WO2012037546A2

WO2012037546A2 - A broadly protective protein of brucella and its use in brucellosis vaccine preparation

Info

Publication number: WO2012037546A2
Application number: PCT/US2011/052126
Authority: WO
Inventors: Ramesh Vemulapalli; Virendra K. Garg; Tracy H. Vemulapalli; Nammalwar Sriranganathan
Original assignee: Purdue Research Foundation; Virginia Tech Intellectual Properties, Inc.
Priority date: 2010-09-17
Filing date: 2011-09-19
Publication date: 2012-03-22
Also published as: WO2012037546A3

Abstract

A vaccine has a Brucella adhesin (BrAd) polypeptide. The vaccine is capable of conferring immunity against a plurality of Brucella species, including but not limited to B. abortus, B. melitensis and B. suis. Additionally, an isolated nucleic acid sequence encoding a Brucella adhesin polypeptide is disclosed. The nucleic acid sequence may be inserted into a plasmid and introduced into a Brucella strain to confer immunity against a plurality of pathogenic Brucella species. Further, a method of treating or preventing brucellosis comprising administering a vaccine having a Brucella adhesin (BrAd) polypeptide.

Description

A BROADLY PROTECTIVE PROTEIN OF BRUCELLA AND ITS USE IN BRUCELLOSIS

VACCINE PREPARATION

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Serial. No.

61/383,887, filed September 17, 2010, which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, and drawings.

TECHNICAL FIELD

This disclosure pertains generally to the field of molecular biology and infectious diseases. More specifically, this disclosure pertains to a vaccine for the pathogen

Brucella.

BACKGROUND

Brucellosis, an infectious disease of animals and humans, is caused by certain bacteria of the genus Brucella. In domestic and wild mammals brucellosis often results in abortions and infertility. In humans, brucellosis is primarily a zoonotic disease; humans acquire the infection by consuming infected dairy and meat products or by coming in contact with infected animal tissues and secretions. Human brucellosis manifests itself as a chronic infection with undulant fever and general malaise; other clinical signs vary depending on the affected organ systems. Human brucellosis is one of the most widely present zoonotic diseases in the world and is a significant public health concern in developing countries.

Brucellosis in domestic animals such as cattle, goats, sheep and pigs is controlled through vaccination using live attenuated Brucella strains. The currently available vaccines are B. abortus strains 19 and RB51 for cattle vaccination, and B. melitensis Rev. 1 for sheep and goat vaccination. However, these vaccines provide less than desirable protection against brucellosis in the target animals, especially when the infection is caused by different Brucella species.

Moreover, three Brucella species (B. melitensis, B. suis and B. abortus) are highly virulent to humans and are also considered potential bioterror/bio warfare agents. There is no vaccine currently available for human use. Development of an effective and safe vaccine for human brucellosis requires identification of an antigenic component of the bacteria that can induce robust, broad protection against pathogenic Brucella species.

Accordingly, there is always a need for an improved vaccine against Brucella. It is to this need, among others, that this application is directed. SUMMARY

This disclosure in part pertains to the development of a vaccine for brucellosis in a vertebrate or a subject. Specific embodiments include a vaccine and a method of vaccinating against onset of disease in subjects infected by Brucella strains using a THERAPEUTICALLY EFFECTIVE AMOUNT of an isolated Brucella adhesin (BrAd) polypeptide in combination with a pharmaceutically-acceptable carrier. In some illustrative examples, fragments of the BrAd polypeptide may be used in addition or instead of the complete BrAd polypeptide.

Another specific embodiment includes an isolated Brucella adhesin (BrAd) polypeptide from the Brucella genus. The BrAd polypeptide can have and amino acid sequence substantially similar to the amino acid sequence of SEQ ID NOS: 1-8.

Another specific embodiment includes an isolated nucleic acid molecule encoding the isolated BrAd polypeptide. An isolated polynucleotide encoding a Brucella adhesin (BrAd) polypeptide is also disclosed herein.

Another specific embodiment includes an antibody that selective binds to a BrAd polypeptide.

Another specific embodiment includes a method of treating or preventing brucellosis in vertebrates or subjects susceptible to brucellosis. Susceptible vertebrates include, but are not limited to, cattle, sheep, goats, pigs, bison, elk, camels, and humans. The method comprises administering a vaccine having a Brucella adhesin polypeptide. The vaccine can confer immunity against a plurality of Brucella species. More particularly, the vaccine can confer immunity against a plurality of Brucella species, including but not limited to, B. abortus, B. melitensis, and B. suis.

Another specific embodiment includes a method of manufacturing a vaccine for treating or preventing brucellosis, comprising (a) amplifying a nucleic acid sequence encoding a Brucella adhesion (BrAd) polypeptide; (b) digesting the nucleic acid sequence with restriction enzymes; (c) inserting the digested nucleic acid sequence into a plasmid; and (d) introducing the plasmid into a strain of Brucella. The BrAd polypeptide can then be isolated and can be incorporated into a vaccine.

In another specific embodiment, Brucella adhesin (BrAd) polypeptides could be used with other vaccines to achieve synergic therapeutic value. For example, expression of BrAd protein in vaccine strain B. abortus RB51 enhanced its protective efficacy against three virulent Brucella species in a murine model of brucellosis. In such cases, the Brucella BrAd adhesin protein can be useful for the development of any type of vaccine, including but not limited to, subunit, DNA, and recombinant vector-based vaccines for protection against brucellosis. DEFINITIONS

The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

The term "amino acid" refers to naturally occurring and non-natural 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 encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (by way of example, norleucine) or may have modified peptide backbones, while still retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.

The term "conservatively modified variants" applies to both natural and non- natural amino acid and natural and non-natural nucleic acid sequences, and combinations thereof. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those natural and non-natural nucleic acids which encode identical or essentially identical natural and non-natural amino acid sequences, or where the natural and non-natural nucleic acid does not encode a natural and non-natural amino acid sequence, to essentially identical sequences. By way of example, because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Thus by way of example every natural or non-natural nucleic acid sequence herein which encodes a natural or non-natural polypeptide also describes every possible silent variation of the natural or non- natural nucleic acid. One of skill will recognize that each codon in a natural or non-natural 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 natural and non-natural nucleic acid which encodes a natural and non-natural polypeptide is implicit in each described sequence. The term "effective amount," as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. By way of example, an agent or a compound being administered includes, but is not limited to, a natural amino acid polypeptide, non-natural amino acid polypeptide, modified natural amino acid polypeptide, or modified non-amino acid polypeptide.

Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be administered for prophylactic, enhancing, and/or therapeutic treatments. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study.

The term "nucleic acid sequence" as used herein, refers to the order and identity of the nucleotides comprising a nucleic acid.

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl

ribonucleotides, and peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses "splice variants."

Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. "Splice variants," as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. 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 polymer.

The term "pharmaceutically acceptable", as used herein, refers to a material, including but not limited to, a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The term "prophylactically effective amount," as used herein, refers to that amount of a composition containing at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide prophylactically applied to a patient which will relieve to some extent one or more of the symptoms of a disease, condition or disorder being treated. In such prophylactic applications, such amounts may depend on the patient's state of health, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, a dose escalation clinical trial.

The phrase "substantially similar," in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 75%, preferably at least 85%, more preferably at least 90%, 95% or higher or any integral value therebetween nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm such as those described below for example, or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 10, preferably about 20, more preferable about 40-60 residues in length or any integral value therebetween, preferably over a longer region than 60-80 residues, more preferably at least about 90-100 residues, and most preferably the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence for example.

The term "synergistic", as used herein, refers to a combination of prophylactic or therapeutic effective agents which is more effective than the additive effects of any two or more single agents. A synergistic effect of a combination of prophylactic or therapeutic agents may permit the use of lower dosages of one or more of the agents and/or less frequent administration of the agents to a subject with a specific disease or condition. In some cases, a synergistic effect of a combination of prophylactic or therapeutic agents may be used to avoid or reduce adverse or unwanted side effects associated with the use of any single therapy.

The term "therapeutically effective amount," as used herein, refers to the amount of a composition containing at least one non-natural amino acid polypeptide and/or at least one modified non-natural amino acid polypeptide administered to a patient already suffering from a disease, condition or disorder, sufficient to cure or at least partially arrest, or relieve to some extent one or more of the symptoms of the disease, disorder or condition being treated. The effectiveness of such compositions depends on conditions including, but not limited to, the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. By way of example only, therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows SDS-PAGE analysis (A) and Western blot plots (B & C) that demonstrate purification of the BrAd polypeptide from E. coli.

FIG. 2 shows an ELISA assay demonstrating the overexpression of BrAd in recombinant strain RB51/BrAd.

FIG. 3 shows an ELISA assay of BrAd-specific (A) and strain RB51 -specific (B)

IgG, IgGl, and IgG2a antibodies.

FIG. 4 shows flow cytometry detection of BrAd- and RB51 -specific T cells secreting interferon-gamma at six (6) weeks post-vaccination.

FIG. 5A shows that the BrAd polypeptide can protect mice against B. abortus challenge.

FIG. 5B shows that the BrAd polypeptide can protect mice against a B.

melitensis challenge.

FIG. 6 shows the combination of the BrAd polypeptide with the vaccine RB51 can confer improved protection against pathogenicity compared to RB51 alone.

DETAILED DESCRIPTION

Specific embodiments are directed to a vaccine for the immunization of vertebrates against disease caused by pathogenic Brucella species/strains. The vaccine comprises a polypeptide designated Brucella adhesin (BrAd) and is capable of inducing a protective immune response against the pathogen. Specific embodiments include a vaccine and a method of vaccinating against onset of disease in subjects infected by Brucella strains using a therapeutically effective amount of an isolated Brucella adhesin (BrAd) polypeptide in combination with a pharmaceutically-acceptable carrier. In some illustrative examples, fragments of the BrAd polypeptide may be used in addition or instead of the complete BrAd polypeptide.

BrAd polypeptides and vaccine compositions provided hereby are useful in eliciting Brucella specific immune responses e.g. as vaccines for prophylaxis or therapy of Brucella-associated conditions. The BrAd polypeptide, e.g. vaccines, for use in the prophylaxis or treatment of Brucella-associated diseases can generate immune responses, e.g. responses involving cellular immunity capable of mediating the regression of chronic Brucella infections. Such immune response can be targeted towards T-cells, e.g. CD4+ cells, e.g. by the use of appropriate adjuvants.

Another specific embodiment includes an isolated Brucella adhesin (BrAd) polypeptide from the Brucella genus. The BrAd polypeptide can have an amino acid sequence substantially similar to the amino acid sequence of SEQ ID NOS: 1-8. As can be seen from Table 1, the BrAd polypeptides can have variable character. It is appreciated that the molecular organization of trimeric autotransporter adhesins consists of trimeric surface structures with a head-stalk-anchor architecture. The head and stalk are composed of a small set of domains, building blocks that are frequently arranged repetitively. Functionally, the head mediates binding to the host, the stalk projects the head from the membrane, and the anchor provides the pore for autotransport and attaches the protein to the bacterial surface after export is complete. Table 1 shows examples of BrAd polypeptides.

TABLE 1

As can be seen, the gene encoding a putative trimeric autotransporter adhesin and the protein product designated Brucella adhesin (BrAd) can vary among different Brucella species and also among different strains/isolates of each Brucella species. This polypeptide was in Brucella species/strains.

Another specific embodiment includes an isolated nucleic acid molecule encoding the isolated BrAd polypeptide. An isolated polynucleotide encoding a Brucella adhesin (BrAd) polypeptide is also disclosed herein. Alternatively, a nucleic acid encoding the polypeptide can be incorporated into a suitable recombinant virus vector and introduced into a host organism, such as a human, in order that expression of the nucleic acid can give rise to the polypeptide in situ.

Another specific embodiment includes an antibody that selectively binds to a BrAd polypeptide. Procedures for raising polyclonal antibodies are well known in the art.

Typically, such antibodies can be raised by administering a protein, i.e., a BrAd having SEQ ID NOs: 1-8 (or fragment thereof), subcutaneously to rabbits which have first been bled to obtain pre-immune serum. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing a protein according to the present disclosure after SDS-polyacrylamide gel electrophoresis. The rabbits can then be bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Antibodies useful include, but are not limited to polyclonals, monoclonals, chimerics, single chains, Fab fragments, and Fab expression libraries.

Another specific embodiment includes a method of treating or preventing brucellosis in vertebrates or subjects susceptible to brucellosis. Susceptible vertebrates include, but are not limited to, cattle, sheep, goats, pigs, bison, elk, camels, and humans. The method comprises administering a vaccine comprising a Brucella adhesin polypeptide. The vaccine can confer immunity against a plurality of Brucella species. In one example, the vaccine can confer immunity against a plurality of Brucella species, including but not limited to, B. abortus, B. melitensis, or B. suis.

Another specific embodiment includes a method of manufacturing a vaccine for treating or preventing brucellosis, comprising (a) amplifying a nucleic acid sequence encoding a Brucella adhesion (BrAd) polypeptide; (b) digesting the nucleic acid sequence with restriction enzymes; (c) inserting the digested nucleic acid sequence into a plasmid; and (d) introducing the plasmid into a strain of Brucella or an attenuated strain. The BrAd polypeptide can then be isolated and incorporated into a vaccine. In another specific embodiment, Brucella adhesin (BrAd) protein could be used with other vaccines to achieve synergic therapeutic value. For example, expression of BrAd protein in vaccine strain B. abortus RB51 enhanced its protective efficacy against three virulent Brucella species in a murine model of brucellosis. In such cases, the Brucella BrAd adhesin protein can be useful for the development of any type of vaccine, including but not limited to, subunit, DNA, and recombinant vector-based vaccines for protection against brucellosis.

In another embodiment, the BrAd polypeptides may be given for therapeutic or prophylactic purposes. Routes and procedures of administration can include, but are not limited to, standard intramuscular, subcutaneous, intradermal, intravenous, oral or rectal routes and procedures. Such immunogenic compositions, e.g. for use as a therapeutic or prophylactic vaccine in humans or non-human animals, can comprise an adsorption complex conventionally used as vaccine adjuvant having adsorbed thereon a polypeptide obtainable as mentioned above.

It is understood that fragments of the above polypeptides are encompassed by this disclosure. The polypeptides can be produced in purified form by conventional techniques. For example, to isolate the proteins, a protocol involving a host cell such as Escherchia coli may be used, in which the E. coli host cell carrying a recombinant plasmid is propagated, homogenized, and the homogenate is centrifuged to remove bacterial debris. Alternatively, fragments of the proteins of the present disclosure can be produced by digestion of a full-length protein with proteolytic enzymes. Alternatively, fragments of the genes encoding the proteins of the present invention may be synthesized by using a PCR technique together with specific sets of primers chosen to represent particular portions of the protein of interest. Chemical synthesis can also be used to make suitable fragments.

An antigenic determinant of a BrAd polypeptide can for example be represented and provided in connection with specific embodiments either by the full sequence of the BrAd protein concerned, or by such sub-sequences as may be desired, e.g. a sequence fragment comprising at least 25%, e.g. at least 50%> or 75% of the full sequence of the protein concerned, e.g. a N-terminal or C-terminal sequence fragment. Sequences can be taken from isolates or published sequences or mutants thereof.

In further detail, another embodiment provides for exemplary polypeptides comprising antigenic determinants from each of at least two different BrAd proteins. For example, in B. melitensis, the BrAd polypeptide can be expressed as two proteins - one corresponding to the amino terminus half and the other carboxy terminus half of the full length BrAd.

One specific embodiment is directed to a method of immunizing a vertebrate against Brucella, comprising (a) preparing a Brucella vaccine by expressing an immunogenic fragment of BrAd in an expression system; (b) purifying the immunogenic fragment; (c) formulating the immunogenic fragment in a Brucella vaccine; and (d) administering the vaccine to the vertebrate.

It is understood that the BrAd polypeptide, either soluble or aggregated, may be used as a vaccine directly or may be administered as a pharmaceutical composition comprising also a pharmaceutically acceptable vehicle, buffer, adjuvant or other acceptable material.

Certain specific embodiments provide a vaccine or pharmaceutical composition comprising a BrAd polypeptide in combination with a suitable carrier or excipient.

The BrAd polypeptide, either soluble or aggregated, may be used as a vaccine directly or may be administered as a pharmaceutical composition comprising also a

pharmaceutically acceptable vehicle, buffer, adjuvant or other acceptable material. Specific embodiments further provide a vaccine or pharmaceutical composition which comprises a BrAd polypeptide as described above in combination with a suitable carrier or excipient.

It is understood that BrAd polypeptides can be prepared using recombinant DNA techniques. Nucleic acids which encode the BrAd polypeptides, cloning and expression vectors incorporating them and parts of them, and transfected and transduced host cells incorporating such nucleic acids are able to express them as protein. In one example, the nucleic acid comprises a gene comprising for example the genes isolated.

In another specific embodiment, Brucella adhesin (BrAd) polypeptide could be used with other vaccines to achieve synergic therapeutic value. For example, expression of BrAd protein in vaccine strain B. abortus RB51 enhanced its protective efficacy against three virulent Brucella species in a murine model of brucellosis. In such case, the Brucella BrAd adhesin protein can be useful for the development of any type of vaccine, including but not limited to, subunit, DNA, or recombinant vector-based vaccines for protection against brucellosis. Certain brucellosis vaccines, which can be used with the BrAd polypeptides, are described in U.S. Pat. No. 6,149,920, the disclosure of which is hereby incorporated by reference.

The examples which follow are set forth to aid in understanding the invention, but are not intended to, and should not be construed as, limiting the scope of the invention in any manner.

Example 1 - PCR amplification

A pair of primers was designed for the PCR amplification of the coding sequences of the gene. The nucleotide sequences of the primers are given in Table 2 below. Table 2. Primers used for PCR amplification of the gene encoding BrAd.

(The engineered Bam HI and Pst I restriction endonuclease sites in BrAd-F and BrAd-R primers, respectively, are underlined. The Nco I restriction endonuclease site in BrAd-F is in bo Id- face.)

PCR was performed using a HOTSTAR High Fidelity PCR kit (Qiagen, Valencia, CA). The genomic DNA of B. abortus strain 2308 was used as the template. The PCR reaction contained 1 μΜ of each of the primers. The thermal cycle parameters were initial denaturation at 95oC for 10 minutes followed by 35 cycles of 95oC for 30 seconds, 62oC for 45 seconds and 68oC for 2 minutes, and a final extension step at 72oC for 10 minutes. Separation of the PCR products by electrophoresis on a 0.75% agarose gel indicated the successful amplification of the expected 4 kb size DNA fragment. The PCR product was first cloned in pGEM-T Easy vector (Promega, Madison, WI) and the nucleotide sequence integrity was confirmed by sequence analysis.

Example 2 - Expression and Purification of BrAd in E. coli

As shown in FIGs. 1 A & IB, the expressed His-BrAd protein was purified by metal affinity chromatography using Ni2+ resin and dialyzed extensively against phosphate- buffered saline. Lane 1, molecular weight marker; lanes 2 to 5, purified fractions of His-BrAd protein. (A) The gel was stained with Coomassie brilliant blue. (B) Western blot showing reaction with monoclonal antibody against His-tag epitope. The BrAd gene was excised from pGEM-T Easy by digestion with Bam HI and Pst I restriction endonucleases and cloned in the same sites of pRSET A vector (Invitrogen, Carlsbad, CA). The resulting pRSET/BrAd plasmid was used to transform E. coli BL21(DE3) and expression of the BrAd protein in fusion with a His-tag was obtained as per the protocol suggested by the manufacturer (Invitrogen).

Example 3 - Raising antibodies to His-BrAd

FIG. 1C shows the Western blot analysis of serum containing antibodies that reacted with purified His-BrAd protein. A group of 4 female, BALB/c mice were immunized by 3 subcutaneous inoculations at 2-week intervals with 25 μg of purified His-BrAd mixed with alum adjuvant (IMJECT, Pierce/Thermo Fisher Scientific, Rockford, IL). Two weeks after the last immunization, the mice were bled for serum collection. The antiserum was used to demonstrate the expression of BrAd in B. abortus RB51. The Western blot shows a reaction with monospecific sera raised in mice against the purified His-BrAd protein. The Arrows indicate the full length His-BrAd protein; the lower molecular size bands were because of the protein degradation. Numbers at the right to each panel indicate approximate molecular masses in kilodaltons.

Example 4 - Expression in B. abortus RB51

The expression of BrAd in strain RB51/BrAd occurred on the surface of the bacterial cells. For expression in strain RB51 , the BrAd gene was excised from pGEM-T Easy by digestion with Nco I and Pst I restriction endonucleases and cloned in the same sites of pBB4Trc vector to obtain pBB4/BrAd. Strain RB51 was electroporated with pBB4/BrAd, and the recombinant colonies were selected on agar plates containing 100 μg/ml of ampicillin. A colony was selected for further studies and it was designated strain RB51/BrAd.

FIG. 2 shows whole-cell ELISA assays using antiserum to His-BrAd and strain

RB51 or normal serum. ELISA plate was coated with 1 x 10⁸ live recombinant strain

RB51/BrAd or strain RB51 per well. Sera from mice immunized with either the purified His- BrAd, live RB51 or inoculated with saline were diluted 1/200 and reacted with the bacteria. Peroxidase conjugated anti-mouse IgG was used to detect the bound antibody. Results are shown as the mean ± standard deviation of OD450 of the color developed.

Example 5 - Vaccination of mice with strain RB51/BrAd

The ability of strain RB51/BrAd to induce better protective immunity against virulent Brucella challenges was determined in a mouse model. BALB/c mice were vaccinated by one intraperitoneal inoculation with 108 CFU of strain RB51/BrAd or a control RB51 strain. As a negative control, another group of mice were inoculated with saline solution. For antibody response analysis, serum from four mice from each group was obtained on week 3 and 6 post vaccination. For cell-mediated immunity analysis, four mice from each group were euthanized on week 6 after vaccination and their spleens were collected aseptically. As a protection study, 5 mice in each group were challenged with 104 CFU of B. abortus 2308, B. melitensis 16 M, or B. suis 1330, and two weeks later, the bacterial burden the spleens of challenged mice was determined. Mice immunized with strain RB51/BrAd, but not RB51 or inoculated with saline, produced significant levels of BrAd- specific antibodies and interferon-gamma secreting CD4+ and CD8+ T cells (Figs. 3 and 4).

More particularly, Fig. 3 shows ELISA analysis of BrAd-specific (A) and strain RB51-specific (B) IgG, IgGl, and IgG2a antibodies in serum of mice vaccinated with recombinant strain RB51/BrAd and RB51 or inoculated with saline alone. Sera collected from four mice of each group at 3 and 6 weeks post- vaccination were diluted 1/200 and assayed for the presence of specific antibodies. The results are shown mean ± standard deviation of OD450 of the color developed. Further, Fig. 4 shows flow cytometry detection of BrAd- and RB51 -specific T cells secreting interferon-gamma at 6 weeks post-vaccination. Splenocytes from the control and vaccinated mice were cultured with or without specific antigen or mitogen stimulation and then stained with fluorochrome-labeled antibodies specific to CD4, CD8 and intracellular interferon- gamma and then analyzed by a flow cytometer.

Example 6 - Protection against B. abortus and B. melitensis

From data obtained from a method similar to that discussed in Example 5, Fig. 5A and Fig. 5 B shows that the BrAd polypeptide provides protection against B. abortus and B. melitensis challenge in vaccinated mice, respectively. Groups of 5 mice each were vaccinated with 108 CFU of RB51 or RB51/BrAd, or inoculated with saline alone. After 6 weeks, all mice were challenged with 104 CFU of either B. abortus 2308 or B. melitensis 16 M. Two weeks after the challenge, mice were euthanized and the bacterial burden in spleens of each group was determined. Solid horizontal line above the x-axis: the lower limit of detection was 20

CFU/spleen.

Example 7 - Protection Against B. suis.

From data obtained from a method similar to that discussed in Example 5, Fig. 6 shows that the BrAd polypeptide or the combination of the BrAd polypeptide with RB51 provides protection against a B. suis challenge in vaccinated mice. Groups of 5 mice each were vaccinated with 108 CFU of RB51/L7-L12 or RB51/BrAd, or inoculated with saline alone. After 6 weeks, all mice were challenged with 104 CFU of B. suis 1330. Two weeks after the challenge, mice were euthanized and the bacterial burden in spleens of each group was determined. Solid horizontal line above the x-axis: the lower limit of detection was 20

CFU/spleen.

While the invention has been illustrated and described in detail in the foregoing description, such an illustration and description is to be considered as exemplary and not restrictive in character, it being understood that only the illustrative embodiments have been described and that all changes and modifications that come within the spirit of the invention are desired to be protected. Those of ordinary skill in the art may readily devise their own implementations that incorporate one or more of the features described herein, and thus fall within the spirit and scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. An immunogenic composition comprising:

a purified Brucella adhesin (BrAd) polypeptide from the Brucella genus; and adjuvant.

2. The composition as claimed in Claim 0, wherein the polypeptide is substantially similar to the polypeptide of Seq. Id. No. 1.

3. The composition as claimed in Claim 0, wherein the polypeptide has a sequence selected from the group consisting of: Seq. Id. Nos. 1-8.

4. The composition as claimed in Claim 0, wherein the polypeptide is a fragment of the polypeptide selected from the group consisting of: Seq. Id. Nos. 1-8.

5. The composition as claimed in Claim 0, wherein the protein has a molecular organization of trimeric autotransporter adhesins consisting of trimeric surface structures with a head-stalk-anchor architecture.

6. The composition as claimed in Claim 0, wherein the composition is adapted for intraperitoneal administration.

7. The composition as claimed in Claim 1 , further comprising RB51.

8. An isolated polypeptide encoding a Brucella adhesin (BrAd).

9. The polypeptide as claimed in Claim 0, wherein the protein has a molecular organization of trimeric autotransporter adhesins consisting of trimeric surface structures with a head-stalk-anchor architecture.

10. The polypeptide as claimed in Claim 0, wherein the head and stalk are composed of a small set of domains, building blocks that are frequently arranged repetitively.

11. An isolated nucleic acid encoding the protein of Claim 0.

12. A method for inducing a protective immune response against brucellosis in a subject susceptible to brucellosis comprising: administering to a subject an effective amount of a composition of a Brucella adhesin (BrAd) polypeptide, wherein the administration is sufficient to stimulate production of specific antibodies or stimulate a cellular immune response by the subject and induce a protective immune response.

13. The method as claimed in Claim 0, wherein the brucellosis is caused by a Brucella species selected from B. abortus, B. melitensis, and B. suis.

14. The method as claimed in Claim 0, wherein the subject is selected from the group consisting of goat, sheep, pigs, bison, elk, camels, and cattle.

15. The method as claimed in Claim 0, wherein the subject is a human.

16. A method of manufacturing a vaccine for brucellosis comprising: amplifying a nucleic acid sequence encoding a Brucella adhesion (BrAd) polypeptide or fragment thereof;

digesting the nucleic acid sequence

inserting the digested nucleic acid sequence into a plasmid; and introducing the plasmid into a strain of Brucella bacteria.

17. The method as claimed in Claim 0, wherein the Brucella bacterium is attenuated.