Abstract:
This invention comprises a recombinant protein comprising the maltose binding protein (MBP) of  Escherichia coli  fused to amino acids 5–337 of the FlaA flagellin of  Campylobacter coli  VC167 which has provided evidence of immunogenicity and protective efficacy against challenge by a heterologous strain of  campylobacter, Campylobacter jejuni  81–176 in mammals. The invention further comprises a recombinant DNA construct encoding the immunodominant region (region I through III) of flagellin from  Campylobacter  spp. for use as a component of a vaccine against  Campylobacter  diarrhea. The invention therefore represents an effective treatment against  Campylobacter  but avoids inducing the autoimmune Guillain Barre Syndrome (GBS), a post-infection polyneuropathy caused by  Campylobacter  molecular mimicry of human gangliosides which has hampered the development of vaccines heretofore.

Description:
CROSS REFERENCE TO RELATED PATENTS AND APPLICATIONS 
     This application is related to the Provisional Application for Patent entitled, “A Recombinant Polypeptide For Use In The Manufacture of Vaccines Against  Campylobacter  Induced Diarrhea filed Nov. 12, 1998 by the inventors Patricia Guerry, Edward Burg, Lanfong H. Lee, and Trevor J. Trust, and having Ser. No. 60/108,114, and is entitled to the benefit of the Nov. 12, 1998 filing date for the matter disclosed therein. That Provisional Application for Patent is incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a construct of a recombinant DNA containing a fragment of a bacterial gene and the expression of the constructs for use as a vaccine or a component of a vaccine. Moreover, the invention relates to a recombinant protein comprising the maltose binding protein (MBP) of  Escherichia coli  fused to amino acids 5–337 of the FlaA flagellin of  Campylobacter coli  VC167. 
     2. Description of the Prior Art 
     The genus  Campylobacter  are gram-negative, curved, spiral or S-shaped or, in some cases, coccoid, bacteria.  Campylobacter  have a single polar, unsheathed flagellum at one or both ends which imparts a characteristic darting or cork-screw motility.  Campylobacter  are a major cause of gastroenteritis in both developed and developing countries (1,2). The major enteric pathogens in the genus are  C. jejuni  and  C. coli . All can be normally present in the gastrointestinal tract of domestic and wild animals which act as a major reservoir for infection in humans. Human infection with  Campylobacter  occurs via:
         1) animal to human contact especially farm animals such as poultry;   2) human to human transmission especially from infected children;   3) food contamination;   4) water contaminated with excreta from animals.       

       Campylobacter  infection can produce both an inflammatory diarrhea and a non-inflammatory diarrhea. The infection is more likely to be of the non-inflammatory type, without fever or bloody diarrhea. However, severe bloody diarrhea resembling bacillary dysentery can occur and frequently is seen in travelers to developing countries. 
     Although  Campylobacter  diarrhea is treatable with antibiotics, an effective vaccine against the organism is much preferred. This is especially true for travelers to regions where the disease is endemic and for use by developing nations where antibiotics are not always available or where their cost prohibits their use by the general population. There are, however, no currently licensed vaccines available against these organisms. 
     An important, possible contraindication of whole cell  Campylobacter  vaccine is the potential for development of Guillain-Barre Syndrome (GBS) (3), a post-infectious polyneuropathy, in vaccinated individuals. There are several reports indicating that prior infection with  C. jejuni  can result in acquisition of immunity (10,11). However, development of vaccines has been hampered by a lack of understanding of the basic virulence mechanisms and by the antigenic complexity of these organisms. For example, the serotyping scheme developed by Lior (12) is based on heat labile (HL) antigens and has over 100 recognized serogroups. The heat stable serotyping scheme of Penner (41), which is thought to be based on lippolysaccharides (LPS), has over 70 serotypes. The LPS cores of many serotypes have been shown to contain sialic acid in structures which resemble human gangliosides (13). This molecular mimicry has been implicated in development of autoantibodies leading to GBS, although the specific structure or structures which enable a given  campylobacter  strain to cause GBS are not clear. Strain 81–176 belongs to Serogroup O:23,36. Strains of O:23 and O:36 have been shown to contain ganglioside-like structures in their lipopolysaccharides. Although some O serotypes of  C. jejuni  are implicated with inducing GBS, O serotyping alone is insufficient in determining the potential for a given strain to induce GBS. Also, there is insufficient information to determine definitively the ability of any  Campylobacter  strain to lead to development of GBS. 
     A formalin fixed whole cell vaccine of  C. jejuni  81–176 adjuvanted with mutant  E. coli  heat labile enterotoxin (LT  R192G ; 12) is currently in human testing (14,15). This formulation appears to offer protection against homologous challenge in animal models (23,9), but the ability to protect against multiple serotypes of  C. jejuni  remains to be determined. Moreover, given the lack of understanding about the pathogenesis of  Campylobacter  associated GBS, there are concerns about use of whole cell preparations of  campylobacter  as vaccines. This concern becomes more compelling if multiple strains, which are less well characterized than 81–176, were to be combined in order to generate broad cross-serotype specific protection. An alternate approach would be to utilize a single  campylobacter  protein, either as a recombinant subunit vaccine or expressed in a carrier vaccine strain, to elicit protection against multiple serotypes of  Campylobacter . Therefore, there exists in the current state-of-the-art, the question whether specific  Campylobacter  strains, used in whole-cell vaccines or whole-cell vaccine candidates, could potentiate GBS and therefore be safe for vaccine use. One candidate for inclusion into such vaccines is flagellin. 
     Flagellin is a component of flagella, which provides swimming motility on many bacterial species including  Campylobacter . Flagellin is the immunodominant antigen recognized during human and experimental animal infections (16,17,18) with  Campylobacter . The structure of flagellin has been determined experimentally using the  Campylobacter coli  strain VC167 as a model. The flagella of this organism is complex, composed of multiple species of flagellin subunits, FlaA and FlaB (4–6). The FlaA and FlaB subunits are encoded by two genes, flaA and flaB, that are located adjacent to one another in a tandem orientation ( FIG. 1 ). The expression of these genes is concomitant and unit length rather than polycistronic. The flaA flagellin gene, which encodes the major flagellin subunit in the complex flagellar filament, has been divided into five regions (5) based on restriction enzyme mapping. Regions I–III encode the most highly conserved regions of the protein among different  Campylobacter  flagellin genes and are also the most immunodominant region of the protein (7). 
     Because of the potentially harmful effects of using whole-cell  Campylobacter  vaccines, it was concluded that an effective vaccine against this organism was needed that, at the same time, did not induce the deleterious autoimmune responses. 
     SUMMARY OF THE INVENTION 
     Accordingly, an object of this invention is a recombinant construct and expressed protein possessing highly immunogenic regions of the flagellar subunit but which did not contain antigenic moieties which induce GBS. 
     Another object of this invention is a recombinant protein, encoded by a portion of the flaA flagellin gene of  Campylobacter coli  VC167 consisting essentially of nucleotides 1–999 of SEQ ID NO.: 1 and corresponding to amino acid residues 1–333 of the amino acid sequence of SEQ ID NO: 238. 
     Yet another object of the invention is a product that can be expressed in different host backgrounds of bacterial strains belonging to the family Enterobacteriaceae for use in different vaccine formulations against  Campylobacter.    
     An additional object of this invention is the induction of a host immune response by purified recombinant flagellin expressed in  E. coli.    
     A further object is the cross-reactivity of antibodies, induced by purified recombinant flagellin expressed in  E. coli , with flagellins from  C. coli  (VC167) and other strains of  Campylobacter  spp. 
     Another object is that the purified recombinant flagellin expressed in  E. coli  is capable of protecting animals from disease and intestinal colonization by a heterologous strain,  Campylobacter jejuni  81–176. 
     A still further object is the use of the construct in vaccine preparations against diarrhea yet reducing the potential for GBS. 
     Another object is the use of the contstruct in vaccine preparations in order to reduce intestinal colonization. 
     These and other objects of the invention are accomplished by a recombinant DNA construct encoding the immunodominant region (regions I through III) of flagellin from  Campylobacter  spp. for use as a component of a vaccine against  Campylobacter  disease. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       A more complete appreciation of the invention will be readily obtained by reference to the following Description of the Preferred Embodiments and the accompanying drawings in which like numerals in different figures represent the same structures or elements. The representations in each of the figures is diagrammatic and no attempt is made to indicate actual scales or precise ratios. Proportional relationships are shown as approximations. 
         FIG. 1  is a schematic representation of the flaA and flaB genes of  C. coli , VC167, which encodes the major flagellin subunits in the flagellar filament. 
         FIG. 2  is a schematic representation of the regions I through V of flaA. The size, in nucleotide base pairs, of each region is shown. The immunodominant regions are regions I, II and III. 
         FIG. 3  is a comparison of the amino acid sequences of flagellins flaA of  Campylobacter coli  VC167T2 (SEQ ID NO:6) and  Campylobacter jejuni  81–176 (SEQ ID NO:5). 
         FIG. 4  is a graph of the experiment evaluating the ability of MBP-flaA to protect against colonization following oral feeding of mice. 
     
    
    
     DETAILED DESCRIPTION 
     Currently, no efficacious vaccine exists for  Campylobacter  disease. The potential for GBS complicates using attenuated strains of  Campylobacter  as a vaccine. It is critical therefore, that a vaccine be produced that is both capable of eliciting a vigorous protective immune response but does not lead to GBS. 
     Sub-unit vaccines hold a great deal of promise in fulfilling both these important criteria. One of the most highly immunogenic exposed regions of  Campylobacter  is the flagellin proteins, in particular the products of the genes flaA and flaB. The flaA gene of  Campylobacter  has been divided into five regions (5) based on restriction enzyme maps. This region of the flaA gene contains both highly conserved and variable regions among  Campylobacter  species. The preferred embodiment of this invention encompasses the gene sequence and expression from 13 through 1,015 base pairs of the flaA gene of  C. coli  (6), covering regions I, II and III as shown in  FIG. 2 , and segments within this region. This gene sequence is used to produce a recombinant  Campylobacter  polypeptide, which if introduced into a host is capable of producing an immunological response. The FlaA flagellin is the major protein subunit comprising the flagella filament or locomotory organelle of  Campylobacter  spp. 
     Region I of the flaA gene represents the highly conserved N terminal region, and regions II and III represent two regions which are more variable among different sequenced flagellin genes. Regions II and III are not, however, as variable as region IV. The construct was made by amplifying the regions I, II and III using the primer flaA-11 (5′ACCAATATTAACACAAATGTTGCAGCA3′) (Seq. ID no. 3) and flaA-2 (5′TTATCTAGACTAATCTCTACCATCATTTTTAAC3′) (Seq. ID no.4). The PCR product is digested with the appropriate restriction enzymes in order to insert the product into an expression vector. Any plasmid expression vector, e.g. PET™ (Novogen, Madison Wis.) or PMAL™ (New England Biolabs, Beverly, Mass.) and viral expression vectors (e.g. adenovirus, M13, herpesvirus, vaccinia, baculovirus, etc) expression systems can be used as long as the polypeptide is able to be expressed. The PET™ vector is used for the cloning and over-expression of recombinant proteins in  E. coli . In the PET™ system, the cloned gene is expressed under the control of a phage T7 promotor. In the PMAL™ protein fusion and purification system, the cloned gene is inserted into a PMAL™vector downstream from the MALE™ gene, which encodes maltose-binding protein (MBP). This results in the expression of an MBP-fusion protein. The technique uses the strong P tac  promotor and the translation initiation signals of LMBP to express large amounts of the fusion protein. The PMAL-C2™ series of vectors have an exact deletion of the MALE™ signal sequence, resulting in cytoplasmicexpression of the fusion protein. The PMAL-P2™ series of vectors contain the normal MALE™ signal sequence, which directs the fusion protein through the cytoplasmic membrane, resulting in periplasmic expression. The preferred expression system is the PMAL-C2™ vector (New England Biolabs, Beverly, Mass.). For insertion into this system the PCR product is digested with SspI and XbaI, purified by agarose gel electrophoresis, and cloned in a commercially available plasmid vector, PMAL-P2™ or PMAL-C2™ (New England Biolabs, Beverly, Mass.) which had been digested with XmnI and XbaI. This vector allows for fusion of the fifth codon of the flaA gene to an  Escherichia coli  gene encoding maltose binding protein (MBP). The MBP-FlaA fusion is transcriptionally regulated by a P tac  promotor and is induced by growth in isopropylthiogalactoside (IPTG). Several transformants of  E. coli  DH5-alpha, containing plasmids with the appropriate size insert, were sequenced with the MALE™ primer (New England Biolabs). The MALE™ primer is used for sequencing downstream from the malE gene across the polylinker. One plasmid with the expected fusion-protein in the correct reading frame to MALE™, termed pEB11-2, was purified. 
     The MBP-FlaA fusion protein was purified on the basis of the ability of the MBP portion of the molecule to bind to an amylose affinity column. 
     Having described the invention, the following examples are given to illustrate specific applications of the invention including the best mode now known to perform the invention. These specific examples are not intended to limit the scope of the invention described in this application. 
     EXAMPLE 1 
     Expression of Recombinant flaA Gene in PMAL-P2/C2™ Plasmid. 
     Purification: 
     The purification scheme of the construct was as recommended by the commercial supplier of pMal plasmid (New England Biolabs). DH5 alpha containing the flagellin-MBP fusion was grown overnight in 100 ml Rich medium (10 g tryptone, 5 g yeast extract, 5 g NaCl, 2 g glucose/liter) supplemented with 100 microgram/ml, and used to inoculate a fresh 1 liter of the same medium. This culture was grown with shaking at 37° C. to OD 600  0.5 and IPTG (isopropyl-B-thiogalactoside) was added to a final concentration of 0.3 mM. Cells were grown for an additional 2 hours and harvested by centrifugation. Cells were resuspended in 10 ml column buffer (20 mM Tris-Cl, 200 mM NaCl, 1 mM EDTA). The cells were frozen at −20° C. overnight, thawed on ice, and sonicated in short pulses for 2 min using a Branson sonicator (Branson, Danbury, Conn.). The sonicated solution was centrifuged for 30 min at 9000 g in a Sorvall RC5-B centrifuge, and the supernatant was diluted 1:5 with column buffer and loaded onto a 2.5×10 cm glass column packed with 15 ml of amylose resin (New England Biolab) at a flow rate of 1 ml/min. The column was washed with 12 volumes of column buffer and the fusion protein was eluted with column buffer containing 10 mM maltose. Protein containing fractions (as determined by BioRad protein assay) were pooled, concentrated using a vacuum concentrator, and stored in aliquots at −20° C. 
     Characterization: 
     The apparent M r  of the protein produced by DH5 alpha (pEB1 1–2) after IPTG induction was approximately 80,000, as determined by SDS-PAGE electrophoresis. This is consistent with the predicted M r  of 79,687 for the fusion protein, which includes M r 34,678 of FlaA and M r  45,009 from MBP. The fusion protein was immunoreactive with two hyperimmune rabbit antiserum made against flagellin purified from two strains of  Campylobacter : antiserum E288 which was made against purified flagellin from the homologous strain,  C. coli  VC167 (8) and antiserum SML2 which was made against purified flagellin from  C. jejuni  strain VC74 (9). 
     Antibody from natural infections was shown to recognize the native protein better than the recombinant protein as shown in Table 2. 
     Electrophoresis and Western Blotting. SDS-PAGE was performed with a mini-slab gel apparatus (Pharmacia, Piscataway, N.J.) by the method of Laemmli (19). Proteins samples solubilized in sample buffer (21) were separated in 12.5% acrylamide (150V) and either stained with Coomasie brilliant blue or transferred to nitrocellulose for immunological detection (20). Rabbit hyperimmune sera E288 against SDS-denatured VC167 T2 flagellin has been described previously (7). Immunodetection was described in Power et al. (7). The secondary antibody for rabbit antisera was alkaline phosphatase tagged goat anti-rabbit IgG (Caltag, Burlingame, Calif.) used at a final dilution of 1:5000; the secondary antibody for ferret antisera was horse radish peroxidase (HRP) labelled goat anti-ferret IgG (Kirkegaard and Perry, Gaithersburg, Md.) used at a dilution of 1:500. Alkaline phosphatase tagged antibodies were developed with NBT-BCIP (Nitro Blue Tetrazolium, 5-bromo-4chloro-3-indolylphosphate; Promega, Madison, Wis.) and peroxidase labelled antibodies were developed with TMB (3,3′,5,5′ tetramethyl benzidine; Sigma, St. Louis, Mo.). 
     Purification of flagellin. Flagellins were purified from  Campylobacter  spp. by the method of Power et al. (37). 
     Immune animal sera. Immune ferret sera were obtained from a collection of sera at NMRC/FDA from experiments in which ferrets were fed either VC167 T2 or 81–176 and subsequently developed diarrhea (21,22) as shown in Table 2. 
     Immune human sera. Immune human sera from volunteers fed 81–176 was the generous gift of David Tribble of NMRC. 
     Hyperimmune rabbit antiserum. Antiserum against the MBP-FlaA in 1 ml Freund&#39;s incomplete adjuvant 2 weeks later. The animal was exsanguinated two weeks after the second injection and the resulting antiserum was called LL 1. 
     ELISA. MaxiSorp 96-well immunoplates were coated with MBP-FlaA or flagellins purified from campylobacters (0.3 micrograms/mL 100 ul/well). ELISA&#39;s were performed as previously described (23). 
     DNA sequence analysis of the flaA gene of  C. jejuni  81–176. The flaA gene of  C. jejuni  81–176 has been cloned previously and the 5′end partially sequenced (24). The intact flaA gene of 81–176 as cloned in pK2–32 (24) was sequenced in order to determine the extend of similarity between the VC167 FlaA protein and that of 81–176 which would represent the challenge strain in protection experiments. The results indicate that the flaA gene of 81–176 encodes a protein of 574 amino acids with a predicted M r  of 59,240. Overall the two proteins are 92% identical and 94% similar. VC167 T2 flagellin has 573 amino acids with a predicted M r  of 59,047. The region of the VC167 T2 FlaA which is included in the MBP-FlaA recombinant protein is amino acids 5–337. The region includes those amino acids which appeared to be the most immunogenic by memeotope analysis. The VC167 and 81–176 flagellins are 98.1% identical and 98.7% similar in this region. The homology is lowest between amino acids 382 and 471. In this region, which includes an additional amino acid in the 81–176 protein, the two flagellins are 73% identical and 84% similar. Comparison of the region of VC167 T2 flagellin in the MBP-FlaA fusion protein with 11 other  C. jejuni  flagellins revealed a range of 82–90% similarity. See  FIG. 3 . 
     EXAMPLE 2 
     Use of Recombinant Flagellin to Induce an Immune Response in Rabbits. 
     Antibodies against the recombinant MBP-FlaA fusion protein are generated in rabbits by injecting rabbits intramuscularly with two (2) doses of 80 micrograms/rabbit for each dose of MBA-FlaA, the first with Freund&#39;s complete adjuvant and the second with freund&#39;s incomplete adjuvant, two (2) weeks apart. This anti-MBP-FlaA anti-serum reacted with the MBP-FlaA, MBP alone, and with flagellins isolated from both  C. coli  strain VC167 and the  C. jejuni  strain 81–176. 
     EXAMPLE 3 
     Use of Recombinant flaA Construction for Production of Subunit Vaccine in the Protection of Mice against  Campylobacter  Disease with or without  E. Coli  Enterotoxin (LT) as Adjuvant. 
       Campylobacter  spp. are primarily pathogens of primates and does not cause diarrheal disease when fed to mice. However, when infected intranasally with  Campylobacter  mice develop a lung infection, often with bacteremia, and swallow enough bacteria to become colonized in their gastrointestinal tract (9). This model has been used previously to evaluate vaccines against  campylobacter.    
     BALB/c mice (6–8 weeks old) were purchased from Jackson Laboratory (Bar Harbor, Me.) and housed in laminar flow cages for a minimum of 8 days before being used in experiments. Standard laboratory chow and water were provided ad libitum. Mice were anaesthetized with methoxyflurane (Metofane, Pitman-Moore, Mundelein Ill.) and immunized intranasally with 30–35 micrograms/l of fusion protein using a micropipet. The doses used were 0, 3, 6, 12, 25 or 50 micrograms of fusion protein in PBS, either with or without 5 ug of heat labile  E. coli  enterotoxin (LT) as adjuvant. A second dose was administered 8 days after the first vaccination. Intestinal lavage was collected 15 days after the first vaccination and blood was collected 29 days after the first vaccination. The mice were challenged intranasally with  C. jejuni  81–176 (2×10 9 /mouse) 34 days after the first vaccination, and the animals were monitored for sickness and death for 5 days. An illness was determined by assigning a score of 0 (healthy), 1 (ill as determined by a hunched back, ruffled fur, lethargy) or 2 (death) for each mouse daily. For each observation day the total score within each group was divided by the number of mice observed to yield the daily index. Fecal pellets were collected daily for 10–14 days after challenge, homogenized in PBS, and plated onto a  campylobacter  selective agar (CVA; Remel, Lenexa, Kans.). Putative  campylobacter  colonies were confirmed by morphology and oxidase reactions. Vaccine efficacy was calculated as (rate for control mice)−(rate for vaccinated mice)/(rate for control mice)×100. 
     The effect of the vaccine on disease symptoms and colonization are summarized in Table 1. The mean disease index on day 5 of mice which received no vaccine was 0.924 and for mice which received only LT was 0.859. The disease index of mice receiving MBP-FlaA without adjuvant decreased as the dose of vaccine increased up to 50 micrograms (disease index=0.371, reflecting 55.3% efficacy). In all cases the addition of LT decreased the disease index compared to the corresponding dose of MBP-FlaA without LT. A dose of 50 micrograms of MBP-FlaA achieved an 81% efficacy in protection against disease. In previous experiments, infection of mice with live 81–176 resulted in 71% efficacy against disease symptoms following a second challenge with the same. 
     There was no effect on the numbers of mice colonized with 81–176 at any dose of MBP-FlaA without LT, except at the highest dose (50 micrograms) which showed 47.6% efficacy against colonization. Similarly, mice receiving 3 micrograms, 6 micrograms or 12 micrograms of MBP-FlaA+LT showed no significant reduction in colonization. However, doses of 25 micrograms or 50 micrograms of MBP-FlaA+LT resulted in 61% and 84% efficacy against colonization, respectively. In previous experiments, infection of mice with live 81–176 resulted in 91% efficacy against colonization following a second challenge with the same strain. 
     The FlaA component of the MBP-FlaA fusion protein is capable of eliciting an immune response which can give significant protection against disease symptoms by a heterologous strain of  Campylobacter  as measured in this animal model, as well as against colonization of the intestine. Protection against colonization would preclude development of diarrheal disease. The regions of the FlaA protein from  C. coli  VC167 which are conserved in the portion of the protein used in these experiments are apparently sufficient to elicit protection against heterologous challenge by  C. jejuni  81–176. This recombinant construction could be (1) used as a fusion protein, as in the example given here, to MBP, (2) be purified via another recombinant construction as a protein of approximate M r  of 35,000, expressed in another expression system, such as a histidine-tag vector system, or (3) be expressed under control of an appropriate promoter in a carrier vaccine strain such as  Salmonella  or  Shigella  attenuated live vaccines, for use as a bivalent vaccine. 
     Genbank accession number. The DNA sequence of the 81–176 flaA gene has been deposited in Genbank under accession number AF15052. 
     EXAMPLE 4 
     Evaluation of Immunogenicity and Efficacy of the MBP-FlaA Protein Against Heterologous Challenge in the Mouse Intranasal Model. 
     Mice were immunized intranasally with 2 doses of 3–50 micrograms of MBP-FlaA with or without 5 micrograms of LT R192G  as adjuvant. Table 3 shows the intestinal IgA and serum IgG response to MBP-FlaA as measured by ELISA. The full range of MBP-FlaA doses elicited significant antigen-specific serum IgG responses in vaccinated animals and these responses were enhanced by adjuvant use, with the exception of the highest dose (50 micrograms). In contrast, stimulation of FlaA-specific intestinal secretory IgA (sIgA) responses required immunization with higher does of MBP-FlaA (≧25 micrograms) or co-administration of lower doses with adjuvant. When given with the adjuvant, as little as 3 micrograms of the MBP-FlaA protein was capable of stimulating a significant antigen-specific sIgA response in immunized animals. In addition, the magnitude of intestinal sIgA responses to the recombinant protein were significantly enhanced in those animals receiving the adjuvanted protein compared to those given MBP-FlaA alone, with the exception of the highest dose. 
     The mice were challnged intranasally with  C. jejuni  81–176 (2×10 9  bacteria/mouse) 26 days after the second immunization. The effects of the vaccine on disease symptoms and colonization on day 7 are summarized in Table 3. The mean disease index of mice which had received no vaccine or LT R192G  alone was 0.92 and 0.85, respectively. The disease index of mice receiving MBP-FlaA without adjuvant decreased as the dose of vaccine increased up to 50 μg (disease index=0.37, reflecting 55.3% efficacy), although the results were not statistically significant. In all cases, except the 3 microgram dose, the addition of LT R192G  decreased the disease index compared to the corresponding dose of MBP-FlaA without LT R192G . A dose of 50 microgram of MBP-FlaA+LT R192G  achieved 81.1% efficacy in protection against disease (P&lt;0.0001). In previous experiments, when mice which had been infected intranasally with live 81–176 were rechallenged 26 days later with the same strain, there was 71% efficacy against disease symptoms. 
     There was no effect on the numbers of mice colonized with 81–176 at any dose of MBP-FlaA without LT, except at the highest dose (50 micrograms) which showed 47.6% efficacy in protecting against colonization. Similarly, mice receiving the lower doses of MBP-FlaA+LT R192G  showed little to no reduction in colonization. However, a dose of 50 micrograms MBP-FlaA+LT R192G  resulted in 84.1% efficacy against colonization (P&lt;0.05). In previous experiments using this model, infection of mice with live 81–176 resulted in 91% efficacy against colonization following a second challenge with the same strain. 
     EXAMPLE 5 
     Evaluation of the Ability of MBP-FlaA to Protect Against Colonization Following Oral Feeding of Mice. 
     To better examine the ability of MBP-FlaA to protect against intestinal colonization, additional mice were vaccinated with two doses each of 50 micrograms MBP-FlaA with and without 5 micrograms LT R192G  adjuvant, 8 days apart. Twenty-six days after the second immunization, groups of 7–8 mice were challenged orally with 3 different doses of 81–176: 8×10 10 , 8×10 9  and 8×10 8 . Control animals immunized with either PBS (open triangles) or LT R192G  (closed triangles) alone were colonized throughout the course of the experiment regardless of challenge dose. These results are shown in  FIG. 4A  for the highest challenge group only. Animals immunized with MBP-FlaA alone showed an apparent transient and insignificant reduction in total numbers colonized at days 5 and 6 (71.4% of the animals were culture positive) in the high dose challenge group only  FIG. 4A . (open circles) However, on day 7 100% of the mice immunized with MBP-FlaA alone were colonized. When animals immunized with MBP-FlaA+LT R192G  were challenged with 8×10 10  organisms, there was a marked difference between the controls at days 5–7, with only 40% of the animals being colonized on days 5 and 6 (P&lt;0.05), and 20% colonized on day 7 (P&lt;0.001;  FIG. 4A  (closed circles). This corresponds to 55.2% efficacy for days 5 and 6, and 71.4% efficacy on day 7. The efficacy improved when the animals were challenged with 8×10 9  bacteria ( FIG. 4B ). In this case, a significant difference between MBP-FlaA+LT R192G  (closed circles) and MBP-FlaA alone (open circles) and control groups was apparent by day 5, with the MBP-FlaA+LT R192G  vaccine giving 55.1% efficacy (P&lt;0.05). By day 7 only 28.6% of the animals in this group remained (71.4% efficacy; P&lt;0.001). Challenge of the MBP-FlaA+LT R192G  group with 8×10 8  bacteria showed a significant reduction in colonization by day 4 (P&lt;0.05) and a drop in bacterial counts throughout the course of the experiment ( FIG. 3C  (closed circles). By day 6 the MBP-FlaA+LT R192G  vaccine resulted in 78% efficacy against colonization (P 0.001), and by day 7 no campylobacters could be detected in the stools under the sampling conditions used (P&lt;0.001). 
     The data presented here indicate that MBP-FlaA, when adjuvanted with LT R192G  is capable of eliciting a protective immune response against a heterologous strain of  campylobacter  as measured in two mouse models involving oral and nasal challenge. At the highest dose (50 micrograms MBP-FlaA+5 micrograms LT R192G ) the vaccine showed 81% protective efficacy against disease and 84% efficacy against colonization of the intestine in the mouse intranasal challenge model (9). In this model immunization with live 81–176, followed by a second infection with the same strain, resulted in 71% efficacy against disease and 91% efficacy against colonization (9). Although the mouse intranasal model uses an unnatural route of infection, it is the only mouse model for  campylobacter  which consistently results in disease symptoms, generally pneumonia and bacteremia (9). Intestinal colonization presumably occurs in this model when the mice swallow some portion of the infecting bacteria. To more directly measure the protection against colonization, we also challenged mice which had undergone the same immunization regimen (50 micrograms MBP-FlaA+LT R192G ) with different oral doses of 81–176. The results showed that when challenged with 8×10 8  bacteria, there was a reduction in colonization as early as 3 days after infection and that no campylobacters could be detected in stools 7 days post-feeding. 
     Flagella are a key virulence determinant of  Campylobacter  spp. since motility is essential for establishment of colonization in the mucus lining of the gastrointestinal tract (25, 26, 27, 28). Moreover, flagellin is an immunodominant antigen recognized during infection (16, 17,11,18), and it has been suggested that development of antibodies against flagellin correlates with development of protection (29, 11, 18). The observation that feeding of one strain of  campylobacter  protects against disease from the homologous, but not heterologous strains, (10) is consistent with the idea that the major protective antigen shows variation among strains. Although there is no flagellar serotyping scheme for campylobacters comparable to the H antigen typing scheme of the  Enterobacteriaceae , there is serological diversity among  campylobacter  flagellins (34, 35, 7). In  Salmonella  and  E. coli  it has been demonstrated that the amino and carboxy ends of flagellins are involved in transport of the monomer and assembly into the filament, and these regions are highly conserved among serotypes. The central region of the flagellin protein, which lacks functional constraints, is the antigenically diverse region responsible for H serospecificity, and is also the region which is surface exposed in the flagellar filament. Based on comparison of DNA sequence analyses of flagellin genes from several strains of  C. jejuni , including that of 81–176 reported here, and one strain of  C. coli  (4,30, 8, 3, 18, 32), the overall structure of  campylobacter  flagellins appears similar to those of the enterics. Thus, the amino and carboxy terminal regions are highly conserved among  campylobacter  flagellins, and the central regions are more variable (36). Moreover, Power et al. (7) have shown that antibodies to the amino and carboxy regions are not surface exposed in the flagella filament of  campylobacter . The only antibodies found in that study to be surface exposed in the filament were those which recognize a glycosyl posttranslational modification (33, 37, 38). These modifications alter the apparent M, of flagellins on SDS-PAGE gels. For example, the masses of the flagellins of VC167 and 81–176 are predicted to differ by only 207, but their apparent difference on SDS-PAGE is greater (see  FIG. 1 ). Moreover, Alm et al. (39) showed that the apparent M r  of flagellin can vary when expressed in different  campylobacter  hosts. The presence of a carbohydrate moiety on a bacterial flagellin is highly unusual and has been shown to confer serospecificity to the flagellin (33). Thus, antisera which recognize the posttranslational modifications on the flagellar filament of VC167 (Lior 8) also react with flagellins of other strains of Lior 8, but not strains of other Lior serogroups (39). Although flagellin is not the serodeterminant of Lior 8 (i.e. non-flagellated mutants of Lior 8 strains still serotype), flagellins appear to be conserved antigenically within the serogroup. Moreover, more recent studies have suggested that glyc syl modifications on flagellin, as well as other  campylobacter  proteins, are immunodominant (38). 
     One would expect that any protective epitopes would be surface exposed on the flagellar filament. The role of these surface exposed posttranslational modifications on protection has been addressed in only one study using the removable intestinal tie adult rabbit diarrhea (RITARD) model. In this model protection against colonization appeared to be limited to strains of the same Lior serotype. In other words, immunization by feeding with VC167 protected rabbits against subsequent colonization following RITARD challenge with the homologous strain, as well as two other  C. jejuni  strains of the Lior 8 serogroup, but not against strains of other serogroups (37). A site-specific mutant defective in a gene required for biosynthesis of the posttranslational modification in VC167 was capable of protecting against a challenge of wildtype VC167, but not the other  C. jejuni  Lior 8 strains, suggesting that the posttranslational modifications are responsible for this Lior 8 serospecific protection. Given this data, one would not expect that recombinant flagellin that lacked the posttranslational modifications, which are encoded by other  campylobacter  genes, would be protective, but the data presented here suggest otherwise. 
     In this regard, it is interesting that antibodies generated during natural infection in ferrets by either 81–176 or VC167 appeared to react more strongly to glycosylated flagellins isolated from  Campylobacter  spp. than to unglycosylated, recombinant flagellins isolated from  E. coli . Similar analysis using serum from a human volunteer who had been infected with 81–176 (15) also suggested a stronger immune response to native flagellin than recombinant flagellin. This is documented in (40) and is incorporated by reference. Although the recombinant constuction used contains a truncated FlaA, this region was selected based on its high immunogenicity in a mimeotope mapping study (7). Thus, the lack of immune response to this region with antisera from experimentally infected humans and animals was surprising, and suggests that during natural gastrointestinal infection the immunodominant epitopes are those of the posttranslational modifications rather than the primary amino acids. Immunization with the recombinant fusion protein lacking these posttranslational modifications may lead to antibody production against epitopes which are less immunogenic in the native molecule due to differences in folding and/or masking by the carbohydrate moiety, but are, nonetheless, capable of eliciting a protective immune response. We are currently further evaluating this recombinant flagellin as a vaccine in a ferret diarrheal disease model (21, 22). 
     
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Resistance to  C. jejuni  81-176 challenge following vaccination with 
               
               
                 recombinant FlaA with or without LT. 
               
             
          
           
               
                   
                 Fecal Excretion 
               
               
                   
                 (day 7) 
               
             
          
           
               
                 Dose of 
                   
                   
                 Disease 
                 % 
                 % Col- 
                 % 
               
               
                 MBP-FLaA 
                 LT 
                 n 
                 Index 
                 Efficacy 
                 onization 
                 Efficacy 
               
               
                   
               
             
          
           
               
                 none 
                 − 
                 13 
                 0.924 +/− 0.214 
                 0 
                 82 
                 0 
               
               
                 none 
                 + 
                 11 
                 0.859 +/− 0.297 
                 7 
                 67 
                 18.2 
               
               
                  3 ug 
                 − 
                 7 
                 0.771 +/− 0.373 
                 16.6 
                 100 
                 0 
               
               
                  6 ug 
                 − 
                 7 
                 0.771 +/− 0.281 
                 16.6 
                 100 
                 0 
               
               
                 12 ug 
                 − 
                 7 
                 0.743 +/− 0.433 
                 18.1 
                 100 
                 0 
               
               
                 25 ug 
                 − 
                 12 
                 0.483 +/− 0.410 
                 44.1 
                 92 
                 0 
               
               
                 50 ug 
                 − 
                 7 
                 0.371 +/− 0.511 
                 55.3 
                 43 
                 47.6 
               
               
                  3 ug 
                 + 
                 6 
                 0.793 +/− 0.365 
                 14.1 
                 60 
                 26.8 
               
               
                  6 ug 
                 + 
                 7 
                 0.429 +/− 0.378 
                 53.6 
                 100 
                 0 
               
               
                 12 ug 
                 + 
                 6 
                 0.667 +/− 0.391 
                 27.8 
                 83.0 
                 0 
               
               
                 25 ug 
                 + 
                 12 
                 0.333 +/− 0.445 
                 64.0 
                 77.0 
                 61.0 
               
               
                 50 ug 
                 + 
                 8 
                 0.175 +/− 0.244 
                 81.1 
                 13.0 
                 84.0 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Serum IgG responses measured by ELISA of ferrets infected with 
               
               
                   Campylobacter  spp. to  campylobacter  flagellins and MBP-FlaA.* 
               
             
          
           
               
                   
                 Number (%) of animals responding to: 
               
             
          
           
               
                 Infecting Strain 
                 VC167 flagellin 
                 81-176 flagellin 
                 MBP-FlaA 
               
               
                   
               
               
                 81-176 
                 8/8 (100%) 
                 8/8 (100%)  
                 3/8 (37.5%) 
               
               
                 VC167 T2 
                 6/8 (75%)  
                 7/8 (87.5%) 
                 3/8 (37.5%) 
               
               
                   
               
               
                 *Animals were infected with between 10 9 –10 10  cells of the indicated strains and serum samples were taken 1 week after infection. An animal was considered to respond to the antigen if there was a greater than 4-fold increase in titer compared to the pre-immune sera. 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Immunogenicity and efficacy of MBP-FlaA given with and without adjuvant in the mouse intranasal model. 
               
             
          
           
               
                   
                 Immunogenicity 
                 Efficacy at day 7 
               
             
          
           
               
                 Immunization Regimen 
                 geometric mean titer #   
                 Disease Symptoms 
                 Fecal Excretion (%) 
               
             
          
           
               
                 μg MBP-FlaA 
                 LT R192G   
                 n 
                 Lavage IgA 
                 Serum IgG 
                 Illness Index 
                 % Efficacy 
                 % Colonized 
                 % Efficacy 
               
               
                   
               
             
          
           
               
                 — 
                 − 
                 13 
                 0.9 ± 0 
                  5.7 ± 1.2 
                 0.92 ± 0.21 
                 NA 
                  82* 
                 NA 
               
               
                 — 
                 + 
                 11 
                 0.9 ± 0 
                  4.3 ± 1.3 
                 0.86 ± 0.29 
                 7.0 
                  67* 
                 18.3 
               
               
                  3 
                 − 
                 7 
                 1.0 ± 0.3 
                  9.9 ± 0.8 a   
                 0.77 ± 0.37 
                 16.6 
                 100  
                 0 
               
               
                 6 
                 − 
                 7 
                 1.3 ± 0.7 
                 10.6 ± 1.2 a   
                 0.77 ± 0.28 
                 16.6 
                 100  
                 0 
               
               
                 12 
                 − 
                 7 
                 1.4 ± 0.5 
                 10.2 ± 1.6 a   
                 0.74 ± 0.43 
                 18.1 
                 100  
                 0 
               
               
                 25 
                 − 
                 12 
                 3.5 ± 1.0 a   
                 11.9 ± 1.9 a   
                 0.48 ± 0.41 
                 44.1 
                 92 
                 0 
               
               
                 50 
                 − 
                 7 
                 5.5 ± 1.1 a   
                 14.0 ± 1.5 a   
                 0.37 ± 0.51 
                 55.3 
                 43 
                 47.6 
               
               
                 3 
                 + 
                 6 
                 3.5 ± 0.8 a,c   
                 13.2 ± 0.6 a,c   
                 0.79 ± 0.37 
                 14.1 
                  60** 
                 26.8 
               
               
                 6 
                 + 
                 7 
                 3.4 ± 0.8 a,c   
                 13.1 ± 0.5 a,c   
                 0.43 ± 0.38 b   
                 53.6 
                 100  
                 0 
               
               
                 12 
                 + 
                 6 
                 3.4 ± 1.3 b,d   
                 12.8 ± 1.0 a,d   
                 0.67 ± 0.39 
                 27.8 
                 83 
                 0 
               
               
                 25 
                 + 
                 12 
                 5.7 ± 12 a,c   
                 14.1 ± 1.7 a,d   
                 0.33 ± 0.44 b   
                 64.0 
                 75 
                 8.5 
               
               
                 50 
                 + 
                 8 
                 6.6 ± 0.6 a,d   
                 14.8 ± 0.7 a   
                 0.17 ± 0.24 a   
                 81.1 
                     13 b   
                 84.1 
               
               
                   
               
               
                   # geometric mean titer is expressed as natural log transformed values. 
               
               
                   a P &lt; 0.001 compared to animals immunized with PBS; 
               
               
                   b P &lt; 0.05 compared to animals immunized with PBS; 
               
               
                   c P &lt; 0.001 compared to animals immunized with a comparable dose of MBP-FlaA without adjuvant; 
               
               
                   d P &lt; 0.05 compared to animals immunized with a comparable dose of MBP-FlaA without adjuvant. 
               
               
                 *2 animals died following challenge; 
               
               
                 **1 animal died following challenge. 
               
             
          
         
       
     
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Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described. Accordingly, the recombinant construct can be used as a fusion protein with not only MBP, described here, but with other protein expression systems. The recombinant construct can also be expressed in carrier vaccines such as  Salmonella  or  Shigella  attenuated, live vaccines, for use as a bivalent vaccine.