WO2019237119A1 - New compositions and detection methods for onchocerca volvulus infection - Google Patents

Abstract

The present invention relates to novel Onchocerca volvulus (Ov) antigens and therefore provides new compositions and methods useful for effectively detecting Ov infection and monitoring efficacy of Onchocerciasis treatment.

Description

NEW COMPOSITIONS AND DETECTION METHODS FOR

ONCHOCERCA VOLVULUS INFECTION

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/682,678, filed June 8, 2018, the contents of which are hereby incorporated by reference in the entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] Onchocerciasis, also known as river blindness, is a disease caused by infection with the parasitic worm Onchocerca volvulus {Ov). The parasitic nematode is transmitted by Simulium (blackfly) bites. Ov infection leads to severe and disfiguring skin disease, lymphadenopathy, and visual impairment including blindness. The disease-transmitting flies breed in fast-flowing streams and rivers, increasing the risk of blindness to individuals living nearby, hence the commonly known name of "river blindness." Within the human body, the adult female worm (macrofilaria) produces thousands of baby or larval worms (microfilariae) which migrate in the skin and the eye of the host. The death of microfilariae is very toxic to the skin and the eye, producing sever itching and various eye manifestations (lesions). After years of repeated exposure, these lesions may lead to irreversible blindness and disfiguring skin diseases sometimes named "leopard" skin and "lizard" skin. The adult female worms can survive within the body of a host for over 10 years and directly contribute to the damaging symptoms of onchocerciasis, they are therefore a focal point in the efforts of detecting onchocerciasis and monitoring treatment status. Onchocerciasis affects resource- limited rural communities and leads to serious economic losses. Globally an estimated 120 million people are at risk, with 18 million infected with Ov in Africa. Over 95% of Ov cases are in Africa as well as some regions in Latin America and the Arabian Peninsula. In some West African communities, about 50% of men over the age of 40 years have been blinded by the disease, resulting in significant human suffering and detriment to local economic. The annual economic losses are estimated in the hundreds of millions of dollars.

[0003] Thanks to the availability of ivermectin, considerable success has been achieved in the control of onchocerciasis. Yet, because ivermectin kills microfilariae only but not adult female worms of Onchocerca volvulus (OvAF), which can have a very long reproductive life, it is crucial to monitor the presence of OvAF in order to ensure elimination of Ov. Therefore, there exists a pressing need for developing new methods for sensitive and convenient detection of OvAF in patients for disease diagnosis as well as monitoring/confirming therapeutic effectiveness. The present invention addresses this and other related needs.

BRIEF SUMMARY OF THE INVENTION

[0004] The application provides the first disclosure of Ov antigens that are adult-specific and therefore can be used as markers for macrofilaricidal activity. Using proteomics, comparative transcriptomics, and genomics, the present inventors have identified 14 novel antigens as biomarkers reflective of viable OvAF, which can be used in immunoassays to detect active onchocerciasis and to follow responses to macrofilaricidal agents. All of these newly identified antigens are highly Ov-specific and do not have significant homology to other filariae (e.g., B. malayi, L. loa , and W. bancrofti ), nor do they have homologues in humans or mice. These antigens are OVOC12265, OVOC4422, OVOC585, OVOC2705, OVOC3624, OVOC486, OVOC12838, OVOC8995, OVOC1751, OVOC12539, OVOC5398, OVOC8934, OVOC6769, and OVOC835, and their amino acid sequences are provided herein as SEQ ID NOs: l-l4. Furthermore, four peptides (SEQ ID NOs: 15-18) derived from two of these antigens have been identified as particularly useful, e.g. , for the purpose of generating Ov-specific antibodies and therefore for the specific detection of this pathogen.

[0005] As such, in a first aspect, this invention provides new OvAF-specific antigens having the amino acid sequences set forth in SEQ ID NOs:l-l4 and SEQ ID NOs: l5-l8.

Each of these antigens in some cases may be an isolated polypeptide, or may be a part of a polypeptide conjugate comprising any one amino acid sequence selected from SEQ ID NOs: 1-14 and SEQ ID NOs: 15-18 conjugated to a heterologous moiety. In some cases, the heterologous moiety is a heterologous peptide sequence (for example, an affinity peptide tag such as 6 x His or an peptide antigen, or an enzyme such as glutathione s-transferases (GST) or b-galactosidase), making the polypeptide conjugate a fusion protein.

[0006] In some embodiments, the heterologous moiety is a detectable label, such as a radioactive or a fluorescent molecule. In some embodiments, the heterologous moiety is a solid substrate. For example, two or more of polypeptides, each comprising a different amino acid sequence selected from SEQ ID NOs: 1-14 and SEQ ID NOs: 15-18, may be conjugated to the solid substrate, e.g. , at a preassigned location for ready identification, to form an array. [0007] In a second aspect, the present invention provide a nucleic acid encoding (1) an Ov antigen comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-14 and SEQ ID NOs: 15-18, or (2) a fusion protein described above, a fusion protein comprising or consisting of any one of Ov antigen selected from SEQ ID NOs: 1-14 and SEQ ID NOs: 15- 18 fused with a heterologous peptide sequence. In some cases, the nucleic acid is in the form of an expression cassette, which comprises a polynucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: l-l4 and SEQ ID NOs:l5-l8 operably linked to a heterologous promoter. In some embodiments, the polynucleotide sequence in the expression cassette encodes a fusion protein comprising a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-14 and SEQ ID NOs: 15-18 fused to a heterologous peptide sequence. In some cases, the nucleic acid is in the form of a vector (e.g., recombinant vector made based on a bacterial or viral vector) comprising the expression cassette described above and herein. In some cases, the vector is present in a host cell, either transiently or permanently.

[0008] Related to this aspect of the present invention, a method is further provided for producing a recombinant protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-14 and SEQ ID NOs: 15-18. The method includes the step of culturing the host cell containing the vector described above and herein under conditions permissible for the expression of a polypeptide encoded by the expression cassette.

[0009] In a third aspect, the present invention provides an antibody that specifically binds an Ov antigen comprising or consisting of the amino acid sequence of any one selected from SEQ ID NOs: 1-14 and SEQ ID NOs: 15-18. The antibody may be polyclonal or monoclonal antibody. It may be subject to further modification or labeling, e.g., with a detectable label such as a radioactive or chemi-luminescent label attached to the antibody for ready detection.

[0010] In a fourth aspect, the present invention provides a method for detecting

Onchocerca volvulus infection by detecting the presence of OvAF in a subject. This method includes the steps of (1) contacting a sample taken from the subject with a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-14 and SEQ ID NOs: 15-18; (2) detecting the presence of an antibody in the sample that specifically binds to the polypeptide; and (3) determining the presence of Onchocerca volvulus infection in the subject. [0011] In some embodiments, the polypeptide used in this method is conjugated to a solid substrate. In some embodiments, the sample in step (1) of the method is contacted with two or more polypeptides each comprising or consisting of a different amino acid sequence independently selected from SEQ ID NOs: l-l4 and SEQ ID NOs: 15-18. In some

embodiments, the sample is contacted with an array comprising two or more polypeptides each comprising or consisting of a different amino acid sequence independently selected from SEQ ID NOs: 1-14 or SEQ ID NOs: 15-18 immobilized on a solid substrate, for example, one of the two or more polypeptides comprises or consists of the amino acid sequence of SEQ ID NO:7, and another of the two or more polypeptides comprises or consists of the amino acid sequence of SEQ ID NO:8; or in another example, one of the two or more polypeptides comprises or consists of the amino acid sequence of SEQ ID NO: 11, and another of the two or more polypeptides comprises or consists of the amino acid sequence of SEQ ID NO: 12. In some embodiments, the presence of antibodies in the sample that specifically binds to at least half of the two or more polypeptides is detected in step (2) before Ov infection is deemed to be present in the subject. In some embodiments, the sample is a blood sample (including whole blood or blood fractions such as plasma or serum sample), skin sample, or urine sample. In some embodiments, the subject is a human patient who has previously received treatment for Onchocerca volvulus infection, e.g., ivermectin administration for at least 6 months or a year.

[0012] In a fifth aspect, the present invention provides a method for detecting Onchocerca volvulus infection by detecting the presence of OvAF in a subject. This method includes the steps of (1) contacting a sample taken from the subject with an antibody that specifically binds a polypeptide comprising or consisting the amino acid sequence of any one of SEQ ID NOs: l-l4 or SEQ ID NOs: l5-l8; (2) detecting the presence of a polypeptide in the sample that specifically binds to the antibody; and (3) determining the presence of Onchocerca volvulus infection in the subject.

[0013] In some embodiments, the antibody used in the method is immobilized to a solid substrate. In some embodiments, in step (1) of the method the sample is contacted with two or more antibodies each specifically binding a different amino acid sequence independently selected from SEQ ID NOs: 1-14 or SEQ ID NOs: 15-18. In some embodiments, the sample is contacted with an array comprising two or more antibodies each specifically binding a different amino acid sequence independently selected from SEQ ID NOs: 1-14 or SEQ ID NOs: 15-18 immobilized on a solid substrate, for example, one of the two or more antibodies specifically binds the amino acid sequence of SEQ ID NO: 7, and another of the two or more antibodies specifically binds the amino acid sequence of SEQ ID NO:8; or in another example, one of the two or more polypeptides comprises or consists of the amino acid sequence of SEQ ID NO: 11, and another of the two or more polypeptides comprises or consists of the amino acid sequence of SEQ ID NO: 12. In some embodiments, the presence of polypeptides in the sample that specifically bind to at least half of the two or more antibodies is detected in step (2) before Ov infection is deemed present in the subject. In some embodiments, the sample used in the method is a blood sample (including whole blood or blood fractions such as plasma or serum sample), skin sample, or urine sample. In some embodiments, the subject is a human patient who has previously received treatment for Onchocerca volvulus infection, e.g., ivermectin administration for at least 6 months or a year.

[0014] In a sixth aspect, the present invention provides a kit for detecting Onchocerca volvulus infection by detecting the presence of OvAF in a subject. The kit typically includes a first container containing a first composition comprising a first antibody that specifically binds a polypeptide comprising or consisting of one amino acid sequence selected from SEQ ID NOs: l-l4 or SEQ ID NOs: 15-18, and a second container containing a second composition comprising a sample taken from a patient confirmed to have Onchocerca volvulus infection and have OvAF in his body. In some embodiments, the kit further incldues one or more additional containers, each containing a composition comprising an antibody that specifically binds a different polypeptide comprising or consisting of a different amino acid sequence independently selected from SEQ ID NO: 1-14 or SEQ ID NOs: 15-18. In some

embodiments, the kit may further include a negative control sample taken from a subject confirmed to not have an active Onchocerca volvulus infection and is without detectable level of OvAF. In some embodiments, the sample type includes a blood sample (including whole blood or blood fractions such as plasma or serum sample), skin sample, or urine sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1: Analytic sensitivity of immunoassays to measure OVOC12838 (top panel) and OVOC8995 (bottom panel) in polyclonal antibody-based sandwich capture ELISAs.

[0016] Figure 2: Antigen levels of OVOC8995 (left panel) and OVOC12838 (right panel) were quantitated in mf positive Ov-infected (‘infected’) and in Ov-uninfected. “All uninfected” included those with other filariae (e.g, L. loa , W. bancrofti ) and“uninfected NA contols” were unexposed North American healthy volunteers. Small dashed line is the cutoff for 100% specificity against all uninfected controls.

[0017] Figure 3: Heat map of positivity (red) and negativity (green) of Ov-infected patients reactive to OVOC8995 (left column) or OVOC12838 (right column). As seen, there are individuals with measurable quantities of both antigens as well as individuals with measurable quantities of either OVOC8995 or OVOC12838.

[0018] Figure 4: OVOC8995 levels in serum from 6 mf positive Ov-infected individuals before (PreTx) and following definitive treatment (PostTx). Dashed line is the cutoff for the positivity in the immunoassay.

[0019] Figure 5: Purified IgG antibodies can detect full-length proteins. Purified antibodies to Pep-l and Pep-2 of OVOC5398 (A) and OVOC8938 (B) detect the full-length OVOC8934 protein better than the peptides to which the anti-sera were raised.

[0020] Figure 6: Configuring antigen-detection immunoassay. Analytic sensitivity of immunoassays to measure OVOC5398 (A) and OVOC8934 (B) in combinations of polyclonal antibody -based sandwich capture ELISAs.

[0021] Figure 7: Testing pooled sera with antibodies generated against two OVOC8934 peptides. Antigen levels of OVOC8934 in pooled sera from Ov-infected (OV) and uninfected blood bank controls (BB) in sandwich ELISA using combinations of polyclonal antibodies, with capture antibody being anti -Pep 1 (A), or anti-Pep2 (B), and detecting with biotinylated anti-Pep2 or anti -Pep 1 respectively.

[0022] Figure 8: OVOC8934 elicits IgG responses in infected individuals (OV) compared to blood bank normal (BB). IgG4 levels were minimal to none (data not shown).

[0023] Figure 9: Testing of individual samples. Antigen levels of OVOC8934 in sera from Ov-infected (OV) and uninfected blood bank controls (BB) in sandwich ELISA. The samples were testes at 1 :4 and 1 :20 dilutions.

DEFINITIONS

[0024] The term“nucleic acid” or“polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof ( e.g ., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0025] The term“gene” means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

[0026] The term“amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.“Amino acid mimetics” refers to chemical compounds having a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0027] There are various known methods in the art that permit the incorporation of an unnatural amino acid derivative or analog into a polypeptide chain in a site-specific manner, see, e.g, WO 02/086075.

[0028] Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. [0029] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences,“conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule.

Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0030] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0031] The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins , W. H. Freeman and Co., N. Y. (1984)).

[0032] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0033] In the present application, amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild- type polypeptide sequence.

[0034] “Polypeptide,”“peptide,” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0035] An "antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad

immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0036] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.

Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0037] Antibodies exist, e.g, as intact immunoglobulins or as a number of well

characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH! by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, NY (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.

[0038] Further modification of antibodies by recombinant technologies is also well known in the art. For instance, chimeric antibodies combine the antigen binding regions (variable regions) of an antibody from one animal with the constant regions of an antibody from another animal. Generally, the antigen binding regions are derived from a non-human animal, while the constant regions are drawn from human antibodies. The presence of the human constant regions reduces the likelihood that the antibody will be rejected as foreign by a human recipient. On the other hand, "humanized" antibodies combine an even smaller portion of the non-human antibody with human components. Generally, a humanized antibody comprises the hypervariable regions, or complementarity determining regions (CDR), of a non-human antibody grafted onto the appropriate framework regions of a human antibody. Antigen binding sites may be wild type or modified by one or more amino acid substitutions, e.g. , modified to resemble human immunoglobulin more closely. Both chimeric and humanized antibodies are made using recombinant techniques, which are well- known in the art (see, e.g, Jones et al. (1986) Nature 321 : 522-525).

[0039] Thus, the term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or antibodies synthesized de novo using recombinant DNA methodologies (e.g, single chain Fv, a chimeric or humanized antibody).

[0040] The term“recombinant” when used with reference, e.g, to a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. [0041] A“promoter” is defined as an array of nucleic acid control sequences that direct transcription of a polynucleotide sequence. As used herein, a promoter includes necessary polynucleotide sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A“constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An“inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a polynucleotide expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second polynucleotide sequence, wherein the expression control sequence directs transcription of the polynucleotide sequence corresponding to the second sequence.

[0042] An“expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified polynucleotide elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter.

[0043] The term "heterologous" as used in the context of describing the relative location of two elements, refers to the two elements such as polynucleotide sequences ( e.g ., a promoter or a protein/polypeptide-encoding sequence) or polypeptide sequences (e.g., an Ov antigen sequence selected from SEQ ID NOs: l-l4 or another peptide sequence serving as a fusion partner with an Ov antigen sequence) that are not naturally found in the same relative positions. Thus, a“heterologous promoter” of a gene refers to a promoter that is not naturally operably linked to that gene. Similarly, a“heterologous polypeptide” or“heterologous polynucleotide” to an Ov antigen or its encoding sequence is one derived from a non-Ov origin or derived from Ov but not naturally connected to the Ov antigen in the same fashion. The fusion of an Ov antigen (or its coding sequence) with a heterologous polypeptide (or polynucleotide sequence) does not result in a longer polypeptide or polynucleotide sequence that can be found naturally in Ov.

[0044] A "label," "detectable label," or "detectable moiety" is a composition detectable by radiological, spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include radioisotopes such as ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g, as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins that can be made detectable, e.g, by incorporating a radioactive component into a polypeptide or used to detect antibodies specifically reactive with the polypeptide. Typically a detectable label is a heterologous moiety attached to a probe or a molecule (e.g, a protein or nucleic acid) with defined binding characteristics (e.g, a polypeptide with a known binding specificity or a polynucleotide), so as to allow the presence of the probe/molecule (and therefore its binding target) to be readily detectable. The heterologous nature of the label ensures that it has an origin different from that of the probe or molecule that it labels, such that the probe/molecule attached with the detectable label does not constitute a naturally occurring composition.

[0045] The phrase“specifically hybridize(s) to” refers to the binding, duplexing, or hybridization of one polynucleotide sequence to another polynucleotide sequence based on Watson-Crick nucleotide base-pairing under stringent hybridization conditions when that sequences are present in a complex mixture (e.g, total cellular or library DNA or RNA). The phrase“stringent hybridization conditions” refers to conditions under which a nucleic acid (e.g, a polynucleotide probe) will hybridize to its target nucleotide sequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5- l0°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For high stringency hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary high stringency hybridization conditions include: 50% formamide, 5x SSC and 1% SDS incubated at 42° C or 5x SSC and 1% SDS incubated at 65° C, with a wash in 0.2x SSC and 0.1% SDS at 65° C.

[0046] The term“immunoassay” describes an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to identify, isolate, target, and/or detect the presence or quantity of the antigen.

[0047] The phrase“specifically binds,” when used to describe the binding relationship between an antibody and its target antigen, refers to a binding reaction that is determinative of the presence of the antigen ( e.g ., a polypeptide) in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular polypeptide at least two times the background and do not substantially bind in a significant amount to other polypeptides or other antigens present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to an Ov antigen having the amino acid sequence of any one of SEQ ID NOs: 1-14, can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with that specific Ov antigen and not with other proteins, e.g., proteins derived from other filariae (e.g, B. malayi, L. loa, and W. bancrofti ) or host animals including mammals such as human. This selection may be achieved by subtracting out antibodies that cross-react with molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g, Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific binding reaction will yield at least twice of the background signal or noise and more typically more than 10, 20, 50, or up to 100 times the background.

[0048] By“host cell” is meant a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa and the like, e.g, cultured cells, explants, and cells in vivo.

[0049] A“biological sample,” as used herein, is a sample of biological tissue or fluid that potentially contains an Ov antigen of any one of SEQ ID NOs: 1-14, or nucleic acid encoding such Ov antigen, or an antibody specifically binding the antigen. Such samples include, but are not limited to, bodily fluids such as blood (including whole blood or any fraction of blood such as serum or plasma) or urine, small segments isolated from relevant organs or tissues (e.g, skin or eyes), and secretion such as sweat or tear taken from an animal (especially a mammal, including human) susceptible to Ov infection, including humans. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

[0050] Onchocerca volvulus (Ov) is the pathogen responsible for onchocerciasis or river blindness. The particular life cycle and the nature of currently available treatment for Ov infection requires monitoring of the presence of adult female Onchocerca volvulus (OvAF) in a patient’s body to ascertain the disease status and to ensure complete eradication of the parasite.

[0051] The present inventors have identified 14 new antigens that are specific for OvAF and can serve as effective and sensitive markers for the presence of OvAF in patients, including those who have been undergoing treatment for Ov infection. This disclosure thus relates to the production and use of these novel antigens as well as their antibodies.

II. Production of New Ov Antigens

A. General Recombinant Technology

[0052] Basic texts disclosing general methods and techniques in the field of recombinant genetics include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel el a/., eds., Current Protocols in Molecular Biology (1994).

[0053] For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.

[0054] Oligonucleotides that are not commercially available can be chemically synthesized, e.g. , according to the solid phase phosphoramidite triester method first described by

Beaucage & Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al. , Nucleic Acids Res. 12: 6159-6168 (1984). Purification of oligonucleotides is performed using any art-recognized strategy, e.g ., native acrylamide gel electrophoresis or anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255: 137-149 (1983).

[0055] The sequence of a polynucleotide encoding an Ov antigen and related fusion protein can be verified after cloning or subcloning using, e.g. , the chain termination method for sequencing double-stranded templates of Wallace et al. , Gene 16: 21-26 (1981).

B. Cloning and Subcloning of Coding Sequences for Ov Antigens

[0056] Polynucleotide sequences encoding Ov antigens can be determined based on their amino acid sequences (e.g, any one of SEQ ID NOs: l-l4) and available information of the Ov genome. They can be isolated from an Ov cDNA or genomic library or can be synthesized by a commercial supplier.

[0057] A nucleic acid sequence encoding an Ov antigen of this invention can be isolated from an Ov cDNA or genomic DNA library using standard cloning techniques such as polymerase chain reaction (PCR). Most commonly used techniques for this purpose are described in standard texts, e.g, Sambrook and Russell, supra.

[0058] cDNA libraries suitable for obtaining a coding sequence for an Ov antigen may be commercially available or can be constructed. The general methods of isolating mRNA, making cDNA by reverse transcription, ligating cDNA into a recombinant vector, transfecting into a recombinant host for propagation, screening, and cloning are well known (see, e.g,

Gubler and Hoffman, Gene, 25: 263-269 (1983); Ausubel etal, supra). Upon obtaining an amplified segment of nucleotide sequence by PCR, the segment can be further used as a probe to isolate a longer length polynucleotide sequence encoding the Ov antigen from the cDNA library. A general description of appropriate procedures can be found in Sambrook and Russell, supra.

[0059] Based on sequence homology, degenerate oligonucleotides can be designed as primer sets and PCR can be performed under suitable conditions (see, e.g, White et al, PCR Protocols: Current Methods and Applications, 1993; Griffin and Griffin, PCR Technology, CRC Press Inc. 1994) to amplify a segment of nucleotide sequence from a cDNA or genomic library. Using the amplified segment as a probe, a longer length nucleic acid encoding an Ov polypeptide antigen is obtained. [0060] Upon acquiring a nucleic acid sequence encoding an Ov antigen, the coding sequence can be modified as appropriate ( e.g . , adding a coding sequence for a heterologous tag, such as an affinity tag, for example, 6 x His tag or GST tag) and then be subcloned into a vector, for instance, an expression vector, so that a recombinant Ov peptide antigen can be produced from the resulting construct, for example, after transfection and culturing host cells under conditions permitting recombinant protein expression directed by a promoter operably linked to the coding sequence.

C. Modification of Nucleic Acids for Preferred Codon Usage in a Host Organism

[0061] The polynucleotide sequence encoding an Ov polypeptide antigen can be further altered to coincide with the preferred codon usage of a particular host. For example, the preferred codon usage of one strain of bacterial cells can be used to derive a polynucleotide that encodes an Ov antigen of the invention and includes the codons favored by this strain. The frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage in a large number of genes expressed by the host cell (e.g., calculation service is available from web site of the Kazusa DNA Research Institute, Japan). This analysis is preferably limited to genes that are highly expressed by the host cell.

[0062] At the completion of modification, the coding sequences are verified by sequencing and are then subcloned into an appropriate expression vector for recombinant production of the Ov polypeptide antigens.

D. Chemical Synthesis of Ov Antigens

[0063] The amino acid sequences of the Ov polypeptide antigens of this invention are provided in SEQ ID NOs: 1-14. These antigens in some cases may be presented in the form of fusion proteins containing at least one, perhaps two (e.g, one on each of the N-terminus and C-terminus) peptides from a heterologous origin, such as affinity tag for the ease of detection and/or isolation of the antigens. Aside from recombinant production, these polypeptides can also be synthesized chemically using conventional peptide synthesis or other protocols well known in the art.

[0064] Polypeptides may be synthesized by solid-phase peptide synthesis methods using procedures similar to those described by Merrifield et al, J. Am. Chem. Soc., 85:2149-2156 (1963); Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Gross and Meienhofer (eds.), Academic Press, N.Y., vol. 2, pp. 3-284 (1980); and Stewart et al. , Solid Phase Peptide Synthesis 2nd ed., Pierce Chem. Co.,

Rockford, Ill. (1984). During synthesis, N-a-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to a solid support, i.e ., polystyrene beads. The peptides are synthesized by linking an amino group of an N-a-deprotected amino acid to an a-carboxy group of an N-a-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation. The most commonly used N-a-protecting groups include Boc, which is acid labile, and Fmoc, which is base labile.

[0065] Materials suitable for use as the solid support are well known to those of skill in the art and include, but are not limited to, the following: halomethyl resins, such as chloromethyl resin or bromomethyl resin; hydroxymethyl resins; phenol resins, such as 4-(a-[2,4- dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin; tert-alkyloxycarbonyl-hydrazidated resins, and the like. Such resins are commercially available, and their methods of preparation are known by those of ordinary skill in the art.

[0066] Briefly, the C-terminal N-a-protected amino acid is first attached to the solid support. The N-a-protecting group is then removed. The deprotected a-amino group is coupled to the activated a-carboxylate group of the next N-a-protected amino acid. The process is repeated until the desired peptide is synthesized. The resulting peptides are then cleaved from the insoluble polymer support and the amino acid side chains deprotected.

Longer peptides can be derived by condensation of protected peptide fragments. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art and so are not discussed in detail herein (See, Atherton et al. , Solid Phase Peptide Synthesis: A Practical Approach , IRL Press (1989), and Bodanszky, Peptide Chemistry, A Practical Textbook , 2nd Ed., Springer-Verlag (1993)).

IV. Expression and Purification of Ov Antigens

[0067] Following verification of the coding sequence, the Ov antigens or fusion proteins of the present invention can be produced using routine techniques in the field of recombinant genetics, relying on the polynucleotide sequences encoding the polypeptides disclosed herein.

A. Expression Systems

[0068] To obtain high level expression of a nucleic acid encoding an Ov polypeptide antigen or fusion polypeptide of the present invention, one typically subclones a polynucleotide encoding the polypeptide into an expression vector that contains a strong promoter (typically heterologous, i.e., of non-Ov origin) to direct transcription, a

transcription/translation terminator and a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g ., in Sambrook and Russell, supra, and Ausubel et al, supra. Bacterial expression systems for expressing a recombinant polypeptide are available in, e.g. , E. coli, Bacillus sp., Salmonella , and

Caulobacter. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. In one embodiment, the eukaryotic expression vector is an adenoviral vector, an adeno-associated vector, or a retroviral vector.

[0069] The promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is optionally positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

[0070] In addition to the promoter, the expression vector typically includes a transcription unit or expression cassette that contains all the additional elements required for the expression of the Ov antigen or fusion polypeptide in host cells. A typical expression cassette thus contains a promoter operably linked to the coding sequence and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation

termination. The nucleic acid sequence encoding the Ov antigen or fusion polypeptide is typically linked to a cleavable signal peptide sequence to promote secretion of the

recombinant polypeptide by the transformed cell. Such signal peptides include, among others, the signal peptides from tissue plasminogen activator, insulin, and neuron growth factor, and juvenile hormone esterase of Heliothis virescens. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

[0071] In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. [0072] The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g ., c-myc.

[0073] Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g. , SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺, rMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0074] Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.

Alternatively, high yield expression systems not involving gene amplification are also suitable, such as a baculovirus vector in insect cells, with a polynucleotide sequence encoding the Ov antigen or fusion polypeptide under the direction of the polyhedrin promoter or other strong baculovirus promoters.

[0075] The elements that are typically included in expression vectors also include a replicon that functions in E. coli , a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable.

The prokaryotic sequences are optionally chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary. Similar to antibiotic resistance selection markers, metabolic selection markers based on known metabolic pathways may also be used as a means for selecting transformed host cells.

[0076] When periplasmic expression of a recombinant protein (e.g, an Ov antigen or fusion polypeptide of the present invention) is desired, the expression vector further comprises a sequence encoding a secretion signal, such as the E. coli OppA (Periplasmic Oligopeptide Binding Protein) secretion signal or a modified version thereof, which is directly connected to 5' of the coding sequence of the protein to be expressed. This signal sequence directs the recombinant protein produced in cytoplasm through the cell membrane into the periplasmic space. The expression vector may further comprise a coding sequence for signal peptidase 1, which is capable of enzymatically cleaving the signal sequence when the recombinant protein is entering the periplasmic space. More detailed description for periplasmic production of a recombinant protein can be found in, e.g., Gray et al, Gene 39: 247-254 (1985), U.S. Patent Nos. 6,160,089 and 6,436,674.

B. Transfection Methods

[0077] Standard transfection methods are used to produce bacterial, mammalian, yeast, insect, or plant cell lines that express large quantities of a recombinant polypeptide, which are then purified using standard techniques (see, e.g. , Colley el al., J. Biol. Chem. 264: 17619- 17622 (1989); Guide to Protein Purification, m Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g, Morrison, J. Bact. 132: 349-351 (1977); Clark-Curtiss & Curtiss , Methods in Enzymology 101: 347-362 (Wu et al, eds, 1983).

[0078] Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, e.g, Sambrook and Russell, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the recombinant polypeptide.

C. Detection of Recombinant Expression of Ov Antigen in Host Cells

[0079] After the expression vector is introduced into appropriate host cells, the transfected cells are cultured under conditions favoring expression of the Ov antigen or fusion

polypeptide. The cells are then screened for the expression of the recombinant polypeptide, which is subsequently recovered from the culture using standard techniques (see, e.g,

Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al, supra, and Sambrook and Russell, supra). [0080] Several general methods for screening gene expression are well known among those skilled in the art. First, gene expression can be detected at the nucleic acid level. A variety of methods of specific DNA and RNA measurement using nucleic acid hybridization techniques are commonly used ( e.g . , Sambrook and Russell, supra). Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA and Northern blot for detecting RNA), but detection of DNA or RNA can be carried out without electrophoresis as well (such as by dot blot). The presence of nucleic acid encoding an Ov antigen or fusion polypeptide in transfected cells can also be detected by PCR or RT-PCR using sequence- specific primers.

[0081] Second, gene expression can be detected at the polypeptide level. Various immunological assays are routinely used by those skilled in the art to measure the level of a gene product, particularly using polyclonal or monoclonal antibodies that react specifically with an Ov antigen of the present invention (e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988; Kohler and Milstein, Nature, 256: 495-497 (1975)). Such techniques require antibody preparation by selecting antibodies with high specificity against the Ov antigen. The methods of raising polyclonal and monoclonal antibodies are well established and their descriptions can be found in the literature, see, e.g, Harlow and Lane, supra ; Kohler and Milstein, Eur. J. Immunol., 6: 511-519 (1976). More detailed descriptions of preparing antibodies against the Ov antigens of the present invention and conducting immunological assays detecting the polypeptides containing the antigens are provided in a later section.

D. Purification of Recombinantly Produced Ov Antigens

[0082] Once the expression of a recombinant Ov antigen or fusion polypeptide in transfected host cells is confirmed, the host cells are then cultured in an appropriate scale for the purpose of purifying the recombinant polypeptide.

1. Purification of Recombinantly Produced Polypeptide from Bacteria

[0083] When the Ov antigen or fusion polypeptides of the present invention are produced recombinantly by transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the polypeptides may form insoluble aggregates. There are several protocols that are suitable for purification of protein inclusion bodies. For example, purification of aggregate proteins (hereinafter referred to as inclusion bodies) typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells, e.g, by incubation in a buffer of about 100-150 pg/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent. The cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al. and Sambrook and Russell, both supra , and will be apparent to those of skill in the art.

[0084] The cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g. ,

20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible. The remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g, 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers will be apparent to those of skill in the art.

[0085] Following the washing step, the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a

combination of solvents each having one of these properties). The proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents that are capable of solubilizing aggregate-forming proteins, such as SDS (sodium dodecyl sulfate) and 70% formic acid, may be inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine

hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of the immunologically and/or biologically active protein of interest. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques. For further description of purifying recombinant polypeptides from bacterial inclusion body, see, e.g., Patra et al, Protein Expression and Purification 18: 182- 190 (2000).

[0086] Alternatively, it is possible to purify recombinant polypeptides, e.g, an Ov antigen or fusion polypeptide, from bacterial periplasm. Where the recombinant protein is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see e.g, Ausubel et al. , supra). To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice- cold 5 mM MgSCri and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.

2. Standard Protein Separation Techniques for Purification

[0087] When a recombinant polypeptide, e.g ., an Ov antigen or fusion polypeptide of the present invention, is expressed in host cells in a soluble form, its purification can follow the standard protein purification procedure described below. This standard purification procedure is also suitable for purifying an Ov antigen or fusion polypeptide obtained from chemical synthesis. i. Solubility Fractionation

[0088] Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20- 30%. This will precipitate the most hydrophobic proteins. The precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, through either dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures. ii. Size Differential Filtration

[0089] Based on a calculated molecular weight, a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of a protein of interest, e.g ., an Ov antigen or fusion polypeptide. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below. iii. Column Chromatography

[0090] The proteins of interest (such as an Ov antigen or fusion polypeptide of the present invention) can also be separated from other proteins on the basis of their size, net surface charge, hydrophobicity, or affinity for ligands. In addition, antibodies raised against an Ov antigen can be conjugated to column matrices and the Ov antigen immunopurified. All of these methods are well known in the art.

[0091] It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g, Pharmacia Biotech).

V. Immunoassays for Detection of Ov Antigens

[0092] One aspect of this invention provides immunoassays used in the detection of OvAF- specific antigens in order to detect the presence of OvAF in a patient’s body. Antibodies against the Ov antigens described herein are useful for carrying out these immunological assays.

A. Production of Antibodies Against An Ov Antigen

[0093] Methods for producing polyclonal and monoclonal antibodies that react specifically with an immunogen of interest are known to those of skill in the art (see, e.g, Coligan, Current Protocols in Immunology Wiley/Greene, NY, 1991; Harlow and Lane, Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY, 1989; Stites el al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, NY, 1986; and Kohler and Mil stein Nature 256: 495-497, 1975). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors (see, Huse et al, Science 246: 1275-1281, 1989; and Ward et al, Nature 341: 544-546, 1989). [0094] In order to produce antisera containing antibodies with desired specificity, the polypeptide of interest ( e.g ., an Ov peptide antigen of the present invention) or an antigenic fragment thereof can be used to immunize suitable animals, e.g., mice, rabbits, or primates.

A standard adjuvant, such as Freund’s adjuvant, can be used in accordance with a standard immunization protocol. Alternatively, a synthetic antigenic peptide derived from that particular polypeptide can be conjugated to a carrier protein and subsequently used as an immunogen.

[0095] The animal’s immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the antigen of interest. When appropriately high titers of antibody to the antigen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich antibodies specifically reactive to the antigen and purification of the antibodies can be performed subsequently, see, Harlow and Lane, supra, and the general descriptions of protein purification provided above.

[0096] Monoclonal antibodies are obtained using various techniques familiar to those of skill in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976). Alternative methods of immortalization include, e.g., transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and the yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.

[0097] Additionally, monoclonal antibodies may also be recombinantly produced upon identification of nucleic acid sequences encoding an antibody with desired specificity or a binding fragment of such antibody by screening a human B cell cDNA library according to the general protocol outlined by Huse et al, supra. The general principles and methods of recombinant polypeptide production discussed above are applicable for antibody production by recombinant methods.

B. Immunoassays for Detecting Ov Antigens

[0098] Once antibodies specific for an Ov peptide antigen of the present invention are available, the amount of the relevant polypeptide in a sample, e.g, a blood/serum/plasma sample, or a skin or urine sample, can be measured by a variety of immunoassay methods providing qualitative and quantitative results to a skilled artisan. For a review of

immunological and immunoassay procedures in general see , e.g., Stites, supra; U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168.

1. Labeling in Immunoassays

[0099] Immunoassays often utilize a labeling agent to specifically bind to and label the binding complex formed by the antibody and the target protein. The labeling agent may itself be one of the moieties comprising the antibody/target protein complex, or may be a third moiety, such as another antibody, that specifically binds to the antibody/target protein complex. A label may be detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Examples include, but are not limited to, magnetic beads (e.g, Dynabeads™), fluorescent dyes (e.g, fluorescein

isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g, ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g, horse radish peroxidase, alkaline phosphatase, and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g, polystyrene, polypropylene, latex, etc.) beads.

[0100] In some cases, the labeling agent is a second antibody bearing a detectable label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second antibody can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.

[0101] Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G, can also be used as the label agents. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non- immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally, Kronval, et al. J. Immunol., Ill: 1401-1406 (1973); and Akerstrom, et al,

J. Immunol., 135: 2589-2542 (1985)).

2. Immunoassay Formats

[0102] Immunoassays for detecting a target protein of interest (e.g. , a polypeptide comprising an Ov antigen of any one of SEQ ID NOs: 1-14) from samples may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured target protein is directly measured. In one preferred“sandwich” assay, for example, the antibody specific for the target protein can be bound directly to a solid substrate where the antibody is immobilized. It then captures the target protein in test samples. The antibody/target protein complex thus immobilized is then bound by a labeling agent, such as a second or third antibody bearing a label, as described above.

[0103] In competitive assays, the amount of target protein in a sample is measured indirectly by measuring the amount of an added (exogenous) target protein displaced (or competed away) from an antibody specific for the target protein by the target protein present in the sample. In a typical example of such an assay, the antibody is immobilized and the exogenous target protein is labeled. Since the amount of the exogenous target protein bound to the antibody is inversely proportional to the concentration of the target protein present in the sample, the target protein level in the sample can thus be determined based on the amount of exogenous target protein bound to the antibody and thus immobilized.

[0104] In some cases, western blot (immunoblot) analysis is used to detect and quantify the presence of an Ov antigen or an Ov polypeptide containing the antigen in the samples. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support (such as a nitrocellulose filter, a nylon filter, or a derivatized nylon filter) and incubating the samples with the antibodies that specifically bind the target protein. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies ( e.g ., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies against an Ov antigen.

[0105] Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see, Monroe et al, Amer. Clin. Prod. Rev., 5: 34-41 (1986)).

[0106] In practicing the method of the present invention for detecting OvAF in a patient, a protein or an antibody array may be used for performing an immunoassay. For example, a plurality of the OvAF-specific antigens (e.g, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 selected from SEQ ID NOs: l-l4, especially SEQ ID NO:7 or 8) or fusion polypeptides each comprising one of the antigens are immobilized to a solid substrate at a predetermined location to form an array, which then may be used in an immunoassay for detecting in a sample taken from a patient the presence of antibodies that specifically bind to the antigens. Typically, positive results with multiple antigens, e.g, at least 10%, 25%, 33%, or 50% of all antigens on the array, indicate the presence of OvAF in the patient.

[0107] Similarly, a plurality of antibodies that each specifically binds an OvAF-specific antigen (e.g, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 selected from SEQ ID NOs: l-l4, especially SEQ ID NO:7 or 8) are immobilized to a solid substrate at a

predetermined location to form an array, which then may be used in an immunoassay for detecting in a sample taken from a patient the presence of the OvAF-specific antigens.

Typically, positive results with multiple antibodies, e.g, at least 10%, 25%, 33%, or 50% of all antibodies on the array, indicate the presence of OvAF in the patient. Suitable antibodies may be polyclonal or monoclonal in nature.

[0108] For these immunoassays, the patient being tested may be one who is at risk of Ov infection but has not demonstrated any symptoms of an active infection or may be one who has previously been diagnosed with Ov infection and has received treatment (e.g, ivermectin administration).

VI. KITS

[0109] The invention also provides kits for detecting Onchocerca volvulus infection, especially the presence of OvAF, in a subject according to the method of the present invention. The kits typically include a first container that contains a composition including a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 1-14, and a second container containing a positive control sample that is taken, optionally processed, from a patient who has been confirmed to have active Onchocerca volvulus infection with OvAF presence in his body. Optionally, the kit may include a negative control sample, which is taken (optionally processed) from a subject who has been confirmed to be without Onchocerca volvulus, i.e., without any detectable OvAF in his body. The polypeptide may be one of a plurality of polypeptides, each comprising or consisting of a different amino acid sequence selected from SEQ ID NOs: 1-14. The polypeptide(s) maybe in form of an array with the polypeptide(s) immobilized to a solid substrate, and the array may be one suitable for use in an immunoassay such as ELISA.

[0110] An alternative version of the kit contains, instead of a composition comprising a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 1-14 in the first container, a composition comprising an antibody that specifically binds an amino acid sequence selected from SEQ ID NO: 1-14. The kit may contain the positive control sample and negative control sample as described above. Similarly, the antibody may be one of a plurality of antibodies (which may be monoclonal or polyclonal antibodies), each specifically binding a different amino acid sequence selected from SEQ ID NOs: 1-14. The antibody or antibodies maybe in form of an array with the antibody or antibodies immobilized to a solid substrate, and the array may be one suitable for use in an immunoassay such as ELISA.,

[0111] In addition, the kit may be further include informational material containing instructions for a user on how to use the kit for performing an assay and determining whether OvAF is present in a patient sample.

EXAMPLES

[0112] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.

Example 1: Ov adult specific genes OVOC8995 and OVOC12838

INTRODUCTION

[0113] There has been considerable success in the control of onchocerciasis, commonly known as“river blindness.” As efforts shift toward disease elimination in Africa and particularly toward the end-game, better tools are needed to identify viable adult female worms of Onchocerca volvulus (OvAF), so as to be able to verify elimination (by their absence).

[0114] Onchocerciasis, commonly known as river blindness, is caused by a parasitic nematode, Onchocerca volvulus {Ov). It is transmitted by Simulium (blackfly) vectors. Infection leads to severe and disfiguring skin disease, lymphadenopathy, and visual impairment (including blindness). Onchocerciasis especially affects resource limited rural communities and leads to serious economic losses. An estimated 120 million people are at risk, with 39 million infected with Ov [1]

[0115] Although more than 99 percent of onchocerciasis infections are found in Africa, there are isolated foci of onchocerciasis outside of Africa, most notably in countries in Central and South America. The Onchocerciasis Elimination Program for the Americas (OEPA) was launched in 1992 and has taken advantage of the ivermectin (Mectizan®) donation program of Merck & Co. Because ivermectin is a microfilaricide, individuals must take ivermectin (annually or semi-annually) for 15-20 years, the time considered necessary for adult worms to die (or be beyond their reproductive life). The adult female worm (OvAF) has a reproductive lifespan of up at least 11 years [2,3] OEPA has made significant progress in the Americas in controlling onchocerciasis and is now moving toward elimination of the disease and interruption of transmission from the region [4] In Africa, efforts focused primarily on disease control. The strategic objective of the African Programme for

Onchocerciasis Control (APOC) is to protect hypoendemic communities at risk in the 19 APOC countries through the establishment of community-directed annual treatment with ivermectin [5] There are significant epidemiological and entomological differences that make onchocerciasis control more difficult in Africa than in the Americas. Factors that hamper control efforts include geographic coverage, human and fly migration, vector efficiency, and co-endemicity with Loa loa infections [6] Recent results from Senegal and regions in Mali, however, suggest that elimination of onchocerciasis in Africa in some regions using ivermectin is possible [7]

[0116] In response to these promising results, APOC has adopted a shift in strategy, moving from purely control efforts toward assessment of the possibility for disease elimination [8] As APOC refines its strategy, the development of better diagnostic tools is greatly needed, especially for post-treatment surveillance where transmission of infection has been brought under control, the certification phase, and for meso- and hypo-endemic areas that had heretofore been ivermectin-naive.

[0117] Currently available tools for the diagnosis for Ov infection are far from ideal and have largely relied on the skin snip as the gold standard. Although it has high specificity, skin snip has low sensitivity in areas of low prevalence and in communities undergoing ivermectin treatment or other control efforts. In such communities, microfilariae (MF) are often absent in skin snips. Furthermore, skin snip microscopy cannot detect infection during the prepatent period (estimated to be 9-15 months following the bite of an infected black fly). Furthermore, because the skin snip is a relatively invasive procedure (and there is also concern of transmission of infectious agents [ e.g ., HIV, Hepatitis B/C)] by the corneoscleral punch biopsy instrument itself), there is growing resistance to its use. PCR on skin snips, however, probably has the highest degree of sensitivity and specificity, but its utility is limited by cost and not being point of care [9]

[0118] Although there have been many efforts attempted to identify an Ov-specific antigen test that could be used in the blood or urine of patients with Ov infection, to date all of these attempts have been unsuccessful. Antibody-based immunoassays, and particularly the Ov- 16 based tests that were developed by the inventors’ group have shown promise for use in Africa as affordable, sensitive, and field friendly [10, 11] In an enzyme-linked immunosorbent assay (ELISA) format, the absence of ()v- 16 specific antibodies has been one of the criteria used to certify areas free of Ov transmission [12] Such an approach has been used successfully by OEPA in the Americas [12-16] (and now by others in parts of ETganda, Tanzania and Yemen). More recently, multiantigen-based serology assays using the luciferase immunoprecipitation systems (LIPS) platform have been developed [17] The analytical performance of these LIPS-based assays appears to be very good, especially for distinguishing Ov infection from other filarial infections such as W. bancroiti and L. loa. However, this technology requires sophisticated equipment and reagents that are not universally available.

[0119] Rapid format Ov- 16 platforms are under development and almost ready for deployment and validation (for a second time). However, because this assay (and others like it) was configured to detect early infections and thus early (L3 and L4-specific) responses in transmission surveillance assessments, additional tools are needed as transmission in communities is reduced and it becomes necessary to identify any (and all) individuals with viable adult female worms. This requires the development of new approaches for the identification of humans with viable adult females of Ov. The ultimate objective of the present study is to identify host- and parasite-specific biomarker(s) present in human subjects with viable adult females of Ov (hereafter OvAF) and to develop and configure rapid point of care methods to detect (or sense) these biomarkers.

[0120] Over the past 3 years, using a variety of techniques (including proteomics, comparative transcriptomics, genomics) the present inventors have identified 14 biomarkers that could be used a sensitive indicators of active onchocerciasis or to assess macrofilaricidal activity of new or repurposed drugs.

RESULTS AND DISCUSSIONS

[0121] Having identified 3 O. volvulus (Ov) adult specific genes (OVOC8995,

OVOC12838 and OVOC12448) through comparative transcriptomics based on RNAseq data from adult females, each of these was synthesized by solid-phase synthesis as full-length proteins or immunogenic peptides. Antisera were raised against immunogenic peptides derived from OVOC8995 (SEQ ID NO:8) and OVOC12838 (SEQ ID NO:7) in rabbits. Rabbit IgG was affinity purified over agarose coupled to each of the peptides. These antibodies were used to configure sandwich immunoassays for the detection of OVOC12838 and OVOC8995. As seen in Figure 1, the assays configured were able to detect 350 pg/ml of OVOC12838 and 3.5 ng/ml OVOC8995.

[0122] Having shown that antibody-based immunoassays could be used to detect antigen in serum (Figure 1 above), the inventors optimized concurrently 2 sandwich ELISA assays and showed that either OVOC8995 or OVOC12838 could be detected in selected Ov-infected serum and serum pools. Next, Ov antigen detection immunoassays for both antigens were used to screen Ov-infected sera (n=204) as well as control sera that consisted of: 1) healthy North American volunteers (n=38); 2) African and South American control sera from Ov- non-endemic regions (n=l2); 3) sera from patients with W. bancrofti (from India and the Cook Islands; n=20) and 4) sera from patients with L. loa (from Ov non-endemic regions of Benin, Gabon, Central African Republic, and Cameroon; n=44). Using ROC analyses, cutoffs at 100% Ov-specificity were obtained. Then using these cutoffs, archived serum from mf positive Ov-infected patients from the Americas (Guatemala, Ecuador), West and Central Africa (Ghana, Cameroon, Nigeria), and East Africa (Uganda), were used to quantify OVOC8995 and OVOC12838 antigen. As can be seen in Figure 2, OVOC8995 and

OVOC12838 antigen could be measured in most of the mf positive Ov-infected sera.

[0123] Thus, using these 2 immunoassays based on monospecific polyclonal antibodies, it was possible to detect one or both antigens in all but 22/204 mf positive sera (Figure 3). This gives a sensitivity of 89% at 100% specificity with a positive predictive value of 100%.

[0124] Using sera from travelers who acquired O. volvulus and were definitively treated with semi-annual ivermectin for at least 5 years (and who never traveled back to Ov-endemic regions of the world) obtained before (PreTx) and 5-10 years following treatment (PostTx), OVOC8995 levels were measured. As can be seen in Figure 4, all patients responded to definitive treatment with a drop in antigen levels, some to levels below the cutoff for positivity.

[0125] The generation of monoclonal antibodies to OVOC8995 will provide additional sensitivity to this assay. Further, monoclonal antibodies to OVOC12838 can also provide better sensitivity without loss of specificity. Both of these immunoassays are easily transferrable to lateral flow assays given that there are two distinct antibodies, each specifically binding to a distinct antigen.

[0126] As no one antigen detection system (detection of a single antigen) is likely to meet all of the proposed criteria (and because the above assays may not perform as well in all settings,) also being considered for use are 12 additional antigens, which are supported by empirical proteomic data (statistically validated with multiple replicates and appropriate controls) obtained from sera/urine collected from Ob’- infected humans (h=10 each) and serum/urine from humanized mice following Ov infection or post-implant of L5s (_juvenile adults). The properties that make each of these good possible candidates are listed below in Table 1. Each is highly rib-specific and have little to no significant homology to other filariae (e.g., B. malayi, L. loa, W. bancrofti ); nor do they have homologues in humans or mice.

Example 2: Ov adult specific genes OVOC5398 and OVOC8934

[0127] Two predicted immunogenic peptides were synthesized for O. volvulus (rib) adult specific genes OVOC5398 (SEQ ID NO: 11) and OVOC5398 (SEQ ID NO: 12). Their amino acid sequences are provided in SEQ ID NO: 15-18. The peptides were conjugated with KLH for raising antisera. In addition, full-length proteins OVOC5398 and OVOC8934 were also synthesized using solid phase synthesis. Rabbits were immunized and hyper-immune sera raised against each KLH-conjugated peptide. Total IgG to each peptide was affinity purified over Protein A/G column. The purified antibodies were conjugated with long chain biotin or horseradish peroxidase (HRP).

[0128] As shown in Figure 5, the purified IgG raised against each peptide was able to detect the corresponding full-length proteins, most of the time better than the peptides in a direct ELISA.

[0129] As shown in Figure 6, the antigen detection assay is optimized: for each of the proteins, a-Pep 1 or a-Pep 2 were used as capture antibodies and the other antibody conjugated to biotin (Bt) or HRP were used as detection antibody. ETsing either combination (a-Pep 1 <> a-Pep 2-Bt; or a-Pep 2 <> a-Pep 1 -Bt), OVOC8934 detectability was much better than OVOC5398. a-Pep 1 <> a-Pep 2-Bt combination for OVOC8934 was slightly better than the combination of a-Pep 2 <> a-Pep l-Bt. The HRP conjugated antibody had higher background noise compared to biotinylated antibodies (data not shown).

[0130] As shown in Figure 7, pooled sera were tested with either a-Pep 1 <> a-Pep 2-Bt or a-Pep 2 <> a-Pep l-Bt combinations for OVOC8934. 4 pools of 6-sera each from O.

volvulus infected individuals (OV) and healthy blood bank individuals (BB) were tested for OVOC8934. The graphs in panels A and B depict the signal to noise values using both combinations. [0131] As shown in Figure 8, infected individuals were found to have detectable levels of IgG to OVOC8934. The graph depicts the signal to noise ratio of total IgG in the sera/plasma of infected (OV) and blood bank normal (BB).

[0132] As shown in Figure 9, individual samples can be tested using the antibodies raised against peptides from OVOC5398 or OVOC8934. Because the 1 :4 dilution of samples gave background noise in the blood bank samples, increasing the dilution to 1 :20 resulted in losing all but one of the blood bank normals (blue circles), with little to no loss in the ov-infected individuals (red circles).

[0133] All patents, patent applications, and other publications, including GenBank

Accession Numbers, cited in this application are incorporated by reference in the entirety for all purposes.

References

1) Winthrop KL, Furtado JM, Silva JC, Resnikoff S, Lansingh VC (2011) River blindness: an old disease on the brink of elimination and control. J Glob Infect Dis 3: 151-155.

2) Boatin BA, Richards FO, Jr. (2006) Control of onchocerciasis. Adv Parasitol 61 : 349-394.

3) Habbema JD, Alley ES, Plaisier AP, van Oortmarssen GJ, Remme JH (1992)

Epidemiological modelling for onchocerciasis control. Parasitol Today 8: 99-103.

4) Cupp EW, Sauerbrey M, Richards F (2011) Elimination of human onchocerciasis: history of progress and current feasibility using ivermectin (Mectizan((R))) monotherapy. Acta Tropl20 Suppl 1 : S 100- 108.

5) World Health Organization. African Programme for Onchocerciasis Control (APOC). WHO; 2012. Available at website: www.who.int/apoc/en/ Accessed November 25, 2012.

6) Gardon J, Gardon-Wendel N, Demanga N, Kamgno J, Chippaux JP, Boussinesq M

(l997)Serious reactions after mass treatment of onchocerciasis with ivermectin in an area endemicfor Loa loa infection. Lancet 350: 18-22.

7) Diawara L, Traore MO, Badji A, Bissan Y, Doumbia K, Goita SF, Konate L, Mounkoro K, Sarr MD, Seek AF, Toe L, Touree S, Remme JH (2009) Feasibility of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: first evidence from studies in Mali and Senegal. PLoS Negl Trop Dis 3: e497.

8) African Programme for Onchocerciasis control. Addendum for the plan of action and budget 2008- 2015. Ouagadougou, Burkina Faso: African Programme for Onchocerciasis Control, World Health Organization; 2010.

9) Zimmerman PA, Guderian RH, Aruajo E, Elson L, Phadke P, Kubofcik J, Nutman TB (1994) Polymerase chain reaction-based diagnosis of Onchocerca volvulus infection:

improved detection of patients with onchocerciasis. J Infect Dis 169: 686-689.

10) Lobos E, Weiss N, Karam M, Taylor HR, Ottesen EA, Nutman TB (1991) An

immunogenic Onchocerca volvulus antigen: a specific and early marker of infection. Science 251 : 1603-1605. 11) Weil GJ, Steel C, Liftis F, Li BW, Mearns G, Lobos E, Nutman TB (2000) A rapid- format antibody card test for diagnosis of onchocerciasis. J Infect Dis 182: 1796-1799.

12) Lindblade KA, Arana B, Zea-Flores G, Rizzo N, Porter CH, Dominguez A, Cruz-Ortiz N, Unnasch TR, Punkosdy GA, Richards J, Sauerbrey M, Castro J, Catu E, Oliva O, Richards FO,Jr. (2007) Elimination of Onchocercia volvulus transmission in the Santa Rosa focus of Guatemala. Am J Trop Med Hyg 77: 334-341.

13) Gonzalez RJ, Cruz-Ortiz N, Rizzo N, Richards J, Zea-Flores G, Dominguez A, Sauerbrey M, Catu E, Oliva O, Richards FO, Lindblade KA (2009) Successful interruption of transmission of Onchocerca volvulus in the Escuintla-Guatemala focus, Guatemala. PLoS Negl Trop Dis 3: e404.

14) Rodriguez-Perez MA, Unnasch TR, Dominguez- Vazquez A, Morales-Castro AL, Pena- Flores GP, et al. (2010) Interruption of transmission of Onchocerca volvulus in the Oaxaca focus, Mexico. Am J Trop Med Hyg 83: 21-27.

15) Rodriguez-Perez MA, Unnasch TR, Dominguez- Vazquez A, Morales-Castro AL, Richards F, Jr., Pena-Flores GP, Orozco- Algarra ME, Prado-Velasco G (2010) Lack of active Onchocerca volvulus transmission in the northern Chiapas focus of Mexico. Am J Trop Med Hyg 83: 15- 20.

16) Cruz-Ortiz N, Gonzalez RJ, Lindblade KA, Richards FO, Jr., Sauerbrey M, Zea-Flores G, Dominguez A, Oliva O, Catu E, Rizzo N (2012) Elimination of Onchocerca volvulus

Transmission in the Huehuetenango Focus of Guatemala. J Parasitol Res 2012: 638429.

17) Burbelo PD, Leahy HP, Iadarola MJ, Nutman TB (2009) A four-antigen mixture for rapid assessment of Onchocerca volvulus infection. PLoS Negl Trop Dis 3: e438.

18) Fink DL, Kamgno J, Nutman TB (2011) Rapid molecular assays for specific detection and quantitation of Loa loa microfilaremia. PLoS Negl Trop Dis 5: el299.

19) Bennuru S, Meng Z, Ribeiro JM, Semnani RT, Ghedin E, Chan K, Lucas DA, Veenstra TD Nutman TB (2011) Stage-specific proteomic expression patterns of the human filarial parasite Brugia malayi and its endosymbiont Wolbachia. Proc Natl Acad Sci U S A 108: 9649-9654.

20) Henry NL, Law M, Nutman TB, Klion AD (2001) Onchocerciasis in a nonendemic population: clinical and immunologic assessment before treatment and at the time of presumed cure. J Infect Dis 183: 512-516.

21 Billerbeck E, Barry WT, Mu K, Dorner M, Rice CM, Ploss A (2011) Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor-, granulocyte-macrophage colonystimulating factor-, and interleukin-3 -expressing NOD-SCID IL2Rgamma(null) humanized mice. Blood 117: 3076-3086.

22) Martinez-Santamaria L, Guerrero-Aspizua S, Del Rio M (2012) Skin bioengineering: preclinical and clinical applications. Actas Dermosifiliogr 103: 5-11.

23) Weiss N, van Den Ende MC, Albiez EJ, Barbiero VK, Forsyth K, Prince AM. Detection of serum antibodies and circulating antigens in a chimpanzee experimentally infected with Ov. Trans R Soc Trop Med Hyg. 1986;80(4):587-91.

24) Lustigman S, Brotman B, Huima T and Prince AM (1991). Characterization of an Ov cDNA clone encoding a genus specific antigen present in infective larvae and adult worms. Mol. Biochem. Parasitol, 45:65-76. Table 1

Ov-specific biomarker candidates based on proteomic data and data mining

OvAF- Ov Adult Female; OvAFES - Ov Adult Female Excretory Secretory Products; MF - Microfilariae; EMB - Embryonic stages; L2/L3 - early larval stages; Exos; exosomes

SEQUENCE LISTING

SEQ ID NO: 1 Amino acid sequence OVOC12265

>OVOC12265

MKIKKTKKRKRKLLLTDEAENTTPEKILRNKKMEEETGSVADDFIPDVMPTRVAYREQLEIG IRNRLLILLQGPIGCGKTS IVREVAHNMKLPCRAVQMGEQIDSKTLFGTFHCLDVPGEFCWK QSTFTKYILDKGI ILLEDIDCASVDLISQI IDLCNNREARVTSGETIRMHDEAYIVATMRCT KEERSFRSADIELLLTS IPFEISLPPFTNKELHRAICILCKRVAPIAQKLINLFDELINSSA NQINGRKLGATDLLKACKRINDLDDLSDPVAVFHELIDCWAFHCRRQEDI IALSEI IANNLS LNHEQLTYQLNLRLPEISITQTSMKCGRVSLQRNRSTITRESKVINFGITRNVCQLLEQIAV CVNRMEPVLLVGETGVGKTAIVQLLADRIGTTLRWNLSQHSDSSDLIGGYKPVSIPYLLKP LKKE FDELFAS T FDSARNEKFLRHLEMCLSNGRYSDYAKL IMETARRTLKMSKEHKSLKLWA NLFVRAERCAESLKASAVSFAYVRGAVAEAAERGEWLLVDEINLATPECLDSWRLIEDPET RHSDFRLFACMNPASDVGKRNLPAGIRSRFTEI FVHETTEMEQLHI IAQAYLPSFDVAKLSA VLELYQTLRITFPGKYSLRTLCRALAFTAENI FGSDARNIYEAVSMSEMSDLTAEAQIWGK LIQSKMSKVTLGKMKTKFTDNRIEVEGYWIEKGNVEPQDDPSYVCTASVRKNLAQLARWCS GKFPVLLEGETSCGKTAMVIHLAKITGNTI IRINNHEHTDLQEYIGSYVPDGDGRFVFVEGP LVKAARNGHWIILDELNLAPTDVIEALNRLLDDNRELFISETNTWKANPQFRLFATQNPVC TYAGRKRLSRALLNRFWLRFDQLPYNELAKMVIVSCKIAPSAAQAMVSVFCDLRAQRSAVG VFSASDGLMTLRDLFRWGKRLAGSDQNDWRQCLAEHGFLLLGARCRNAVDVECVKKILEKNL KYQINVERLFANDSPYLPLEFRSS IAVNNIVLTDTIRRMLVLVSQSWHFDEPVLI IGETGCG KTTVAQMLAKEKLLSLNCHQRTEAADFLGSLRPVGEGKFKWIDGIWQAMKEGRPLLIDEIS LASDSVLERLNPLLEPSRSLFLNDGGLSGGEVRAKSGFNWATMNPGGDYGKKELSKALRNR FTEIWCPSNVSGDDLIAIVDQLLSKAALLDTNDLRLKISKCIVQFVLWFDSKFSHVLRSTMT IRDIVAISHLVTATLSRSNSPSVS IYHAFSATLFDAFGTLSTRISLDCDVLKKKATDKLYQI MQQELGNICDVASLLLPVALHFDEKESRSLIVGDFS IPFGPSKREMPKNFTFKAPTCKQNI F RLARGLTVDKPILLEGPPGCGKSSTWALAAVTGHALTRLNLSEQTDLADLFGCDVPIVSSD GTPSFIWRDGPILHAIKTGQWVLLDEMNLASQSVLEGLNACFDHRHHLFIPELNRTFDIGVG DNKTRFFACQNPCSQGGNRRKLPKSFVNRFTS IYVAEMDGSDFFEI IRSSFGSVLVDDVIQC MVDVNSS IANLMAEDPQFLRRGSPFE FNLRDLLRWAQLTVDNNGDVAYGFDLLYIWRLRSNI DRQKMRSLFHKCFKFECIEAVASLSLIDEYVRIGKVELKRSDAFHGRQNMKLLSSQMKLLDK LAVCVKMNWLTLIVGKSGSGKSTTIGILASLLGKELYTIHLTSESDSLELLGSFEQVTDYIN FAS IKQHCLNLLQQFPDLSNDVNNAEDILTLRMALQSAMLLVDEDIATKLSKYDNELSNSKM RFEWLNSRFVDAFINGYWILIENVNCCSAAVLDRLNACLESNGELNLQECGDGTS IVKAHND FRVFFTMDDDYGSVSRAMRNRSVELYLLADDSAWFKVAQDLVNMVMDTENQDIVRVTSAVMQ ACEKLSCWELLKLKTFLKEKHMDFENAIKHFKITMNEEVMEIDENEEETEMVYPTVDVDFAT TYSKWKVQVWSHVSHRDWILGVLWATFCIPFKQLKIAEMVELFKYADKKTKDKVIETIQNLK QNMTLDYDERFDGSLTLRITKSPDTYVDNDRFI IGMCALWAKFSLGKIS IEKGSAADMSNLF AKKKIRSDQLPSSAISYLKELLNELYANISKFSSANSAVSDSCS ILWNIVLFVKTCHQRMDL RSACAPLHLAWQRLNDPSYSNIWRQWSTGLCSAATAIDKVWMTDKKASERYSNFHKSCSFFE LFRSYDEWRKYQDELENLTINSATVDKENCS IEQDIDQIGKCITDKSSVLSVALKLMRDIYC GFAILNNRNGKQILLEFLSISAHHPIDLCRLAWLNIKDENWSQIACFTQYLCSDFWGEIAI ACKNSDLAWVTLGTELFSSVWRSMGFQLSSQEITLAQHGNFPMEQKHLCAYFWKIGVHLKSF KLKYREKLLTMIDHFESLLDSPSSLNSSSDVTLLSLMRRLEI ITCALLPLSVPAKNCIDPVM NDEMNFNYLMNKKKLLENIIGVLQSYQGIISGQTDLALFSNSMHPFISSLLNTYNSWKELE NYAHNTVSFRPRYTDFPLLATTMNSFLETVLEQHRTFSVIDLDHLDCLSDDNVRMTVAQIDT FLTS IQSFYNKI FSEYSAFTDIVCPFEMFLNILVATVSYKRFHMIQLMNLRNLI IAYNFPAK FPIEWNCLNNLPESDILYKWCISEKTIMPKRLQKELIALCLQKYKRDMECFADPKEIMVKEI IEWIRNEWSKWFEANTEKQDKTYIYRKSRKHEEELDEDDIKVRELLPDFSTFDDEQDSCEEP AFFAPPSTSFIDSDSLTEILYQLVSEKYICGYDGHMALAWMMDTASSCGLVNDLVDSKI FVY NLHALNQLDLSEDIQIVDVYRKNSRSELGKCIVAMKLLIKRVYWLKEKWPEMTVLDGILQRV HKILSTSMATPQMQFAALLERLLAEADLWEKVADRNHSLVNELEELRHLLVKWRKMEVLCWN NLLEQVQTDCRAQTLLMSWPLFEALDKVDKSDDEILAMTIEWMQNSTI IDFKARLVTAELLI KFIKLTKNCMRDKLCQRLKSWAYFRFFDTIIEKKLDAQKEPIEKQLNDFVKIMKYNDLNLW SVKASAQKAHKQLFRLLKQFRLSGNDLIAPLLDEIPPMLPTTDHETKVTFANDRI IPCNDFY AKRAVDLTLKIATHEMDDMHFDDIQELTEFVKCCNDLIRKDIRYEGDDSEKEKQQGRALSER QKAIARLFKNSVAVGLSSRKGLTVNAEQLTANWAGMSEDDTMLTKFIRHGGASRNIVLKNF HKPNTQIGAKTMSQLKGLTEFILNELYLCHQTVTSLRETLKTMDKAKKMLLNYLENVKDVKQ PYLCGQQWLHRLQQLKYSTFKFNSFLEEMRTKLENAPECNTDSEENMI IKFSGIQDTQLSQL YKQHQEYDVTLDAIRKTQDAAVRILETIKWSLFNSVDCENIAIWKSSDISRTIDIVKSESTD INSHLSRISSVMAEEVEEAMGILHEILEALPNPTNVQETIQNISWERIQALLI I IQNTYKQA MS IDLDNSREMDILRQLMNLLRNLSFDKPTEAIWNLCVQLSDDKGSMECDSLEQVIS INSTL CELLS FIVDWENSAIELAKYYMHFEAMTAALLEKGYVNPIPKPQKESSEKDEGELQSSEDD IAGLGDAKGEKDVGDDIDETGQVEGLKGDEENVDEGTDKNGDDSTPLDMDDDFGGCLEDIDR EQHGDSEESGEDEDNEPDTNMEMGDVNQSEEDKLDPDLWDQNDDNEKELVEGSEGANKATDN MGANNDEGDSENDKNNKDANNADDDKNNEDVDDDDDDDLENVDERELLDSGVDNQDPKQSDE RNDSNDNENKLEDDMENVMNDKLDEDEDQSDDNIEEELQTEMTEKNDEEMQENLLNETTDAT VEELNKNDDEVLKGGAGGMEKGAVEDNMDKNS I SNPNVMDEDEENMKENDSRTEMDNEGKNG CGESKDDDI SNEKPENVKKDDKKSEKEKKLADDI TDI T IQE ILPSEGEQKDEEGPEFGYNNE NNSSLREQIVIDKSSVEEARNSKGNRDQLKDLKNISAEGDEKMMEDDNTEDRQEVNENDLID ADIWNSDFHNSIIHSSTDFYHLVEQSVSTSNLLDAEEWSSSADAEERWNRISDSISVLAAE LSENLRMI IEPTVASRFEGDYRTGKRLNMRRLIAYIASGYRKDKIWLRRTKKAQHNYQILIA VDDSASMHDNQIKLKACQSVAMIESGLRRLEIGQLAICKFGASVKMISDFGNYGDSGLGGKL INELNFDQDRTDLVNLLKCSKKI FERARGRERNNQMLI IVSDGRGVLADGVETTKKALAELH ADQVTVLFVAIDNGEKS IVDMKVAEFT IDGNVNLI PYLQKFPFPFYWVGHVAMLPAT IGDA VRQWFELTARDS

SEQ ID NO:2 Amino acid sequence of OVOC4422

>OVOC4422

MSDS I IVNIEPAKQKLAQLIEEVKGLDLSPLDEQLPNNELFKQCEARRRVINQKIKLLELYI GVIESNNEKWLEFIQKVSTSLKKKEEEEKYAMVIEEKDDVEAI IQELGRAQIKLPDPHQTSQ TVPQTVNLPQLPLPTFSGDPRLWRQFWSGFEAAVHTHPIPVIQKLNYLLSCLRGEALLAVRG FDIAPENYEVIRDILINKYGKSST IKKSLYKELES IKRNEKDWKTT IET IERVLRQLEALGE NLEHSS IENI IETRLPNWIMDKVYQQKEEQQVWSVAKLRSFLGKLVERSQEVARNQLSSSGK TKYSDEGKPHKFPYNTRGETSALVAFKQPSTKNQKEGNDSKKVAVYKPRRLCI FCIKKHWDT DCQEYPNLKQGLESLGVNKACVNLI SSPFLLSATLNHHLE 11GTKLALE IRKNLYVDNI ILS AMDTEEALWKYEETKS I FGDAAMNIREFLSNDKNFNTKIAEQDRANMEVKKILGINWDHVKD TIQLAAKPWTGKELTKRAVLQFVASQYDPLGFLVPVMIRVKLFLQNLWKKNYAWDQLISSED TEIWNSLISDWPTNIKELPRFVTDYSSQIQVHVITDASSVAYSAAVYLRSQGSQGIETLLVF AKSRITPIKGMI IPVLELLAILIGMQAARFVINEDYNLAELILIKQAQAEGITEEERKKWNL YRDDQSLWRSLSRLENSELEEGSKHPIYLPRHNPVTELLIQQYHEDLFHAGIAHTLAEMRRR FWIPKGRSEVKRVLNKCRACKRWTTKPFKLPDMPNLPENRVIRSRTFAHVGLDYMGPLSVKI DSGLTKRWVALFTCFTTRAVHLEIAENLSAECFLHI FRRFIARRGCPESVLSDNASQFQLVF KTMKEQDIKLTNFFAKKGMVWENIVPRAPWSGGVYERI IGLTKRAMRRAIGRRLLWERELIT LITEIEGILNTRPLTYVNFDDYVI IRPIDFITPNASLVTPLINDDDQEEFIPHRLNNKEKLI RHWLNTLKVLDI FWETWKNEYLTSLRERTQRQHISPRNVETRIPREKEIVLVNEPDIPRGMW KLARIKEIKKGTDGEVRNAI IEMPHGRFLVRPINSLCPLEVDDVSNSRSQSPVFEKPMNKSK QEEPIARRTRSATRNRVPEATPEQNFTSTTVAVYTRTHVRIPAIRCNNITRTVCTKAFLRTM

VTFRLGLTQQSTVKEHYSALRLSQELWCHEPGSNRLPLDGFES

SEQ ID NO:3 Amino acid sequence of OVOC585

>OVOC585

MREKEKDEQNGVEDQTDRVIKDTSKSKDWAAADLEKVTDFNEDSDIGREVSNEKLDSLISGP

SGSLRENRKIVNVRKEDVLLIMDELELPKIRAEKKLIEHNGDVIAATKDLLGF

SEQ ID NO:4 Amino acid sequence of OVOC2705

>OVOC2705

MNFHSRLQLFRTCFVWFILHAFSTSFEYAKLQINRNSMEGFVLNVLSWRNHLFRHTNEFYIP CGRDKPIEKEETKYI FLRRGDYVAKYTGFVN

SEQ ID NO:5 Amino acid sequence of OVOC3624

>OVOC3624

MVKTALTPLGLDIEAIIISWLFITGGVIIFACCFVITKSYLFYKRACLKDKRKAIEIKYIW

KDFSSDELNVFFFSKENDLWTHFNDFSSSKIRFLA

SEQ ID NO:6 Amino acid sequence of OVOC486

>OVOC486

MELEDTTRRPSDQTTNITILCSNLRS IRTIGPSHTPLLIQDLWKKGMSWDEKLEPEEMRRCL ELEKAFNQFDVIEVPRWTPQEYSEIHVFVDASEKALTRTILYVLRFLAKVSKEKIRNLKEFS KENSTSREYEKATQLLIKMAQSNITQKEIEHWGLRKDQNGNWRCVGRLRRTMPQIEDFPYFI NKGKFAELIVKYYHEKLFHASAHYTWTKMRQRYWIPRGRAYIKKILRKICKGCAMWWTPFE QPDFPPYPTARVTATRPFETVGVNLFGPI I IMENYAKTKRWVALFTCLATRAVHLEVMETMS TQQCVQAFRRFIG

SEQ ID NO:7 Amino acid sequence of OVOC12838

>OVOC12838

MLPGFLEYSLAGKALEKKIWNLEWDIRFFAEDKHSTVDDVPYGGGAGMIMRPDVIGSAVDS VFSAHKNTRFIYMTPSGTKFNQ

SEQ ID NO:8 Amino acid sequence of OVOC8995

>OVOC8995

MNAGSKHAFVMKIYRCKSQLAFLDTVILSVPTWGSMDEEVHGKEYLGLSTPCCVGTCWECM

HGNVPVP SEQ ID NO:9 Amino acid sequence of OVOC1751

>OVOC1751

MNPPSSSSTSASNSSPKLIWDMES IKEGLQQIEHMDELKSTNELKELIKNCNERQLIRQNLS NEINNLAATLTQEVKEMDEDLIEIDYMNKLIWENECRES ISNRNIKKKNSLRILFRRKMKKN HSTITNATTDTIATNSTRNSFDMRAMEKEMEKIEKGIGKKSELAGKKRTISVIVFRPHNIRK

TTHFKYYRHQSQEMNSTLKNLAMIRYNHCCRRS

SEQ ID NO: 10 Amino acid sequence of OVOC12539

>OVOC12539

MMRSTWIVLVLLLELS IVIAAIEASVGSNDSVS IGEKEIALRSHRVKRFCCCPPKCLVILLC CGGGQHGRRRQRSGGATVQKEMFRNWWLNIPLLLLPMSMSWMKS I FY

SEQ ID NO: 11 Amino acid sequence of OVOC5398

>OVOC5398

MLINSDVLKFEKIKLVIQRLVIPNNVTRKIQQDSSALLNTRGHKERQEEIRITNFTAPESNK CFIMYKKKKKKGKTKVKLRMEEINIPITSLSSGFT SEQ ID NO: l2 Amino acid sequence of OVOC8934

>OVOC8934

MLILKTLMLILHISVMYCNWISS IADSDQPCDNDRKKNVNCGGKNCEPMRVPNIRKS IKSSQ PHQ

SEQ ID NO: 13 Amino acid sequence of OVOC6769

>OVOC6769

MGDNIPKYTPSSSGINGQPYMGASGGYVPPMQYAGGTEASTTAPPQQQPQMQFVQGSVQHGI TAEEVAIAKLDSS IASMEEQQMTADPRYAQMVLLKQKITGAPPTEVTKHHHQMDTSVKEPPE STFTSEQLEQLKAQIGAYKQLAAQEPVAATLIASSVSKPSSLLPEPYEFPVETENGEKLPYD LMKILTLHQQRANRSTCLPPPPGVDPQTVLKEREYRIQNRIGARIQWLSNLPANLSKRLLLK AEIELRALRLLNLQTQVRNEVLSQLKKDTTLETALNPYAYRRTKRQSLREARVTEKLEKQQK VEQERRRRQKHNDLLQAILQHGKEFKEYHRNNQVKQSKIKKAVLTYHANSEKERKKDELRNE RMRMQKLMQEDEEGYRQLLDEKKDKRLVFLLQQTDEYVESLTGLVKQHQATEKRRKRNERRE QKEKEKMQESGESEIRLRIRDATTGEILPIEEMPKSEDIDTWIEAHPGHEWSREEYSESED SESDEPIPEPIEQKKDDEFEGMDEETRNRKI IEKARNEEDEYDQKNRRQMESYYATAHKIKE KIVAQHSSLGGGNPALQLKPYQLKGLEWMVSLYNNNLNGILADEMGLGKTIQTIALVTYLME VKKLNGPYLIIVPLSTIANWSLELEKWAPHWSIVYKGNKEARKKLEASIRRNAFNVLLTTY DYVLKEKGLLGKIRWKYMI IDEGHRMKNHNCKLTLVLNGYFTAQHRLLLTGTPLQNKLPELW ALLNFLLPS I FSSCGTFEQWFNAPFATTGEKVELNQEETMLI IRRLHKVLRPFLLRRLKKEV ESQLPEKTEYVIKCDMSALQRILYQHMQKGLLIDSKHAGGRALMNTWHLRKLCNHPFLFEN VEDECREFWKVSDVSGKDLYRVSGKFELLDRVLPKLKASGHRILMFCQMTSLMTIMEDYLNY REFKYLRLDGSTKPDERGQLLELYNAPNSEYFI EMLSTRAGGLGLNLQTADTVI I FDSDWNP HQDMQAQDRAHRIGQSREVRVLRLVTVNS IEEKILAAARYKLNVDEKVIQAGKFDQRSTGAE RRQMLEQI IRAESEDDDEDEVPDDETINQMVARSEDEFDLFQRMDIERRRQEAAEYRRKPRL IEDSEIPEGIVKASQHFIDEEKEPQKSKLAFEPVGRRQRKEVDYSQDLMSDRDWLKS IDEDV DEDDDVDEEEKKRKKGKKDRGRKKRQIDDDDDEPPKRRKVSPEITSFLNKLYEALIKYKTSS GKELAAAFEQLPSRRELPDYYEI IEKPMDLNKVKRKIKDGKYHSVQDMGIDIRLLCANARKY NIDGSEI FNDSVLLEVLWTKISGDEQQATTSKSFLSAEPKTEKTFSEPRDTGKDSSKDTGKD SKESTATSSRSSPNLEDDKESGRRKKSHPVKRS ISPDD

SEQ ID NO: 14 Amino acid sequence of OVOC835

>OVOC835

LQIYSDKIKIRLLRLKKKTKHLIVVPNVDALQNPES ILTFQKFSAANGMHTENSRKFGEPLN KES IKRVRIKDERWEDKLESRPSSNSSKIERLSYVS ISSGTSTWVQRRKTL SEQ ID NO: 15 Amino acid sequence of OVOC5398-Pepl :

CIPNNVTRKIQQDSSALLNTRGHKERQEEIR

SEQ ID NO: 16 Amino acid sequence of OVOC5398-Pep2:

CNFTAPESNKCFIMYKKKKKKGKTKVKLRMEEINI

SEQ ID NO: 17 Amino acid sequence of OVOC8934-Pepl :

CS IADSDQPCDNDRKKNVNCGGKNCEPMR

SEQ ID NO: 18 Amino acid sequence of OVOC8934-Pep2:

CEPMRVPNIRKS IKSSQPHQ

Claims

WHAT IS CLAIMED IS: 1. A polypeptide conjugate comprising a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-18 conjugated to a heterologous moiety.

2. The polypeptide conjugate of claim 1, consisting of the amino acid sequence of any one of SEQ ID NOs: 1-18 conjugated to a heterologous moiety.

3. The polypeptide conjugate of claim 1, wherein the heterologous moiety is a heterologous peptide sequence, and the polypeptide conjugate is a fusion protein.

4 The polypeptide conjugate of claim 1, wherein the heterologous moiety is a detectable label.

5. The polypeptide conjugate of claim 1, wherein the heterologous moiety is a solid substrate.

6. The polypeptide conjugate of claim 5, wherein two or more polypeptides, each comprising a different amino acid sequence selected from SEQ ID NOs: 1- 18, are conjugated to the solid substrate.

7. A nucleic acid comprising a polynucleotide sequence encoding a fusion protein comprising a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-18 fused to a heterologous peptide sequence.

8. The nucleic acid comprising a polynucleotide sequence encoding a fusion protein consisting of the amino acid sequence of any one of SEQ ID NOs: 1-18 fused to a heterologous peptide sequence.

9. An expression cassette comprising a polynucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1-18 operably linked to a heterologous promoter.

10 The expression cassette of claim 9, wherein the polynucleotide sequence encodes a fusion protein comprising a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-18 fused to a heterologous peptide sequence.

11. The expression cassette of claim 9, wherein the polynucleotide sequence encodes a fusion protein consisting of the amino acid sequence of any one of SEQ ID NOs: l-l8 fused to a heterologous peptide sequence.

12. A vector comprising the expression cassette of any one of claims 9-11.

13. A host cell comprising the vector of claim 8.

14. A method for producing a recombinant protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-18, comprising the step of culturing the host cell of claim 13 under conditions permissible for the expression of a polypeptide encoded by the expression cassette.

15. An antibody that specifically binds the amino acid sequence of any one of SEQ ID NOs: 1-18.

16. The antibody of claim 15, which is a monoclonal antibody.

17. A method for detecting Onchocerca volvulus infection in a subject, comprising the steps of:

(1) contacting a sample taken from the subject with a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-18;

(2) detecting the presence of an antibody in the sample that specifically binds to the polypeptide; and

(3) determining the presence of Onchocerca volvulus infection in the subject.

18. The method of claim 17, wherein the polypeptide is conjugated to a solid substrate.

19. The method of claim 17, wherein in step (1) the sample is contacted with two or more polypeptides each comprising a different amino acid sequence selected from SEQ ID NOs: 1-18.

20. The method of claim 17, wherein in step (1) the sample is contacted with an array comprising two or more polypeptides each comprising a different amino acid sequence selected from SEQ ID NOs: l-l8 immobilized on a solid substrate.

21. The method of claim 20, wherein one of the two or more polypeptides comprises the amino acid sequence of SEQ ID NO:7, and another of the two or more polypeptides comprises the amino acid sequence of SEQ ID NO:8; or wherein one of the two or more antibodies specifically binds the amino acid sequence of SEQ ID NO: 11, and another of the two or more antibodies specifically binds the amino acid sequence of SEQ ID NO: 12.

22. The method of claim 19 or 20, wherein the presence of antibodies in the sample that specifically binds to at least half of the two or more polypeptides is detected in step (2).

23. The method of claim 17, wherein the sample is a blood sample, skin sample, or urine sample.

24. The method of claim 21, wherein the subject is a human patient who has previously received treatment for Onchocerca volvulus infection.

25. A method for detecting Onchocerca volvulus infection in a subject, comprising the steps of:

(1) contacting a sample taken from the subject with an antibody that specifically binds a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: l-l8;

(2) detecting the presence of a polypeptide in the sample that specifically binds to the antibody; and

(3) determining the presence of Onchocerca volvulus infection in the subject.

26. The method of claim 25, wherein the antibody is immobilized to a solid substrate.

27. The method of claim 25, wherein in step (1) the sample is contacted with two or more antibodies each specifically binds a different amino acid sequence selected from SEQ ID NOs: 1-18.

28. The method of claim 25, wherein in step (1) the sample is contacted with an array comprising two or more antibodies each specifically binds a different amino acid sequence selected from SEQ ID NOs: 1-18 immobilized on a solid substrate.

29. The method of claim 28, wherein one of the two or more antibodies specifically binds the amino acid sequence of SEQ ID NO: 7, and another of the two or more antibodies specifically binds the amino acid sequence of SEQ ID NO:8; or wherein one of the two or more antibodies specifically binds the amino acid sequence of SEQ ID NO: 11, and another of the two or more antibodies specifically binds the amino acid sequence of SEQ ID NO: 12.

30. The method of claim 27 or 28, wherein the presence of polypeptides in the sample that specifically binds to at least half of the two or more antibodies is detected in step (2).

31. The method of claim 25, wherein the sample is a blood sample, skin sample, or urine sample.

32. The method of claim 25, wherein the subject is a human patient who has previously received treatment for Onchocerca volvulus infection.

33. A kit for detecting Onchocerca volvulus infection, comprising a container containing a first composition comprising a first polypeptide comprising a first amino acid sequence selected from SEQ ID NOs: l-l8, and a second container containing a second composition comprising a sample taken from a patient confirmed to have Onchocerca volvulus infection.

34. The kit of claim 32, further comprising one or more additional containers, each containing a composition comprising a different polypeptide comprising a different amino acid sequence selected from SEQ ID NO: 1-18.

35 The kit of claim 32, further comprising a negative control sample taken from a subject confirmed to not have Onchocerca volvulus infection.

36. A kit for detecting Onchocerca volvulus infection, comprising a container containing a first composition comprising a first antibody that specifically binds a polypeptide comprising one amino acid sequence selected from SEQ ID NOs: l-l8, and a second container containing a second composition comprising a sample taken from a patient confirmed to have Onchocerca volvulus infection.

37 The kit of claim 36, further comprising one or more additional containers, each containing a composition comprising an antibody that specifically binds a different polypeptide comprising a different amino acid sequence selected from SEQ ID NO: l-l8.

38 The kit of claim 36, further comprising a negative control sample taken from a subject confirmed to not have Onchocerca volvulus infection.