# Patent

## Title
Bordetella outer-membrane protein antigens and methods of making and using the same

## 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.

## Background
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 NABH4, 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, 3rd 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'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., 35S, 125I, 131I), 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 5×105 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. 1Score obtained using the Antigenic program


(http://bioinfo.bgu.ac.il/cgi-bin/emboss.pl?_action = input_&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
Score1,2



















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. 1Score 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. 2Minimum 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 5×105 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.

## Claims

1. A composition comprising an isolated Bordetella colonization factor A (BcfA) protein fragment consisting of 20 to 500 consecutive amino acids of either SEQ ID NO: 2 or SEQ ID NO: 3 in combination with an adjuvant and/or a binder.

2. The composition of claim 1 in a pharmaceutically acceptable carrier.

3. The composition of claim 1 comprising said adjuvant.

4. The composition of claim 1, wherein said BcfA protein fragment consists of 20 to 500 consecutive amino acids of SEQ ID NO: 2.

5. The composition of claim 1, wherein said protein is produced by a synthetic or recombinant process.

6. A method of producing an immune response in a mammalian subject in need thereof, comprising administering said subject an isolated BcfA protein fragment consisting of 20 to 500 consecutive amino acids of either SEQ ID NO: 2 or SEQ ID NO: 3 in an amount effective to produce an immune response.

7. The method of claim 6, wherein said immune response is a protective immune response to Bordetella infection.

8. The method of claim 7, wherein said Bordetella infection is Bordetella bronchiseptica or Bordetella pertussis infection.