabilising, so as to assess the presence of a fluorescent signal on the surface of cells. In an alternative protocol, instead of fixing cells with 3.7% formaldehyde, cells were fixed by bathing for at least 24 hours in SAF fixative (Fronine Lab Supplies. Australia) in a 1:1 ratio of culture supcrnalant Co SAF. The cells were then immediately bathed in a large volume of PBS and centrifuged once more to remove any residual formaldehyde or SAF.
[0113] For preparation of slides, 40μ1 of Mayers albumin was smeared on the slide. The cell pellet was suspended in a small volume of PBS, placed on the slide and allowed to dry. Slides were then blocked for one hour with 5% skim milk or 3% BSA followed by three washes in a solution of 0.2% BSA in PBS (wash solution). The slides were then incubated in a solution of polyclonal antibody (testing different dilutions) for one hour followed by three washes. Finally, the slides were incubated in a solution of dilute (1:80) FITC conjugated anti-rabbit IgG (Sigma Aldrich. product number F0382) for one hour followed by an additional three washes. The slides were cover slipped and trophozoites were examined for fluorescence using an Olympus BXS1 fluorescent microscope under oil emersion at 1000X magnification.
[0114] Of the seven polyclonal antibody preparations evaluated, antibody preparations A and B exhibited a very strong reaction to the surface of Dientamoeka trophozoites (Figures 2 and 3) after fixation with 3.7% formaldehyde in PBS. All other antibody preparations resulted in an extremely weak to completely absent fluorescent signal. Interestingly, each of antibody preparation A and B gave rise to slightly different fluorescent patterns. Antibody preparation A produced a di fuse, even fluorescent signal across the surface of Dieniamaeba cells (Figure 2). Staining with antibody preparation B produced a distinct ring pattern of fluorescence characterized by a densely stained fluorescent ring on the periphery of cells and a lighter fluorescent signal within the ring (Figure 3). Negative controls (cultured ceils treated with only the secondary antibody) did not fluoresce. Examination of fixed, unstained Dieniamaeba trophozoites demonstrated that they are not auto-fluorescent. Cells from control slides stained with antibody preparations A and B, though without the application of the FITC conjugated anti-rabbit IgG, also failed to fluoresce. The same staining profile was observed with the SAF fixation protocol.
Example 6. Indirect fluorescent antibody staining of other parasites
Γ0115] The same staining protocol described in Example 5 was used to determine whether the antibodies in the preparations A and B could bind to Entamoeba histolytica, E. tnoshkovskii and Giardia trophozoites and Blastocysts cells, thereby assessing specificity of the antibodies.
[0116] The use of antibody preparation A in an indirect immunofluorescence assay with E. moshkovskii or E. histolytica trophozoites resulted in a strong fluorescent signal (Figures 4 and 6. respectively , indicating that antibodies directed to Peptide A cross-reacted with Entamoeba trophozoites. A sparse speckling fluorescent signal throughout cells was also observed when Giardia trophozoites were stained with antibody preparation A (Figure 10). although no fluorescence of Blastocysts cells was observed (Figure 8).
Γ0117] When antibody preparation B was used, no fluorescence of either species of the Entamoeba trophozoites (Figures 5 and 7), the Giardia trophozoites (Figure 11) or Blastocysts cells (Figure 9) was observed, indicating that antibodies directed to Peptide B were specific for D.fragilis and did not cross-react with any of the other parasites tested.

Claims

CLAIMS:
1. An isolated antibody or antigen-binding fragment thereof that specifically binds to an antigenic determinant within the D. fragilis enolasc polypeptide comprising the sequence set forth in SEQ ID NO: l or a variant thereof having at least about 85% sequence identity with the sequence set forth in SEQ ID NO: 1.
2. The antibody or antigen-binding fragment of claim I wherein the variant has at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%. 95%. 96%, 97%, 98% or 99% sequence identity with the sequence set forth in SEQ ID NO:l.
3. The antibody or antigen-binding fragment of claim 1 or claim 2 that is a monoclonal antibody.
4. The antibody or antigen-binding fragment of claim 1 or claim 2 that is a polyclonal antibody.
5. The antibody or antigen-binding fragment of any one of claims 1 to 4, that is linked to a detectable label.
6. The antibody or antigen-binding fragment of claim 5, wherein the detectable label is selected from among a fluorescent label, a chemiluminescent label, an enzymatic label, gold particle, biotin and streptavidin.
7. The antibody or antigen-binding fragment of any one of claims 1 to 6. wherein the antigenic determinant comprises, or is located at or within, amino acids 453-467 of SEQ ID NO: I or corresponding amino acids in the variant.
8. The antibody or antigen-binding fragment of any one of claims .1 to 6, wherein the antigenic comprises, or is located at or within, amino acids 264-272 of SEQ ID NO:l or corresponding amino acids in the variant.
9. The antibody or antigen-binding fragment of any one of claims 1 to 6. wherein the antigenic determinant comprises, or is located at or within, amino acids 177- 189 of SEQ ID NO: 1 or corresponding amino acids in the variant.
10. The antibody or antigen-binding fragment of any one of claims .1 to 6, wherein the antigenic determinant comprises, or is located at or within, amino acids 212-222 of SEQ ID NO: 1 or corresponding amino acids in the variant.
1 1. The antibody or antigen-binding fragment of any one of claims 1 to 6. wherein the antigenic determinant comprises, or is located at or within, amino acids 294-302 of SEQ JD NO: I or corresponding amino acids in the variant.
12. The antibody or antigen-binding fragment of any one of claims .1 to 6, wherein the antigenic determinant comprises, or is located at or within, amino acids 319-328 of SEQ ID NO: 1 or corresponding amino acids in the variant.
13. The antibody or antigen-binding fragment of any one of claims 1 to 6. wherein the antigenic determinant comprises, or is located at or within, amino acids 409-419 of SEQ D NO: 1 or corresponding amino acids in the variant.
14. The antibody or antigen-binding fragment of any one of claims 1 to 13, for use in detecting the presence oi& D.fmgilis enolase polypeptide in a sample.
15. The antibody or antigen-binding fragment of any one of claims 1 to 13. for use in detecting the presence of a D. fragilis parasite in a sample.
16. The antibody or antigen-binding fragment of any one of claims 1 to 13, for use in diagnosing D. fragilis infection of a subject.
17. A method for the detection of a D. fragilis enolase polypeptide in a sample, comprising contacting the sample with an antibody or antigen binding fragment of any one of claims 1 to 1 . and detecting binding of the antibody or antigen binding fragment to the enolase polypeptide.
18. A method for the detection of a D. fragilis parasite in a sample, comprising contacting the sample with an antibody or antigen binding fragment of any one of claims I to 8, and detecting binding of the antibody or antigen binding fragment to the D. fragilis parasite.
19. A method for the diagnosis of D. fragilis infection in a subject, comprising contacting a sample from the subject with an antibody or antigen binding fragment of any one of claims 1 to 8, and detecting binding of the antibody or antigen binding fragment to a D.fragilis parasite in the sample.
20. The method of claim 18 or claim 19, wherein the sample comprises blood, serum, stool, gastric lavage or urine.
21. The method of claim 18 or claim 19, wherein Che sample is a stool sample.
22. The method of any one of claims 17 to 21. wherein detection is performed by direct immunofluorescence.
23. The method of any one of claims 17 to 21, wherein detection is performed by indirect immunofluorescence.
24. A kit, comprising the antibody or antigen binding fragment of any one of claims 1 to 13.
25. The kit of claim 24, when used for detecting the presence of a D. fragtiis enolasc polypeptide in a sample.
26. A kit, comprising the antibody or antigen binding fragment of any one of claims 1 to 13, when used for detecting the presence of a D.fragilis parasite in a sample.
27. A kit, comprising the antibody or antigen binding fragment of any one of claims 1 to .13, when used for diagnosing D.fragilis infection in a subject.
28. The kit of any one of claims 24 to 27, further comprising a labelled secondary antibody.
29. An isolated polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:l or 2, or a sequence of amino acids having at least about 85% sequence identity with the sequence set forth in SEQ ID NO:l or 2.
30. The isolated polypeptide of claim 29, comprising a sequence of amino acids having at least about 86%, 87%. 88%, 89%. 90%, 91 %, 92%. 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence set forth in SEQ ID NO: I or 2.
31. An isolated polypeptide, comprising the sequence of amino acids set forth in SEQ ID NO:3 or 10.
32. An isolated polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:3 or the sequence of an antigenic variant of the polypeptide set forth in SEQ ID ΝΌ:3, wherein the variant has at least 75%. 80%. 85% or 90% sequence identity with the sequence set forth in SEQ ID NO:3, and wherein the isolated polypeptide is less than 300 amino acids in length.
33. The polypeptide of claim 32, comprising Che sequence of amino acids set forth in SEQ JD NO:3 or 10.
34. The polypeptide of claim 32, consisting of the sequence set forth in SEQ ID NO:3 or 10.
35. An isolated polypeptide, comprising the sequence of amino acids set forth in SEQ ID NO:4 r 1 1.
36. An isolated polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:4 or the sequence of an antigenic variant of the polypeptide set forth in SEQ ID NO:4. wherein the variant has at least 75%, 80% or 85% sequence identity with the sequence of amino acids set forth in SEQ ED NO:4, and wherein the isolated polypeptide is less than 300 amino acids in length.
37. The polypeptide of claim 36. comprising the sequence of amino acids set forth in SEQ ID O:4 or l 1.
38. The polypeptide of claim 36. consisting of the sequence set forth in SEQ ID NO:4 or 11.
39. The polypeptide of any one of claims 29 to 38 that is linked to at least one moiety.
40. The polypeptide of claim 39, wherein the moiety is selected from among another antigenic determinants, a T helper cell epitope or polypeptide that comprises a T helper cell epitope, an immunos mulatory molecule, a targeting agent, a polymer, a multimcrisation domain, a detectable label and an affinity tag.
41. The polypeptide of claim 40, wherein the moiety a T cell epitope or polypeptide that comprises a T cell epitope.
42. A nucleic acid molecule, encoding the polypeptide of any one of claims 29 to 41.
43. The polypeptide of any one of claims 29 to 41, when used to elicit antibodies that specifically bind to a D.fr gilis enolase polypeptide.
44. The polypeptide of any one of claims 29 to 41, when used to elicit antibodies that specifically bind to a D.fragilis parasite.
45. A method of eliciting antibodies that, specifically bind to a D. fragilis enolase polypeptide, comprising administering a polypeptide of any one of claims 29 to 41 to a subject, thereby eliciting antibodies that specifically bind to a D. fragilis cnolase polypeptide.
46. A method of eliciting antibodies that specifically bind to a D. fragilis parasite, comprising administering a polypeptide of any one of claims 29 to 41 to a subject, thereby eliciting antibodies that specifically bind to a D. fragilis parasite.
PCT/AU2014/050035 2013-05-13 2014-05-13 Antigenic polypeptides of dientamoeba fragilis and antibodies thereto WO2014183166A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5334509A (en) * 1992-10-22 1994-08-02 Riordan Neil H Method for detecting intestinal pathogen dientamoeba fragilis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5334509A (en) * 1992-10-22 1994-08-02 Riordan Neil H Method for detecting intestinal pathogen dientamoeba fragilis

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Title
DATABASE EMBL 20 February 2007 (2007-02-20), accession no. AY05605.1 *
DATABASE GENBANK 5 December 2008 (2008-12-05), accession no. AF48679.1 *
MUNDODI, V. ET AL.: "Immunogenic and Plasminogen-Binding Surface-Associated c Enolase of Trichomonas vaginalis", INFECTION AND IMMUNITY, vol. 76, no. 2, February 2008 (2008-02-01), pages 523 - 531 *

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