Vaccine for brucellosis

Abstract

Provided herein are methods and compositions for the detection of Brucella antigen, and more particularly, to a vaccine or immunogenic composition comprising: Brucella strain from which at least a portion of the immunogenic protein DUF883 has been deleted provided in an amount sufficient to trigger an immune response against the immunogenic protein; and a pharmaceutically acceptable vaccine carrier.

Description

VACCINE FOR BRUCELLOSISTECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates in general to methods and compositions for the treatment of Brucella induced diseases and disorders in mammals, and more particularly, to a novel antigenic target for immunization against Brucella.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 vaccines for brucellosis.

[0005] One such vaccine is taught in U.S. Patent No. 11,707,515, issued to Grillo Dorset, et al., entitled “Modified Brucella Vaccine Strain for The Treatment Of Brucellosis”. These inventors are said to teach a modified Brucella strain, its use as a medicament, and its use as a medicament for the treatment and / or prevention of brucellosis, in which the Brucella strain has been modified through an inactivation of the wzm gene, pharmaceutical compositions, and a kit that comprises the modified Brucella strain and a pharmaceutically acceptable carrier or diluent and its use for the treatment and / or prevention of brucellosis.

[0006] Another such vaccine is taught in U.S. Patent Publication No. 20220023408, filed by Curtin, entitled, “Protective Immunity Enhanced Salmonella Vaccine (PIESV) Against Brucella spp ”. This applicant is said to teach live attenuated bacterial vaccine vectors that confer a virulence and safety, but which decrease the ability of the vaccine to invade cells in the MALT to colonize and persist in internal effector lymphoid tissues.

[0007] Despite these advances, a need remains for novel immunizations that prevent or treat a Brucella infection in mammals.SUMMARY OF THE INVENTION

[0008] 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.

[0009] As embodied and broadly described herein, an aspect of the present disclosure relates to a vaccine composition comprising: Brucella strain from which at least a portion of the DUF883 protein has been deleted and the Brucella strain is provided in an amount sufficient to trigger an immune response against the immunogenic protein; and a pharmaceutically acceptable vaccine carrier. In one aspect, the composition further comprises an adjuvant. In one aspect, the adjuvant is selected from at least one of:dimethyldioctadecyl ammonium bromide, oil-in-water emulsion, alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, cytosine-guanosine oligonucleotide (CpG-ODN) sequence, granulocyte macrophage colony stimulating factor (GM-CSF), monophosphoryl lipid A (MPL), poly(I:C), MF59, Quil A, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), FIA, montanide, poly (DL-lactide- coglycolide), squalene, virosome, AS03, ASO4, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL- 12, IL-15, IL-17, IL-18, STING, CD40L, pathogen-associated molecular patterns (PAMPs), damage- associated molecular pattern molecules (DAMPs), Freund's complete adjuvant, Freund's incomplete adjuvant, transforming growth factor (TGF)-beta antibody or antagonists, A2aR antagonists, lipopolysaccharides (LPS), Fas ligand, Trail, lymphotactin, Mannan (M-FP), APG-2, Hsp70 and Hsp90, pattern recognition receptor ligands, TLR3 ligands, TLR4 ligands, TLR5 ligands, TLR7 / 8 ligands, or TLR9 ligands. In one aspect, the DUF883 protein is ElaB from a genome of Brucella melitensis 16M BMEI0805. In one aspect, the DUF883 protein is 104 amino acids comprising a transmembrane alpha helix domain, a long cytosolic tail, and a short extracellular sequence. In one aspect, the DUF883 protein is deleted from a live attenuated vaccine. In one aspect, the vaccine composition further comprises a diagnostic gene knockout comprises a differentiation of infected animals from vaccinated animals (DIVA) mutant that includes AvirB12, Abcsp31, or Aasp24. In one aspect, an attenuating gene knockout is Brucella strain 16MA.vjbRADUF883, A / 6R / DIVA, or 16MAvjbR / M)UF883 / T)INA . In one aspect, the at least a portion of the DUF883 protein that is deleted, is by deletion of sequences that are 5’-, 3’-, or within a coding sequence of a DUF883 gene, selected from deleting sequences, frameshift, insertions, early termination or equivalent thereof, elimination of promoter sequences, or sequences that control expression of the D UF883 gene, such that one or more mutations prevent expression of some or all of the DUF883 protein or portions thereof, or transcription of the DUF883 gene, or expression of the DUF883 gene is silenced. In one aspect, the vaccine is used for a prophylaxis, an amelioration of symptoms, a treatment, or any combinations thereof against brucellosis in an animal subject or a human. In one aspect, the vaccine is administered by an oral, an intranasal, a parenteral, an intradermal, an intramuscular, an intraperitoneal, an intravenous, a subcutaneous, an epidural, a mucosal, a rectal, a vaginal, a sublingual, or a buccal route. In one aspect, the vaccine composition further comprises an encapsulating agent that is an alginate bead or a microsphere.

[0010] As embodied and broadly described herein, an aspect of the present disclosure relates to an immunogenic formulation comprising a live attenuated Brucella that is \ 6A[ \v / hR \1)UF883. wherein the immunogenic formulation has higher safety and efficacy for animals against different Brucella species than live attenuated Brucella strain 16MAv / W?. In one aspect, the composition further comprises an adjuvant. In one aspect, the Brucella species affects livestock and companion animals selected from B. abortus, B. melitensis, B. suis, and B. canis. In one aspect, the adjuvant is selected from at least one of: dimethyldioctadecyl ammonium bromide, oil-in-water emulsion, alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, cytosine-guanosine oligonucleotide (CpG-ODN) sequence, granulocyte macrophage colony stimulating factor (GM-CSF), monophosphoryl lipid A (MPL), poly(LC), MF59, Quil A, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), FIA, montanide, poly (DL-lactide-coglycolide), squalene, virosome, AS03, ASO4, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL- 12, IL-15, IL-17, IL-18, STING, CD40L, pathogen-associated molecular patterns (PAMPs), damage- associated molecular pattern molecules (DAMPs), Freund's complete adjuvant, Freund's incomplete adjuvant, transforming growth factor (TGF)-beta antibody or antagonists, A2aR antagonists, lipopolysaccharides (LPS), Fas ligand, Trail, lymphotactin, Mannan (M-FP), APG-2, Hsp70 and Hsp90, pattern recognition receptor ligands, TLR3 ligands, TLR4 ligands, TLR5 ligands, TLR7 / 8 ligands, or TLR9 ligands. In one aspect, the immunogenic protein DUF883 is ElaB from a genome of Brucella melitensis 16M BMEI0805. In one aspect, the immunogenic protein DUF883 is 104 amino acids comprising a transmembrane alpha helix domain, a long cytosolic tail, and a short extracellular sequence. In one aspect, the immunogenic formulation further comprises a diagnostic gene knockout comprises a differentiation of infected animals from vaccinated animals (DIVA) mutant that includes AvirB12, Abcsp31, or Aasp24. In one aspect, the live attenuated Brucella further comprises Av’ / 7? / / DIVA. l 6MAv’ / 7? / / DIVA. or any combinations thereof. In one aspect, the live attenuated Brucella is used for a prophylaxis, an amelioration of symptoms, a treatment, or any combinations thereof against brucellosis in an animal or a human. In one aspect, the live attenuated Brucella is administered by an oral, an intranasal, a parenteral, an intradermal, an intramuscular, an intraperitoneal, an intravenous, a subcutaneous, an epidural, a mucosal, a rectal, a vaginal, a sublingual, or a buccal route. In one aspect, the at least a portion of the DUF883 protein that is deleted, is by deletion of sequences that are 5’-, 3’-, or within a coding sequence of a DUF883 gene, selected from deleting sequences, frameshift, insertions, early termination or equivalent thereof, elimination of promoter sequences, or sequences that control expression of the DUF883 gene, such that one or more mutations prevent expression of some or all of the DUF883 protein or portions thereof, or transcription of the DUF883 gene, or expression of the DUF883 gene is silenced. In one aspect, the immunogenic formulation further comprises an encapsulating agent that is an alginate bead or a microsphere.

[0011] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of immunizing the mammal with an immunogenic formulation comprising a live attenuated Brucella that is 16MAvjbR and wherein at least a portion of a Brucella DUF883 protein or immunogenic portions thereof has been deleted or inactivated, and an adjuvant, wherein the immunogenic formulation has higher safety and efficacy for animals against a different Brucella species than the live attenuated Brucella strain 16MAvjbRA.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

[0013] 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.

[0014] FIGS. 2A. Quantitative Analysis of Dipstick Results with field samples. Data interpretation using Image J Software. FIG. 2B ROC Curve Analysis. Assessing dipstick sensitivity and specificity when using field samples.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] FIG. 8 shows the experimental design and timeline. Evaluation of Brucella melitensis \6MAvjbR / Candidate A DIVA capability in guinea pigs.

[0021] 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 comparingthe mean of the groups using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison test.

[0022] FIG. 10 shows the experimental design and timeline . Evaluation of the efficacy of different vaccine formulations of the candidate Brucella melitensis 16MAy / 7?R / DUF883 in mice.

[0023] FIG. 11 are graphs that shows the evaluation of vaccine efficacy in mice vaccinated with 16MBvJbrR / DUF883 in combination with five different adjuvants (designated #1, #2, #3, #4, and #5). Control groups included 16M / lv / 7? / 7 alone and naive, non-vaccinated mice. Bacterial burden 4 weeks postchallenge in the spleen, liver, and lung per organ are depicted. The horizontal bar indicates the mean. P values were determined by unpaired t-test with Welch’s correction. Values that are significantly different are indicated by bars and asterisks (*, P < 0.05, * *, / > < 0.01, ** *, / > < 0.001, *** *, P < 0.0001).

[0024] FIG. 12 is a graph that shows mti-Brucella total IgG antibodies detected in serum samples of mice vaccinated with 16MBvjbrR / DUF883 in combination with five different adjuvants (designated #1, #2, #3, #4, and #5), Statistical analysis was performed by comparing the mean of the groups using two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Values that are significantly different against the control group (c) and the vaccinated group without adjuvant (na) are indicated in asterisks (*, P < 0.05, * *, < 0.01, ** *, / > < 0.001, *** *, P < 0.0001).

[0025] FIGS. 13A to 13F shows representative H&E-stained section of the spleen (left column FIG. 13A, FIG. 15D), liver (middle column FIG. 13B, FIG. 13E), and lung (right column FIG. 13C, FIG. 13F) 4- week post-challenge with IxlO4CFU / ml B. melitensis 16M. No microscopic lesions were observed in any tissue from animals vaccinated with Treatment Group 1 (FIG. 13D, FIG. 13E, FIG. 13F). In contrast, unvaccinated mice had intense neutrophilic and histiocytic splenitis (FIG. 13 A) multifocal random foci of neutrophilic and histiocytic inflammation in the liver parenchyma (FIG. 13B), and multifocal BALT hyperplasia (FIG. 13C). Hematoxylin and eosin (H&E).

[0026] FIG. 14 shows graphs that demonstrate the efficacy of the vaccine formulation compared to Rev- 1 (Commercially available vaccine). The only approved B. melitensis vaccine Rev-1 for commercial use is known to be highly effective in preventing the disease, however two main drawbacks include: 1) inability to differentiate infected from vaccinated (DIVA compatibility) and 2) significant safety concerns both for animals and humans that has resulted in prohibition of its use in many countries around the world. The new vaccine formulation has been compared utilizing the same methodology described above to compare protective efficacy. Results demonstrate that the efficacy of the vaccine candidate is similar to the Rev-1 while being safe, providing a significant advantage.

[0027] FIG. 15 is a graph that showing the bactericidal effect of the five different adjuvants (designated #1, #2, #3, #4, and #5), mixed with the live attenuated vaccine 6MAvjbrR / DUF883 after 0, 1, 2, 3, 4, 5, 6 after the inoculants were prepared and used for the vaccination in mice.

[0028] FIG. 16 is a graph that showing the bactericidal effects of the five different adjuvants (designated #1 and #5), mixed with the live attenuated vaccine 16M vjbrR / DUF883 after 0, 1, 2, and 7 weeks after the inoculants were prepared and used for the vaccination in mice.DETAILED DESCRIPTION OF THE INVENTION

[0029] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0030] To facilitate the understanding of this invention, 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 invention. 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 embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] As used herein, the term “subject” as used herein refers to any mammal, preferably livestock, wild animals, or domestic animals, or even a human patient. 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, hooved animals, as well as humans.

[0036] As used herein, the term “immunogenically-effective amount” refers to that amount of an immunogen required to generate an immune response (e.g. invoke a cellular response and / or the production of protective levels of antibodies in a host upon vaccination).

[0037] 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.

[0038] As used herein, the term “carrier” as used herein refers to a diluent, adjuvant, excipient or vehicle with which the active compound is administered. Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. When administered to a subject, the pharmaceutically acceptable vehicles are preferably sterile. Water can be the vehicle when the active compound is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0039] 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.

[0040] 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 within the 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.

[0041] 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, andan 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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 nucleic acids (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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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)).

[0051] 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 (z.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of 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.

[0052] 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 acomparison 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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 produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH- 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 DNAmethodologies (e.g., single chain Fv) orthose identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0057] 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. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. 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).

[0058] 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.

[0059] 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 tospecific 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.

[0060] 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.

[0061] 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.

[0062] 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 or pathogen) 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.

[0063] As used herein, the term “effective amount” or “effective dose” refers to that amount of the peptide or protein T cell epitopes of the invention sufficient to induce immunity, to prevent and / or ameliorate an infection or to reduce at least one symptom of an infection and / or to enhance the efficacy of another dose of peptide or protein T cell epitopes. An effective dose may refer to the amount of peptide or protein T cell epitopes sufficient to delay or minimize the onset of an infection. An effective dose may also refer to theamount of peptide or protein T cell epitopes that provides a therapeutic benefit in the treatment or management of an infection. Further, an effective dose is the amount with respect to peptide or protein T cell epitopes of the invention alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of an infection. An effective dose may also be the amount sufficient to enhance a mammals own immune response against a subsequent exposure to an infectious agent. Levels of immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and / or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay. In the case of a vaccine, an “effective dose” is one that prevents disease and / or reduces the severity of symptoms. A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). A “prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms, in this case, an infectious disease, and more particularly, a Brucella infection. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, for the given parameter, an effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2 -fold, 5-fold, or more effect over a control. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1 or 2, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins), relevant portions incorporated herein by reference.

[0064] As used herein, in certain embodiments, the term “protective immune response” or “protective response” refers to an immune response mediated by antibodies against an infectious agent, which is exhibited by a mammal, which prevents or ameliorates an infection or reduces at least one symptom thereof. Peptide and protein T cell epitopes of the invention can stimulate the production of antibodies that, for example, neutralize infectious agents, blocks infectious agents from entering cells, blocks replication of said infectious agents, and / or protect host cells from infection and destruction. In other embodiments, the term can also refer to an immune response that is mediated by T-lymphocytes and / or other white blood cells against an infectious agent, exhibited by a mammal, that prevents or ameliorates Brucella infection or reduces at least one symptom thereof. Peptide and protein T cell epitopes of the invention can stimulate the T cell responses that, for example, neutralize infectious agents, kill bacteria, blocks infectious agents fromentering cells, blocks replication of said infectious agents, and / or protect host cells from infection and destruction.

[0065] 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.

[0066] In addition, in certain embodiments, “treatment,” “treat,” or “treating” refers to a method of reducing the effects of one or more symptoms of infection with Brucella. Thus, in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established infection, disease, condition, or symptom of the infection, disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition and / or complete prevention of infection. Further, as used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination.

[0067] 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.

[0068] 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.

[0069] The peptide(s) or protein(s) of the present invention can also be used in binding assays including, but are not limited to, 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, fluorescent 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).

[0070] As used herein, the term “administering” means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini -osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. The compounds of the invention can be administered alone or can be co-administered to the patient. Co-administration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). The compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0071] As used herein, the term “adjuvant” refers to a compound that when administered in conjunction with the compositions provided herein including embodiments thereof, augments the composition’s immune response. Generally, adjuvants are non-toxic, have high-purity, are degradable, and are stable.

[0072] Adjuvants can augment an immune response by several mechanisms including lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. The adjuvant increases the titer of induced antibodies and / or the binding affinity of induced antibodies relative to the situation if the immunogen were used alone. A variety of adjuvants can be used in combination with the agents provided herein including embodiments thereof, to elicit an immune response. Adjuvants augment the intrinsic response to an immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. Adjuvants for use with the present invention include EMULSIGEN® (oil and water emulsion where the oil phase is broken into small globules (2 microns or less) to absorb antigensand disperse them in a stable emulsion), aluminum hydroxide and aluminum phosphate, 3 De-O-acylated monophosphoryl lipid A (MPL™) (see GB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now part of Corixa). STIMULON™ QS-21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US Patent No. 5,057,540), (Aquila BioPharmaceuticals, Framingham, MA). Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers, and killed mycobacteria. Another adjuvant is CpG (WO 98 / 40100). Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before, concurrently with, or after administration of the therapeutic agent.

[0073] Other adjuvants contemplated for the invention are saponin adjuvants, such as Stimulon™ (QS-21, Aquila, Framingham, MA) or particles generated therefrom such as ISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvants include RC-529, GM-CSF and Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA). Other adjuvants include cytokines, such as interleukins (e.g., IL-1 a and [3 peptides, IL-2, IL-4, IL-6, IL-12, IL-13, and IL-15), macrophage colony stimulating factor (M-CSF), granulocyte -macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), chemokines, such as MIPloc and P and RANTES. Another class of adjuvants is glycolipid analogues including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immuno-modulators or adjuvants (see US Pat. No. 4,855,283). Heat shock proteins, e.g., HSP70 and HSP90, may also be used as adjuvants.

[0074] A further embodiment of the present invention involves the use of knockout, live Brucella that is delivered, e.g., orally, alone or using microencapsulation-mediated controlled release compositions. These compositions are storage-stable and compatible with a number of pharmaceutical agents. At this time and following decades of testing, the only effective vaccines for the prevention of brucellosis are living Brucella cells that stimulate the immune system through limited infection. Alternatives to the use of live, attenuated Brucella vaccines, including subunit vaccines and killed Brucella, have thus far proven non-efficacious. Live Brucella vaccines have been applied for decades to prevent brucellosis in cattle and sheep. Previous reports have postulated that use of attenuated strains appropriate for use in animals is difficult, since spontaneously derived strains retain some level of virulence and are genetically undefined as disclosed in Spink et al. (1962) Bulletin of the WHO 26, 409-19, incorporated herein by reference. Recent evaluation of attenuated mutants has confirmed the superiority of long-term survival in stimulating a protective immune response, yet the added safety of short-term survival cannot be overlooked in the development of animal vaccines. The present invention combines the optimal features of prolonged interaction with the immune system with enhanced safety of highly attenuated, single and / or double gene deletion Brucella mutants as a composition that is safe, free of side effects and efficacious in animals.

[0075] Brucella organisms can be delivered via aerosol to infect animals. 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 incorporated herein by reference. Infection incapacitates 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 animals. No vaccine for animals is available against this threat. Expected market and commercial need: The primary need for animal brucellosis vaccines is for specialty protection of military personnel, public health workers and veterinarians with the cross-over opportunity for extensive markets in the high-risk zones that occur throughout the world, particularly in the Middle East, Central Asia, Latin-America, Africa and the Far East. While there is a huge need for a animal brucellosis vaccine world-wide, the question is whether or not major biologies manufacturers will recognize these needs as a profitable market, thus it is more plausible that federal government subsidized stockpiles, e.g., Bioshield I and Bioshield II, to protect the general public and military personnel represent a more likely market. The potential reluctance of the general population to use live vaccines is based on a limited trust of scientists and government, and such thinking must not be used to deter the development of products based on otherwise sound scientific principles. The use of such vaccines in animals is expected under extreme circumstances, such as stockpiling large reserves for protection against biological terrorism or biological warfare. Starting with the work of Louis Pasteur, live vaccines have offered the best possible solution for immune protection. Use in animals requires that safety be determined beyond a shadow of a doubt. Questions concerning the preliminary production under Good Manufacturing Procedures and safety testing of such products warrant studies in animal models.

[0076] The present invention provides for controlled release compositions further comprising attenuated, live mutant vaccines. Drug delivery materials have historically been derived from many sources including commodity plastics and textile industries and have been incorporated into vehicles as diverse as pH responsive hydrogels and polymer microparticles or implants designed for surface or bulk erosion as disclosed in Langer RaP, N. A. (2003) Bioengineering, Food and Natural Products 49, 2990-3006, incorporated herein by reference. In the case of controlled release formulations, a drug is typically released by diffusion, erosion or solvent activation and transport. In most cases, the desired polymer characteristicsinclude biocompatibility, lack of immunogenicity, capability of breakdown by the body and water solubility. Many of the processes used to entrap pharmaceuticals involve harsh organic solvents which are bacteriocidal and capable of denaturing proteins. When considering controlled release vehicles for the entrapment of active enzymes or living cells, new alternatives are needed. A number of milder processes based on established technologies and variations have recently been applied to the delivery of active protein agents such as insulin, erythropoietins and chemokines as provided for in Marschutz et al (2000) Biomaterials 21, 1499-07. Takenaga et al (2002) J Control Release 79, 81-91. and Qiu et al (2003) Biomaterials 24, 11-18., all of which are incorporated by reference, or as encapsulants for living cells to permit transplantation as disclosed in Young et al (2002) Biomaterials 23, 3495-3501, hereby incorporated by reference. The technologies cover a wide range of materials including gelatin-based hydrogels, protein- PEG microparticles, novel PEG copolymers, biodegradable PLGA particles, PLG / PVA microspheres and surface modified nanospheres. Alginate, a naturally occurring biopolymer, is especially well suited to the entrapment of living cells. Alginate is a linear unbranched polysaccharide composed of l-4’-linked [3-D- mannuronic acid and a-L-guluronic acids in varying quantities. Alginate polymers are highly water-soluble and easily crosslinked using divalent cations such as Ca2+ or polycations such as poly-L-lysine as provided for in Wee & Gombotz (1998) Adv Drug Deliv Rev 31, 267-285, hereby incorporated by reference. The relatively mild conditions required to produce either an alginate matrix or particle is compatible with cell viability. Entrapment in alginate has been shown to greatly enhance viability and storage as provided for in Cui et al (2000) Int J Pharm 210, 51-59 and Kwok et al (1989) Proc. Int. Symp. Contol. Release Bioact. Mater. 16, 170-171, both of which are incorporated by reference. The physical properties such as porosity, rate of erosion, and release properties may be modulated through mixing alginates of different guluronic acid composition and through applying different coatings to the matrix as provided for in Wee & Gombotz (1998) Adv Drug Deliv Rev 31, 267-285. While in no way limiting the scope of the present invention, it is generally thought that release of a biomolecule from alginate matrices generally occurs through i) diffusion through pores of the polymer or ii) erosion of the polymer network. In general, the alginate matrix is stabilized under acidic conditions, but erodes slowly at pH of 6.8 or above.

[0077] The present invention exploits the performance and safety of live Brucella strains, which have been rendered safe by the elimination of certain genes that attenuate the Brucella.

[0078] There is strong support for oral vaccination with alginate and alginate / protein encapsulated strains as disclosed in Arenas-Gamboa et al. Infect Immun (2008) vol. 76, 2448-55, Kahl-McDonagh et al (2007) Infect Immun 75, 4923-32, Suckow et al (2002) J Control Release 85, 227-235, Kim et al (2002) J Control Release 85, 191-202., all of which are hereby incorporated by reference. In addition, lyophilization of bacteria in alginate beads extends their viability. Embodiments of the present invention include a storagestable delivery system that may be administered orally and is generally applicable to a number of select agents.

[0079] Pharmaceutical Formulations: The present compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable vehicle is a capsule (see e.g., U.S. Patent No. 5,698,155). In one embodiment, the vaccine is encapsulated using materials described in U.S. Patent Application Publication No. 2005 / 0260258, hereby incorporated by reference.

[0080] In a preferred embodiment, the active compound and optionally another therapeutic or prophylactic agent are formulated in accordance with routine procedures as pharmaceutical compositions adapted for administration to animal beings. Typically, the active compounds for administration are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions can also include a solubilizing agent. Compositions for administration can optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the active compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the active compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0081] Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, where in tablet or pill form, the compositions can be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for an oral administration of the active compound. In these later platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profde as opposed to the spiked profdes of immediate release formulations. A time delay material such as glycerol monostearate or glycerol stearate can also be used. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such vehicles are preferably of pharmaceutical grade.

[0082] Further, the effect of the active compound can be delayed or prolonged by proper formulation. For example, a slowly soluble pellet of the active compound can be prepared and incorporated in a tablet or capsule. The technique can be improved by making pellets of several different dissolution rates and fdling capsules with a mixture of the pellets. Tablets or capsules can be coated with a fdm that resists dissolution for a predictable period of time. Even the parenteral preparations can be made long acting, bydissolving or suspending the compound in oily or emulsified vehicles, which allow it to disperse only slowly in the serum.

[0083] Compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.

[0084] Thus, the compound and optionally another therapeutic or prophylactic agent and their physiologically acceptable salts and solvates can be formulated into pharmaceutical compositions for administration by inhalation or insufflation (either through the mouth or the nose) or oral, parenteral or mucosal (such as buccal, vaginal, rectal, sublingual) administration. In some embodiments, the administration is ophthalmic (e.g., eyes drops applied directly to the eye). In one embodiment, local or systemic parenteral administration is used.

[0085] For oral administration, the compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

[0086] Preparations for oral administration can be suitably formulated to give controlled release of the active compound. The microencapsulated vaccine gives a controlled release or continual boosting effect. Those formulations with vpB and alginate are described in U.S. Patent Application Publication No. 2005 / 0260258, hereby incorporated by reference.

[0087] For buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner.

[0088] For administration by inhalation, the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0089] The compositions can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0090] In addition to the formulations described previously, the compositions can also be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the pharmaceutical compositions can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0091] The compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

[0092] In certain preferred embodiments, the pack or dispenser contains one or more unit dosage forms containing no more than the recommended dosage formulation as determined in the Physician's Desk Reference (62nd ed. 2008, herein incorporated by reference in its entirety).

[0093] Methods of administering the active compound and optionally another therapeutic or prophylactic agent include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal, rectal, vaginal, sublingual, buccal or oral routes). In a specific embodiment, the active compound and optionally other prophylactic or therapeutic agents are administered intramuscularly, intravenously, or subcutaneously. The active compound and optionally other prophylactic or therapeutic agents can also be administered by infusion or bolus injection and can be administered together with other biologically active agents. Administration can be local or systemic. The active compound and optionally the prophylactic or therapeutic agent and their physiologically acceptable salts and solvates can also be administered by inhalation or insufflation (either through the mouth or the nose). In a preferred embodiment, local or systemic parenteral administration is used.

[0094] Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the active compound can be formulated as a suppository, with traditional binders and vehicles such as triglycerides.

[0095] Selection of a particular effective dose can be determined (e.g., via clinical trials) by a skilled artisan based upon the consideration of several factors, which will be known to one skilled in the art. Such factors include the disease to be treated or prevented, the symptoms involved, the subject's body mass, the subject's immune status and other factors known by the skilled artisan.

[0096] The dose of the active compound to be administered to a subject, such as an animal, is rather widely variable and can be subject to independent judgment. It is often practical to administer the daily dose at various hours of the day. However, in any given case, the amount administered will depend on such factors as the viability of the active component, the formulation used, subject condition (such as weight), and / or the route of administration.

[0097] Improving Diagnosis and DIVA capabilities for brucellosis in animals.

[0098] 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 LPS, 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).

[0099] 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.

[0100] Development of a new Point-of-Care lateral flow assay for the detection of bovine brucellosis using DUF883.

[0101] 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), ormolecular 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.

[0102] 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 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).

[0103] 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.

[0104] 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 (Figure 4). Further optimization to increase these parameters is underway. However, this novel dipstick test appears to be a promising candidate for the development of a rapid point-of-care diagnostic test for the diagnosis of bovine brucellosis.

[0105] FIG. 2A. Quantitative Analysis of Dipstick Results with field samples. Data interpretation using Image J Software. FIG. 2B. ROC Curve Analysis. Assessing dipstick sensitivity and specificity when using field samples.

[0106] Generation of a DIVA-compatible vaccine candidate for brucellosis. The objective is to perform a gene knockout targeting an immunodominant protein (DUF883, Candidate A) into the vaccine candidate B. melitensis to allow for differentiation between vaccinated and unvaccinated animals (DIVA) when using Candidate A as the antigen in the diagnostic assay.

[0107] Generation of a B. melitensis 16M AvjbR ACandidate A knockout mutantSteps1: 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.

[0108] Step 1: Primer Design.

[0109] Primers were designed to amplify regions upstream and downstream of the sequence encoding for 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.

[0110] 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.

[0111] 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.

[0112] Step 2: Generation of Deletion Cassette.

[0113] 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.

[0114] 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.

[0115] Step 3: Transformation of E. coli with deletion cassette.

[0116] Mach-1 E. coli 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.

[0117] Transformants that successfully grew on selective media.

[0118] Steps 4 and 5: Plasmid extraction and B. melitensis 16M AvjbR transformation by electroporation.

[0119] 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.

[0120] 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.

[0121] Screening for plasmid suicide and loss of kanamycin resistance.

[0122] 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.

[0123] 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.

[0124] Selected colonies.

[0125] Step 6: Confirmation of gene knockout through PCR and sequencing.

[0126] PCR: Colonies that were able to grow on selective media were subsequently chosen for PCR analysis to confirm deletion of the gene.

[0127] Figure 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.

[0128] Results: Four B. melitensis AvjbR ACandidate A (D883) deletion mutants were obtained (colonies 1, 2, 5 and 6).

[0129] 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.

[0130] 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.

[0131] Assessment of DIVA capability of the vaccine candidate.

[0132] Objective: To assess the DIVA capability of the vaccine candidate B. melitensis 16M vjbRAcandidateA vs. 16MA / W?. The trial consisted of the ability to distinguish vaccinated animalsutilizing 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 (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 16MAvjbRADUF883.

[0133] FIG. 8 shows the experimental design and timeline. Evaluation of Brucella melitensis FAAAvjbR / Candidate A DIVA capability in guinea pigs.

[0134] Animal protocol .

[0135] Facilities and procedures involving the use of the genetically modified strain of B. melitensis \ ('M \vjbR 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).

[0136] 2) Bacterial strains and vaccine seed stock.

[0137] B. melitensis biovar 1 strain 16M was originally acquired from the lung of an aborted goat.[4]The B. melitensis FAAAvjbR utilized was from the same stock described in STUDIES# 1-8. 16MAvjbRACandidate A master stock was created at Texas A&M University (TAMU) following the SOPs herein. 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.

[0138] 3) Animal selection, identification, and vaccination.

[0139] 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 inguinal region with 100 pl of 1 x 109CFU of Brucella melitensis \6W^vjbR or Brucella melitensis \ 6W^vjbR / ^Candidate A (D883).

[0140] Treatment groups:1) 16MAvjW? at 1x109CFU (sub-Q) (n=4)2) 16MAvjbRADUF883 at IxlO9CFU (sub-Q) (n=4)

[0141] 4) Analysis of the potential DIVA capabilities of Brucella melitensis FAAAvjbR / Candidate A.

[0142] Blood was collected at T=0-, 3-, 14-, and 28-days post-vaccination. Indirect ELISA (iELISA) for anti-Hrace / Za-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. Anti-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 (FIGS. 9A and 9B). However, a cleardistinction 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.

[0143] FIGS. 9A and 9B are graphs that show the detection of antibodies. FIG. 9A shows the detection of anti-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 mti-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 / hB 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.

[0144] Studies conducted in guinea pigs clearly demonstrate the DIVA capability when a second mutation is introduced to the 16MAvjbR. Studies to demonstrate the DIVA capability in the natural host are still a subject of investigation.

[0145] Efficacy studies in the mouse model of the 16MAvjbRACandidateA (D883).

[0146] The inventors assessed the adjuvanticity effect of 5 different vaccine formulations in an attempt to further enhance the immunogenicity and protective efficacy (i.e., the ability of the vaccine to prevent bacterial colonization) of the vaccine candidate B. melitensis 16MAvjbR / DUF883. Parameters studied during efficacy trial included: (1) the ability of the vaccine to prevent tissue colonization; (2) the ability of the vaccine to induce a strong and robust humoral response; and / or (3) the ability of the vaccine to prevent tissue pathology: (histopathologic examination of spleen, liver, lung).

[0147] Species: C57BL / 6J female mice 6-8 week. Type of Study: Animal efficacy in female mice. Condition: Laboratory study in Biocontainment 2 and 3 facilities at Texas A&M University. Duration of the Study: 5 months. Vaccine: Single dose of IxlO5of B. melitensis 16MAvjbR / DUF883 + adjuvant candidates (see below for a complete set of groups).

[0148] FIG. 10 shows the experimental design and timeline. Evaluation of the efficacy of different vaccine formulations of the candidate Brucella melitensis 16M vjbR / DUF883 in mice.

[0149] Protocols and Results. Facilities and procedures involving the use of the genetically modified strain of B. melitensis 16MAvjbR / DUF883 and the mouse animal model were conducted under protocols approved by Texas A&M University (IACUC 2021-0038, IBC2018-013 and IBC2019-019).

[0150] Bacterial strains and vaccine seed stock. B. melitensis \ <FF\ \vjbR and B. melitensis I6MA / WL 'DUF883 strains were acquired and developed at Texas A&M University following the required approvals and protocols. The B. melitensis biovar 1 strain 16M was originally acquired from the lung of an aborted goat at Texas A&M University[5]. This strain has been tested in multiple animal species and has been demonstrated to be highly virulent. It is also routinely tested in vitro in the J774A.1 macrophage forbehavior. All wild-type and vaccine strains were grown on tryptic soy agar (TSA) or tryptic soy broth (TSB) at 37°C for 72 hours. Immunization and inoculum doses were verified by retrospective serial dilution, plating, and enumeration of colonies.

[0151] Animal selection, identification, and vaccination. Total number of animals used for the study: Thirty-five (35). Age and sex: C57BL / 6J female mice 6-8 week. Animal source: Mice were acquired from the Texas A&M Institute for Genomic Medicine. Animal housing conditions and vaccination dose / route: Mice were grouped-housed (n=5 per treatment) in micro isolator cages at a BSL-2 animal facility. Following an acclimation period of 3 days, all animals were vaccinated via subcutaneous injection cranial to the scapular area with a single dose containing IxlO5CFU of Brucella melitensis 16M vjbR / DUF883 with our without different adjuvants 10-50% (v / v) in a total of lOOul inoculum. Brucella melitensis 16MAv / W? was used as a control.

[0152] Treatment groups:1) IxlO5\6M vjbR / Candidate A + adjuvant #1 (n=5)2) IxlO5\6MAvjbR / Candidate A + adjuvant #2 (n=5)3) IxlO5\6MAvjbR / Candidate A + adjuvant #3 (n=5)4) IxlO5\6MAvjbR / Candidate A + adjuvant #4 (n=5)5) IxlO5\6M vjbR / Candidate A + adjuvant #5 (n=5)6) IxlO516MAvjW? (n=5)7) Control (n=5)

[0153] Ability of the vaccine to prevent tissue colonization. Following vaccination, all mice were challenged at 8 weeks post- vaccination with 5xl04CFU of B. melitensis 16M via intranasal inoculation in a 30 pl inoculum (15ul on each nostril). At 4 weeks post-challenge, mice were euthanized via carbon dioxide inhalation and necropsied, and efficacy was determined by enumerating bacterial colonization of the spleen, liver, and lung. Briefly, organs were homogenized in 1 ml of sterile PBS using an Omni® Tissue homogenizer. After serial dilutions, 100 pl of each dilution was plated on Farrell’s media and incubated at 37°C for 72 hours, and colonies were enumerated.

[0154] There was a significant reduction of tissue colonization in animals vaccinated with the vaccine candidate alone, however, two of the treatment groups (Treatment groups 1 and 5) containing different adjuvants in their formulation were able to significantly enhance the protective efficacy, demonstrating their adjuvanticity effect. Excitingly, the vaccine formulation from Treatment group #1 was able to induce sterile immunity in 100% of the mice in the examined target organs (FIGS. 13A-13F). These results highly suggest that the formulation in Treatment group # 1 had a potent immunostimulant effect that enhances and confers a significant protective efficacy to a degree never seen in our efficacy trials over the past years. This formulation will be further tested both in vivo and in vitro.

[0155] FIGS. 11A, 1 IB include graphs that shows the evaluation of vaccine efficacy in mice vaccinated with 16MBvjbrR / DUF883 in combination with five different adjuvants (designated #1, #2, #3, #4, and #5). Control groups included \6MAvjbrR alone and naive, non-vaccinated mice. Bacterial burden 4 weeks postchallenge in the spleen, liver, and lung per organ are depicted. The horizontal bar indicates the mean. P values were determined by unpaired t-test with Welch’s correction. Values that are significantly different are indicated by bars and asterisks (*, P < 0.05, * *, P < 0.01, ** *, / > < 0.001, *** *, < 0.0001).

[0156] Ability of the vaccine to induce a strong and robust humoral response. Blood was collected 0 at 4, 6, 8 weeks post-vaccination and 2 weeks (68 days) and 4 weeks (82 days) following challenge. An indirect ELISA (iELISA) for anti-5race / / a-specific total IgG was performed to compare the immune response between the different treatment groups and to assess the ability of the different formulations to induce a humoral response. . vF\-Briicella IgG antibodies utilizing LPS in all treatment groups were detected at 4 weeks post- vaccination with peak levels observed between 4 weeks and 6 weeks post vaccination (FIG. 14). No significant differences were found between all vaccinated groups except in Treatment Group 1, Treatment Group 2, and Treatment Group 5 (FIG. 14). However, all mice in Treatment Group 1 had a significantly stronger and robust humoral response compared to other animals (Figure 3). The strong humoral response coincided with sterile protective efficacy.

[0157] FIG. 12 is a graph that shows wB-Brucella total IgG antibodies detected in serum samples of mice vaccinated with \6MAvjbrR / DUF883 in combination with five different adjuvants (designated #1, #2, #3, #4, and #5), Statistical analysis was performed by comparing the mean of the groups using two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Values that are significantly different against the control group (c) and the vaccinated group without adjuvant (na) are indicated in asterisks (*, P < 0.05, * *, < 0.01, ** *, / > < 0.001, *** *, < 0.0001).

[0158] Ability of the vaccine to prevent tissue pathology: (histopathologic examination of target organs). Tissue pathology was evaluated following challenge with 16M B. melitensis to determine if vaccination prevented or reduced the inflammatory response secondary to the infection. No significant lesions were noted in animals vaccinated in the Treatment Group 1 group following challenge in any tissue and correlated with the absence of recoverable bacteria. The most significant histologic findings in mice from the unvaccinated control were random foci of neutrophilic inflammation and lymphoplasmacytic periportal infiltrates in the liver (FIG. 1 IB), infiltration and expansion of the white pulp with neutrophils and macrophages in the spleen (FIG. 11A) and focal BALT hyperplasia (FIG. 14). All of the histopathologic changes described in the control mice are those typically observed in infected animals. This demonstrates that not only that the vaccine formulation in Treatment Group 1 is not only capable of completely preventing tissue colonization in the organs examined, induced a stronger humoral response but equally important prevented the development of any type of tissue pathology.

[0159] FIGS. 13A to 13F shows representative H&E-stained section of the spleen (left column FIG. 13A, FIG. 13D), liver (middle column FIG. 13B, FIG. 13E), and lung (right column FIG. 13C, FIG. 13F) 4- week post-challenge with IxlO4CFU / ml B. melitensis 16M. No microscopic lesions were observed in anytissue from animals vaccinated with Treatment Group 1 (FIG. 13D, FIG. 13E, FIG. 13F). In contrast, unvaccinated mice had intense neutrophilic and histiocytic splenitis (FIG. 13 A) multifocal random foci of neutrophilic and histiocytic inflammation in the liver parenchyma (FIG. 13B), and multifocal BALT hyperplasia (FIG. 13C). Hematoxylin and eosin (H&E).

[0160] FIG. 14 shows graphs that demonstrate the efficacy of the vaccine formulation compared to Rev- 1 (Commercially available vaccine). The only approved B. melitensis vaccine Rev-1 for commercial use is known to be highly effective in preventing the disease, however two main drawbacks include: 1) inability to differentiate infected from vaccinated (DIVA compatibility) and 2) significant safety concerns for animals that has resulted in prohibition of its use in many countries around the world. The new vaccine formulation has been compared utilizing the same methodology described above to compare protective efficacy. Results demonstrate that the efficacy of the vaccine candidate is similar to the Rev-1 while being safe, providing a significant advantage.

[0161] Evaluation of toxicity effect on the Live Attenuated Vaccine candidate of the different vaccine formulations. Previous studies have demonstrated that the live attenuated vaccine candidate can persist for up to 4 weeks following vaccination in the mouse model (see previous study reports). This is in contrast with the prolonged persistence of up to 18-20 weeks typically observed with 16M. Although, this provides strong evidence that attenuation is important for safety, it is well known that the best vaccines in terms of protective efficacy are those that can persist for some time in the host to mount a strong protective immunity. It is our expectation that the final vaccine formulation does not affect the survival characteristics of the organism specially at the initial stages of vaccination, which are known to be necessary to mount the strong immune response. This experiment was designed to demonstrate any potential bactericidal effect caused by the vaccine formulations described above (Treatment Groups 1-5).

[0162] Protocol: Following manufacturing, a subset of doses from all the vaccine formulations were subsequently plated onto TSA at 0, 1, 2, 3, 4 and 5 days post-vaccination. All doses were kept at 4°C for storage.

[0163] Assessment of potential bactericidal effects of the adjuvants. To assess the potential bactericidal effects of the adjuvants to the live attenuated vaccine Brucella melitensis 16M vjbR / DUF883, the full dose of each vaccine formulation was plated and subsequently monitored in vitro for 5 days. There was no evidence of a bactericidal effect caused by the formulation, demonstrating that specifically that the final formulation in Treatment Group 1 does not affect vaccine viability at the early stages (FIG. 15).

[0164] Subsequently our attention was focused on assessing the viability of the best two formulations (Treatment Group 1 and 5) for an extended time. Similarly, there seems to be no significant bactericidal effect at 2 weeks post vaccination (FIG. 16), however by 7 weeks post infection there was no recoverable viable bacteria in the formulation from Treatment Group 1, in contrast to the vaccine in Treatment Group 5 or the vaccine alone (FIG. 16). However, despite this difference in the bacterial viability based on the formulation, it seems that this does not affect protective efficacy since the formulation in Treatment Group 1 is far superior.

[0165] FIG. 15 is a graph that showing the bactericidal effect of the five different adjuvants (designated #1, #2, #3, #4, and #5), mixed with the live attenuated vaccine 16MBvjbrR / DUF883 after 0, 1, 2, 3, 4, 5, 6 after the inoculants were prepared and used for the vaccination in mice.

[0166] FIG. 16 is a graph that showing the bactericidal effects of the five different adjuvants (designated #1 and #5), mixed with the live attenuated vaccine 16MBvjbrR / DUF883 after 0, 1, 2, and 7 weeks after the inoculants were prepared and used for the vaccination in mice.

[0167] The newly designed vaccine formulation described as “Treatment Group 1” significantly outperforms the previously described formulation. Superiority is observed via 3 parameters: (1) ability to prevent tissue colonization and in this case providing sterile immunity, something rarely achieved when testing improved Brucella vaccines; (2) ability to mount a stronger humoral immune response and (3) ability to prevent the development of tissue pathology in targeted organs. These results provide us with strong evidence for the selection of this vaccine formulation for the efficacy studies in the target host.

[0168] In vitro studies demonstrate that the formulation in Treatment Group 1 does not affect the initial vaccine viability for the first week, however viability starts to be reduced at two weeks with no recoverable bacteria at 50 days post-formulation. It is important to highlight that during the manufacturing process, addition of the adjuvant is only done prior to vaccination and this should not affect vaccine shelf stability. Nonetheless, it is important to mention that the decrease in viability later does not affect protective efficacy or overall vaccine performance.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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 thisapplication, 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.

[0173] 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.

[0174] 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.

[0175] 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 vaccine composition comprising: a Brucella bacterial strain from which at least a portion of a DUF883 protein has been deleted, wherein the Brucella bacterial strain is provided in an amount sufficient to trigger an immune response to a Brucella infection; and a pharmaceutically acceptable vaccine carrier.

2. The vaccine composition of claim 1, wherein the composition further comprises an adjuvant.

3. The vaccine composition of claim 2, wherein the adjuvant is selected from at least one of: dimethyldioctadecyl ammonium bromide, oil-in-water emulsion, alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, cytosine-guanosine oligonucleotide (CpG-ODN) sequence, granulocyte macrophage colony stimulating factor (GM-CSF), monophosphoryl lipid A (MPL), poly(I:C), MF59, Quil A, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), FIA, montanide, poly (DL-lactide- coglycolide), squalene, virosome, AS03, ASO4, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL- 12, IL-15, IL-17, IL-18, STING, CD40L, pathogen-associated molecular patterns (PAMPs), damage- associated molecular pattern molecules (DAMPs), Freund's complete adjuvant, Freund's incomplete adjuvant, transforming growth factor (TGF)-beta antibody or antagonists, A2aR antagonists, lipopolysaccharides (LPS), Fas ligand, Trail, lymphotactin, Mannan (M-FP), APG-2, Hsp70 and Hsp90, pattern recognition receptor ligands, TLR3 ligands, TLR4 ligands, TLR5 ligands, TLR7 / 8 ligands, or TLR9 ligands.

4. The vaccine composition of claim 1, wherein the DUF883 protein is ElaB from a genome of Brucella melitensis 16M BMEI0805.

5. The vaccine composition of claim 1, wherein the DUF883 protein is 104 amino acids comprising a transmembrane alpha helix domain, a long cytosolic tail, and a short extracellular sequence.

6. The vaccine composition of claim 1, wherein the DUF883 protein is deleted from a live attenuated vaccine.

7. The vaccine composition of claim 6, further comprising a diagnostic gene knockout comprises a differentiation of infected animals from vaccinated animals (DIVA) mutant that includes AvirB12, Abcsp31, or Aasp24.

8. The vaccine composition of claim 7, wherein an attenuating gene knockout is Brucella strain 16MAvjbRADUF883, Av / DIVA, or 16M vJbR / )UF883 / DWA .

9. The vaccine composition of claim 1, wherein the at least a portion of the DUF883 protein that is deleted, is by deletion of sequences that are 5’-, 3’-, or within a coding sequence of a DUF883 gene, selected from deleting sequences, frameshift, insertions, early termination or equivalent thereof, elimination of promoter sequences, or sequences that control expression of the DUF883 gene, such thatone or more mutations prevent expression of some or all of the DUF883 protein or portions thereof, or transcription of the DUF883 gene, or expression of the DUF883 gene is silenced.

10. The vaccine composition of claim 1, wherein the vaccine is used for a prophylaxis, an amelioration of symptoms, a treatment, or any combinations thereof against brucellosis in an animal subject or a human.

11. The vaccine composition of claim 1, wherein the vaccine is administered by an oral, an intranasal, a parenteral, an intradermal, an intramuscular, an intraperitoneal, an intravenous, a subcutaneous, an epidural, a mucosal, a rectal, a vaginal, a sublingual, or a buccal route.

12. The vaccine composition of claim 1, further comprising an encapsulating agent that is an alginate bead or a microsphere.

13. An immunogenic formulation comprising a live attenuated Brucella that is \ \ \vjbR \DU 883. wherein the immunogenic formulation has higher safety and efficacy for animals against different Brucella species than live attenuated Brucella strain 16M. \v / bR.

14. The immunogenic formulation of claim 13, wherein the formulation further comprises an adjuvant.

15. The immunogenic formulation of claim 13, wherein the Brucella species affects livestock and companion animals selected from B. abortus, B. melitensis, B. suis, and B. canis.

16. The immunogenic formulation of claim 14, wherein the adjuvant is selected from at least one of: dimethyldioctadecyl ammonium bromide, oil-in-water emulsion, alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, cytosine-guanosine oligonucleotide (CpG-ODN) sequence, granulocyte macrophage colony stimulating factor (GM-CSF), monophosphoryl lipid A (MPL), poly(I:C), MF59, Quil A, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), FIA, montanide, poly (DL-lactide- coglycolide), squalene, virosome, AS03, ASO4, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL- 12, IL-15, IL-17, IL-18, STING, CD40L, pathogen-associated molecular patterns (PAMPs), damage- associated molecular pattern molecules (DAMPs), Freund's complete adjuvant, Freund's incomplete adjuvant, transforming growth factor (TGF)-beta antibody or antagonists, A2aR antagonists, lipopolysaccharides (LPS), Fas ligand, Trail, lymphotactin, Mannan (M-FP), APG-2, Hsp70 and Hsp90, pattern recognition receptor ligands, TLR3 ligands, TLR4 ligands, TLR5 ligands, TLR7 / 8 ligands, or TLR9 ligands.

17. The immunogenic formulation of claim 13, wherein the DUF883 protein is ElaB from a genome of Brucella melitensis 16M BMEI0805.

18. The immunogenic formulation of claim 13, wherein the DUF883 protein is 104 amino acids comprising a transmembrane alpha helix domain, a long cytosolic tail, and a short extracellular sequence.

19. The immunogenic formulation of claim 13, further comprising a diagnostic gene knockout comprises a differentiation of infected animals from vaccinated animals (DIVA) mutant that includes AvirB12, Abcsp31, or Aasp24.

20. The immunogenic formulation of claim 13, wherein the live attenuated Brucella further comprises A / ^R / DIVA, 16MAvj ?R / DIVA, or any combinations thereof.

21. The immunogenic formulation of claim 13, wherein the live attenuated Brucella is used for a prophylaxis, an amelioration of symptoms, a treatment, or any combinations thereof against brucellosis in an animal or a human.

22. The immunogenic formulation of claim 13, wherein the live attenuated Brucella is administered by an oral, an intranasal, a parenteral, an intradermal, an intramuscular, an intraperitoneal, an intravenous, a subcutaneous, an epidural, a mucosal, a rectal, a vaginal, a sublingual, or a buccal route.

23. The immunogenic formulation of claim 13, wherein the at least a portion of the DUF883 protein that is deleted, is by deletion of sequences that are 5’-, 3’-, or within a coding sequence of DUF883 gene, selected from deleting sequences, frameshift, insertions, early termination or equivalent thereof, elimination of promoter sequences, or sequences that control expression of the DUF883 gene, such that one or more mutations prevent expression of some or all of the DUF883 protein or portions thereof, or transcription of the DUF883 gene, or expression of the DUF883 gene is silenced.

24. The immunogenic formulation of claim 13, further comprising an encapsulating agent that is an alginate bead or a microsphere.

25. A method of immunizing a mammal against Brucella comprising: immunizing the mammal with an immunogenic formulation comprising a live attenuated Brucella that is 16MAvjbR and wherein at least a portion of a Brucella DUF883 protein or immunogenic portions thereof has been deleted or inactivated, and an adjuvant, wherein the immunogenic formulation has higher safety and efficacy for animals against a different Brucella species than the live attenuated Brucella strain 16M\vjbR \.

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