Diagnostic test for brucellosis

Abstract

Provided herein are methods, kits and systems for the detection of Brucella antigen, method for detecting brucellosis in a mammal comprising the steps of: obtaining or having obtained a biological sample suspected being infected with Brucella sp.; and detecting in the biological sample immunogenic protein DUF883 or anti-DUF883 antibodies, wherein the presence of DUF883 or anti-DUF883 antibodies are indicative that the mammal has been exposed to Brucella, e.g., to a vaccine or immunogenic composition comprising: immunogenic protein DUF883 provided in an amount sufficient to trigger an immune response against the immunogenic protein; and a pharmaceutically acceptable vaccine carrier.

Description

DIAGNOSTIC TEST FOR BRUCELLOSISTECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates in general to methods and compositions for the detection of Brucella induced diseases and disorders in mammals, and more particularly, to new diagnostic markers that allow the detection of specific antibodies in serum in animals exposed to brucellosis.STATEMENT OF FEDERALLY FUNDED RESEARCH

[0002] Not applicable.REFERENCE TO A SEQUENCE LISTING

[0003] Not applicable.BACKGROUND OF THE INVENTION

[0004] Without limiting the scope of the invention, its background is described in connection with diagnostic tests for brucellosis.

[0005] Current diagnostic tests use Lipopolysaccharide (LPS) as the antigen to detect the presence of antibodies against brucellosis in serum. This is not very specific due to the presence of LPS in all Gramnegative bacteria.

[0006] Another such diagnostic is taught in U.S. Patent Publication No. 20060246086, filed by Lestrate and Letesson, entitled “Virulence Genes and Proteins From Brucella Melitensis, And Their Use”. These applicants are said to teach a series of genes from Brucella spp that are shown to encode products which are implicated in virulence and genes to produce attenuated microorganisms and the use of the genes or their encoded products in the manufacture of vaccines for therapeutic application.

[0007] Another diagnostic is taught in U.S. Patent Publication No. 20020160457, filed by Schurig, entitled “Rapid Diagnostic Test to Identify Animals Vaccinated With Brucella Abortus RB 51”. This applicant is said to teach a kit to detect whether animals, particularly cattle, have been vaccinated with B. abortus RB 51, a brucellosis inhibitor in cattle, and a kit that includes latex beads which are used, in the latex agglutination test or enzyme-linked immunosorbent assay, to determine whether an animal has, in fact, been vaccinated.

[0008] Despite these advances, a need remains for novel diagnostic methods, kits, and systems for the rapid detection of antigens and antibodies against those antigens that are indicative of a Brucella infection.SUMMARY OF THE INVENTION

[0009] The present invention relates to methods and compositions for the detection of Brucella induced diseases and disorders in mammals, and more particularly, to new diagnostic markers that allow the detection of specific antibodies in serum in animals exposed to brucellosis.

[0010] As embodied and broadly described herein, an aspect of the present disclosure relates to a method for detecting brucellosis in a mammal comprising the steps of: obtaining or having obtained a biologicalsample suspected being infected with Brucella sp:, and detecting in the biological sample a DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies or antigen binding fragments thereof, wherein the presence of the DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies or antigen binding fragments thereof antibodies are indicative that the mammal has been exposed to Brucella. In another aspect, an assay to detect the DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies or antigen binding fragments thereof, is selected from a immunoassay selected from at least one of: an enzyme-linked immunosorbent assay (ELISA), a Lateral Flow Assay (LFA), an immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western blot, a competitive immunoassay, a non-competitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or an immunofluorescence reporter-assay. In another aspect, the method comprises a sample collection device comprises a needle, an inoculation loop, capillary transfer pipette, absorbent pad, swab, nasopharyngeal swab, sponge or pipette. In another aspect, the biological sample is a blood or serum sample. In another aspect, the method further comprises a vessel that comprises at least one of: a substrate or strip, running sample / running buffer, an ampoule, bottle or sachet, a sample pad comprises cellulosic material, glass fiber pads treated with a blocking buffer containing protein, detergents and salts, or a conjugate pad comprises Protein A-Colloidal Gold. In another aspect, the test results are obtained in 1 to 45, 1 to 30, or 1 to 20 minutes. In another aspect, the method further comprises instructions for use provide details concerning collection of a bodily fluid, introduction of the bodily fluid to a test kit, application of a biological sample to a cassette, operation of the test kit, interpretation of results, and effective disposal of the test kit. In another aspect, detecting a presence or absence of the DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies or antigen binding fragments thereof is an immunoassay selected from at least one of: an enzyme-linked immunosorbent assay (ELISA), a Lateral Flow Assay (LFA), an immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay, a sandwich assay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western blot, a competitive immunoassay, anon-competitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or a immunofluorescence reporter-assay.

[0011] As embodied and broadly described herein, an aspect of the present disclosure relates to a test kit for detecting Brucella infection, anti-Brucella antibodies or antigen binding fragments thereof, in a bodily fluid, the test kit comprising: (a) a sample collection device; (b) a vessel containing sample / running buffer; (c) a cassette comprising a substrate, strip, an absorbing / wicking pad, striped processed membrane, conjugate pad sample pad, and backing card that comprises a DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies or antigen binding fragments thereof; and (d) instructions for use. In one aspect, the sample collection device comprises a needle, an inoculation loop, capillary transfer pipette, absorbent pad, swab, nasopharyngeal swab, sponge or pipette. In another aspect, the biological sample is a blood or serum sample. In another aspect, the vessel comprises at least one of: a substrate or strip, running sample / running buffer, an ampoule, bottle or sachet, a sample pad comprises glass fiber pads treated witha blocking buffer containing protein, detergents and salts, or a conjugate pad comprises Protein A-Colloidal Gold. In another aspect, the test results are obtained in 1 to 45, 1 to 30, or 1 to 20 minutes. In another aspect, the test kit further comprises instructions for use provide details concerning collection of a bodily fluid, introduction of the bodily fluid to a test kit, application of a biological sample to a cassette, operation of the test kit, interpretation of results, and effective disposal of the test kit. In another aspect, detecting a presence or absence of the DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies or antigen binding fragments thereof is an immunoassay selected from at least one of: an enzyme-linked immunosorbent assay (ELISA), a Lateral Flow Assay (LFA), an immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay, a sandwich assay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western blot, a competitive immunoassay, a non-competitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or a immunofluorescence reporter-assay.

[0012] As embodied and broadly described herein, an aspect of the present disclosure relates to a system for detecting brucellosis in a mammal comprising: a test kit for detecting Brucella infection, anti-Brucella antibodies or antigen binding fragments thereof, in a bodily fluid, the test kit comprising: (a) a sample collection device; (b) a vessel containing sample / running buffer; (c) a cassette comprising an absorbing / wicking pad, striped processed membrane, conjugate pad sample pad, and backing card that comprises a DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies or antigen binding fragments thereof; and (d) instructions for use, wherein the instructions comprise: obtaining or having obtained a biological sample suspected being infected with Brucella sp:, and detecting in the biological sample the DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies or antigen binding fragments thereof, wherein the presence of DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies or antigen binding fragments thereof, are indicative that the mammal has been exposed to Brucella. In another aspect, the system further comprises an assay detects the immunogenic protein DUF883 or anti-DUF883 antibodies is selected from a immunoassay selected from at least one of: an enzyme-linked immunosorbent assay (ELISA), a Lateral Flow Assay (LFA), an immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western blot, a competitive immunoassay, a non-competitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or an immunofluorescence reporter-assay. In another aspect, the biological sample is a blood or plasma sample. In another aspect, the system further comprises a sample collection device comprises a needle, an inoculation loop, capillary transfer pipette, absorbent pad, swab, nasopharyngeal swab, sponge or pipette. In another aspect, the system further comprises a vessel that comprises at least one of: running sample / running buffer, an ampoule, bottle or sachet, a conjugation, sample or running pad, optionally, treated with a blocking buffer containing protein, detergents and salts, or a conjugate pad comprises Protein A-Colloidal Gold. In another aspect, the test results are obtained in 1 to 45, 1 to 30, or 1 to 20 minutes. In another aspect, the system further comprises instructions for use provide details concerning collection of a bodily fluid, introduction of the bodily fluid to a test kit,application of a biological sample to a cassette, operation of the test kit, interpretation of results, and effective disposal of the test kit. In another aspect, detecting a presence or absence of the DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies or antigen binding fragments thereof is an immunoassay selected from at least one of: an enzyme-linked immunosorbent assay (ELISA), a Lateral Flow Assay (LFA), an immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay, a sandwich assay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western blot, a competitive immunoassay, anon-competitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or a immunofluorescence reporter-assay.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] For a more complete understanding of the features and advantages of the present disclosure, reference is now made to the detailed description of the disclosure along with the accompanying figures and in which:

[0014] FIG. 1 shows a lateral flow assay (LFA) design: Serum applied to the sample pad interacts with the 40nm gold nanoparticle, the detection agent, as it flows through the conjugation pad. The immune- dominant protein bond at the test line captures the 40nm GNP-antibody complex as it moves through the membrane, facilitating brucellosis detection through a visual signal.

[0015] FIG. 2. Left: Quantitative Analysis ofDipstick Results with field samples. Data interpretation using ImageJ Software. Right: ROC Curve Analysis. Assessing dipstick sensitivity and specificity when using field samples.

[0016] FIG. 3 is a diagram that shows the upstream and downstream primer pairs were designed to amplify a 960bp and 994bp sequence above and below the target gene respectively. Restriction sites were added to the primers to facilitate the recombination of the sequences within a deletion cassette (plasmid). Junctional (564pb) and within gene (18 Ibp) primers were designed as controls to ensure successful deletion of the target.

[0017] FIG. 4 is a diagram that shows gel purification, the purified DNA fragments were subjected to digestion by their respective enzymes (see above). The vector was linearized using EcoRI and PstI enzymes. The digested fragments and linearized plasmid vector were ligated together utilizing DNA ligase, resulting in the creation of a suicide plasmid deletion cassette.

[0018] FIG. 5 shows the deletion of the gene is accomplished through homologous recombination. The process takes places inside the host cell and results in the replacement of the original sequence of the gene with its shorter version contained inside the plasmid.

[0019] FIG. 6 shows the suicide plasmid provides resistance to Kanamycin and susceptibility to sucrose. Enabling the growth of only the transformed cells on kanamycin-containing media. Splitting of the colonies, creating overnight stocks, and then plating on sucrose and kanamycin plates allows for identifying cells that have lost resistance to Kanamycin and susceptibility to sucrose. These colonies are then subjected to PCR confirmation of the knockout.

[0020] FIG. 7 is a gel that shows the confirmation of Gene Knockout through Conventional PCR: DNA extracted from all presumed knockout colonies was subjected to PCR using junctional primers as described previously. The amplified segment with the gene is 564bp, whereas the segment without it is 339bp.

[0021] FIG. 8 shows the experimental design and timeline. Evaluation of Brucella melitensis \6MAvjbR / Candidate A DIVA capability in guinea pigs.

[0022] FIGS. 9A and 9B are graphs that show the detection of antibodies. FIG. 9A shows the detection of wB-Brucella specific IgG antibodies in the serum of guinea pigs vaccinated with the two different vaccine candidates when utilizing LPS as the coating antigen. There is a strong response from both groups starting at 14 days post-vaccination. However, no significant differences between groups were noted. FIG. 9B shows the detection of wB-Brucella specific IgG antibodies in the serum of guinea pigs utilizing DUF883 as the detection antigen. Note the distinction between the reactivity of the single vs double deletion mutant. Only animals that received the 16MAy / 7?R had detectable antibodies against the DUF883 protein. The results are expressed as the mean of OD values (450nm). Statistical analysis was performed by comparing the mean of the groups using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison test.

[0023] FIG. 10 shows the results of field testing of the diagnostic test of the present invention using bovine samples from Cameroon.DETAILED DESCRIPTION OF THE INVENTION

[0024] While the making and using of various aspects of the present disclosure are discussed in detail below, it should be appreciated that the present disclosure provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific aspects discussed herein are merely illustrative of specific ways to make and use the disclosure and do not delimit the scope of the invention.

[0025] To facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure. Terms such as “a”, “an,” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific aspects of the disclosure, but their usage does not delimit the invention, except as outlined in the claims.

[0026] As used herein, “Brucella” refers to a genus of Gram-negative bacteria. They are small, non-motile, encapsulated coccobacilli. While not limiting the scope of the present invention, Brucella is often transmitted by ingesting infected food, direct contact with an infected animal, or inhalation of aerosols.

[0027] As used herein, the term “brucellosis” refers to a disease caused by ingestion of milk or meat and / or contact with the bodily fluids or secretions of animals infected with Brucella bacterial species. While not limiting the scope of the present invention, symptoms of brucellosis include but are not limited to acute undulating fever, headache, night sweats, fatigue, sterility and anorexia.

[0028] As used herein, the terms “prevent” and “preventing” include the prevention of the recurrence, spread or onset of a disease or disorder. It is not intended that the present invention be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease or disorder is reduced.

[0029] As used herein, the terms “treat” and “treating” are not limited to the case where the subject (e.g. patient) is cured and the disease is eradicated. Rather, the present invention also contemplates treatment that merely reduces symptoms, improves (to some degree) and / or delays disease progression. It is not intended that the present invention be limited to instances wherein a disease or affliction is cured. It is sufficient that symptoms are reduced.

[0030] As used herein, the term “subject” as used herein refers to any mammal, preferably a human patient, livestock, or domestic pet. It is intended that the term “subject” encompass both human and non-human mammals, including, but not limited to canine, bovine, caprine, ovine, equine, porcine, feline, canine, etc., as well as humans.

[0031] In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals or humans.

[0032] In one aspect, the present disclosure includes assays that detect the presence of the antigen or an antibody that specifically binds the antigen. For example, a rapid diagnostic test (RDT) apparatus can be used to measure the intensity of a detectable signal from test and control samples on, e.g., one or more liner flow assay (LFA) test substrates or strips to the presence of the antigen or antibodies to the antigen. An RDT is a test that is quick and easy to perform. RDTs are suitable for preliminary screening, for use in the field with limited resources, and offer a useful alternative to complex assays using complex and expensive equipment that requires highly trained personnel. The assay can also be a point-of-care (POC) testing in primary care in situations where only a laboratory test could provide a diagnosis. RDTs do not require clinical diagnostic methods, such polymerase chain reaction (PCR), and can be performed independent of laboratory equipment by minimally trained personnel, and deliver results quickly. An RDT can even be a dipstick or cassette format into which a biological specimen (such as a blood) collected from a mammal is applied to a sample pad on the test strip (or card) along with certain reagents. After a length of time (depending on the test), the presence of specific bands in or on the test substrate or strip indicates whether a certain antigen (or antibody against the antigen) of interest is present in the biological sample. Typically, a drop of sample (e.g., blood) is added to the RDT through one hole (sample well), and then a number of drops of buffer are usually added to flow and wash the sample. The buffer carries the sample along the length of the RDT.

[0033] Non-limiting examples of assay techniques such as immunoassays, sandwich assays and competitive binding assays can be used to identify the presence and amount of the antigen or antibody against the antigen. In one such embodiment, a species that binds the antigen or antibody against the antigen (e.g., a complimentary or anti-biomolecule) is deposited on a matrix. The antigen or antibody against the antigen binds to an indicator that has a binding species (e.g., a complimentary or anti-biomolecule) on oneside and an indicator species. Next, a detectable label such as metal, polymer nanoparticle, fluorescence label, enzyme that induces a detectable signal. The antigen or antibody against the antigen that is bound to the detectable label reacts with the binding species in the detection zone, and in this way forms a “sandwich” on the membrane. In certain embodiments, the detectable signal forms a visible line or spot formed in the detection zone which indicates presence of the antigen or antibody against the antigen. In other embodiments, the detectable signal is not visible as a colorimetric line, but instead can be detected via fluorescence or chemiluminesence. For example, the antigen or antibody against the antigen can be detected using binding assays, with an immobilized the antigen or antibody against the antigen. Other methods of detection can be used, such as electrochemical or capacitive detection.

[0034] As the person of ordinary skill in the art will appreciate, the devices and methods described herein can be used to perform a variety of assays. Generally, a substate or strip can include in the first fluid pathway a detection zone. The detection zone can be configured to provide a variety of types of measurements. For example, the detection zone can include a detectable substance that will interact (e.g., via reaction, binding or coordination) with the analyte to provide some detectable change, e.g., a color change, chemi- or bioluminescence, a darkening, or a change that is detectable using any of a variety of instrumental techniques, e.g., colorimetric, spectrophotometric, fluorescence or electrochemical detection). The detectable substrate can be, for example, anchored to the material of the substate or strip (e.g., through chemical bonding), as would be apparent to the person of ordinary skill in the art. Of course, in other embodiments, the detection substance is not anchored to substate or strip. The detectable substrate can be, for example, a small molecule, a colorimetric reagent, a metal, a nanoparticle, an oligo- or polypeptide (e.g., a protein), or an oligo- or polynucleotide. The detection zone can thus be configured from an assay such as an immunoassay, an ELISA, a sandwich assay or a antigen or antibody binding assay. In other embodiments, no particular detection substance need be present; in such embodiments, a variety of techniques can be used to detect the analyte (e.g., colorimetric, spectrophotometric, fluorescence or electrochemical detection). The person of ordinary skill in the art will appreciate that virtually any detection scheme useful in lateral flow assays or microfluidic -based assays can be applied to the present devices.

[0035] The system may be a linear flow assay (LFA) strip. In lateral flow assays, the liquid sample moves through a substrate, strip, matrix or material by lateral flow or capillary action. The sample is applied at a first point of the substrate, strip, matrix or material and then the sample moves through that so-called “sample application zone” to a detection zone, which comprises regions with detectable agents or substrates.

[0036] As used herein, the term “gene” means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a “protein gene product” is a protein expressed from a particular gene.

[0037] As used herein, the word “expression” or “expressed” as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present withinthe cell or the amount of protein encoded by that DNA produced by the cell. The level of expression of non-coding nucleic acid molecules (e.g., sgRNA) may be detected by standard PCR or Northern blot methods well known in the art. See, Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1- 18.88.

[0038] As used herein, 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, y-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 that have 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. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0039] 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.

[0040] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may, in embodiments, be conjugated to a moiety that does not consist of amino acids. The 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. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0041] Proteins and peptides include isolated and purified forms. Proteins and peptides also include those immobilized on a substrate, as well as amino acid sequences, subsequences, portions, homologues, variants, and derivatives immobilized on a substrate.

[0042] Proteins and peptides can be included in compositions, for example, a pharmaceutical composition. In particular embodiments, a pharmaceutical composition is suitable for specific or non-specific immunotherapy, or is a vaccine composition.

[0043] As used herein, the term “nucleic acid” refers to any compound and / or substance that comprise a polymer of nucleotides, referred to herein as polynucleotides. Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleicacids (LNAs), including diastereomers of LNAs, functionalized LNAs, or hybrids thereof. Isolated nucleic acid (including isolated nucleic acid) encoding the proteins and peptides are also provided. Cells expressing a protein or peptide are further provided. Such cells include eukaryotic and prokaryotic cells, such as mammalian, insect, fungal and bacterial cells.

[0044] Methods and uses and medicaments of proteins and peptides of the invention are included. Such methods, uses and medicaments include modulating immune activity of a cell against Brucella.

[0045] As used herein, the term “peptide mimetic” or “peptidomimetic” refers to protein-like chain designed to mimic a peptide or protein. Peptide mimetics may be generated by modifying an existing peptide or by designing a compound that mimic peptides, including peptoids and [3-peptides.

[0046] As used herein, the phrase “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. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will 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 which 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 which encodes a polypeptide is implicit in each described sequence.

[0047] 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 disclosure. 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 (1984)).

[0048] As used herein, the phrase “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignmentof the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0049] As used herein, the terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0050] An amino acid or nucleotide base “position” is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N- terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0051] As used herein, the terms “numbered with reference to” or “corresponding to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0052] As used herein, the term “multimer” refers to a complex comprising multiple monomers (e.g., a protein complex) associated by noncovalent bonds. The monomers be substantially identical monomers, or the monomers may be different. In embodiments, the multimer is a dimer, a trimer, a tetramer, or a pentamer.

[0053] As used herein, the term “antibody” is used according to its commonly known meaning in the art. Antibodies exist, e.g., as intact immunoglobulins or as a number of well -characterized fragments producedby 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- CHI 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 Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 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 using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) orthose identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0054] As used herein, the phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein or peptide, often in a heterogeneous population of proteins and other biologies. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other 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, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0055] Antibodies are large, complex molecules (molecular weight of -150,000 or about 1320 amino acids) with intricate internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system. The light and heavy chain variable regions come together in 3 -dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions (“CDRs”). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3 -dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework (“FR”), which forms the environment for the CDRs.

[0056] 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. The Fc (i.e., fragment crystallizable region) is the “base” or “tail” of an immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0057] As used herein, the term “antigen” and the term “epitope” refers to a molecule or substance capable of stimulating an immune response. In one example, epitopes include but are not limited to a polypeptide and a nucleic acid encoding a polypeptide, wherein expression of the nucleic acid into a polypeptide is capable of stimulating an immune response when the polypeptide is processed and presented on a Major Histocompatibility Complex (MHC) molecule. Generally, epitopes include peptides presented on the surface of cells non-covalently bound to the binding groove of Class I or Class II MHC, such that they can interact with T cell receptors and the respective T cell accessory molecules. However, antigens and epitopes also apply when discussing the antigen binding portion of an antibody, wherein the antibody binds to a specific structure of the antigen.

[0058] Proteolytic Processing of Antigens. Epitopes that are displayed by MHC on antigen presenting cells are cleavage peptides or products of larger peptide or protein antigen precursors. For MHC I epitopes, protein antigens are often digested by proteasomes resident in the cell. Intracellular proteasomal digestion produces peptide fragments of about 3 to 23 amino acids in length that are then loaded onto the MHC protein. Additional proteolytic activities within the cell, or in the extracellular milieu, can trim and process these fragments further. Processing of MHC Class II epitopes generally occurs via intracellular proteases from the lysosomal / endosomal compartment.

[0059] As used herein, the term an “immunogenic composition” and “vaccine” refer to a composition that comprises an antigenic molecule where administration of the composition to a subject or patient results in the development in the subject of a humoral and / or a cellular immune response to the antigenic molecule of interest. “Vaccine” or “immunization” are used interchangeably and refer to a composition that can provide active acquired immunity to and / or therapeutic effect (e.g., treatment) of a particular disease or a pathogen. A vaccine or immunization typically contains one or more agents that can induce an immune response in a subject against a pathogen or disease, i.e., a target pathogen or disease. The immunogenic agent stimulates the body’s immune system to recognize the agent as a threat or indication of the presence of the target pathogen or disease, thereby inducing immunological memory so that the immune system can more easily recognize and destroy any of the pathogen on subsequent exposure. Vaccines or immunizations can be prophylactic (e.g., preventing or ameliorating the effects of a future infection by any natural orpathogen) or therapeutic (e.g., reducing symptoms or aberrant conditions associated with infection). The administration of a vaccine or immunization is referred to as vaccination or immunization, respectively.

[0060] As used herein, a “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test condition, e.g., in the presence of a test compound, and compared to samples from known conditions, e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control). A control can also represent an average value gathered from a number of tests or results. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.

[0061] As used herein the terms “diagnose” or “diagnosing” refers to recognition of an infection, disease or condition by signs and symptoms. Diagnosing can refer to determination of whether a subject has an infection or disease. Diagnosis may refer to determination of the type of disease or condition a subject has or the type of bacteria the subject is infected with.

[0062] Diagnostic agents provided herein include any such agent, which are well-known in the relevant art. Among imaging agents are fluorescent and luminescent substances, including, but not limited to, a variety of organic or inorganic small molecules commonly referred to as “dyes,” “labels,” or “indicators.” Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, and cyanine dyes. Enzymes that may be used as imaging agents in accordance with the embodiments of the disclosure include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, [3-galactosidase, [3- glucoronidase or [3-lactamase. Such enzymes may be used in combination with a chromogen, a Anorogenic compound or a luminogenic compound to generate a detectable signal.

[0063] The peptide(s) or protein(s) of the present invention can also be used in binding assays including, but are not limited to, lateral How assays, immunoassays such as competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, Meso Scale Discovery (MSD, Gaithersburg, Md.), immunoprecipitation assays, ELISPOT, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, Huorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, relevant portions incorporated herein by reference).

[0064] Brucella organisms can be delivered via aerosol to infect humans. The use of Brucella as a weapon was calculated to pose a substantial financial risk as disclosed in Kaufmann et al. (1997) Emerging Infectious Diseases 3, 83-94 and Pappas et al. Cell Mol Life Sci (2006) 63, 2229-36, both incorporatedherein by reference. Infection incapacitates human hosts with mostly flu-like symptoms, but will result in death if left untreated as provided for in Young EJ (1995) Clin Inf Dis 21, 283-290, hereby incorporated by reference. Bioengineering poses the additional risk of introducing antibiotic resistance, rendering ineffective the most successful form of treatment. The transposon Tn 10 encoding tetracycline resistance has been used to obtain stable transformants. The financial impact study did not attempt to determine the threshold at which financial risk may pose a risk to national security. Nor did the study outline scenarios in which the use of one organism might be favored over the use of others. The study did underscore the need to invest in research in all understudied organisms to prevent their use in this manner and suggested that decreased study of these organisms increase the potential consequences resulting from their use as weapons. Brucella spp. have been weaponized by several countries, including the former Soviet Union, Japan and the USA, and thus is a recognized biological warfare threat that can cause illness and death in humans.

[0065] Improving Diagnosis and DIVA capabilities for brucellosis in animals.

[0066] One hurdle facing control of brucellosis is the difficulties in achieving a diagnosis. Sera is typically first analyzed using a screening test that detects agglutinating antibodies against the EPS, with the most widely available test being the Rose Bengal Test. Unfortunately, the sensitivity can range between 70-90% and the specificity can be as low as 83% due to cross reactivity with other Gram negative bacteria. The gold standard for diagnosis, culture, has a low sensitivity due to fluctuation of bacteremia in infected animals over time. Similarly, while PCR is essentially confirmatory, this test also relies on the presence of bacteria in the blood and shares the same poor sensitivity of culture and it is not currently utilized as a standardized or approved method for diagnosis. Other serological assays such as Indirect ELISA has shown to increase the specificity in some instances, however all commercially available kits are based on the same principle: utilization of LPS as the detection antigen. Nevertheless, cross reactions with other bacteria remain a concern for any serologic assay utilizing LPS or whole-cell lysates for brucellosis (Corbel et al, 1983). This issue has led to an expansion in development new alternatives to detect antibodies against specific Brucella proteins for diagnosis with the intention of 1) increasing sensitivity and specificity, 2) avoid the use of multiple tests to increase diagnostic confidence, 3) avoid use of culture due to technical difficulties and biorisk associated with this practice, 4) reduce the time from sample collection to diagnosis 5) deploy easy to use and cost effective tests that could be used with minimal infrastructure around the world and 6) develop a test that could differentiate infected from vaccinated animals (DIVA capability).

[0067] Both to develop a more sensitive and specific serologic diagnostic assay for brucellosis and to complement vaccine development, the present inventors identify immunodominant proteins for Brucella. and assess their diagnostic ability. The present invention includes novel antigens that are not only highly effective in detecting infection but show DIVA compatibility in a live attenuated vaccine.

[0068] Identification of new immunodominant proteins with promissory diagnostic capacities in brucellosis.

[0069] Table 1. Area under the curve, sensitivity and specificity, and predictive values of the indirect ELISA for different Brucella recombinant proteins. AUC, area under the curve; TP, true positives; TN, true negatives; FP, false positives; FN, false negatives; PPV, positive predictive value (TP / TP+FP); NPV, negative predictive value (TN / TN+FN); Sen, sensitivity; Spe, specificity.

[0070] Development of anew Point-of-Care lateral flow assay for the detection of bovine brucellosis using DUF883 (Candidate A).

[0071] Currently used serodiagnostic tests rely on antibody recognition of the O-polysaccharide (O-PS) side chain of the Lipopolysaccharide (LPS) which can lead to cross-reactivity with other Gram-negative organisms. Other diagnostic approaches including bacterial culture (considered the gold standard), or molecular detection by PCR, are impractical in resource-limited settings where brucellosis is endemic. Therefore, there is a pressing need for new diagnostic tests that are easy to implement, economically viable, and user-friendly.

[0072] A lateral flow assay (LFA) to detect 5race / / a-specific antibodies in bovine serum is being developed as an alternative to current serological tests. The LFA utilizes a newly discovered immunodominant antigen specific for 5race / / a-denominated DUF883 (candidate A) on the test line and anti-bovine antibodies (IgG) on the control line (FIG. 1). Colloidal gold nanoparticles (40 nm GNP), conjugated with protein G, served as the detector reagent for visual determination (FIG. 1). The skilled artisan will recognize that other detectable agents and / or reagents can be used. For example, the colloidal gold particle size can be varied, but the detection could be any detectable agent or method, as described hereinabove.

[0073] FIG. 1 shows a lateral flow assay (LFA) design: Serum applied to the sample pad interacts with the 40nm gold nanoparticle, the detection agent, as it flows through the conjugation pad. The immune- dominant protein bond at the test line captures the 40nm GNP-antibody complex as it moves through the membrane, facilitating brucellosis detection through a visual signal.

[0074] The assay was evaluated using: 1) national standards, 2) experimentally infected bovine serum samples, and 3) naturally infected field samples from a 5race / / a-endemic area in Africa achieving a sensitivity of 66.7% and a specificity of 89.4% utilizing experimentally infected serum, and a sensitivity of 93.3% and a specificity of 85% using naturally infected field samples (FIG. 2). This novel dipstick test was used and can be a rapid point-of-care diagnostic test for the diagnosis of bovine brucellosis.

[0075] FIG. 2. Left: Quantitative Analysis of Dipstick Results with field samples. Data interpretation using ImageJ Software. Right: ROC Curve Analysis. Assessing dipstick sensitivity and specificity when using field samples.

[0076] Generation of a DIVA-compatible vaccine candidate for brucellosis. The objective is to perform a gene knockout targeting an immunodominant protein (DUF883) into the vaccine candidate B. melitensis 16M \v / 7? / to allow for differentiation between vaccinated and unvaccinated animals (DIVA) when using Candidate A as the antigen in the diagnostic assay. While B. melitensis was used in this example, any Brucella species can be used, e.g., B. amis, B. abortus, B. suis. etc.

[0077] Generation of a B. melitensis 16M AvjbR ACandidate A knockout mutant.Steps1: Primer design.2: Generation of deletion cassette.3: Transformation of E. coli with deletion cassette.4: Plasmid extraction and purification & Transformation of Brucella.5 : Screening for successfully transformed Brucella.7: Confirmation through conventional PCR and sequencing.

[0078] Step 1: Primer Design.

[0079] Primers were designed to amplify regions upstream and downstream of the sequence encoding for DUF883 / Candidate A in the B. melitensis genome. The amplified regions were flanked by restriction sites to facilitate its insertion in a deletion cassette used to knockout the target gene from the 16M AvjbR strain.

[0080] FIG. 3 is a diagram that shows the upstream and downstream primer pairs were designed to amplify a 960bp and 994bp sequence above and below the target gene respectively. Restriction sites were added to the primers to facilitate the recombination of the sequences within a deletion cassette (plasmid). Junctional (564pb) and within gene (18 Ibp) primers were designed as controls to ensure successful deletion of the target.

[0081] The primers amplified the targeted sequences as expected using B. melitensis genomic DNA, obtaining two amplicons: one upstream of the coding region of target gene containing restriction sites for EcoRI and BamHI and the other a sequence downstream of the target gene containing restriction sites for BamHI and Pstl.

[0082] Step 2: Generation of Deletion Cassette.

[0083] The plasmid vector used forthe deletion cassette was pNPTS138. This plasmid provides resistance to kanamycin and susceptibility to sucrose (sacB gene). Allowing for subsequent plating on kanamycin for identification of transformed colonies. Deletion cassette was successfully generated.

[0084] FIG. 4 is a diagram that shows gel purification, the purified DNA fragments were subjected to digestion by their respective enzymes (see above). The vector was linearized using EcoRI and Pstl enzymes. The digested fragments and linearized plasmid vector were ligated together utilizing DNA ligase, resulting in the creation of a suicide plasmid deletion cassette.

[0085] Step 3: Transformation of E. coli with deletion cassette.

[0086] Mach-1 E. col were transformed with the newly constructed pNTPTS138 deletion cassette. Competent Mach- 1 cells were thawed over ice, and pNTPTS 138 DNA was added. After a brief incubation on ice the cells were heat shocked at 42°C for 30 seconds, followed by rapid cooling on ice. Transformed cells were incubated in SOC media for Ih and plated on selective media containing kanamycin. The next day individual colonies from the plates were inoculated into LB broth containing kanamycin and grown overnight at 37°C.

[0087] Transformants that successfully grew on selective media.

[0088] Steps 4 and 5: Plasmid extraction and B. melitensis 16M AvjbR transformation by electroporation.

[0089] Plasmid was extracted from liquid culture of transformed Mach-1 cells using the Miniprep Kit (Qiagen) following the manufacturer's protocol. Purity and concentration were assessed by NanoDrop.

[0090] Electrocompetent cells were prepared by washing B. melitensis 16M AvjbR in cold, sterile water. The pNTPTS 138 deletion cassette was added to the electrocompetent cells and transferred to a 1mm cuvette . The cuvette was inserted into a Gene Pulser Xcell Electroporation System (Bio-Rad) and shocked with 2500 volts. After electroporation, cells recovered in a warm SOC medium for 7 hours at 37°C with shaking. Transformed cells were then plated onto selective media containing kanamycin and then on selective media containing sucrose to enable the identification and isolation of successfully transformed colonies.

[0091] Screening for plasmid suicide and loss of kanamycin resistance.

[0092] FIG. 5 shows the deletion of the gene is accomplished through homologous recombination. The process takes places inside the host cell and results in the replacement of the original sequence of the gene with its shorter version contained inside the plasmid.

[0093] FIG. 6 shows the suicide plasmid provides resistance to Kanamycin and susceptibility to sucrose. Enabling the growth of only the transformed cells on kanamycin-containing media. Splitting of the colonies, creating overnight stocks, and then plating on sucrose and kanamycin plates allows for identifying cells that have lost resistance to Kanamycin and susceptibility to sucrose. These colonies are then subjected to PCR confirmation of the knockout.

[0094] Selected colonies.

[0095] Step 6: Confirmation of gene knockout through PCR and sequencing.

[0096] PCR: Colonies that were able to grow on selective media were subsequently chosen for PCR analysis to confirm deletion of the gene.

[0097] FIG. 7 shows the confirmation of Gene Knockout through Conventional PCR: DNA extracted from all presumed knockout colonies was subjected to PCR using junctional primers as described previously. The amplified segment with the gene is 564bp, whereas the segment without it is 339bp.

[0098] Results: Four B. melitensis AvjbR ACandidate A deletion mutants were obtained (colonies 1, 2, 5 and 6).

[0099] Sequencing: DNA from colonies 2 and 5 was amplified using the upstream forward and downstream reverse primers by PCR. The resulting amplicons were purified by gel purification and sent for sequencing using the junctional primers.

[0100] Results : NCBI Align Sequences Nucleotide BLAST of Candidate A Gene and Sequencing. 193bp were deleted from the Candidate A gene of B. melitensis AvjbR \D883 confirming successful knockout of the target gene.

[0101] Assessment of DIVA capability of the vaccine candidate.

[0102] Objective: To assess the DIVA capability of the vaccine candidate B. melitensis 16M vjbRAcandidateA vs. 16MAv / 7? / ?. The trial consisted of the ability to distinguish vaccinated animals utilizing LPS vs. single antigen detection. Species: Guinea pigs, adults. Type of Study: Kinetics of humoral response utilizing LPS-based antigen vs. newly discovered immunodominant antigen (DUF883 / Candidate A). Condition: Laboratory study in Biocontainment 3 facilities at Texas A&M University. Duration of the Study: 1 month. Vaccine: Single dose at IxlO9of encapsulated B. melitensis and 16M vjbRADUF883.

[0103] FIG. 8 shows the experimental design and timeline. Evaluation of Brucella melitensis \6MAvjbR / Candidate A DIVA capability in guinea pigs.

[0104] Animal protocol.

[0105] Facilities and procedures involving the use of the genetically modified strain of B. melitensis I 6M \v / / ? / ? in guinea pigs was approved by CDC. All animal research was conducted under a protocol approved by Texas A&M University (IACUC 2021-0038, IBC2018-013 and IBC2019-019).

[0106] 2) Bacterial strains and vaccine seed stock.

[0107] B. melitensis biovar 1 strain 16M was originally acquired from the lung of an aborted goat.[4]The B. melitensis \6MAvjbR utilized was from the same stock described in STUDIES# 1-8. 16MAvjbR Candidate A master stock was created at TAMU following the SOPs included in Section 5. All wild-type and vaccine strains were grown on tryptic soy agar (TSA) or tryptic soy broth (TSB) at 37°C in an atmosphere containing 5% (vol / vol) CO2 for 72 hours. Immunization dose was verified by retrospective serial dilution, plating, and enumeration of colonies.

[0108] 3) Animal selection, identification, and vaccination.

[0109] Hartley guinea pigs of -900-1200 grams were individually housed in micro isolator cages at a BSL-2 animal facility. Following an acclimation period of 5 days, guinea pigs were randomly assigned into two groups (n=4 each) and were subsequently vaccinated by subcutaneous injection in the right inguinalregion with 100 pl of 1 x 109CFU of Brucella melitensis \6MAvjbR or Brucella melitensis \ W^vjbR ^Candidate A.

[0110] Treatment groups :1) 16MAvjW? at IxlO9CFU (sub-Q) (n=4)2) 16MAvjbRADUF883 at IxlO9CFU (sub-Q) (n=4)

[0111] 4) Analysis of the potential DIVA capabilities of Brucella melitensis \6MAvjbR / Candidate A.

[0112] Blood was collected at T=0-, 3-, 14-, and 28-days post-vaccination. Indirect EUISA (iEUISA) for anti-5race / / a-specific total IgG was performed to compare the immune response between the single mutant and the double mutant utilizing LPS as the antigen. A second iELISA utilizing whole protein A as an antigen was used to compare and assess DIVA capabilities. nB-Brucella IgG antibodies utilizing LPS in both treatment groups were detected at 14 days post vaccination with peak levels observed at 28 days of vaccination. As expected, no significant differences were found between both vaccine groups when utilizing LPS as the coating antigen since both strains are smooth (Figure 10). However, a clear distinction was evident when the antigen instead of the LPS was used for detection, starting at 14 days post-vaccination with a significant difference at 28 days.

[0113] FIGS. 9A and 9B are graphs that show the detection of antibodies. FIG. 9A shows the detection of wB-Brucella specific IgG antibodies in the serum of guinea pigs vaccinated with the two different vaccine candidates when utilizing LPS as the coating antigen. There is a strong response from both groups starting at 14 days post-vaccination. However, no significant differences between groups were noted. FIG. 9B shows the detection of wB-Brucella specific IgG antibodies in the serum of guinea pigs utilizing DUF883 as the detection antigen. Note the distinction between the reactivity of the single vs double deletion mutant. Only animals that received the I 6M \v / bR had detectable antibodies against the DUF883 protein. The results are expressed as the mean of OD values (450nm). Statistical analysis was performed by comparing the mean of the groups using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison test.

[0114] FIG. 10 shows the results of field testing of the diagnostic test of the present invention using bovine samples from Cameroon. Lateral flow assays were conducted on multiple field samples from Cameroon. Test line: purified candidate protein - Immobilize antibodies of interest from serum sample. Control line: a-target species antibody - Make sure that the conjugate did flow properly through membrane.

[0115] Table 2. Summary of results from multiple field samples from Cameroon.

[0121] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0122] It may be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0123] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0124] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0125] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0126] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0127] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it may be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for detecting brucellosis in a mammal comprising the steps of: obtaining or having obtained a biological sample suspected being infected with Brucella sp:, and detecting in the biological sample an immunogenic DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies, wherein a presence of DUF883 protein or anti-DUF883 antibodies are indicative that the mammal has been exposed to Brucella.

2. The method of claim 1, wherein an assay to detect the DUF883 protein, antigenic fragments thereof, or anti-DUF883 antibodies is selected from a immunoassay selected from at least one of: an enzyme-linked immunosorbent assay (ELISA), a Lateral Flow Assay (LFA), an immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western blot, a competitive immunoassay, a non-competitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or an immunofluorescence reporter-assay.

3. The method of claim 1, further comprising a sample collection device comprises a needle, an inoculation loop, capillary transfer pipette, absorbent pad, swab, nasopharyngeal swab, sponge or pipette.

4. The method of claim 1, wherein the biological sample is a blood or serum sample.

5. The method of claim 1, further comprising a vessel that comprises at least one of: a substrate or strip, running sample / running buffer, an ampoule, bottle or sachet, a sample pad comprises glass fiber pads treated with a blocking buffer containing protein, detergents and salts, or a conjugate pad comprises Protein A-Colloidal Gold.

6. The method of claim 1, wherein test results are obtained in 1 to 45, 1 to 30, or 1 to 20 minutes.

7. The method of claim 1, further comprising instructions for use provide details concerning collection of a bodily fluid, introduction of the bodily fluid to a test kit, application of a biological sample to a cassette, operation of the test kit, interpretation of results, and effective disposal of the test kit.

8. The method of claim 1, wherein detecting a presence or absence of the DUF883 protein, anti- DUF883 antibodies, or anti-DUF883 antigen-binding fragment is an immunoassay selected from at least one of: an enzyme-linked immunosorbent assay (ELISA), a Lateral Flow Assay (LFA), an immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay, a sandwich assay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western blot, a competitive immunoassay, a non-competitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or a immunofluorescence reporter-assay.

9. A test kit for detecting Brucella infection, anti-Brucella antibodies or antigen binding fragments thereof, in a bodily fluid, the test kit comprising:(a) a sample collection device;(b) a vessel containing sample / running buffer;(c) a cassette comprising a substrate, strip, an absorbing / wicking pad, striped processed membrane, conjugate pad sample pad, and backing card that comprises a DUF883 protein or antigenic fragments thereof, or anti-DUF883 antibodies or antigen binding fragments thereof; and(d) instructions for use.

10. The test kit of claim 9, wherein the sample collection device comprises a needle, an inoculation loop, capillary transfer pipette, absorbent pad, swab, nasopharyngeal swab, sponge or pipette.

11. The test kit of claim 9, wherein a biological sample is a blood or serum sample.

12. The test kit of claim 9, wherein the vessel comprises at least one of: a substrate or strip, running sample / running buffer, an ampoule, bottle or sachet, a sample pad comprises glass fiber pads treated with a blocking buffer containing protein, detergents and salts, or a conjugate pad comprises Protein A-Colloidal Gold.

13. The test kit of claim 9, wherein test results are obtained in 1 to 45, 1 to 30 or 1 to 20 minutes.

14. The test kit of claim 9, further comprising instructions for use provide details concerning collection of a bodily fluid, introduction of the bodily fluid to a test kit, application of a biological sample to a cassette, operation of the test kit, interpretation of results, and effective disposal of the test kit.

15. The test kit of claim 9, wherein detecting a presence or absence of the DUF883 protein, antigenic fragments thereof, anti-DUF883 antibodies, or anti-DUF883 antigen-binding fragment is an immunoassay selected from at least one of: an enzyme-linked immunosorbent assay (ELISA), a Lateral Flow Assay (LFA), an immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay, a sandwich assay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western blot, a competitive immunoassay, anon-competitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or a immunofluorescence reporter-assay.

16. A system for detecting brucellosis in a mammal comprising: a test kit for detecting Brucella infection, anti-Brucella antibodies or antigen binding fragments thereof, in a bodily fluid, the test kit comprising:(a) a sample collection device;(b) a vessel containing sample / running buffer;(c) a cassette comprising an absorbing / wicking pad, striped processed membrane, conjugate pad sample pad, and backing card that comprises a DUF883 protein or antigenic fragments thereof or anti- DUF883 antibodies or antigen binding fragments thereof; and(d) instructions for use, wherein the instructions comprise: obtaining or having obtained a biological sample suspected being infected with Brucella sp:, and detecting in the biological sample a DUF883 protein, antigenic fragments thereof, anti-DUF883 antibodies, or anti-DUF883 antigen-binding fragment, wherein a presence of DUF883 protein, antigenicfragments thereof, anti-DUF883 antibodies, or anti-DUF883 antigen-binding fragments are indicative that the mammal has been exposed to Brucella.

17. The system of claim 16, wherein an assay to detect the DUF883 protein, antigenic fragments thereof, or anti-DUF883 antibodies is selected from a immunoassay selected from at least one of: an enzyme-linked immunosorbent assay (ELISA), a Lateral Flow Assay (LFA), an immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western blot, a competitive immunoassay, a non-competitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or an immunofluorescence reporter-assay.

18. The system of claim 16, wherein the biological sample is a blood or serum sample.

19. The system of claim 16, further comprising a sample collection device comprises a needle, an inoculation loop, capillary transfer pipette, absorbent pad, swab, nasopharyngeal swab, sponge or pipette.

20. The system of claim 16, further comprising a vessel that comprises at least one of: running sample / running buffer, an ampoule, bottle or sachet, a conjugation, sample or running pad, optionally, treated with a blocking buffer containing protein, detergents and salts, or a conjugate pad comprises Protein A-Colloidal Gold.

21. The system of claim 16, wherein test results are obtained in 1 to 45, 1 to 30, or 1 to 20 minutes.

22. The system of claim 16, further comprising instructions for use provide details concerning collection of a bodily fluid, introduction of the bodily fluid to a test kit, application of a biological sample to a cassette, operation of the test kit, interpretation of results, and effective disposal of the test kit.

23. The system of claim 16, wherein detecting apresence or absence of the DUF883 protein, antigenic fragments thereof, anti-DUF883 antibodies, or anti-DUF883 antigen-binding fragment is an immunoassay selected from at least one of: an enzyme-linked immunosorbent assay (ELISA), a Lateral Flow Assay (LFA), an immunoprecipitation, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay, a sandwich assay, a chemiluminescent assay, an agglutination assay, a nephelometric assay, a turbidimetric assay, a Western blot, a competitive immunoassay, anon-competitive immunoassay, a homogeneous immunoassay, a heterogeneous immunoassay, a bioassay, or a immunofluorescence reporter-assay.

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