WO2010002989A2

WO2010002989A2 - Vaccines comprising pasteurella multocida recombinant filamentous hemagglutinin peptides

Info

Publication number: WO2010002989A2
Application number: PCT/US2009/049401
Authority: WO
Inventors: Robert E. Briggs; Fred M. Tatum
Original assignee: Biotechnology Research And Development Corporation; Department Of Agriculture
Priority date: 2008-07-03
Filing date: 2009-07-01
Publication date: 2010-01-07
Also published as: AU2009266920A1; WO2010002989A3; CA2729818A1; EP2313427A2

Abstract

Vaccines comprising one or more P. multocida FHAB2 peptides of the invention can be used to achieve broad cross protection against different P. multocida capsular serotypes.

Description

VACCINES COMPRISING PASTEURELLA MULTOCIDA RECOMBINANT FILAMENTOUS HEMAGGLUTININ PEPTIDES

[01] This application claims the benefit of and incorporates by reference Serial No. 60/077,948 filed July 3, 2008.

FIELD OF THE INVENTION

[02] The invention relates to protection against different P. multocida capsular and serotypes through immunization with recombinant FHAB2 peptides.

BACKGROUND OF THE INVENTION

[03] Pasteurella multocida (P. multocida) is associated with a variety of diseases. As a commensal in cats and dogs P. multocida causes zoonotic abcesses arising from bites or scratches. P. multocida also causes shipping fever in cattle. Pasteurella multocida also is the causative agent of fowl cholera (FC), a highly contagious disease that affects all species of birds. The turkey industry is the most severely affected sector of commercial importance. Predominately, P. multocida serotypes A: l, A:3, and A:3,4 are responsible for most fowl cholera outbreaks in poultry flocks. It is thought that P. multocida principally enters host tissues through mucus membranes of the pharynx or upper air passage resulting in septicemia accompanied with high morbidity and mortality.

[04] Current commercial vaccines against FC include bacterins and live attenuated strains. The adjuvanted-bacterins generally provide limited protective immunity and do not control disease by different serotypes (Davis, Poult. ScL 20, 434-460, 1987). The commercial attenuated live vaccines are recognized as providing broad protection across serotypes but their precise genetic lesions are uncharacterized and outbreaks associated with such vaccines have occurred (Christensen et ah, Rev Sci Tech. 2000 Aug;19(2):626-37). There is, therefore, a need in the art for improved vaccines with broad efficacy against diseases caused by P. multocida. BRIEF DESCRIPTION OF THE FIGURES

[05] FIGS. 1-C. FIG. IA, schematic representation of the fhaB2 gene of P. multocida strain P-1059. The arrow indicates the length and direction of the gene. FIG. IB, representation of the 311 kDa protein encoded by fhaB2. FIG. 1C, fhaB2 fragments cloned into pGEMEX^®-l to generate rFHA peptides 1, 2, and 3 respectively.

[06] FIGS. 2A-B. Expression and purification of recombinant FHAB (r-FHAB) peptides in E. coli. FIG. 2A, Coomassie blue-stained SDS-PAGE of whole cells lysates expressing the recombinant FHAB2 peptides. FIG. 2B, purified samples of r-FHAB2 peptides.

[07] FIGS. 3A-B. FIG. 3A, Western blot analysis of pooled sera from vaccinated turkeys, after immunization and prior to challenge, reacting to wild-type (wt) or filamentous hemagglutinin mutant (ΔfhaB2) whole cell lysates. FIG. 3B, Western blot analysis using the same pooled sera as in FIG. 2A reacting to supernatants of wild-type (wt) or filamentous hemagglutinin mutant (ΔfhaB2) cultures.

[08] FIGS. 4A-B. FIG. 4A, Western blot analysis of pre-immune pooled turkey sera reacting to identical loadings of wild-type (wt) or filamentous hemagglutinin (ΔfhaB2) mutant whole cell lysates as shown in FIG. 2A. FIG. 4B, Western blot analysis using pre-immune pooled turkey sera reacting to identical loadings of supernatants of wild-type (wt) or filamentous hemagglutinin mutant (ΔfhaB2) cultures as shown in FIG. 2B.

DETAILED DESCRIPTION OF THE INVENTION

[09] Embodiments of the invention provide peptides derived from P. multocida filamentous hemagglutinin (fhab2) that can be used in vaccine compositions to provide broad efficacy against diseases caused by P. multocida. fhab2 proteins of a bovine (A:3; NCBI AAK61595.1; SEQ ID NO: 1) and an avian (F:3; NP_244996; SEQ ID NO:2) strain of P. multocida are highly conserved (>99% identity). Thus, vaccines comprising one or more P. multocida FHAB2 peptides of the invention can be used to achieve broad cross protection against different P. multocida capsular and serotypes. Vaccines of the invention can be administered to animals (including livestock, ungulates, and companion animals) and birds (including poultry) to provide protective immunity against wild-type P. multocida, e.g., to prevent or reduce the severity of diseases such as hemorrhagic septicemia or pneumonia in livestock, ungulates, and companion animals and to prevent or reduce the severity of fowl cholera in birds, especially poultry, respectively.

FHAB2 Peptides

[10] Peptides of the invention are derived from approximately the first N-terminal 1050 amino acids of P. multocida filamentous hemagglutinin. Preferred peptides, as referred to in the Examples below, include "peptide 1" (SEQ ID NO: 3), "peptide 2" (SEQ ID NO:4), and "peptide 3" (SEQ ID NO:5). In some embodiments peptides consist of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In other embodiments peptides can include additional amino acids at either the N- or C-terminus. These include amino acids which are contiguous to SEQ ID NOS:3, 4, or 5 in the full-length FHAB2 protein, up to full-length FHAB2, as well as amino acid sequences which are not part of FHAB2. In some embodiments the peptides are about 330-400 amino acids in length.

Nucleic Acid Molecules and Expression ofFHAB2 Peptides

[11] The invention also provides nucleic acid molecules which encode peptides of the invention. Nucleotide sequences which encode peptides 1, 2, and 3 are provided in SEQ ID NOS:6, 7, and 8, respectively, although any nucleotide sequences which encode the peptides can be used. In some embodiments a nucleic acid molecule of the invention is an expression plasmid comprising elements which permit expression of the encoded peptide. Any suitable expression system can be used to produce peptides of the invention. See the specific Examples, below.

Vaccine Compositions

[12] Vaccine compositions of the invention comprise one or more peptides of the invention and a pharmaceutically acceptable vehicle. Vaccines of the invention can comprise any combination of peptides of the invention, including peptides that comprise SEQ ID NO:3 (peptide 1), peptides that comprise SEQ ID NO:4 (peptide 2), peptides that comprise SEQ ID N0:5 (peptide 3), peptides that consist of SEQ ID N0:3 (peptide 1), peptides that consist of SEQ ID NO:4 (peptide 2), peptides that consist of SEQ ID NO:5 (peptide 3), or any combinations thereof. In some embodiments a vaccine comprises at least one of each of peptides 1, 2, and 3. In other embodiments a vaccine comprises two or more of peptides 1, 2, and 3 (e.g., peptides 1 and 2; peptides 2 and 3; peptides 1 and 3; or peptides 1, 2, and 3). In other embodiments a vaccine of the invention comprise a single type of peptide (e.g., peptide 1, peptide 2, or peptide 3). Vaccines of the invention also can comprise full-length FHAB2.

Adjuvants

[13] If desired, an adjuvant such as TITERMAX r® Gold, which contains block copolymer CRL-8300 (US 2006/0134221), squalene, and a sorbitan monooleate, can be added to a vaccine. Other useful adjuvants include, without limitation, surfactants (e.g., hexadecylamine, octadecylanine, lysolecithin, di-methyldioctadecylammonium bromide, N,N-dioctadecyl-n'-N-bis(2-hydroxyethyl-propane diamine), methoxyhexadecylglycerol, and pluronic polyols); polyanions (e.g., pyran, dextran sulfate, poly IC, polyacrylicacid, carbopol), peptides (e.g., muramyl dipeptide, dimethylglycine, tuftsin), oil emulsions, alum, and mixtures thereof.

Polyvalent Vaccines

[14] Vaccines comprising one or more peptides of the invention can be given alone or as a component of a polyvalent vaccine, i.e., in combination with other vaccines such as the Mannheimia haemolytica lktA in-frame mutant vaccine. Insertion of the protective FHAB2 peptide(s) into the lktA deletion site Mannheimia haemolytica would create a bivalent cattle vaccine expressing a protective chimeric peptide.

Pharmaceutically Acceptable Vehicles and Carriers [15] Vaccines of the invention comprise a pharmaceutically acceptable vehicle, such as saline, Earle's balanced salt solution (EBSS) without phenol red, Tris buffer, and the like.

[16] Vaccines comprising peptides of the invention also can comprise one or more pharmaceutically acceptable carriers. Such carriers are well known to those in the art and include, but are not limited to, large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Pharmaceutically acceptable salts can also be used in the vaccine, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as the salts of organic acids such as acetates, proprionates, malonates, or benzoates. Vaccines also can contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents.

Administration of Vaccine Compositions

[17] Vaccines of the invention can be used to confer immunity against Pasteurella multocida to both animals and birds.

Administration to Animals

[18] "Animals" include livestock (domestic animals raised for food, milk, or fiber such as hogs, sheep, cattle, donkeys, lambs, sheep, and horses) and companion animals (e.g., dogs, cats). "Ungulates" include, but are not limited to, cattle (bovine animals), water buffalo, bison, sheep, swine, deer, elephants, and yaks. Each of these includes both adult and developing forms (e.g., calves, piglets, lambs, etc.). Vaccines of the invention can be administered either to adults or developing animals, preferably livestock, ungulates, or companion animals.

[19] A convenient method of delivering a bacterium of the invention to animals (such as livestock, ungulates, or companion animals) is by oral administration (e.g., in the feed or drinking water or in bait). Typically, large animals (e.g., livestock/ungulates such as cattle) are dosed with about 250 μg. Doses can be adjusted for smaller livestock/ungulates such as sheep (e.g., about 100 μg). Analogous dosing regimens can be readily deduced for companion animals.

[20] Although the oral route is preferred for ease of delivery, other routes for vaccination can also be used. These include without limitation, subcutaneous, intramuscular, intravenous, intradermal, intranasal, intrabronchial, etc.

Administration to Birds

[21] "Birds" include wild (e.g., game fowl) and domesticated (e.g., poultry or pet) birds and includes both adult and developing forms (e.g., hatchlings, chicks, poults, etc.). "Poultry" or "poultry birds" include all birds kept, harvested, or domesticated for meat or eggs, including chicken, turkey, ostrich, game hen, squab, guinea fowl, pheasant, quail, duck, goose, and emu.

[22] Vaccines of the invention can be administered to a bird by any known or standard technique, including mucosal or intramuscular injection. In a hatchery, techniques such as in ovo vaccination, spray vaccination, and subcutaneous vaccination can be used. On the farm, vaccines can be administered using techniques such as scarification, spray vaccination, eye drop vaccination, in-water vaccination, in-feed vaccination, wing web vaccination, subcutaneous vaccination, and intramuscular vaccination.

[23] Effective doses depend on the size of the bird. Doses range and can vary, for example, from about 10-100 μg of purified protein.

[24] Dosage regimens include 1 or 2 injections of recombinant protein. Vaccines can conveniently be provided in kits, which also can comprise appropriate labeling and instructions for administering a vaccine to an animal (e.g., cattle, sheep, lambs, donkeys, swine, cats, dogs, horses) or a bird (e.g., poultry, particularly turkeys).

[25] All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLE 1

Materials and Methods

Bacterial strains

[26] P. multocida strain P- 1059 (A:3) was obtained from the collection of Drs. R. Rimler, R.E. Briggs, and F. M. Tatum maintained at the National Animal Disease Center, Ames, IA, USA. Wild type P. multocida P-1059, and a P. multocida P-1059 fhaB2 (filamentous hemagglutinin) mutant used in this study were cultured on dextrose starch agar containing 5% sheep blood and cells were harvested into Columbia broth containing 10 glycerol to produce stock cultures stored at -80 ⁰C. Before use in this study, the wild-type P-1059 challenge strain was passed through a turkey that developed peracute fowl cholera following intravenous challenge. Pure P. multocida culture was obtained from liver of the infected bird and liver tissue was stored at -80 0C. The challenge inoculum was generated by first swabbing the stored liver sample onto a blood agar plate and incubating it overnight. The pure P. multocida growth was amplified in Columbia broth to an optical density of 0.4 at 600 nm and diluted in Earle's balanced salt solution without phenol red (EBSS, Gibco) to obtain the intended challenge dose. The exact CFU in the challenge solution was determined by colony plate counts of serial dilutions.

Recombinant DNA Techniques

[27] The fhaB2 specific primer pairs were designed by sequence analysis of the fhaB2 gene obtained from P. multocida strain P-1059. The primer pair nucleotides depicted in capital letters annealed to specific nucleotides on fhaB2 while the nucleotides (lower case) at the 5' termini contained EcoRl (forward primers) and HinDIII (reverse primers) recognition sites which were included to facilitate cloning of the products. All primers described in this work were custom synthesized with an oligonucleotide synthesizer (Applied Biosystems Inc., Integrated DNA Technologies Inc. Coralville, IA).

[28] The fhaB2 fragments were amplified using whole cells of strain P- 1059 as template. Primer pairs are listed in Table 1. Primers were added to EASYSTART™ PCR mix in a tube protocol of Molecular BioProducts (San Diego, CA). Reaction conditions were 30 cycles, with 30 seconds at 95 ⁰C, 30 seconds at 58 ⁰C, and 90 seconds at 72 0C per cycle. The reaction products were purified with QIA QUICK^® spin columns (Qiagen Inc. Valencia, CA) then they were subjected to digestion by EcoRl and HinDIII. Each fragment was ligated into the like digested pGEMEX^®-l (Promega Corp. Madison, WI) and transformed into E. coli DE-3 cells. The identities and coding frames of the fhaB2 fragments inserted into pGEMEX^®-l were verified by sequence analysis. BL21 (DE3) cells were transformed with each of the fhaB2 fragments inserted into pGEMEX^®-l and spread onto Columbia blood agar media supplemented with 50 μg/ml ampicillin. Plates were incubated overnight at 37 ⁰C. Individual colonies were transferred to 40 ml LB-medium, supplemented with 50 μg/ml ampicillin and either with or without 0.5 mM IPTG when the cell density reached an A₆oo of 0.7. After 4 h, the cells were harvested by centrifugation and recombinant protein was extracted using a mild, nonionic detergent containing 20 mM Tris HCl, pH 7.5 (B-PER^® Bacterial Protein Extraction Reagent; Pierce) according to the manufacturer's directions for midi-scale protein extractions. The pGEMEX^®-l plasmid expressed the recombinant FHAB2 peptides as chimeric proteins with T7 gene 10. These recombinant products were contained primarily in inclusion bodies. Further purification of the peptides within the inclusion bodies was accomplished using B-PER^® Bacterial Protein Extraction Reagent according to the manufacturer's directions. The purified recombinant products were resuspended in phosphate buffered saline and concentrations were determined by comparing them to known amounts of bovine serum aluminates run on a 4-15% SDS PAGE after Coomassie blue staining (FIG. 1). [29] Vaccine was prepared by diluting the recombinant antigens to 100 mg/ml in saline and then mixing them with adjuvant containing TITERMAX^® Gold (CyRx Corp., Norcross, GA) 50:50 (volume:volume) according to the manufacturer's directions.

[30] Six-week old mixed sex broad-breasted white turkeys were injected intramuscularly with the three recombinant FHAB2 fragments (50 μg total) mixed with adjuvant in a volume of 200 μl. At the same time, control birds were injected with T7 gene 10 protein (50 μg) expressed by pGEMEX^®-l mixed with adjuvant in a volume of 200 μl. Two weeks after the primary immunization, a booster immunization identical to the first was given. Serum was collected from each bird at the time of the first vaccination and again three weeks later.

Western blotting

[31] Wild type P. multocida P- 1059 and a P. multocida P- 1059 fhaB2 mutant were grown in 25 ml Columbia broth (Difco) to midlog phase and treated with 250 Units of hyaluronidase type 1-S (Sigma Chem. Co., St. Louis, MO) for 10 minutes to remove capsule and thus aid cell harvesting. The cells were pelleted by centrifugation at 5000 x g for fifteen minutes and growth media was concentrated ten fold using Centriprep 30 concentrators (Amicon, Inc. Beverly, MA). Fifteen μl of concentrated culture media or cells were solubilized in 2X Laemmli buffer, (50 mM Tris, pH 6.8; 100 nM dithiothreitol; 2% SDS; 0.1% bromophenol blue; 10% glycerol) and subjected to SDS/PAGE on a 4-15% polyacrylamide gel. Running buffer consisted of 196 mM glycine, 0.1% SDS, 5OmM Tris-HCl pH 8.3. Upon completion of electrophoresis, the separated proteins were either stained with Coomassie blue or electrophoretically transferred to BA-S nitrocellulose filters (Schleicher and Schuell, Keene, NH). Filters were hybridized overnight at 40 ⁰C with pooled turkey sera diluted 1:200 in Tris buffered saline (TBS) blocking buffer containing 5% Carnation instant milk. After washing 3X in TBS for 10 minutes each wash, filters were hybridized for 2 hours at 25 ⁰C with alkaline phosphatase-conjugated rabbit anti-chicken IgG (Sigma St. Louis, MO) diluted 1 :2000 in TBS containing 5% Carnation instant milk. The filters were washed as before and developed in alkaline phosphatase developing buffer (100 mM Tris, pH 9.5; 5 mM MgC^) containing nitro blue tetrazolium chloride (NBT) and 5- bromo-4-chloro-3'-indolyl phosphate (BCIP).

Vaccination and challenge studies in broad breasted white turkeys

[32] One week after the second boost with the recombinant FHAB2 peptides or with the control peptide all turkeys were challenged intranasally with 1.1 x 10⁷ CFU P. multocida strain P-1059 in a volume of 0.2 ml. The P-1059 strain used in this study was previously passed through a turkey that developed peracute fowl cholera after intravenous challenge. After challenge the birds were observed three times daily at 08:00, 16:00, and 21 :00 hours for signs of disease over the duration of the experiment. Birds showing clinical signs of fowl cholera, such as ruffled feathers, ataxia, or dehydration, were euthanized by intravenous injection of sodium pentobarbital (Sleepaway, Fort Dodge Animal Health, Fort Dodge, IA). Swab specimens were aseptically collected from liver and spleen of dead birds by puncturing the surface with a sterile scalpel and rolling the swab in the opening until saturated. The swabs were rolled onto a quarter of Columbia blood agar base plates then three consecutive quadrants were streaked with a sterile loop for colony isolation. Representative colonies were subjected to PCR analysis using P. multocida specific primers, KMT17T (SEQ ID NO:15) and KMT1SP6 (SEQ ID NO: 16), (Townsend et al, J Clin Microbiol. 1998; 36: 1096-1100) to confirm P. multocida growth on the plates. Seven days after infection, the remaining birds were euthanized by intravenous injections, and tissues were processed and examined as described above. This animal experiment was approved by the Animal Care and Use Committee at the National Animal Disease Center, Ames, IA.

[33] Vaccine trials conducted in this study are summarized in Table 2. The survival rates and the mean times to death were compared by Chi-squared tests.

[34] EXAMPLE 2 Expression and Purification of recombinant FHAB2 peptides

[35] Three gene fragments encompassing the 5' 3,250 bps of fhaB2 were cloned and expressed as recombinant peptides for use as immunogens against P. multocida (FIG. 1). The fhaB2 gene fragments were amplified from P. multocida strain P-1059 (serotype A:3) by PCR using the primers indicated in Table 1. Each fragment was digested with EcoRl and HinDIII prior to insertion into the similarly treated expression vector, pGEMEX^®-l (Promega). The pGEMEX^®-l constructs containing the fhaB2 gene fragments were expressed by E. coli DE-3 cells as fusion proteins joined to a 260 amino acid N-terminal leader of phage T7 gene 10. The calculated molecular masses of the recombinant FHAB2 peptides 1, 2, and 3 were 84, 74, and 72 kDa respectively (FIG. 2A). Western blot analysis using anti-T7 monoclonal antibody was done to confirm that the expressed recombinant peptides were joined to phage T7 genelO were of the predicted mass. Expression of the FHAB2 peptides by pGEMEX^®- 1 was unexpectedly decreased when E. coli DE-3 host cells were grown in the presence of IPTG inducer. Each of the recombinant peptides expressed in E. coli were contained primarily in inclusion bodies and purified using B-PER^® Bacterial Protein Extraction Reagent (FIG. 2B).

EXAMPLE 3

Immunization and challenge studies in turkeys using P. multocida P-1059 (serotype A:3)

[36] This example demonstrates that recombinant peptides of the invention protect turkeys against heterologous challenge with P. multocida P-1059 (serotype A:3).

[37] Seventeen six-week old broad breasted white turkeys were injected intramuscularly with 50 μg of purified recombinant FHAB2 peptides in saline with adjuvant. Also at this time seventeen control turkeys were treated identically with an inoculum consisting of the T7 gene 10 peptide expressed by pGEMEX^®-! mixed in adjuvant. [38] Two-weeks later, each bird was treated as before. One week later, all turkeys were challenged intranasally with 1.1 x 10⁷ CFU of wild type P. multocida strain P- 1059 resuspended in 200 μl Earle's balanced salt solution.

[39] All control birds in this study died of acute fowl cholera with an average survival time of 49 h (Table 2). Pure cultures of P. multocida were obtained from all liver and trachea specimens sampled from this group. PCR analysis of selected colonies using P. multocida specific primers confirmed the recovered bacterial growths were P. multocida serotype A. In contrast, fourteen of the seventeen birds vaccinated with the recombinant FHAB2 peptides survived intranasal challenge with P. multocida P- 1059 (p<0.001) (Table 2). All surviving birds exhibited no clinical signs over the duration of this study. After seven days, the remaining birds (all vaccinates) were euthanized and tissue samples were obtained. No liver samples from the vaccinated survivors were culture positive and only one tracheal sample from this group was positive for P. multocida as determined by culturing and PCR analysis. High levels of P. multocida were recovered from liver and trachea samples taken from the three vaccinates that died following challenge. The average time till death for the vaccinated birds was 105 h which was significantly longer than that of controls (p<0.05).

[40] Western blot analysis of pooled sera taken prior to bacterial challenge showed that turkeys immunized with the purified recombinant FHAB2 peptides developed antibody titers against a large protein of an approximate mass of 170 kDa present in whole cells (FIG. 3A) and multiple reactive high molecular weight bands were present in the concentrated culture media (FIG. 3B). In contrast, blots of P. multocida fhaB2 P-1059 mutant whole cells (FIG. 3A) and culture media (FIG. 3B) were devoid of the respectivel70 kDa protein and the secreted proteins present in the wild-type parent. The blots using pooled pre-immune turkey sera against either wild type or fhaB2 mutant cells (FIG. 4A) and culture supernatants (FIG. 4B) did not recognize the presumed FHAB2 product(s) identified above. A protein, approximately 65 kDa, present in P. multocida wild-type and fhaB2 mutant cell lysates, was recognized by both the preimmune and the vaccinate sera.

Table 1. Primer Pairs

Table 2. Protection against P. multocida challenge conferred in turkeys by recombinant FHAB2 peptides

** ^=0.001 * 77=0.05

EXAMPLE 4

Immunization and challenge studies in turkeys using P. multocida χ73 (serotype A: 1)

[41] This example demonstrates that recombinant peptides of the invention protect turkeys against heterologous challenge with P. multocida χ73 (serotype A: 1).

[42] Forty eight-week old mixed sex turkeys (Willmar Poultry Company, Willmar MN) were injected intramuscularly with pooled recombinant FHAB2 fragments (peptides 1, 2, and 3) (50 μg total) mixed with adjuvant (TITERMAX® Gold) in a volume of 200 μl. Also at this time, forty control birds were injected with 50 μg purified T7 gene 10 protein mixed with adjuvant as above. Two weeks later, booster immunizations identical to the first were given. Sera were collected from each bird at the time of the first vaccination and again three weeks later.

[43] One week after the second boost, 20 vaccinates and 20 controls were challenged intranasally with 2.1 x lO⁶ CFU P. multocida strain P- 1059 in a volume of 0.2 ml. Also at this time, 20 vaccinates and 20 controls were challenged intranasally with 2.2 x lO⁶ CFU P. multocida strain χ73. Afterwards, the birds in each challenge group were housed together in single rooms. The vaccinate and control animals were separated by a wire screen. The birds were observed for signs of disease three times daily at 08:00, 16:00, and 21:00 hours over the duration of the experiment. Birds showing clinical signs of fowl cholera, such as ruffled feathers, ataxia, or dehydration, were euthanized by intravenous injection of sodium pentobarbital (Sleepaway, Fort Dodge Animal Health, Fort Dodge, IA). Tracheal samples were collected by direct swabbing. Liver and spleen specimens were collected aseptically by puncturing the surfaces with a sterile scalpel and rolling a swab in the opening until saturated. The saturated swabs were rolled onto blood agar base plates, then the plates were streaked with a sterile loop for colony isolation. Representative colonies were subjected to PCR analysis using P. multocida specific primers, KMT 17T and KMT1SP6, to confirm P. multocida growth on the plates. Seven days after infection, the remaining birds were euthanized by intravenous injection, and sample specimens were processed and examined as described above. This animal experiment was approved by the Animal Care and Use Committee at the National Animal Disease Center, Ames, IA.

[44] Results from the experiment are tabulated below. All control and vaccinates which died without euthanasia died of acute FC as evidenced by pure cultures of P. multocida obtained from all trachea, liver, and spleen specimens sampled. PCR analysis of selected colonies using P. multocidaspscific primers confirmed that the recovered bacterial growths were P. multocida serotype A. In both challenge groups, birds vaccinated with the recombinant FHAB2 peptides were significantly protected from death (Chi Square Test). The surviving birds were euthanized after seven days, and tissue samples were obtained. No liver or spleen samples from the vaccinated survivors were culture-positive. Of the surviving controls, two challenged with P- 1059 and one challenged with χ73 were culture-positive.

Table 3. Protection conferred by vaccination with the recombinant P. multocida FHAB2 peptides.

Immunization dose Challenge dose (CFU) Survival

2x 50 μg rFHAB2 2.IxIO⁶ P-1059 15/20**

2x 50 μg control protein 2.1xlO^b P-1059 5/20

2x 50 μg rFHAB2 2.2xlO⁶ χ73 14/20**

2x 50 μg control protein 2.2xlO⁶ χ73 2/20

** /Kθ.001

Claims

1. A purified peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, wherein the peptide is less than a full-length P. multocida filamentous hemagglutinin protein.

2. The purified peptide of claim 1 which consists of the amino acid sequence SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

3. A nucleic acid molecule which encodes a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, wherein the peptide is less than a full-length P. multocida filamentous hemagglutinin protein.

4. The nucleic acid molecule of claim 3 which is an expression plasmid.

5. A vaccine composition, comprising: one or more peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; and a pharmaceutically acceptable vehicle.

6. The vaccine composition of claim 5 which comprises a peptide comprising the amino acid sequence SEQ ID NO:3, a peptide comprising the amino acid sequence SEQ ID NO:4 and a peptide comprising the amino acid sequence SEQ ID NO:5.

7. The vaccine composition of claim 5 further comprising an adjuvant.

8. The vaccine composition of claim 5 which is multivalent.

9. The vaccine composition of claim 5 wherein the peptide is less than a full- length P. multocida filamentous hemagglutinin protein.

10. A method of conferring immunity against Pasteurella multocida, comprising administering to an animal or bird in need thereof an effective amount of the vaccine composition of any of claims 5, 6, 7, 8, or 9.

11. The method of claim 10 wherein the vaccine composition is administered to a bird and the bird is a poultry bird.