Strongyloides stercoralis protein and/or corresponding DNA and RNA sequences for application in diagnosis

Abstract

The present invention relates to methods of screening biological samples for the presence of Strongyloides spp. More particularly, the present invention relates to a sensitive and specific screening test for the presence of anti-Strongyloides spp antibodies, protein or nucleic acid in subjects using particular Strongyloides spp L3 stage antigens and nucleic acids encoding same which have diagnostic efficacy.

Claims

1. A method of detecting Strongyloides in a biological sample, comprising the steps of: a) contacting the biological sample with an antigen, wherein the antigen is an isolated or recombinant Strongyloides stercoralis protein selected from the group comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4; and b) detecting specific and selective antigen-antibody binding, wherein binding indicates the presence of Strongyloides stercoralis-specific antibodies present in the biological sample.

2. The method according to claim 1, wherein the antigen-antibody binding is detected using immunoassay.

3. The method according to claim 2, wherein the immunoassay is Western blot, flow-through (vertical flow) test or lateral flow assay.

4. A Strongyloides screening kit to screen an isolated biological sample for the presence of Strongyloides-specific antibody comprising: a) an isolated or recombinant Strongyloides protein comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 capable of binding to Strongyloides-specific antibody present in the biological sample.

5. The kit according to claim 4, further comprising immunoassay reagents.

6. The kit according to claim 4, further comprising a lateral flow assay device to detect the presence of Strongyloides antibodies or protein.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Representative results of sensitivity and specificity evaluation of clone Ss3a by phage immunoblot. PC: positive control serum; NC: negative control serum; Pos: serum from patients with strongyloidiasis; Neg: serum from a healthy individual.

(2) FIG. 2A: The 894 nucleotide sequence of the insert of the phage clone Ss1a of Strongyloides stercoralis L3 larvae stage is represented by SEQ ID NO: 1. The nucleotides encoding the predicted protein sequence are underlined.

(3) FIG. 2B: The predicted 297 amino acid sequence encoded by the insert of the phage clone Ss1a of Strongyloides stercoralis L3 larvae stage is represented by SEQ ID NO: 2.

(4) FIG. 3A: The 818 nucleotide sequence of the insert of the phage clone Ss3a of Strongyloides stercoralis L3 larvae stage is represented by SEQ ID NO: 3. The nucleotides encoding the predicted protein sequence are underlined.

(5) FIG. 3B: The predicted 224 amino acid sequence encoded by the insert of the phage clone Ss3a of Strongyloides stercoralis L3 larvae stage is represented by SEQ ID NO: 4.

(6) FIG. 4: Alignment of the DNA sequence of the insert of clone Ss1a (SEQ ID NO: 1) with GenBank database shows a high level of identity (99%) with a region of Strongyloides stercoralis genome assembly S_stercoralis_PV0001, scaffold SSTP_contig0000018; Sequence ID: emb LL999076.1. There are two gaps in the clone sequence; at 299 and 731; and a substitution of T/G at position 779.

(7) FIG. 5: Blastp alignment of rSsIa clone predicted protein sequence (SEQ ID NO: 2) shows 76% identity and 91% homology with Strongyloides ratti Immunoglobulin-binding protein 1 (Accession emb CEF66010.1). There is one gap in the clone sequence at amino acid position 130.

(8) FIG. 6: Alignment of the DNA sequence of the insert of clone Ss3a (SEQ ID NO: 3) with GenBank database shows a high level of identity (99%) with a reverse complementary region of Strongyloides stercoralis genome assembly S_stercoralis_PV0001, scaffold SSTP_scaffold0000002. Sequence ID: There is a substitution of T/A at position 759.

(9) FIG. 7: Blastp alignment of rSs3a clone predicted protein sequence (SEQ ID NO: 4) shows 67% identity and 76% homology with Strongyloides ratti hypothetical protein SRAE_2000383300 (Accession emb CEF69183.1). There is one gap in the clone sequence at amino acid position 218 to maximise alignment.

(10) FIG. 8A-B: SDS-PAGE profile of E. coli-expressed rSsIa protein A) and rSs3a protein B).

(11) FIG. 9A-B: Western blot profiles of rSsIa antigen (A) and rSs3a antigen (B) probed with anti-S. stercoralis antibody-positive and negative human serum samples. Lane M: Low molecular weight marker; Lanes 1-3: Antibody-positive serum samples; Lanes 4-6: Antibody-negative serum samples.

(12) FIG. 10A-B: Representative images of dot-dipstick tests using A) rSsIa and B) rSs3a and probed with positive and negative serum samples.

DETAILED DESCRIPTION

Definitions

(13) Certain terms employed in the specification, examples and appended claims are collected here for convenience.

(14) The terms amino acid or amino acid sequence, as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. The term Strongyloides protein sequence, as used herein, refers to an antigenic polypeptide used to identify and or generate Strongyloides protein-specific antibodies.

(15) In this context fragments refers to a Strongyloides protein according to the invention which has been reduced in length by one or more amino acids and which retains antigenic activity sufficient to raise and or detect antibodies specific to Strongyloides Immunoglobulin-binding protein 1 or hypothetical protein SRAE. For example, the rSs1a antigen described in the present application is produced from the Ss1a DNA clone of SEQ ID NO: 1 and has the amino acid sequence defined in SEQ ID NO: 2, and the aligned sequence in FIG. 5 may be considered a fragment of the protein of SEQ ID NO: 2. It would be understood that the rSs1a antigen polypeptide could be further reduced in length (i.e. fragmented) and retain a Strongyloides Immunoglobulin-binding protein 1 epitope that binds to serum IgG from subjects with strongyloidiasis.

(16) As used herein, the terms specific binding or specifically binding refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigenic determinant or epitope). For example, if an antibody is specific for epitope A, the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody. In the context of the invention, specificity refers to the lack of reactivity of the Strongyloides protein of SEQ ID NO: 2 or 4, or an antigenic fragment thereof, with serum from healthy individuals or from patients with other infections. Thus if 9 serum samples are not reactive out of 10 healthy sera, the specificity of the antigen in the detection of anti-Strongyloides antibodies is 90%.

(17) As used herein, the term sensitive binding or sensitivity_refers to the number of serum samples from Strongyloides infected individuals who are reactive with the Strongyloides protein of SEQ ID NO: 2 or 4, or antigenic fragments thereof. Thus if 8 serum samples are reactive out of 10 infected sera, the sensitivity of the antigen in the detection of anti-Strongyloides antibodies is 80%.

(18) An antibody is any immunoglobulin, including antibodies and fragments thereof that bind to a specific epitope. The antibody according to the invention may be prepared against a polypeptide having the amino acid sequence of at least one of SEQ ID NOS: 2 or 4 or an antigenic fragment thereof. Such antibodies include, but are not limited to, isolated and/or recombinant polyclonal, monoclonal, chimeric, humanised, single chain, Fab, Fab{hacek over ()}, F(ab) {hacek over ()} fragments and/or F(v) portions of the whole antibody.

(19) The term :variant{hacek over ()}, as used in the context of the present invention is intended to describe variations to the amino acid sequence of the Strongyloides protein of SEQ ID NO: 2 or 4 that do not remove the antigenicity of the polypeptide in terms of eliciting antibodies which bind to the Strongyloides protein. Variants include conservative amino acid substitutions, and additions or deletions of amino acids that do not affect antigenicity. A variant may include a homologous sequence from a Strongyloides species other than S. stercoralis, such as S. ratti.

(20) A conservative amino acid substitution as used herein is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a Strongyloides protein comprising, essentially consisting of, or consisting of the amino acid sequence encoded by the nucleic acid sequence defined by SEQ ID NO: 1 or 3, or a fragment thereof, may be replaced with one or more other amino acid residues from the same side chain family without significantly reducing the antigenicity of the polypeptide or deviating significantly from the scope of the present invention.

(21) The term treatment, as used in the context of the invention refers to prophylactic, ameliorating, therapeutic or curative treatment.

(22) The term comprising as used in the context of the invention refers to where the various components, ingredients, or steps, can be conjointly employed in practicing the present invention. Accordingly, the term comprising encompasses the more restrictive terms consisting essentially of and consisting of. With the term consisting essentially of_it is understood that the epitope/antigen of the present invention substantially_comprises the indicated sequence as essential_element Additional sequences may be included at the 5{hacek over ()} end and/or at the 3{hacek over ()} end. Accordingly, a polypeptide consisting essentially of_sequence X will be novel in view of a known polypeptide accidentally comprising the sequence X. With the term consisting of_it is understood that the polypeptide, polynucleotide and/or antigen according to the invention corresponds to at least one of the indicated sequence (for example a specific sequence indicated with a SEQ ID Number or a homologous sequence or fragment thereof).

(23) Accordingly, in a first aspect, the present invention provides the use of an isolated or recombinant Strongyloides stercoralis protein comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or a fragment or variant thereof, to detect Strongyloides spp in an isolated biological sample.

(24) In a preferred embodiment, the Strongyloides spp may be selected from the group comprising Strongyloides stercoralis, Strongyloides fuelleborni and Strongyloides kellyi.

(25) In a preferred embodiment of the invention the Strongyloides stercoralis protein or fragment or variant thereof is encoded by an isolated or recombinant nucleic acid molecule comprising the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or a fragment thereof. The fragment thereof may comprise or consist of the nucleic acids of clones Ss1a and/or Ss3a represented by nucleotides 1-891 or 98-770, respectively. The skilled person would understand that these nucleic acids could be further reduced in length and still be capable of encoding an antigenic protein that could be used to detect anti-Strongyloides antibodies in, for example, an isolated biological sample. More particularly, the biological sample may be serum of an infected subject.

(26) In another preferred embodiment the presence of Strongyloides is indicated by specific and selective binding of the isolated or recombinant Strongyloides stercoralis protein, comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or antigenic fragment or variant thereof, to Strongyloides stercoralis-specific antibodies present in the isolated biological sample.

(27) The biological sample may be from any source that may contain Strongyloides. More particularly the sample may be from an animal, preferably a mammal, more preferably a human subject.

(28) According to another aspect of the invention, there is provided a method of detecting Strongyloides in a biological sample, comprising the steps of:

(29) a) providing at least one biological sample;

(30) b) contacting the at least one biological sample with an antigen, wherein the antigen is an isolated or recombinant Strongyloides stercoralis protein, comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or antigenic fragment thereof; and

(31) c) detecting specific and selective antigen-antibody binding, wherein the binding indicates the presence of Strongyloides stercoralis-specific antibodies present in the biological sample.

(32) In a preferred embodiment of the method of the invention, the antigen-antibody binding is detected using immunoassay. The immunoassay may include various formats, for example chip-based immunoassay, ELISA, Western blot, flow-through (vertical flow) test or lateral flow assay. More preferably, the detection method may be a flow-through (vertical flow) test or lateral flow dot dipstick test, because these methods advantageously do not require sophisticated machinery to perform in a point of care scenario.

(33) According to another aspect of the invention, there is provided a method of detecting Strongyloides in a biological sample, comprising the steps of:

(34) a) providing at least one biological sample;

(35) b) detecting the presence of Strongyloides protein in the biological sample, wherein the detection of the Strongyloides protein comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or fragment thereof in the biological sample constitutes detection of Strongyloides.

(36) In another preferred embodiment of the method of the invention, the Strongyloides protein is detected with an antibody that specifically and selectively binds the protein.

(37) In another preferred embodiment of the method of the invention, the Strongyloides protein is detected using immunoassay.

(38) According to another aspect of the invention, there is provided a method of detecting whether a subject has strongyloidiasis, comprising the steps;

(39) a) contacting a test sample from the subject with an antigen, wherein the antigen is an isolated or recombinant Strongyloides protein or a fragment thereof, and detecting specific and selective antibody-antigen binding, wherein the binding indicates the presence of strongyloidiasis in the subject; or

(40) b) detecting the presence of Strongyloides protein in a test sample from the subject, wherein the detection of the Strongyloides protein in the test sample indicates the presence of strongyloidiasis.

(41) In another preferred embodiment of the method of the invention, the Strongyloides protein or fragment or variant thereof is encoded by an isolated or recombinant nucleic acid molecule comprising the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof.

(42) In another preferred embodiment of the method of the invention, the Strongyloides specific antibodies are IgG antibodies.

(43) According to another aspect of the invention, there is provided a vaccine comprising at least one Strongyloides protein or antigenic fragment or variant thereof. In a preferred embodiment, the vaccine comprises at least one protein or antigenic fragment or variant selected from SEQ ID NO: 2 and SEQ ID NO: 4 which may be used separately or in combination.

(44) According to another aspect of the invention, there is provided a method of treatment or prophylaxis of strongyloidiasis, comprising administering to a subject an efficacious amount of a vaccine as defined above.

(45) According to another aspect of the invention, there is provided a use of at least one Strongyloides protein or antigenic fragment or variant thereof for the preparation of a vaccine for the prophylaxis or treatment of strongyloidiasis.

(46) According to another aspect of the invention, there is provided an expression construct comprising a nucleic acid comprising the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or antigen encoding fragment thereof.

(47) According to another aspect of the invention, there is provided a kit for screening a biological sample for the presence of Strongyloides-specific antibody and/or Strongyloides antigen, comprising:

(48) a) an isolated or recombinant Strongyloides protein, or a fragment thereof according to any aspect of the invention, capable of binding to Strongyloides-specific antibody present in the biological sample; and/or

(49) b) an isolated or recombinant Strongyloides protein-specific antibody according to any aspect of the invention, capable of binding to Strongyloides protein present in the biological sample.

(50) In a preferred embodiment, the kit further comprises immunoassay reagents when a) or b) are present.

(51) In another preferred embodiment, the kit further comprises a flow-through (vertical flow) test device or lateral flow assay device to detect the presence of Strongyloides antibodies, protein or nucleic acid. Preferably the device may include a flow-through membrane or lateral flow dipstick dot test strip.

(52) A method of treatment or prophylaxis of strongyloidiasis, comprising administering to a subject an efficacious amount of an isolated or recombinant Strongyloides protein comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or an immunogenic fragment or variant thereof.

EXAMPLES

(53) A person skilled in the art will appreciate that the present invention may be practiced without undue experimentation according to the methods given herein. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology text books such as Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001).

Example 1: Screening for L3 Stage Larvae Antigens

(54) Immunoscreening of S. stercoralis cDNA library was performed to identify cDNA clones representing specific genes expressed in the L3 larvae stage that produce antigenic recombinant proteins that would specifically be recognized by immunoglobulin antibodies i.e. IgG.sub.4 and IgG present in a biological sample. This study has led to the identification of two cDNA clones, designated SsIa and Ss3a.

(55) The diagnostic sensitivity and specificity of the isolated SsIa and Ss3a phage clones was evaluated in an immunoblotting study utilizing a panel of serum samples from patients with strongyloidiasis, healthy individuals or patients infected with other diseases (FIG. 1). Both of the cDNA clones produced recombinant proteins (rSs1a and rSs3a) which demonstrated high diagnostic sensitivity and specificity in detecting anti-Strongyloides antibodies in human serum samples (Table 1).

(56) TABLE-US-00001 TABLE 1 Summary of the reactivity of phage immunoblot using rSsIa and rSs3a Number of serum samples Serum rSsIa rSs3a samples Positive Negative Total Positive Negative Total Positive 14 3 17 12 3 15 (100%) (100%) Negative 0 19 19 0 27 27 (86.4%) (90%) Total 14 22 36 12 30 42 Diagnostic sensitivity of rSs1a: 100% Diagnostic specificity of rSs1a: 86.4% Diagnostic sensitivity of rSs3a: 100% Diagnostic specificity of rSs3a: 90%

(57) These clones were then in vivo excised to convert the phagemids into plasmids, followed by plasmid extraction and sequencing, to determine the DNA sequences.

(58) The 894 nucleotide sequence of the insert of the phage clone SsIa of Strongyloides stercoralis L3 larvae stage is represented by SEQ ID NO: 1 (FIG. 2A). The nucleotides encoding the predicted protein sequence are underlined. The predicted 297 amino acid sequence encoded by the insert of the phage clone Ss1a of Strongyloides stercoralis L3 larvae stage is represented by SEQ ID NO: 2 (FIG. 2B).

(59) The 818 nucleotide sequence of the insert of the phage clone Ss3a of Strongyloides stercoralis L3 larvae stage is represented by SEQ ID NO: 3 (FIG. 3A). The nucleotides encoding the predicted protein sequence are underlined. The predicted 224 amino acid sequence encoded by the insert of the phage clone Ss3a of Strongyloides stercoralis L3 larvae stage is represented by SEQ ID NO: 4 (FIG. 3B).

(60) Bioinformatics analysis of the deduced nucleotide sequence of the DNA insert of SsIa revealed that it has 99% identity to the Strongyloides stercoralis genome assembly S_stercoralis_PV0001, scaffold SSTP_contig0000018 with accession number LL999076.1, where Ss1a is represented by nucleotides 296-894 of SEQ ID NO: 1 (FIG. 4). Analysis of the translated protein of the SsIa cDNA (SEQ ID NO: 2) using Blastp showed the highest similarity to Strongyloides ratti immunoglobulin-binding protein 1 [Identities=188/247 (76%), Positives=225/247 (91%), Gaps=1/247 (0%)] with accession number CEF66010.1 (FIG. 5). Alignment to Strongyloides ratti may be due to the fact that the genome sequencing and annotation of Strongyloides stercoralis has not been completed.

(61) The nucleotide sequence of the phage cDNA clone Ss3a showed the highest identity to Strongyloides stercoralis genome assembly S_stercoralis_PV0001, scaffold SSTP_scaffold0000002 with accession number LL999049.1 [Identities=818/819 (99%), Gaps=0/819 (0%)], where Ss3a is represented by nucleotides 1-818 of SEQ ID NO: 3 (FIG. 6). There is a difference (substitution of T/A) at position 759. Blastp searches demonstrated a high percent of similarity (67% identity; 76% homology) of the Ss3a translated protein sequence (SEQ ID NO: 4) to a hypothetical protein SRAE_2000383300 from Strongyloides ratti with accession number CEF69183.1 (FIG. 7). There is one gap introduced into the clone sequence at amino acid position 218 to maximise alignment.

(62) The SsIa DNA sequence insert of the plasmid was codon optimized for E. coli expression by EPOCH Life Science Inc. (Texas, USA) and custom-cloned into the expression vector pET28b (Novagen, Madison, Wis., USA), followed by transformation into BL21(DE3) host cells (Novagen, Madison, Wis., USA). Expression of the plasmid in Terrific Broth (900 ml H.sub.2O containing 12 g Tryptone, 24 g Yeast extract, 4 ml Glycerol sterilised plus 100 ml solution of 0.17M KH2PO4 and 0.72M K2HPO4) yielded a significant amount of soluble His-tagged rSsIa protein with an observed molecular weight of 37 kDa (FIG. 8A). The evidence of expression of the SsIa-His fusion protein was verified by Western blot analysis using anti-His HRP antibody and validated by MALDI-TOF/TOF analysis.

(63) Meanwhile, the codon-optimized Ss3a cDNA clone was custom-cloned into a pET42a vector (Novagen, Madison, Wis., USA) and transformed into Lemo21(DE3) cells (New England Biolabs, Hitchin, UK), producing a GST-fusion protein designated rSs3a (FIG. 8B). Western blot analysis of the expressed fusion protein using anti-GST HRP antibody determined the molecular weight to be about 45 kDa, which was validated by MALDI TOF/TOF analysis.

(64) Western blot analyses of both recombinant proteins, rSs1a and rSs3a, demonstrated a high diagnostic sensitivity and specificity in detecting anti-Strongyloides IgG.sub.4 in human serum samples (FIG. 9, Table 2), prompting further evaluation of both recombinant proteins for application in the development of a lateral flow dot dipstick (LFA) test.

(65) TABLE-US-00002 TABLE 2 Summary of the diagnostic sensitivity and specificity of a) rSsIa and b) rSs3a by Western blot analysis. Number of serum samples Serum rSsIa rSs3a samples Positive Negative Total Positive Negative Total Positive 11 2 13 21 4 25 (100%) (100%) Negative 0 8 8 0 87 87 (80%) (95.6%) Total 11 10 21 21 91 112 Diagnostic sensitivity of rSs1a: 100% Diagnostic specificity of rSs1a: 80% Diagnostic sensitivity of rSs3a: 100% Diagnostic specificity of rSs3a: 95.6%

(66) In developing the lateral flow dot dipstick test for rSsIa, anti-human IgG.sub.4 conjugated to colloidal gold was used as the conjugate reagent whereas for rSs3a anti-GST conjugated to colloidal gold was used as the conjugate reagent. This was due to the large GST tag fused to the rSs3a protein that was not removed. Both lateral flow strips were then tested with a panel of serum samples obtained from Strongyloides-infected patients and controls (FIG. 10A-B). Results of the evaluation studies showed that both lateral flow dot dipstick tests were highly sensitive and specific in detecting anti-Strongyloides IgG.sub.4 antibody in serum samples of Strongyloides-infected humans (Table 3).

(67) TABLE-US-00003 TABLE 3 Summary of the diagnostic sensitivity and specificity of dipstick dot tests using a) rSsIa and b) rSs3a. Number of serum samples Serum rSsIa rSs3a samples Positive Negative Total Positive Negative Total Positive 27 1 28 10 0 10 (90%) (100%) Negative 3 45 48 0 10 10 (98%) (100%) Total 30 46 76 10 10 20 Diagnostic sensitivity of rSs1a: 90% Diagnostic specificity of rSs1a: 98% Diagnostic sensitivity of rSs3a: 100% Diagnostic specificity of rSs3a: 100%

(68) Further work will be performed to produce rSs3a using a suitable His-tagged vector that can yield soluble recombinant protein with good yield and purity.

(69) We have successfully produced two Strongyloides recombinant proteins, rSsIa and rSs3a, which show high sensitivity and specificity in detecting anti-Strongyloides antibody in the serum of human subjects using western blot and lateral flow dipstick dot tests. The development of a lateral flow test to detect strongyloidiasis is important since this test format is very suitable for use in resource-poor settings where the disease is endemic.

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