Abstract:
The invention relates to the use of  Campylobacter jejuni  gene product jlpA and immunogenic fragments of jlpA as a component of a pharmaceutical formulation capable of eliciting an anti- Campylobacter jejuni  immune response. The invention also relates to a method of inducing an anti- Campylobacter  immune response by the administration of  C. jejuni  jlpA gene products or immunogenic fragments of such gene product to a subject.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims priority to Provisional Application No. 61/239,969 filed Sep. 4, 2009 (now expired), and a divisional application of U.S. patent application Ser. No. 12/874,304 filed on 2 Sep. 2010. 
     
    
     TECHNICAL FIELD 
       [0002]    The invention relates to a  Campylobacter jejuni  gene product and immunogenic fragments of the gene product useful as component(s) of a pharmaceutical formulation capable of eliciting an anti- Campylobacter jejuni  immune response. The invention also relates to a method of inducing an anti- Campylobacter  immune response by the administration of  C. jejuni  jlpA gene products or immunogenic fragments of such gene product. 
         [0003]      Campylobacter jejuni  is a Gram-negative, spiral, micro-aerophilic bacterium that exists as a commensal organism in the intestinal tracts of a variety of wild and domestic animals. It is a leading cause of acute bacterial enterocolitis in humans. Clinical symptoms of campylobacteriosis range from a mild watery diarrhea to bloody diarrhea accompanied by nausea, vomiting, fever, abdominal cramps and the presence of fecal leucocytes. 
         [0004]      Campylobacter  infection also has been associated with Guillain-Barré syndrome, an autoimmune-mediated disorder of the peripheral nervous system causing paralysis and even death. Like many other human enteric bacterial pathogens,  C. jejuni  infects humans by colonizing the mucus layer of the intestine followed by adherence and invasion of epithelial cells. 
         [0005]    Although  Campylobacter  infection is treatable with antibiotics, an effective vaccine formulation against the organism is desired for disease prevention. Vaccine administration is especially beneficial for travelers to regions of the world where  Campylobacter  is endemic. However, there are currently no licensed vaccines for this organism. 
         [0006]    Adherence of  C. jejuni  is a multifactorial event in which multiple binding factors may be required to bind to their respective receptors to achieve an efficient interaction with host cells.  Campylobacter jejuni  surface constituents, including PEB1 (Pei et al., 1998), CadF (Konkel et al., 1997), JlpA (Jin et al., 2001 and Jin et al., 2003), a 43 kDa major outer membrane protein (MOMP) (Moser et al., 1997), lipopolysaccharides (LPS) (Fry et al., 2000), and motility provided by the polar flagellum of the bacterium (Yao et al., 1994), each play a role in promoting bacterial adherence. Non-specific binding of the bacterium to epithelial cell lipids may also mediate adherence (Szymanski and Armstrong, 1996). 
         [0007]    Jin (2001) identified an 1116 base pair open reading frame (ORF), designated jlpA, encoding a novel species-specific lipoprotein of  Campylobacter jejuni  TGH9011, from recombinant plasmid pHIP-O. The jlpA gene encodes a polypeptide (hereafter “JlpA”) of 372 amino acid residues with a molecular mass of 42.3 kDa. JlpA contains a typical signal peptide and lipoprotein processing site at the N-terminus. The presence of a lipid moiety on the JlpA molecule was confirmed by the incorporation of [ 3 H]-palmitic acid. Immunoblotting analysis of cell surface extracts indicated that JlpA is a surface-exposed lipoprotein in  C. jejuni . JlpA is loosely associated with the cell surface, as it is easily extracted from the  C. jejuni  outer membrane by detergents. The adherence of both insertion and deletion mutants of jlpA to HEp-2 epithelial cells was reduced compared with that of parental  C. jejuni  TGH9011. Adherence of  C. jejuni  to HEp-2 cells was inhibited in a dose-dependent manner when the bacterium was preincubated with anti-GST-JlpA antibodies or when HEp-2 cells were preincubated with JlpA protein. A ligand-binding immunoblotting assay showed that JlpA binds to HEp-2 cells, which suggests that the  C. jejuni  surface lipoprotein JlpA mediates adherence of the bacterium to epithelial cells. 
         [0008]    Jin (2003) further demonstrated that JlpA interacts with HEp-2 cell surface heat shock protein (Hsp) 90α and initiates signaling pathways leading to activation of NF-κB and p38 MAP kinase. Gel overlay and GST pull down assays showed that JlpA interacts with Hsp90α. Geldanamycin, a specific inhibitor of Hsp90, and anti-human Hsp90α antibody significantly blocked the interaction between JlpA and Hsp90α, suggesting a direct interaction between JlpA and HEp-2 cell surface-exposed Hsp90α. The treatment of HEp-2 cells with GST-JlpA initiated two signaling pathways: one leading to the phosphorylation and degradation of IκB and nuclear translocation of NF-κB; and another one to the phosphorylation of p38 MAP kinase. The activation of NF-κB and p38 MAP kinase in HEp-2 cells suggests that JlpA triggers inflammatory/immune responses in host cells following  C. jejuni  infection. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0009]      FIG. 1 : Alignment of Amino Acid Sequences of JlpA of different  Campylobacter  strains. 
           [0010]      FIG. 2 : JLPA-specific serum IgG responses in mice. 
           [0011]      FIG. 3 : JLPA-specific fecal IgA responses in mice. 
       
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0012]    JlpA is a  Campylobacter  specific protein and is highly conserved among  Campylobacter  strains ( FIG. 1 ), which make it a valuable vaccine candidate against  C. jejuni . An aspect of this invention is an immunogenic composition comprising  Campylobacter jejuni  protein JlpA or fragments thereof. The composition can be isolated and prepared from natural  C. jejuni  isolates or be recombinantly produced. Another aspect of this invention is a method of inducing an immune response against  Campylobacter  by the administration of JlpA or its immunogenic fragments. 
       Example 1 
     Expression of JlpA as a Recombinant Histidine Tagged Protein in  E. coli    
       [0013]    In an exemplary embodiment of the invention, the jlpA gene from  Campylobacter jejuni  81-176 was amplified by polymerase chain reaction (hereafter “PCR”) with the jlpA forward primer: 
         [0000]                            (SEQ ID. NO. 5)           CCA TAT GTG CGG AAA TTC CAT AGA TGA AAA AAC;            
and jlpA reverse primer:
 
         [0000]    
       
         
               
               
             
           
               
                   
                 (SEQ ID. NO. 6) 
               
               
                   
                 GGG ATC CTT AAA ATG ACG CTC CGC CC.. 
               
             
          
         
       
     
         [0014]    The polymerase used for the amplification in this embodiment was HF2 DNA polymerase (Clontech Laboratories, Inc. Mountain View, Calif.). The primers introduced NdeI and BamHI sites into the amplicon. The amplicon was digested with NdeI and BamHI and ligated to NdeI and BamHI-digested pET19b plasmid (NOVAGEN®, Gibbstown, N.J.). The ligation mixture was used to transform DH5 alpha cells with selection on ampicillin resulting in strain PG2604. The DNA in this plasmid was sequenced to confirm that the appropriate gene had been cloned. The plasmid in PG2604 was transformed into BL21 (DE3) to express recombinant JlpA with a hexahistidine tag. The confirmed DNA sequence of the cloned jlpA gene with a hexahistidine tag was identified in SEQ ID. NO. 4. The predicted protein encoded by this DNA sequence had a predicted mass of 43.33 kDa and a predicted pI of 5.03. The predicted protein sequence was identified in SEQ ID. NO. 3. 
       Example 2 
     Immunogenicity of JIpA During Human Infection with  C. jejuni    
       [0015]    Study subjects were diarrhea patients enrolled in field studies during Cobra Gold 1999, in Thailand. The pathogens isolated are shown in the Table 1. A group of 10 subjects who participated in the Cobra Gold exercise in 2000 in Thailand, but did not develop diarrhea were used as negative controls. 
         [0016]    Plasma samples were collected on study days 7 and 60, following presentation at the clinic. A single blood sample was collected from the control group during the exercise. Samples were kept frozen until assayed for JlpA81-176-specific IgA or IgG using ELISA. Mean+2 standard deviation value of the control group was used to determine responder rates among the infected groups. Mean IgA and IgG titers among the control group were 1,040±1,032 and 3,800±2,437, respectively. Responders were defined as individual having IgA titer of 1:3,200 and IgG titer of ≧1:9,000. Following infection (in a field setting), systemic immune response to JlpA was observed at the time points tested in 50% of the subjects recovering from  Campylobacter  infection. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Systemic immune responses to JlpA antigen in humans 
               
             
          
           
               
                   
                   
                 Serum responses to JlpA (% responders) 
               
             
          
           
               
                 Pathogen Isolated 
                 N 
                 IgA 
                 IgG 
                 Any Isotype 
               
               
                   
               
             
          
           
               
                   Campylobacter  only 
                 18 
                 38.9 
                 16.7 
                 50.0 
               
               
                   Salmonella  only 
                 8 
                 0 
                 30.0 
                 30.0 
               
               
                 Other Pathogens 
                 7 
                 0 
                 14.3 
                 14.3 
               
               
                 None 
                 7 
                 0 
                 0 
                 0 
               
               
                 Control 
                 10 
                 0 
                 0 
                 0 
               
               
                   
               
             
          
         
       
     
       Example 3 
     Immunogenicity and Efficacy Of Protein JlpA of  C. jejuni  Strains in Mice 
       [0017]    A murine study were undertaken in order to evaluate the immunogenicity and potential protective efficacy of an embodiment of the vaccine composition. In the study, adult female BALB/c mice were lightly anesthetized with isoflurane and immunized intranasally with 30 μl of PBS containing recombinant JlpA protein alone or with a mucosal adjuvant, LTR192G. The experimental groups are illustrated in Table 2. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Experimental groups for JLPA vaccination 
               
             
          
           
               
                   
                   
                 Recombinant 
                   
                   
               
               
                   
                 Group 
                 JlpA (μg) 
                 LTR192G (μg) 
                 N 
               
               
                   
                   
               
             
          
           
               
                   
                 1 
                 0 
                 − 
                 8 
               
               
                   
                 2 
                 5 
                 + 
                 8 
               
               
                   
                 3 
                 25 
                 − 
                 7 
               
               
                   
                 4 
                 25 
                 + 
                 7 
               
               
                   
                 5 
                 100 
                 − 
                 7 
               
               
                   
                 6 
                 100 
                 + 
                 7 
               
               
                   
                   
               
             
          
         
       
     
         [0018]    Three doses of the embodiment vaccine composition were delivered at 14 day intervals to the subject, on study days: 0, 14 and 28. Seven days after the last vaccination, on study day 35, antigen-specific secretory IgA was measured from fecal pellets. Antigen-specific serum IgG was determined in tail blood collected 21-22 days after the last vaccination, on study day 49-50. All antigen-specific responses were detected using ELISA. 
         [0019]    On study day 58 (30 days post last vaccination), all animals were challenged intranasally with 3×10 9  cfu of  C. jejuni  81-176. Following the challenge, animals were followed for 6 consecutive days for the development of the infection associated illness. Based on the severity of sickness a score was assigned to each animal as follows.
       0=No apparent illness   1=ruffled fur   2=ruffled fur and hunched back   3=dead
 
Daily sickness indices, and then group average indices were determined and vaccine efficacy was calculated as: (Control—vaccinated)/(Control)×100
       
 
       Immunogenicity of JlpA 
       [0024]    JlpA-specific serum IgG responses in mice were shown in  FIG. 2 . JlpA-specific fecal IgA responses in mice were shown in  FIG. 3 . Data are presented as group geometric mean titer (natural log e ) and standard deviation. 
         [0025]    As illustrated in  FIG. 2 , a robust vaccine dose dependent serum IgG levels were detected in animals immunized with recombinant JlpA alone, which was further enhanced by inclusion of a mucosal adjuvant LTR192G ( FIG. 2 ). Animals immunized with PBS showed low levels of anti-JlpA IgG responses (mean+2 standard deviations≦1:100). Based on this cut off, all animals in all groups immunized with or without LTR192G were categorized as IgG responders. 
         [0026]    Similar to serum IgG response, a robust mucosal IgA response was detected after vaccination. Interestingly, LTR192G had a greater adjuvant effect on mucosal IgA response than observed for serum IgG response, with 5 to 6 fold increase when the same amount of protein was delivered with the adjuvant. Animals immunized with PBS showed no detectable levels anti-JlpA fecal IgA response (mean+2 standard deviations≦1:4). Based on this cut off, 100% of the animals receiving 100 μg of vaccine alone or vaccine doses tested (5 μg, 25 μg, 100 μg) with the adjuvant were categorized as responders for IgA. Five of 6 animals (83%; sample was unavailable for one animal) receiving 25 μg showed detectable levels of JlpA-specific IgA in stool extracts. 
       Protective Efficacy of JlpA 
       [0027]    Following the challenge with  C. jejuni  81-176, animals were observed for the development of sickness. The illness index and calculated JlpA efficacy of different vaccine formulations are presented Table 3. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 JlpA efficacy in mice challenged with  C. jejuni  81-176 
               
             
          
           
               
                   
                 Recombinant 
                   
                 Illness index  
                   
               
               
                 Group 
                 JlpA (μg) 
                 LTR192G (μg) 
                 (mean ± sd) 
                 Efficacy ((%) 
               
               
                   
               
             
          
           
               
                 1 
                 0 
                 − 
                 1.21 ± 0.30 
                 n/a 
               
               
                 2 
                 5 
                 − 
                 nd 
                   
               
               
                 3 
                 5 
                 + 
                 1.23 ± 0.31 
                 −6.8 
               
               
                 4 
                 25 
                 − 
                 1.01 ± 0.24 
                 16.1 
               
               
                 5 
                 25 
                 + 
                 0.78 ± 0.21 
                 35.4 
               
               
                 6 
                 100 
                 − 
                 0.77 ± 0.54 
                 35.6 
               
               
                 7 
                 100 
                 + 
                 0.41 ± 0.25 
                 66.3 
               
               
                   
               
             
          
         
       
     
         [0028]    Twenty four hours following each vaccination, animals receiving 25 μg or higher dose of vaccine with the adjuvant showed a mild transient signs of ruffled fur, which lasted for &lt;24 hours. No apparent signs of vaccine associated side effects were seen among animals receiving PBS or vaccine alone. Animals immunized with 100 μg of the vaccine with the adjuvant showed 66.3% protection. To achieve a moderate (approximately 35%) protection, a 100 μg dose alone or 25 μg with the adjuvant was required. Other doses showed no protection against illness in mice. 
         [0029]    These studies illustrate the utility of the JlpA construct of  C. jejuni  81-176 as components, alone or in combination with other moieties, as vaccines against  Campylobacter . Accordingly, the inventive constructs can be used in methods for induction of protective anti- Campylobacter  immunity 
       Prophetic Example 
     Induction of the Anti- C. jejuni  Mediated Response in Human 
       [0030]    A prophetic method for the induction of the anti- C. jejuni  mediated response in humans contains the following steps:
       a. administration of immunogen comprising JlpA, or immunogenic fragments thereof, with or without a tag, such as histidine. If a boosting dose or doses are to be given this first administration of immunogen is a priming dose. The immunogen can be derived from isolated native polypeptide or recombinantly produced JlpA, or JlpA fragments. The immunogen can be administered orally, nasally, subcutaneously, intradermally, transdermally, transcutaneously intramuscularly, or rectally. The range of a unit dose of immunogen is 25 μg to 1 mg of immunogen. The immunogen is administered in any number of aqueous buffered solutions with or without carrier protein or adjuvant. The adjuvant can be any number of potential adjuvants, including but not limited to LTR 192G, Aluminum hydroxide, RC529E, QS21, E294, oligodeoxynucleotides (ODN), CpG-containing oligodeoxynucleotides, aluminum phosphate, MPL® (GlaxoSmithKline, Middlesex, UK) or combinations of these or other potential adjuvants.   b. The inventive method also contemplates immunization with or without administration of subsequent boosting. The boosting step comprises the administration of, subsequent to a priming dose, 1 to 4 boosting doses with a unit dose range of 50 μg to 1 mg of immunogen in buffered aqueous solutions.       
 
         [0033]    Alternative embodiments of the inventive method include inserting nucleotide constructs encoding JlpA, or fragments thereof, into an expression system capable of expression in mammalian subjects. In this embodiment, the expression system can be a plasmid, viral or DNA expression vector. Another alternative embodiment of the inventive method is to induce immunity by administering a live attenuated carrier strain of bacteria transformed with a suitable viral or DNA expression system that contains one or more nucleic acid sequences encoding one or more of the polypeptides JlpA, or fragments thereof. The expression system contemplated is capable of expressing in the selected bacteria carrier. 
         [0034]    Having described the invention, one skilled in the art will appreciate in the appended claims that many modifications and variations of the present invention are possible in light of the above teaching. It is therefore, to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described. 
       REFERENCES 
       [0000]    
       
         Fry, B. N., Feng, S., Chen, Y., Newell, D. G., Coloe, P. J., and Korolik, V. (2000) The galE gene of  Campylobacter jejuni  is involved in lipopolysaccharide synthesis and virulence. Infect Immun 68: 2594-2601. 
         Jin, S., Joe, A., Lynett, J., Hani, E., Sherman, P., and Chan, V. L. (2001) JlpA, a novel surface-exposed lipoprotein specific to  Campylobacter jejuni , mediates adherence to host epithelial cells. Mol Microbiol 39: 1225-1236. 
         Jin S., Song Y. C., Emili A., Sherman P. M. and Chan V L (2003) JlpA of  Campylobacter jejuni  interacts with surface-exposed heat shock protein 90α and triggers signalling pathways leading to the activation of NF-κB and p38 MAP kinase in epithelial cells. 
         Konkel, M. E., Garvis, S. G., Tipton, S. L., Anderson, D. E. Cieplak, W., Jr (1997) Identification and molecular cloning of a gene encoding a fibronectin-binding protein (CadF) from  Campylobacter jejuni . Mol Microbiol 24: 953-963. 
         Moser, I., Schroeder, W., and Salnikow, J. (1997)  Campylobacter jejuni  major outer membrane protein and a 59-kDa protein are involved in binding to fibronectin and INT 407 cell membranes. FEMS Microbiol Lett 157: 233-238. 
         Pei, Z., Burucoa, C., Grignon, B., Baqar, S., Huang, X., Kopecko, J., et al. (1998) Mutation in the pel1A locus of  Campylobacter jejuni  reduces interactions with epithelial cells and intestinal colonization of mice. Infect Immun 66: 938-946. 
         Szymanski, C. M., and Armstrong, G. D. (1996) Interactions between  Campylobacter jejuni  and lipids. Infect Immun 64: 3467-3474. 
         Yao, R., Burr, D. H., Doig, P., Trust, T. J., Niu, H., and Guerry, P. (1994) Isolation of motile and non-motile insertional mutants of  Campylobacter jejuni : the role of motility in adherence and invasion of eukaryotic cells. Mol Microbiol 14: 883-893.