Patent Abstract:
An isolated protein or peptide selected from the group consisting of  Bordetella  colonization factor A (BcfA) protein and antigenic fragments thereof is described, along with an isolated nucleic acid encoding the same, antibodies that bind to the same, methods of producing an immune response in a mammalian subject in need thereof by administering the proteins, peptides or antibodies, and pharmaceutical compositions comprising the same.

Full Description:
RELATED APPLICATIONS 
     This application is a 35 U.S.C. §371 national phase application of PCT Application PCT/US2008/012051, filed Oct. 23, 2008, and published in English on Jul. 30, 2009, as International Publication No. WO 2009/094006, and which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 60/982,513, filed Oct. 25, 2007, the disclosure of each of which is incorporated herein by reference in its entirety. 
    
    
     GOVERNMENT SUPPORT 
     This invention was made with Government Support under Grant No. NCR-2005-05000 from the USDA. The United States Government has certain rights to this invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention concerns antigens, formulations thereof, and methods of using the same. 
     BACKGROUND OF THE INVENTION 
     Bordetellae are Gram-negative bacteria that colonize the respiratory tracts of humans and animals.  Bordetella bronchiseptica  and  Bordetella pertussis  are well-adapted pathogens of the human and animal respiratory tract, respectively.  Bordetella pertussis  infects only humans and causes the acute respiratory disease known as whooping cough. It is estimated that 20-30% of adolescents and adults who have chronic cough lasting for more than one week are infected with  B. pertussis . The current acellular vaccines, although effective against severe symptoms, are not particularly effective in preventing the carrier state. Adult and adolescent carriers harboring  B. pertussis  in the nasopharynx are responsible for the familial transmission of the bacteria to infants and young children, in whom the disease is severe and sometimes lethal. Thus, the continued presence of  B. pertussis  and  B. parapertussis  and the resurgence of pertussis despite widespread vaccinations, make the development of efficacious vaccines a top priority. 
       B. bronchiseptica  has a broad host range infecting a variety of animals. It typically establishes asymptomatic infections but can cause atrophic rhinitis in pigs, kennel cough in dogs, snuffles in rabbits and bronchopneumonia in guinea pigs. Animals continue to be carriers of  B. bronchiseptica  despite vaccinations and frequently shed bacteria resulting in outbreaks among herds. Since  B. bronchiseptica  can cause respiratory disease in immunocompromized patients, vaccination of pets and food-producing animals with attenuated  B. bronchiseptica  may pose health risks to these patients through zoonotic transmission. Thus, there is a need to develop acellular vaccines for immunizing animals. 
     SUMMARY OF THE INVENTION 
     This invention is based upon our identification of a gene, designated by us as BcfA ( Bordetella  colonization factor A) by a bioinformatics approach. We produced and purified BcfA-T7-tagged fusion protein from  E. coli , and have raised anti-sera against the purified protein in rats. Western-blotting with anti-BcfA antibody indicated that BcfA is localized to the outer membrane and that it is expressed during  Bordetella  infection of rats. By intranasal infection of rats, we have shown that BcfA plays an important role in respiratory colonization of  B. bronchiseptica . We have also found that BcfA is expressed in recent clinical isolates of  B. pertussis  from human patients. Pilot experiments conducted in the laboratory also provide evidence that anti-serum against BcfA is able to protect mice against subsequent challenge with  B. bronchiseptica . These data indicate that BcfA is useful as a vaccine and that anti-BcfA serum has a protective effect in animals. 
     A first aspect of the invention is an isolated protein or peptide selected from the group consisting of  Bordetella  colonization factor A (BcfA) protein and antigenic fragments thereof. In some embodiments, the BcfA protein has the sequence of SEQ ID NO: 2. In some embodiments, the protein or peptide is an antigenic fragment of BcfA from 20 to 500 amino acids in length. In some embodiments, the protein or peptide is an antigenic fragment of BcfA having the sequence given herein as SEQ ID NO: 3 or an antigenic fragment comprising 10 or more contiguous amino acids thereof. 
     A further aspect of the invention is an isolated nucleic acid that encodes a protein or peptide as described herein. The nucleic acid may in some embodiments be operatively associated with a promoter, and in some embodiments may be in a host cell that contains the nucleic acid and expresses the encoded protein or peptide. 
     A further aspect of the invention is a method of producing an immune response in a mammalian subject in need thereof, comprising administering the subject a protein or peptide as described herein in an amount effective to produce an immune response in that subject (e.g., a protective immune response to  Bordetella  infection, such as a  Bordetella bronchiseptica  or  Bordetella pertussis  infection). 
     A further aspect of the present invention is a composition comprising a protein or peptide as described herein in a pharmaceutically acceptable carrier. 
     A further aspect of the invention is an isolated antibody (e.g., a monoclonal antibody or polyclonal antibody) that binds to BcfA protein (e.g., a protein of SEQ ID NO: 2). In some embodiments the antibody may be coupled to a solid support or a detectable group. 
     A further aspect of the present invention is a composition comprising an antibody as described herein in a pharmaceutically acceptable carrier. 
     A further aspect of the present invention is a method of treating a mammalian subject for a  Bordetella  infection (e.g., a  Bordetella bronchiseptica  or  Bordetella pertussis  infection), comprising administering the subject an antibody as described herein in a treatment effective amount. 
     A further aspect of the invention is a method of detecting  Bordetella  (e.g.,  Bordetella bronchiseptica  or  Bordetella pertussis ) in a biological sample, comprising: contacting the sample to an antibody as described herein; and then detecting the presence or absence of specific binding of the antibody to the sample, the presence of specific binding to the sample indicating the presence of  Bordetella  in the sample. 
     A still further aspect of the invention is the use of a protein, peptide, or antibody as described herein for the preparation of a medicament for carrying out a method of treatment as described herein. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the nucleotide sequence encoding BcfA protein. 
         FIG. 2  shows the amino acid sequence of full-length BcfA ( FIG. 2A ), the predicted 508 amino acid residue extracellular domain of BcfA ( FIG. 2B ), as well as fragments of the extracellular domain ( FIG. 2C-2L ). 
         FIG. 3  shows that Immunization with BcfA protects mice against  B. bronchiseptica  challenge. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Subjects to be treated by the methods of the present invention are generally mammalian subjects, including but not limited to human, monkey, chimpanzee, ape, dog, cat, pig, rabbit, goat, cow, cattle, horse, etc. Subjects may be male or female and may be any age including neonate, infant, juvenile, adolescent, adult, and geriatric subjects. 
     “Antibodies” as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al.,  Molec. Immunol.  26, 403-11 (1989). The antibodies may be recombinant monoclonal antibodies, for example produced according to the methods disclosed in Reading U.S. Pat. No. 4,474,893, or Cabilly et al., U.S. Pat. No. 4,816,567. The antibodies may be humanized or chimeric antibodies. The antibodies may also be chemically constructed according to methods such as disclosed in Segal et al., U.S. Pat. No. 4,676,980. 
     “Antigenic fragment” of a protein (e.g., BcfA) as used herein is any portion of the protein that, when administered in accordance with the methods described herein, elicits, in a subject, an immune response that is either a fragment-specific or specific for the protein from which the fragment was obtained. The immune response can be either a humoral or a cell-mediated response. Antigenic fragments are known. See, e.g., U.S. Pat. No. 7,101,987; see also U.S. Pat. Nos. 7,270,816; 7,211,411; 7,163,685; and 7,151,082. Antigenic fragments can be of any suitable length (e.g., from 10, 12 or 20 contiguous amino acids up to 50, 100 or 200 contiguous amino acids or more) and generated by known techniques such as epitope mapping. (e.g., a fragment that includes an epitope region as described below). 
     1. Antigens. 
     The present invention includes  B. bronchiseptica  compositions composed of one or more  B. bronchiseptica  antigens against which it is desired to generate an immune response. The use of bacterial antigens in the production of antigen compositions and vaccines is well-known in the art and described in, for example, U.S. Pat. No. 7,255,867. 
     Compositions of the invention may be composed of BcfA (SEQ ID NO:2), the extracellular domain of BcfA (SEQ ID NO:3), or fragments or epitopes thereof. The instant vaccine can be a monovalent vaccine or multi-valent vaccine. Multi-valent vaccines generally include more than one type of antigen and can be produced by mixing a number of different antigens. 
     The instant antigen(s) can be made using any conventional synthetic or recombinant means. The amino acid sequence of an antigen for use in the invention can be modified to include non-naturally occurring amino acids or to increase the stability of the compound. When the antigen is produced by synthetic means, such amino acids may be introduced during production. The antigen may also be modified following either synthetic or recombinant production. 
     The antigen for use in the invention may also be produced using D-amino acids. In such cases, the amino acids will be linked in reverse sequence in the C to N orientation. This is conventional in the art for producing such peptides: A number of side chain modifications are also known in the art and may be made to the side chains of the antigen for use in the present invention. Such modifications include, for example, modifications of amino acids by reductive alkylation by reaction with an aldehyde followed by reduction with NABH 4 , amidination with methylacetimidate or acylation with acetic anhydride. 
     An antigen for use in the invention can be produced in large scale following purification by high pressure liquid chromatography (HPLC) or other techniques after recombinant expression as described herein. 
     Polynucleotides to produce an antigen for use in the invention can include DNA or RNA. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modifications to polynucleotides are known in the art. These include methylphosphate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3′ and/or 5′ ends of the molecule. Although the techniques mentioned herein are generally well-known in the art, reference may be made in particular to Sambrook and Russell (2001)  Molecular Cloning: A Laboratory Manual,  3 rd  Edition, CSHL Press. 
     An antigen for use in the present invention can be produced by recombinant means by providing a polynucleotide encoding the antigen and, where appropriate, encoding any desired flanking sequences under the control of a promoter and other required sequences. Such a polynucleotide is generally provided in the form of an expression vector. 
     Such vectors can be transformed into a suitable host cell to provide for expression of an antigen of the invention. Thus, an antigen for use according to the invention can be obtained by cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression of the antigen, and recovering the expressed antigen. 
     The vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid. Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. 
     Host cells transformed (or transfected) with the polynucleotides or vectors for the replication and expression of polynucleotides of the invention will be chosen to be compatible with the said vector and preferably will be bacterial, e.g.,  E. coli . Alternatively they may be cells of a human or animal cell line such as CHO or COS cells, or yeast or insect cells. The cells may also be cells of a non-human animal such as a sheep or rabbit or plant cells. 
     2. Antigen Compositions. 
     An antigen composition of the present invention can also include one or more adjuvants. Adjuvants for use in the production of antigenic compositions such as vaccines are well-known and routinely employed by the skilled artisan. See, e.g., U.S. Pat. No. 7,183,402. For example, adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate. The antigen is precipitated with, or adsorbed onto, the aluminum compound according to standard protocols. Other adjuvants, such as RIBI (ImmunoChem, Hamilton, Mont.), are used in parenteral administration. 
     Adjuvants for mucosal administration include bacterial toxins, e.g., the cholera toxin (CT), the  E. coli  heat-labile toxin (LT), the  Clostridium difficile  toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof such as a purified preparation of native cholera toxin subunit B (CTB). Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used. Suitable mutants are described, e.g., in WO 95/17211, WO 96/06627, and WO 95/34323. Other adjuvants, such as a bacterial monophosphoryl lipid A (MPLA) of, e.g.,  E. coli, Salmonella minnesota, Salmonella typhimurium , or  Shigella flexneri ; saponins, or polylactide glycolide (PLGA) microspheres, are also be used in mucosal administration. 
     Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/02415), DC-chol (3 b-(N—(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol (U.S. Pat. No. 5,283,185 and WO 96/14831) and QS-21 (WO 88/09336). 
     The compositions of the present invention may be administered by any suitable route. The compositions can be formulated for delivery by a mucosal, parenteral or transdermal route. Mucosal delivery routes include nasal, oral and oropharangeal routes, whereas parenteral routes include intramuscular, intraperitoneal, or subcutaneous injection. 
     Suitable binders and carriers may also be introduced into the present composition depending on the type of formulation that is provided. Oral formulations typically may include excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, sodium saccharine, cellulose, and magnesium carbonate. In some embodiments, vaccination is carried out by intranasal delivery of a liquid or spray. 
     The compositions are administrated in a manner compatible with the dosage formulation in such an amount as will be prophylactically effective. The quantity to be administered depends on a number of factors. These include the subject to be treated, capacity of the subject&#39;s immune system to synthesize antibodies and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner. In general, the dose per subject may be 5 μg, 50 μg, or 250 μg, up to 10 mg or 100 mg, per dose. 
     The compositions may be given in a single dose schedule or preferably in a multiple-dose schedule. A multiple-dose schedule is one in which a primary course of vaccination may be with 1 or 2 up to 5 or 10 separate doses, followed by other doses given at subsequent time intervals required to maintain and/or reinforce the immune response, for example, at 1 to 4 months for a second dose and if needed, a subsequent dose(s) after several months. 
     3. Antibodies. 
     Polyclonal antibodies used to carry out the present invention may be produced by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen to which a monoclonal antibody to BcfA binds, collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, in accordance with known procedures. 
     Monoclonal antibodies used to carry out the present invention may be produced in a hybridoma cell line according to the technique of Kohler and Milstein,  Nature  265, 495-97 (19.75). For example, a solution containing the appropriate antigen may be injected into a mouse and, after a sufficient time, the mouse sacrificed and spleen cells obtained. The spleen cells are then immortalized by fusing them with myeloma cells or with lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells. The hybridoma cells are then grown in a suitable media and the supernatant screened for monoclonal antibodies having the desired specificity. Monoclonal Fab fragments may be produced in  Escherichia coli  by recombinant techniques known to those skilled in the art. See, e.g., W. Huse,  Science  246, 1275-81 (1989). 
     Antibodies specific to BcfA can also be obtained by phage display techniques known in the art. 
     Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof which may be useful for carrying out the method disclosed herein. See generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see also U.S. Pat. No. 4,727,022 to Skold et al. titled “Methods for Modulating Ligand-Receptor Interactions and their Application,” U.S. Pat. No. 4,659,678 to Forrest et al. titled “Immunoassay of Antigens,” U.S. Pat. No. 4,376,110 to David et al., titled “Immunometric Assays Using Monoclonal Antibodies,” U.S. Pat. No. 4,275,149 to Litman et al., titled “Macromolecular Environment Control in Specific Receptor Assays,” U.S. Pat. No. 4,233,402 to Maggio et al., titled “Reagents and Method Employing Channeling,” and U.S. Pat. No. 4,230,767 to Boguslaski et al., titled “Heterogenous Specific Binding Assay Employing a Coenzyme as Label.” Applicants specifically intend that the disclosures of all U.S. Patent references cited herein be incorporated herein by reference in their entirety. 
     Antibodies as described herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation. Antibodies as described herein may likewise be conjugated to detectable groups such as radiolabels (e.g.,  35 S,  125 I,  131 I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques. The term “antigenic equivalents” as used herein, refers to proteins or peptides which bind to an antibody which binds to the protein or peptide with which equivalency is sought to be established. Antibodies which are used to select such antigenic equivalents are referred to as “selection antibodies” herein. 
     4. Utility. 
     Antigens of the present invention (BcfA and fragments thereof) and formulations of such antigens are useful for producing an immune response against said antigen in a mammalian subject. Such an immune response is useful for the production of antibodies, which antibodies can be used for diagnostic purposes (in detecting the presence of  Bordetella ) or for therapeutic purposes in treating  Bordetella  by passive immunity as described herein. 
     Antigens of the present invention are also useful as vaccines for providing protective immunity in mammalian subjects against  Bordetella  infection. 
     Example 1 
     Passive Immunization 
     Groups of five C57/BL6 mice were separately injected intraperitoneally with 200 μl of sera harvested from wild-type inoculated rats, BcfA-specific polyclonal serum, preimmune sera or sterile phosphate-buffered saline (PBS). Three to four hours after inoculation, these mice were intranasally challenged with 5×10 5  colony forming units (cfus) of wild-type  B. bronchiseptica  strain RB50 in a 25 μl droplet. Seven days post-inoculation, mice were sacrificed and trachea, nasal septum and lungs were harvested in sterile PBS and homogenized. Colonization of these organs was quantified by plating different dilutions of the homogenate in BG blood plates containing 50 of streptomycin and subsequent colony counting. The results of this analysis indicated that anti-serum raised against BcfA was able to protect mice against subsequent challenge with  B. bronchiseptica.    
     Example 2 
     BcfA Epitopes 
     The purified BcfA protein migrates in an SDS-polyacrylamide gel at a mobility corresponding to ≈100 kDa, which is consistent with the annotated length (969 amino acids;  FIG. 2A ) of the BcfA open reading frame. BcfA displays homology to other bacterial proteins including BipA from  Bordetella , invasins from  Yersinia , and intimins from enteropathogenic  E. coli . Based on the known structure of these proteins, the C-terminal 508 amino acid residues of BcfA are expected to encompass the extracellular region of BcfA and thus will interact with the immune system. Accordingly, amino acid residues 461-969 of BcfA (SEQ ID NO:3;  FIG. 2B ), or one or more fragments thereof, are expected to elicit an immune response against  B. bronchiseptica . Exemplary fragments of BcfA are shown in  FIGS. 2C-2L . 
     Additional fragments of BcfA include antigenic regions of the BcfA extracellular domain as well as fragments expected to bind to major histocompatibility complex (MHC) class I and MHC class II molecules. Accordingly, the amino acid sequence of the extracellular region of BcfA was analyzed using two independent web-based algorithms that predict antigenic sites in proteins (Table 1) and potential binding to MHC class I and MHC class II molecules (Table 2). 
     Multiple peptides within the extracellular region of BcfA were predicted to be antigenic and exhibit high binding affinity for human HLA molecules (Table 3). 
     
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                 SEQ ID 
                   
                 Antigenic 
               
               
                 Predicted Epitope 
                 NO: 
                 Location 
                 Socre′ 1   
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 GDYPVTLVLED 
                 14 
                 443 
                 1.200 
               
               
                   
               
               
                 GGPVKRPYHDIFVPVPPTVEVATD 
                 15 
                 165 
                 1.179 
               
               
                   
               
               
                 APTVVLHT 
                 16 
                 91 
                 1.165 
               
               
                   
               
               
                 QTLLGGKIRLLRPVARLLLSP 
                 17 
                 350 
                 1.162 
               
               
                   
               
               
                 SGVVTVTGY 
                 18 
                 277 
                 1.143 
               
               
                   
               
               
                 PQTAALLAAIKLHDPN 
                 19 
                 402 
                 1.137 
               
               
                   
               
               
                 GKAPWPGANGV 
                 20 
                 474 
                 1.128 
               
               
                   
               
               
                 GKPVRRPYVDTVAPTPMKVTID 
                 21 
                 248 
                 1.119 
               
               
                   
               
               
                 GTGVVTVT 
                 22 
                 11 
                 1.110 
               
               
                   
               
               
                 ASGPIVAIA 
                 23 
                 57 
                 1.108 
               
               
                   
               
               
                 TMVLKVTGS 
                 24 
                 463 
                 1.106 
               
               
                   
               
               
                 GGSLLIG 
                 25 
                 495 
                 1.095 
               
               
                   
               
               
                 VGGSTVTVTFP 
                 26 
                 287 
                 1.093 
               
               
                   
               
               
                 RAKVKVDFP 
                 27 
                 200 
                 1.092 
               
               
                   
               
               
                 GGDIVVTQ 
                 28 
                 233 
                 1.089 
               
               
                   
               
               
                 GAVRTH 
                 29 
                 4 
                 1.086 
               
               
                   
               
               
                 LDGIVARF 
                 30 
                 387 
                 1.086 
               
               
                   
               
               
                 GDWAG 
                 31 
                 37 
                 1.081 
               
               
                   
               
               
                 SGRVTVSGK 
                 32 
                 188 
                 1.079 
               
               
                   
               
               
                 KEVVAGP 
                 33 
                 127 
                 1.078 
               
               
                   
               
               
                 RTVQYD 
                 34 
                 76 
                 1.078 
               
               
                   
               
               
                 FTVASKGDV 
                 35 
                 47 
                 1.073 
               
               
                   
               
               
                 PAGPIRVSAR 
                 36 
                 321 
                 1.068 
               
               
                   
               
               
                 DHYLDA 
                 37 
                 341 
                 1.052 
               
               
                   
               
               
                 GAKVRID 
                 38 
                 24 
                 1.051 
               
               
                   
               
               
                 YTVTST 
                 39 
                 312 
                 1.050 
               
               
                   
               
               
                 DITVSGT 
                 40 
                 149 
                 1.037 
               
               
                   
               
               
                 Location is the position of the first residue.  1 Score obtained using the Antigenic program 
               
               
                 (http://bioinfo.bgu.ac.il/cgi-bin/emboss.pl?_action = input_&amp;app = antigenic) which employs 
               
               
                 the method of Kolaskar and Tongaonkar (1990). FEBS Letters 276: 172-174. 
               
             
          
         
       
     
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Predictied 
                 SEQ ID 
                   
                 HLA 
                 BIMAS 
               
               
                 Epitope 
                 NO: 
                 Location 
                 Molecule 
                 Score 1,2   
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 RRTVQYDDR 
                 41 
                 75 
                 HLA-B_2705 
                 3000 
               
               
                   
               
               
                 LRPVARLLL 
                 42 
                 360 
                 HLA-B_2705 
                 2000 
               
               
                   
               
               
                 AREATTMVL 
                 43 
                 458 
                 HLA-B_2705 
                 2000 
               
               
                   
               
               
                 IRLLRPVARL 
                 44 
                 357 
                 HLA-B_2705 
                 2000 
               
               
                   
               
               
                 AREATTMVLK 
                 45 
                 458 
                 HLA-B_2705 
                 2000 
               
               
                   
               
               
                 KRPYHDIFV 
                 46 
                 166 
                 HLA-B_2705 
                 1800 
               
               
                   
               
               
                 RRTVQYDDRV 
                 47 
                 75 
                 HLA-B_2705 
                 1800 
               
               
                   
               
               
                 GPVKRPYHDI 
                 48 
                 163 
                 HLA-B_5102 
                 1320 
               
               
                   
               
               
                 EVATDSSSGR 
                 49 
                 181 
                 HLA-A68.1 
                 1200 
               
               
                   
               
               
                 RPYHDIFVPV 
                 50 
                 167 
                 HLA-B_5102 
                 1100 
               
               
                   
               
               
                 IRLLRPVAR 
                 51 
                 357 
                 HLA-B_2705 
                 1000 
               
               
                   
               
               
                 ARFEPANGA 
                 52 
                 392 
                 HLA-B_2705 
                 1000 
               
               
                   
               
               
                 IRVSARGPR 
                 53 
                 325 
                 HLA-B_2705 
                 1000 
               
               
                   
               
               
                 VRIDFPDGTF 
                 54 
                 27 
                 HLA-B_2705 
                 1000 
               
               
                   
               
               
                 VPVPPTVEV 
                 55 
                 174 
                 HLA-B_5102 
                 660 
               
               
                   
               
               
                 APWPGANGV 
                 56 
                 476 
                 HLA-B_5102 
                 660 
               
               
                   
               
               
                 LESNKMFIYL 
                 57 
                 420 
                 HLA-B60 
                 640 
               
               
                   
               
               
                 GRPGDTIRV 
                 58 
                 111 
                 HLA-B_2705 
                 600 
               
               
                   
               
               
                 MRTDGNSGV 
                 59 
                 271 
                 HLA-B_2705 
                 600 
               
               
                   
               
               
                 RRPYVDTVA 
                 60 
                 252 
                 HLA-B_2705 
                 600 
               
               
                   
               
               
                 VRRPYVDTV 
                 61 
                 251 
                 HLA-B_2705 
                 600 
               
               
                   
               
               
                 YRATSDGDV 
                 62 
                 223 
                 HLA-B_2705 
                 600 
               
               
                   
               
               
                 VRTHPGTGV 
                 63 
                 6 
                 HLA-B_2705 
                 600 
               
               
                   
               
               
                 MRTDGNSGW 
                 64 
                 271 
                 HLA-B_2705 
                 600 
               
               
                   
               
               
                 VRTHPGTGW 
                 65 
                 6 
                 HLA-B_2705 
                 600 
               
               
                   
               
               
                 NRVPNGDYPV 
                 66 
                 438 
                 HLA-B_2705 
                 600 
               
               
                   
               
               
                 ARLLLSPGSM 
                 67 
                 364 
                 HLA-B_2705 
                 600 
               
               
                   
               
               
                 YRLESNKMFI 
                 68 
                 418 
                 HLA-B_2705 
                 600 
               
               
                   
               
               
                 FPGGTSKTV 
                 69 
                 207 
                 HLA-B_5102 
                 586 
               
               
                   
               
               
                 APTPMKVTI 
                 70 
                 260 
                 HLA-B_5101 
                 484 
               
               
                   
               
               
                 GPSLGGSLLI 
                 71 
                 491 
                 HLA-B_5102 
                 484 
               
               
                   
               
               
                 GPSLGGSLLI 
                 71 
                 491 
                 HLA-B_5101 
                 440 
               
               
                   
               
               
                 SPGSMTYTEI 
                 72 
                 369 
                 HLA-B_5101 
                 440 
               
               
                   
               
               
                 GPVKRPYHDI 
                 48 
                 163 
                 HLA-B_5101 
                 440 
               
               
                   
               
               
                 APTPMKVTI 
                 70 
                 260 
                 HLA-B_5102 
                 440 
               
               
                   
               
               
                 SPGSMTYTEI 
                 72 
                 369 
                 HLA-B_5102 
                 440 
               
               
                   
               
               
                 WAGPDGTYR 
                 73 
                 129 
                 HLA-A68.1 
                 400 
               
               
                   
               
               
                 FPGGTSKTV 
                 69 
                 207 
                 HLA-B_5101 
                 381 
               
               
                   
               
               
                 FPDGTTKEW 
                 74 
                 121 
                 HLA-B_5101 
                 381 
               
               
                   
               
               
                 RESPRRTVQY 
                 75 
                 71 
                 HLA-B_4403 
                 360 
               
               
                   
               
               
                 FPDGTTKEV 
                 76 
                 121 
                 HLA-B_5101 
                 346 
               
               
                   
               
               
                 AALLAAIKL 
                 77 
                 405 
                 HLA-B_5102 
                 330 
               
               
                   
               
               
                 MPGAAGKPV 
                 78 
                 243 
                 HLA-B_5101 
                 315 
               
               
                   
               
               
                 FPDGTFGDV 
                 79 
                 31 
                 HLA-B_5101 
                 315 
               
               
                   
               
               
                 VAPTPMKVTI 
                 80 
                 259 
                 HLA-B_5101 
                 315 
               
               
                   
               
               
                 KLHDPNYRL 
                 81 
                 412 
                 HLA-A_0201 
                 307 
               
               
                   
               
               
                 DAWTKQTLL 
                 82 
                 345 
                 HLA-B_5102 
                 303 
               
               
                   
               
               
                 DTMNSDPYNR 
                 83 
                 430 
                 HLA-A68.1 
                 300 
               
               
                   
               
               
                 YRLESNKMF 
                 84 
                 418 
                 HLA-B_2705 
                 300 
               
               
                   
               
               
                 GRVTVSGKGR 
                 85 
                 103 
                 HLA-B2705 
                 300 
               
               
                   
               
               
                 Location is the position of the first residue.  1 Score obtained using the BIMAS program 
               
               
                 (http://www-bimas.cit.nih.gov/molbio/hla_bind/) developed by Parker, et al. (1994) J. 
               
               
                 Immunol. 152:163, which provides the rank potential of 8-mer, 9-mer, or 10-mer peptides 
               
               
                 based on a predicted half-time of dissociation to HLA class I molecules.  2 Minimum scores 
               
               
                 300 on the BIMAS site were used. 
               
             
          
         
       
     
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                   
                 SEQ 
                   
                   
                   
               
               
                   
                 ID 
                   
                 Antigenic 
                 BIMAS 
               
               
                 Predicted Epitope 
                 NO: 
                 Location 
                 Score 
                 Score 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                  RTVQYD 
                 34 
                 76 
                 1.179 
                   
               
               
                 RRTVQYDDR 
                 41 
                 75 
                   
                   
               
               
                 RRTVQYDDRV 
                 47 
                 75 
                   
                 1800 
               
               
                   
               
               
                 GGPVKRPYHDIFVPVPPTVEVATD 
                 15 
                 165 
                 1.179 
                   
               
               
                     KRPYHDIFV 
                 46 
                 166 
                   
                 1800 
               
               
                      RPYHDIFVPV 
                 50 
                 167 
                   
                   
               
               
                             VPVPPTVEV 
                 55 
                 174 
                   
                   
               
               
                   
               
               
                 GKPVRRPYVDTVAPTPMKVTID 
                 21 
                 248 
                 1.119 
                   
               
               
                    VRRPYVDTV 
                 61 
                 251 
                   
                   
               
               
                     RRPYVDTVA 
                 60 
                 252 
                   
                   
               
               
                            VAPTPMKVTI 
                 80 
                 259 
                   
                   
               
               
                             APTPMKVTI 
                 70 
                 260 
                   
                 315 
               
               
                   
               
               
                 QTLLGGKIRLLRPVARLLLSP 
                 17 
                 350 
                 1.162 
                   
               
               
                        IRLLRPVARL 
                 44 
                 357 
                   
                   
               
               
                        IRLLRPVAR 
                 51 
                 357 
                   
                   
               
               
                           LRPVARLLL 
                 42 
                 360 
                   
                 2000 
               
               
                   
               
               
                 PQTAALLAAIKLHDPN 
                 19 
                 402 
                 1.137 
                   
               
               
                    AALLAAIKL 
                 77 
                 405 
                   
                 330 
               
               
                   
               
               
                 GKAPVVPGANGV 
                 20 
                 474 
                 1.128 
                   
               
               
                   APVVPGANGV 
                 56 
                 476 
                   
                 660 
               
               
                   
               
             
          
         
       
     
     Example 3 
     Active Immunization 
       FIG. 3  shows that Immunization with BcfA protects mice against  B. bronchiseptica  challenge. Mice were immunized intraperitoneally at 0 and 3 weeks with either 10 or 30 μg of BcfA adsorbed to alum or alum only. One week after the second immunization, mice were intranasally challenged with 5×10 5  CFU or RB50 in a 25 μl volume. Mice were sacrificed at 1 day ( FIG. 3A ) and 6 days ( FIG. 3B ) post-challenge and the number of CFU was determined in the nasal septum, trachea and lungs. Individual symbols represent a single mouse. The dashed line represents the lower limits of CFU detection. Black bars represent mean colonization of respective groups. A statistical analysis was carried out using an unpaired two-tailed Student t test. The asterisks indicate the range of the different P values (one asterisk, ≦0.05; two asterisks, ≦0.005 and three asterisks, ≦0.0005). 
     The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Technology Classification (CPC): 2