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Campylobacter jejuni

Table of Contents
  1. General Information
    1. NCBI Taxonomy ID
    2. Disease
    3. Introduction
    4. Microbial Pathogenesis
    5. Host Ranges and Animal Models
    6. Host Protective Immunity
  2. Vaccine Related Pathogen Genes
    1. CadF (Protective antigen)
    2. CjaA from C. jejuni (Protective antigen)
    3. CjaA from C. jejuni RM 1221 (Protective antigen)
    4. FlaA from C. jejuni 81-176 (Protective antigen)
    5. FlaA from C. jejuni NCTC 11168 (Protective antigen)
    6. FlaC (Protective antigen)
    7. FspA1 (Protective antigen)
    8. FspA2 (Protective antigen)
    9. Peb1A (Protective antigen)
    10. PorA (Protective antigen)
  3. Vaccine Related Host Genes
    1. IgA
    2. IgG
  4. Vaccine Information
    1. Avirulent Salmonella vaccine strain carrying C. jejuni cjaA gene
    2. C. jejuni DNA vaccine pcDNA3.1(+)-cadF
    3. C. jejuni DNA vaccine pcDNA3.1(+)-peblA
    4. C. jejuni FlaC protein vaccine
    5. C. jejuni FspA1 protein vaccine
    6. C. jejuni FspA2 protein vaccine
    7. C. jejuni MBP-FlaA protein vaccine
    8. C. jejuni PorA protein vaccine
    9. deltaphoP/Q S. typhimurium strains expressing PEB1-ss
    10. Inactivated C. jejuni whole-cell (CWC)
    11. MBP fused on Campylobacter FlaA (MBP-FlaA)
  5. References
I. General Information
1. NCBI Taxonomy ID:
197
2. Disease:
Campylobacterosis
3. Introduction
Campylobacter jejuni is a small gram-negative, microaerophilic, motile, curved-to-spiral rod that has a single polar flagellum. It can be found in the intestinal tract and oral cavity (Smibert, 1978). C. jejuni is among the most frequent causes of bacterial diarrhea worldwide and an important cause of diarrhea in travellers and deployed military personnel. It is also the infectious agent most often associated with Guillain-Barre syndrome (GBS), a postinfectin polyneuropathy, and is increasingly identified as a cause of bacteremia and a variety of systemic and localized infections in immunocompromised hosts. The bacteria is characterized by a lack of understanding of the basic virulence mechanisms and by its antigenic complexity. The LPS cores of many serotypes have been shown to contain structures which resemble human gangliosides and have been implicated in the development of autoantibodies leading to GBS, although the specific structure or structures which enable a given campylobacter strain to cause GBS are not clear (Lee et al., 1999; Pawelec et al., 2000).
4. Microbial Pathogenesis
Most Campylobacter infections are mild, self-limiting diarrheal illnesses, but severe infections do occur. The primary risk factors associated with Campylobacter infection are consuming and handling foods of animal origins, especially poultry products. In addition to epidemiological evidence, microbiological evidence supports poultry as the primary source of human Campylobacter infection (Nelson et al., 2007). The majority of Campylobacter-associated infections are thought to be sporadic cases of food poisoning; contaminated drinking water, including that from public water supplies, is the vehicle of large Campylobacter-associated outbreaks. Although Campylobacter spp. are widely distributed in the environment, the epidemiology of many cases of Campylobacter-associated infections remains unclear (Abulreesh et al., 2006). After incubation of approximately three days, abrupt cramping pain in the abdomen is followed by diarrhea. The mechanism of diarrhea induction is a complex and multifactorial process. The diarrheal stage lasts a few days. Complications are rare, but infection may be followed by the development of reactive arthritis or GBS.

Flagella-mediated motility has been shown to be necessary for Campylobacter to colonize the intestinal tract, and experimental infections in humans and animals, and in vitro analyses of adherence and invasion in cultured human cells have demonstrated that cell invasiveness is necessary in Campylobacter-induced inflammatory diarrhea. Several bacterial components have been shown to have adhesive properties (LPS, flagella, fimbrial filaments, surface-exposed proteins), and a direct role of toxins including of cytolethal distending toxin (CDT) in disease remains to be demonstrated. Host factors are of importance in the pathogenesis of GBS following a Campylobacter infection. It is now clear that specific bacterial genes are crucial for the induction of anti-ganglioside antibodies (Engberg, 2006).
5. Host Ranges and Animal Models
The primary risk factors associated with Campylobacter infection are consuming and handling foods of animal origins, especially poultry products. In addition to epidemiological evidence, microbiological evidence supports poultry as the primary source of human Campylobacter infection (Nelson et al., 2007).
6. Host Protective Immunity
Anti-flagellar serum antibody titres of the dams did not correlate with protection of their young (Dolby et al., 1986). There was a good correlation between high campylobacter-specific IgG response and bactericidal activity. The degree of protection conferred by vaccinated dams on infant mice against colonization by Campylobacter jejuni depended on the bacterial strain, preparation, and route of administration of the vaccine. In some instances of homologous protection, serum bactericidal titres correlated well with protection. However, boiled C. jejuni vaccine, which was non-protective, also elicited a strong bactericidal antibody response. Conversely, bactericidal activity could not be demonstrated against strains capable of cross-protection. (Abimiku et al., 1989).
1. CadF
  • Gene Name : CadF
  • Sequence Strain (Species/Organism) : Campylobacter jejuni
  • VO ID : VO_0011044
  • NCBI Protein GI : 4704601
  • Other Database IDs : CDD:174558
    CDD:143586
  • Taxonomy ID : 197
  • Gene Strand (Orientation) : ?
  • Protein Name : fibronectin binding protein
  • Protein pI : 6.19
  • Protein Weight : 34606.79
  • Protein Length : 287
  • Protein Note : Surface antigen; cl01155
  • Protein Sequence : Show Sequence
    >gi|4704601|gb|AAD28174.1|AF104303_1 fibronectin binding protein [Campylobacter jejuni]
    ASVLFGRDNNVKFEITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLDQLEFGLEHYSDVKYTNTNKT
    TDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDNKSGGFGHYGAGVKFRLSDSLALRLETRDQI
    NFNHANHNWVSTLGISFGFGGKKEKAVEEVADTRPAPQAKCPVPSREGALLDENGCEKTISLEGHFGFDK
    TTINPTFQEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIKTVG
    YGQDNPR
  • Molecule Role : Protective antigen
  • Molecule Role Annotation : Chitosan-DNA vaccines were prepared by embedding pcDNA3.1(+)-cadF and pcDNA3.1(+)-peblA with chitosan respectively. The mice immunized with chitosan-DNA vaccines have generated high levels of IgA and IgG from the sera and IgA from the intestinal secretions and the P/N value went up to 20.58, 30.13 and 6.87 respectively. The chitosan-DNA vaccines induced strongest level of protection in BALB/c mice against challenge with C. jejuni HS:19 strain and the protective efficacies was 93.70 (Zheng et al., 2007).
  • Related Vaccine(s): C. jejuni DNA vaccine pcDNA3.1(+)-cadF
2. CjaA from C. jejuni
  • Gene Name : CjaA from C. jejuni
  • Sequence Strain (Species/Organism) : Campylobacter jejuni
  • VO ID : VO_0010956
  • NCBI Protein GI : 1813949
  • Other Database IDs : CDD:128376
    CDD:29040
    GOA:P94643
    HSSP: Q9PNV7
    InterPro: IPR001638
    UniProtKB/TrEMBL: P94643
  • Taxonomy ID : 197
  • Gene Strand (Orientation) : ?
  • Protein pI : 5.79
  • Protein Weight : 28587.37
  • Protein Length : 279
  • Protein Note : Bacterial periplasmic substrate-binding proteins; smart00062
  • Protein Sequence : Show Sequence
    >gi|1813949|emb|CAA71822.1| cjaA [Campylobacter jejuni]
    MKKMLLSIFTTFVAVFLAACGGNSDSGASNSLERIKQDGVVRIGVFGDKPPFGYVDEKGVNQGYDIVLAK
    RIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQTPERAEQVDFCLPYMKVALGVAVPQDSNI
    SSIEDLKDKTLLLNKGTTADAYFTKEYPDIKTLKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEF
    KMAIKELGNKDVIAPAVKKGDKELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGGKI
  • Molecule Role : Protective antigen
  • Molecule Role Annotation : A total of 840 Light Sussex chickens were used to evaluate a Salmonella enterica serovar Typhimurium DeltaaroA vaccine expressing the C. jejuni amino acid binding protein CjaA. Chickens were given the vaccine at 1-day-old and two weeks later by oral gavage, then challenged after a further two weeks with C. jejuni. Across six biological replicates, statistically significant reductions in caecal C. jejuni of c. 1.4log(10) colony-forming units/g were observed at three and four weeks post-challenge relative to age-matched unvaccinated birds. Protection was associated with the induction of CjaA-specific serum IgY and biliary IgA (Buckley et al., 2010).
  • Related Vaccine(s): Avirulent Salmonella vaccine strain carrying C. jejuni cjaA gene
3. CjaA from C. jejuni RM 1221
  • Gene Name : CjaA from C. jejuni RM 1221
  • Sequence Strain (Species/Organism) : Campylobacter jejuni RM1221
  • VO ID : VO_0010955
  • NCBI Gene ID : 3231573
  • NCBI Protein GI : 57237809
  • Locus Tag : CJE1064
  • Genbank Accession : NC_003912.7
  • Protein Accession : YP_179057.1
  • Taxonomy ID : 197
  • Gene Starting Position : 993663
  • Gene Ending Position : 994502
  • Gene Strand (Orientation) : -
  • Protein Name : surface antigen, CjaA
  • Protein pI : 6.53
  • Protein Weight : 30966.3
  • Protein Length : 279
  • Protein Note : identified by similarity to GB:AAD26123.1; similarity to GB:CAA71822.1; match to protein family HMM PF00497
  • DNA Sequence : Show Sequence
    >GeneID|3231573 [Campylobacter jejuni RM1221 ] 993663..994502
    ttaaatttttccaccttcaatcactacatcatcagccttaacatcatctccaaaatgagcttttaaagtt
    tcatcataagccttgtgaaaaaactgctcttggcctagtttaatgatcaaattatcgataaattctttaa
    gttctttatcgccttttttaaccgctggtgcgataacatctttgttacctaactctttgatacccatttt
    aaaatcaggatgatctttcacccaagcaaaaagcaaggtattatcatgacttaaagcatcgcctctttta
    tccatcaaagcggcaaaggtttcggtattttgatcatattttaaagttttaatattaggataattttgcg
    taaaataagcatctgctgttgtacctttgtttaaaagcaaggttttatcttttaaatcttctacgctagt
    tatattactatcctttggtacagctacgcctaaagctaccttcatataaggcaagcaaaaatcaacctgt
    tctgccctttgcggagtttgagtaaaattagccaaaataatatctactttatttgattttaaaaactcaa
    ccctatttgcagcttcaacaagaacaaattgcaccttattttcatcgccaaaaagttcttttgctatgcg
    tttagctaaagctatatcatagccttgattgtttcctttttcatctacataaccaaaaggtggtttatcg
    ccaaataccccaatcctaacaactccattttgcttgatcttatcaagagaatttaaagttttagagtcag
    aatttcctccacaagcagccaatactactgcaacaaaggtcgttaaaacacttagaagtatttttttcat
  • Protein Sequence : Show Sequence
    >gi|57237809|ref|YP_179057.1| surface antigen, CjaA [Campylobacter jejuni RM1221 ]
    MKKILLSVLTTFVAVVLAACGGNSDSKTLNSLDKIKQNGVVRIGVFGDKPPFGYVDEKGNNQGYDIALAK
    RIAKELFGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQTPQRAEQVDFCLPYMKVALGVAVPKDSNI
    TSVEDLKDKTLLLNKGTTADAYFTQNYPNIKTLKYDQNTETFAALMDKRGDALSHDNTLLFAWVKDHPDF
    KMGIKELGNKDVIAPAVKKGDKELKEFIDNLIIKLGQEQFFHKAYDETLKAHFGDDVKADDVVIEGGKI
  • Molecule Role : Protective antigen
  • Related Vaccine(s): Avirulent Salmonella vaccine strain carrying C. jejuni cjaA gene
4. FlaA from C. jejuni 81-176
  • Gene Name : FlaA from C. jejuni 81-176
  • Sequence Strain (Species/Organism) : Campylobacter jejuni subsp. jejuni 81-176
  • VO ID : VO_0011046
  • NCBI Gene ID : 4682159
  • NCBI Protein GI : 121612545
  • Locus Tag : CJJ81176_1339
  • Genbank Accession : AY102622
  • Protein Accession : YP_001000997
  • Taxonomy ID : 354242
  • Gene Starting Position : 1255199
  • Gene Ending Position : 1256929
  • Gene Strand (Orientation) : -
  • Protein Name : flagellin
  • Protein pI : 5.37
  • Protein Weight : 56366.68
  • Protein Length : 576
  • Protein Note : FlaA; structural flagella protein; in Helicobacter the flagella are composed of flagellin A and flagellin B; the amounts of each seem to be controlled by environmental conditions
  • DNA Sequence : Show Sequence
    >gi|121612099:1255199-1256929 Campylobacter jejuni subsp. jejuni 81-176, complete genome
    ACTATTGTAATAATCTTAAAACATTTTGCTGACTAGAATTTGCTTGAGCCATTGCATAAGAACCACTTTG
    GGCTAAGATGTTAGCTTTAGAGTAGTTTGCACTCTCACTTGCAAAGTCTACATCTCTGATTTGCGATTCT
    GCTGCTTTAACATTAACTTGAGTTACAGTAATGTTATTTATAGTTGATGTAACTTGATTTTGTATAGAAC
    CAATATCTGCTCTGATTTGATCTAGATTTGTTATAGCAGTTTCTGCTATATCCATAACCGCCATTGCACC
    TTTAAGAGTGGTTACACCTGCAGTCTCATCAGTTGTATTAGCAGCAGTAGTTTTAAGGGCTGCAAATTGA
    GAATTTCCTGAGCCAGAAGAAAATCCTGAACCTGCTGAAACAACATAAGTATTGCTCATTGAAGCCGCAC
    TTGAAATAATTTGTATTCCTTGACTCAATCCAACAGATAAATTTTTACCACTACCCACAGAAAATCCTGA
    ACCTCTAGAAAATCCTGAACCTTGTGCACTCATAAATGCAGAAATTGAACTTACATTTTGAGTAAAAACA
    AATTTTCCACCACCTTTATAAGAATTAAATCCCATAGCATCGGCATTGGTTGCTGAAATTTGACCTTTTG
    ATTCTCTTAAAGACACTGAAGATTGAGAAATCATATCTGTTGTACCCATACCTATAGCACTAAGATTGGT
    TCCACTTATATTGATATCTCTACCATCATTTTTAACTAAAGATAATCGCCCATAGTTTTCTTTTTGATTT
    GCCAAAATACCAGAACCAACACCTATATCTCCAGTAATTTTAATACCCCTGCCATCAGCCGATGTAAGAA
    CAAGCTTGCCGTTTTCATCTTTAGAAGCTTGAACTCCTGTGGTATCTTTAACCGCATTGATAGCTGAAAT
    CAAAGAGCCGTTACCATCTCCGTCTTTGTATTCAATTTTTCCTATAGTTACTCCATTAATGGCAAATTCT
    TGAGAAGTAGTTCCTTCTTTTATAGCATAAACGCCAGTTGTTTTTACATCGTAAGTTGCGCGAACTCCTG
    TTTTATCAGCGCTTTTATTGATCTCTTCAGCCAAAGCTCCAAGTCCTGTTCCAACTGAAGTTGAAATCAC
    AACATTATCAAATTTAAAATCTTCTATACCATTGTAGTTTTTAATAGTAAGACCAACCACACCTGAAGTA
    AAACTTTGAGCACCGGTTTCAAATCTTGTAACACCGATTTTAGAAGATTGAGTAGCACCGATAGTTGCTT
    TCACAGTTTGGTTTGAACTTGCGCCGATTTGGAATTCTTGATTGGTAAAATTTCCACTTAAAAGTTGCTT
    ACCATTAAATGAAGTAGTATTTGCGATATTATCAAGCTCTTCCATTAATTTATTAATATCTGCTTGAAGC
    ATAGTTCTTGTTTTTAAACTTTGTCCATCTTGAGCTGCTTGAGTAGCTTTAGTCTTGATAGTATCAAGAA
    TTTTTAATTGCTCATCCATAGCTTTATCTGCGGTTTGTAAGATACCTAAAGCATCATTACCATTTGATAT
    AGCTTGACCTAAAGTATTTGCTTGAGATCTTAAGCTATCTGCTATCGCCATCCCTGAAGCATCATCTGCT
    GCTGAGTTAATTCTAAGACCTGAACTAAGTCTGCTTAAAGAAGCATCTAAACTTTTAGCATTAAGATCAG
    AGTTTGCTTTTGCATTCAATGCTGCAACATTTGTGTTAATACGAAATCCCA
  • Protein Sequence : Show Sequence
    >gi|121612545|ref|YP_001000997.1| flagellin [Campylobacter jejuni subsp. jejuni 81-176]
    MGFRINTNVAALNAKANSDLNAKSLDASLSRLSSGLRINSAADDASGMAIADSLRSQANTLGQAISNGND
    ALGILQTADKAMDEQLKILDTIKTKATQAAQDGQSLKTRTMLQADINKLMEELDNIANTTSFNGKQLLSG
    NFTNQEFQIGASSNQTVKATIGATQSSKIGVTRFETGAQSFTSGVVGLTIKNYNGIEDFKFDNVVISTSV
    GTGLGALAEEINKSADKTGVRATYDVKTTGVYAIKEGTTSQEFAINGVTIGKIEYKDGDGNGSLISAINA
    VKDTTGVQASKDENGKLVLTSADGRGIKITGDIGVGSGILANQKENYGRLSLVKNDGRDINISGTNLSAI
    GMGTTDMISQSSVSLRESKGQISATNADAMGFNSYKGGGKFVFTQNVSSISAFMSAQGSGFSRGSGFSVG
    SGKNLSVGLSQGIQIISSAASMSNTYVVSAGSGFSSGSGNSQFAALKTTAANTTDETAGVTTLKGAMAVM
    DIAETAITNLDQIRADIGSIQNQVTSTINNITVTQVNVKAAESQIRDVDFASESANYSKANILAQSGSYA
    MAQANSSQQNVLRLLQ
  • Molecule Role : Protective antigen
  • Molecule Role Annotation : Mice were immunized intranasally with two doses of 3 to 50 microgram of MBP-FlaA. The protective efficacies of a 50 microgram of MBP-FlaA plus LT(R192G) dose against disease symptoms and intestinal colonization were 81.1 and 84%, respectively. When mice which had been immunized with 50 microgram of MBP-FlaA plus LT(R192G) intranasally were challenged orally with 8 x 10(10), 8 x 10(9), or 8 x 10(8) cells of strain 81-176, the protective efficacies against intestinal colonization at 7 days postinfection were 71.4, 71.4, and 100%, respectively (Lee et al., 1999).
  • Related Vaccine(s): C. jejuni MBP-FlaA protein vaccine
5. FlaA from C. jejuni NCTC 11168
  • Gene Name : FlaA from C. jejuni NCTC 11168
  • Sequence Strain (Species/Organism) : Campylobacter jejuni subsp. jejuni NCTC 11168
  • VO ID : VO_0010942
  • NCBI Gene ID : 905631
  • NCBI Protein GI : 15792662
  • Locus Tag : Cj1339c
  • Genbank Accession : NC_002163.1
  • Protein Accession : NP_282485.1
  • Other Database IDs : BioCyc: CJEJ192222:CJ1339C-MONOMER
    EMBL: AL139078
    InterPro: IPR001029
    InterPro: IPR001492
    InterPro: IPR010810
    KEGG: cje:Cj1339c
    Pfam: PF00669
    Pfam: PF00700
    Pfam: PF07196
    PIR: H81277
    PRINTS: PR00207
    ProDom: PD000316
    UniProt: P56963
  • Taxonomy ID : 197
  • Gene Starting Position : 1269232
  • Gene Ending Position : 1270950
  • Gene Strand (Orientation) : -
  • Protein Name : flagellin
  • Protein pI : 5.36
  • Protein Weight : 59038.1
  • Protein Length : 572
  • Protein Note : flaA, flagellin A, len: 572 aa; 80.7% identity to FLAA_CAMJE flagellin A (575 aa), fasta scores; opt: 2816 z-score: 2717.6 E(): 0, 80.7% identity in 575 aa overlap. 48.3% identity to HP0601. Also similar to Cj1338c (flaA), Cj0720c flaC (24.5% identity in 220 aa overlap), and Cj0887c flaD (20.2% identity in 560 aa overlap). Contains fam match to entry PF00669 Flagellin_N, Bacterial flagellin N-terminus, and Pfam match to entry PF00700 Flagellin_C, Bacterial flagellin C-terminus. The flaA and flaB genes have a GC content of 37% compared to the overall genome GC content of 30.5%
  • Protein Annotation : FUNCTION: Flagellin is the subunit protein which polymerizes to form the filaments of bacterial flagella (UniProt: P56963)

    SUBUNIT: Heteropolymer of flaA and flaB (UniProt: P56963)

    SIMILARITY: Belongs to the bacterial flagellin family (UniProt: P56963)
  • DNA Sequence : Show Sequence
    >GeneID|905631 [Campylobacter jejuni subsp. jejuni NCTC 11168 ] 1269232..1270950
    ctactgtagtaatcttaaaacattttgttgaacagaattagcctgtgccatggcataagagccgctttga
    gctaagatatttgctttagagtagtttgcactctcggctgcaaagtctacatcacggatttgcgattctg
    ctgctttaacgtttacttgagttacggtgatgttgtttatagttgatgtaacttgattttgtaccgaacc
    aatgtcggctctgatttgatcaagatttgttatagctgtttcagctatatccatcacagccatagcgcct
    ttaagtgtggtaacacctgctgtttcatcttttactccaaaagcagttgttttcatagttgcaaactgag
    aaagtgttgaaccacttgaaaaacctgagcctgcagaaacattatataccgtagaaagttgcgaagcagc
    tgaaatagctatagcgtttgcaaaacctgtggaataatttttaccgctacctacagaataacctgaacct
    gaagaaaatccacttcctgcgctactcatataggcactaacagaagaataaccacctaacacaactcctt
    tgtttgcagaaccaaatcccatagcatcagcgatattagcatcaatttgtccttttgactctcttaaaga
    aacagaagcttgagagataaattgagttgcaccaaaacctgcagaagaaagattgctaccgctgattaaa
    atatctttaccatcatttttaactaaagacaagcggccatagttttctttcatatcagcattgataaagg
    cacctccacctatattaccatcgattttaatccctctaccttctcttgaagtaagtaaaagttgtccatt
    agcatcgatcgaagcttcaactccagtggtatctttaaccgaattgattgcagcaactaaggctccatta
    gcatcaccatctttgtaatctactttaccgatttttaccccattgatagcaaaagtatctgaagtagctc
    ctgctctaactgcagctatacctctagtttctactgtaaaagtagctctaacacctgttttatcagcatt
    tttattgatctcatctgctaaagctccaagtcctgttccaactgaagttgaaatcacaactttttgaaac
    tgaaaatcatctataccattgtaatttttaagagtaaattgtacttcgccactagttgaaattcttcctc
    ctgtttcaaagcgtgttaaacctatcttagaagattgagttgctcctatagtagcttttacagtttgatt
    tgaacttgcaccgatttgaaattcttgattgataaaattcccacttaaaagttgtttaccgttaaatgaa
    gtagtatttgcaatattgtcaagttcttccattaaacggttgatatctgcttgaagcatggttcttgttt
    ttaaactttgtccatcttgagccgcttgagttgccttagttttgattgtatctaagattttaagttgctc
    atccatagccttatcagcagtttgtaagatacctaaagcatcattaccattagatatagcttgacctaaa
    gtattagcttgagatcttaaactatctgctatcgccatccctgaagcatcatctgctgcggagttgattc
    taagacctgaactaagtctgcttaaagaagcatctaaacttttactatttaaatcagcgtttgcttttgc
    atttaaagctgcaacattggtgttaatacgaaatcccat
  • Protein Sequence : Show Sequence
    >gi|15792662|ref|NP_282485.1| flagellin [Campylobacter jejuni subsp. jejuni NCTC 11168 ]
    MGFRINTNVAALNAKANADLNSKSLDASLSRLSSGLRINSAADDASGMAIADSLRSQANTLGQAISNGND
    ALGILQTADKAMDEQLKILDTIKTKATQAAQDGQSLKTRTMLQADINRLMEELDNIANTTSFNGKQLLSG
    NFINQEFQIGASSNQTVKATIGATQSSKIGLTRFETGGRISTSGEVQFTLKNYNGIDDFQFQKVVISTSV
    GTGLGALADEINKNADKTGVRATFTVETRGIAAVRAGATSDTFAINGVKIGKVDYKDGDANGALVAAINS
    VKDTTGVEASIDANGQLLLTSREGRGIKIDGNIGGGAFINADMKENYGRLSLVKNDGKDILISGSNLSSA
    GFGATQFISQASVSLRESKGQIDANIADAMGFGSANKGVVLGGYSSVSAYMSSAGSGFSSGSGYSVGSGK
    NYSTGFANAIAISAASQLSTVYNVSAGSGFSSGSTLSQFATMKTTAFGVKDETAGVTTLKGAMAVMDIAE
    TAITNLDQIRADIGSVQNQVTSTINNITVTQVNVKAAESQIRDVDFAAESANYSKANILAQSGSYAMAQA
    NSVQQNVLRLLQ
  • Molecule Role : Protective antigen
  • Related Vaccine(s): MBP fused on Campylobacter FlaA (MBP-FlaA)
6. FlaC
  • Gene Name : FlaC
  • Sequence Strain (Species/Organism) : Campylobacter jejuni subsp. jejuni 81-176
  • VO ID : VO_0011048
  • NCBI Gene ID : 4682885
  • NCBI Protein GI : 121612344
  • Locus Tag : CJJ81176_0743
  • Genbank Accession : CP000538
  • Protein Accession : YP_001000416
  • Taxonomy ID : 354242
  • Gene Starting Position : 671962
  • Gene Ending Position : 672711
  • Gene Strand (Orientation) : -
  • Protein Name : flagellin subunit protein FlaC
  • Protein pI : 4.32
  • Protein Weight : 25481.71
  • Protein Length : 249
  • Protein Note : identified by match to protein family HMM PF00669
  • DNA Sequence : Show Sequence
    >gi|121612099:671962-672711 Campylobacter jejuni subsp. jejuni 81-176, complete genome
    ATTATTGTAATAAATTAGCAATTTTGCTTTGAAGCTGCATGTTGGATTGCGCAGCAACAAAAGCAGCAGC
    ATTTTCTTTTAGATAATTGGCATTAAAATCATTAACATTTTTTGCCATATCATTATTTAGTAAATTATTT
    TCAGCTGCTTTTGAGTTGATGCTATTTTGAACACTTGCATTAATATTTGATGTAATGGCATTGATACCTG
    AACCTATTTCACTTCTTAAACTTCCAAGTTGATCCATAAAATTTGTAATACTATCTTGATTATCTATGCT
    TAATCCACCTGTTGCTAATGGATTTAAATTTGTAGTTTCAGTTCCGCTACCTACTACAAAATTCATAGTT
    TGAAAGACATTTTTTCCATTATAAGTTGCATTATTAAAAGAATTATTGATGGATTCTTGTATGCGTGTTG
    CTTCTGTTCTTAGCATTCCTTTTTGAGAATCATTAAGTGCAGCATTGTTCATTTTTACTGAAAGTTCATT
    AAGTCTATCTGCGCTTTGAGAGATATTGGTAAGGCTAGCATCTGCAATTTGTAAAACCCCTATAGCATCA
    TAAGCATTTGCGACACCTTGATCTATAGTGCTTGATTGAGATCTTAAAGAATCAGCAATAGCTAAATTAG
    CACTATCAACTCCACTTATTGCGCGAACAGCTGCAATATTTTCTAAAGCTTTATCGCTAGCTTTTTGTGC
    ATTATTTAAATAATAATTTTGTTGCATCATAGTTGCATCAGAGATCATCA
  • Protein Sequence : Show Sequence
    >gi|121612344|ref|YP_001000416.1| flagellin subunit protein FlaC [Campylobacter jejuni subsp. jejuni 81-176]
    MMISDATMMQQNYYLNNAQKASDKALENIAAVRAISGVDSANLAIADSLRSQSSTIDQGVANAYDAIGVL
    QIADASLTNISQSADRLNELSVKMNNAALNDSQKGMLRTEATRIQESINNSFNNATYNGKNVFQTMNFVV
    GSGTETTNLNPLATGGLSIDNQDSITNFMDQLGSLRSEIGSGINAITSNINASVQNSINSKAAENNLLNN
    DMAKNVNDFNANYLKENAAAFVAAQSNMQLQSKIANLLQ
  • Molecule Role : Protective antigen
  • Molecule Role Annotation : Immunogenicity and protective efficacy of three Campylobacter jejuni flagellum-secreted proteins, FlaC, FspA1, and FspA2, were compared by use of a mouse model. All three proteins were immunogenic, FlaC provided an 18% protection against disease from C. jejuni 81-176 (Baqar et al., 2008).
  • Related Vaccine(s): C. jejuni FlaC protein vaccine
7. FspA1
  • Gene Name : FspA1
  • Sequence Strain (Species/Organism) : Campylobacter jejuni
  • VO ID : VO_0011047
  • NCBI Protein GI : 116292649
  • Taxonomy ID : 197
  • Gene Strand (Orientation) : -
  • Protein Name : FspA1
  • Protein Length : 142
  • Protein Note : sigma 28 regulated; Campylobacter jejuni-specific virulence factor that is secreted through the flagella filament
  • Protein Sequence : Show Sequence
    >gi|116292649|gb|ABJ97640.1| FspA1 [Campylobacter jejuni]
    MQINNSLNSLSQYVKVNSNEENQNSKNQEQNALAQDPAVEVNISKEAKEKSNASNQNNSQAPAQALNAQN
    NTQQDSSSNSEDKLTELTQKLAEIQAKIVELTAKMSKANEDQIKSIESQIATLNAQASTIQAQIQELQSQ
    QA
  • Molecule Role : Protective antigen
  • Molecule Role Annotation : Immunogenicity and protective efficacy of three Campylobacter jejuni flagellum-secreted proteins, FlaC, FspA1, and FspA2, were compared by use of a mouse model. Immunization with FspA1 resulted in 57.8% protection without adjuvant or 63.8% protection with adjuvant against homologous challenge with 81-176 (Baqar et al., 2008).
  • Related Vaccine(s): C. jejuni FspA1 protein vaccine
8. FspA2
  • Gene Name : FspA2
  • Sequence Strain (Species/Organism) : Campylobacter jejuni
  • VO ID : VO_0011049
  • NCBI Protein GI : 116292677
  • Taxonomy ID : 197
  • Gene Strand (Orientation) : ?
  • Protein Name : FspA2
  • Protein Length : 142
  • Protein Note : sigma 28 regulated; Campylobacter jejuni-specific virulence factor that is secreted through the flagella filament
  • Protein Sequence : Show Sequence
    >gi|116292677|gb|ABJ97654.1| FspA2 [Campylobacter jejuni]
    MKIDTLTKNFSNYQTQINKNNDLASNVHSDNVVKIQISDEARSLSQANSKNEKEYEIKVSQEKIENHKDQ
    NSIQSSKGGGNPALEALIAKLAEILAKIAELTQKMTNANEQMKTTFQKQIDVLISQADVIQAQIQELQSQ
    QA
  • Molecule Role : Protective antigen
  • Molecule Role Annotation : Immunogenicity and protective efficacy of three Campylobacter jejuni flagellum-secreted proteins, FlaC, FspA1, and FspA2, were compared by use of a mouse model. Immunization with FspA2 provided 38.4% (without adjuvant) or 47.2% (with adjuvant) protection against disease from homologous challenge with CG8486 (Baqar et al., 2008).
  • Related Vaccine(s): C. jejuni FspA2 protein vaccine
9. Peb1A
  • Gene Name : Peb1A
  • Sequence Strain (Species/Organism) : Campylobacter jejuni
  • VO ID : VO_0011045
  • NCBI Protein GI : 112360246
  • 3D structure: PDB ID : 2V25
  • Other Database IDs : CDD:171343
    CDD:29040
    GOA:Q0P9X8
    InterPro: IPR015683
    PDB: 2V25
    UniProtKB/UniProt: Q0P9X8
  • Taxonomy ID : 192222
  • Gene Strand (Orientation) : ?
  • Protein Name : aspartate/glutamate-binding ABC transporter protein
  • Protein pI : 9.46
  • Protein Weight : 26094.11
  • Protein Length : 259
  • Protein Note : bifunctional adhesin/ABC transporter aspartate/glutamate-binding protein; Reviewed; PRK11917
  • Protein Sequence : Show Sequence
    >gi|112360246|emb|CAL35041.1| aspartate/glutamate-binding ABC transporter protein [Campylobacter jejuni subsp. jejuni NCTC 11168]
    MVFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGEIKGFEVDVAK
    LLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYNFSEPYYQDAIGLLVLKEKKY
    KSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFPDYPSIKAALDAKRVDAFSVDKSILLGYVDD
    KSEILPDSFEPQSYGIVTKKDDPAFAKYVDDFVKEHKNEIDALAKKWGL
  • Molecule Role : Protective antigen
  • Molecule Role Annotation : Chitosan-DNA vaccines were prepared by embedding pcDNA3.1(+)-cadF and pcDNA3.1(+)-peblA with chitosan respectively. The mice immunized with chitosan-DNA vaccines have generated high levels of IgA and IgG from the sera and IgA from the intestinal secretions and the P/N value went up to 20.58, 30.13 and 6.87 respectively. The chitosan-DNA vaccines induced strongest level of protection in BALB/c mice against challenge with C. jejuni HS:19 strain and the protective efficacies was 93.70 (Zheng et al., 2007).
  • Related Vaccine(s): C. jejuni DNA vaccine pcDNA3.1(+)-peblA
10. PorA
  • Gene Name : PorA
  • Sequence Strain (Species/Organism) : Campylobacter jejuni subsp. jejuni NCTC 11168
  • NCBI Gene ID : 905550
  • NCBI Protein GI : 218562871
  • Locus Tag : Cj1259
  • Genbank Accession : AL111168
  • Protein Accession : YP_002344650
  • Taxonomy ID : 192222
  • Gene Starting Position : 1189120
  • Gene Ending Position : 1190394
  • Gene Strand (Orientation) : ?
  • Protein Name : major outer membrane protein
  • Protein pI : 4.49
  • Protein Weight : 42413
  • Protein Length : 424
  • Protein Note : Original (2000) note: Cj1259, porA, major outer membrane protein (MOMP), len: 424 aa; 94.6% identical to TR:AAC82317 (EMBL: U96452) C. jejuni major outer membrane porin (424 aa), and 98.0% identity to MOMP_CAMJE major outer membrane protein (fragments) (399 aa). No hp match. Also simlar in part to Cj1021c (63 aa, 34.5% identity in 55 aa overlap)~Updated (2006) note: Pfam domain PF05538 Campylobacter major outer membrane protein identified within CDS. Further support given to product function. Characterised within Campylobacter jejuni, so putative not added to product function. Functional classification -Membranes, lipoproteins and porins~PMID:7543469, PMID:9163918
  • DNA Sequence : Show Sequence
    >gi|15791399:1189120-1190394 Campylobacter jejuni subsp. jejuni NCTC 11168 chromosome, complete genome
    CATGAAACTAGTTAAACTTAGTTTAGTTGCAGCTCTTGCTGCAGGTGCTTTTTCAGCAGCTAACGCTACT
    CCACTTGAAGAAGCGATCAAAGATGTTGATGTATCAGGTGTATTAAGATACAGATACGATACAGGTAATT
    TTGATAAAAATTTCGTTAACAACTCAAATTTAAACAACAGCAAACAAGATCACAAATATAGAGCACAAGT
    TAACTTCAGTGCTGCTATAGCTGATAACTTCAAAGCTTTTGTTCAATTTGACTATAATGCTGCTGATGGT
    GGTTATGGTGCTAATGGAATAAAAAATGATCAAAAAGGACTTTTTGTTCGTCAATTATACTTAACTTATA
    CAAATGAAGATGTTGCTACAAGTGTAATCGCTGGTAAACAACAATTAAACCTTATCTGGACGGATAACGC
    TATTGATGGTTTAGTTGGCACAGGTGTTAAAGTAGTAAATAACAGCATCGATGGTTTAACTCTAGCTGCT
    TTTGCTGTAGATAGCTTCATGGCTGCAGAGCAAGGTGCAGATTTATTAGAACATAGTAATATTTCAACAA
    CATCAAATCAAGCTCCTTTTAAAGTAGATTCAGTAGGAAATCTTTACGGTGCTGCTGCTGTAGGTTCTTA
    TGATCTTGCTGGTGGACAATTCAACCCACAATTATGGTTAGCTTATTGGGATCAAGTAGCATTCTTCTAT
    GCTGTAGATGCAGCTTATAGTACAACTATCTTTGATGGAATCAACTGGACACTTGAAGGTGCTTACTTAG
    GAAATAGCCTTGATAGCGAACTTGATGATAAAACACACGCTAATGGCAATTTATTTGCTTTAAAAGGTAG
    CATTGAAGTAAATGGTTGGGATGCTAGCCTTGGTGGTTTATACTACGGTGATAAAGAAAAAGCTTCTACA
    GTTGTAATCGAAGATCAAGGTAATCTTGGTTCTTTACTTGCAGGTGAGGAAATTTTCTATACTACTGGTT
    CAAGACTAAATGGTGATACTGGTAGAAATATCTTCGGTTATGTAACTGGTGGATATACTTTCAACGAAAC
    AGTTCGCGTTGGTGCTGACTTCGTATATGGTGGAACAAAAACAGAAGCTGCTAATCATTTAGGTGGTGGT
    AAAAAACTTGAAGCTGTTGCAAGAGTAGATTACAAATACTCTCCAAAACTTAACTTCTCAGCATTCTATT
    CTTATGTGAACCTAGATCAAGGTGTAAACACTAATGAAAGTGCTGATCATAGCACTGTAAGACTTCAAGC
    TCTTTACAAATTCTA
  • Protein Sequence : Show Sequence
    >gi|218562871|ref|YP_002344650.1| major outer membrane protein [Campylobacter jejuni subsp. jejuni NCTC 11168]
    MKLVKLSLVAALAAGAFSAANATPLEEAIKDVDVSGVLRYRYDTGNFDKNFVNNSNLNNSKQDHKYRAQV
    NFSAAIADNFKAFVQFDYNAADGGYGANGIKNDQKGLFVRQLYLTYTNEDVATSVIAGKQQLNLIWTDNA
    IDGLVGTGVKVVNNSIDGLTLAAFAVDSFMAAEQGADLLEHSNISTTSNQAPFKVDSVGNLYGAAAVGSY
    DLAGGQFNPQLWLAYWDQVAFFYAVDAAYSTTIFDGINWTLEGAYLGNSLDSELDDKTHANGNLFALKGS
    IEVNGWDASLGGLYYGDKEKASTVVIEDQGNLGSLLAGEEIFYTTGSRLNGDTGRNIFGYVTGGYTFNET
    VRVGADFVYGGTKTEAANHLGGGKKLEAVARVDYKYSPKLNFSAFYSYVNLDQGVNTNESADHSTVRLQA
    LYKF
  • Molecule Role : Protective antigen
  • Molecule Role Annotation : Adult BALB/c mice were orally immunized with a recombinant glutathione S-transferase (GST) fused to PorA prepared from Campylobacter jejuni C31 (O:6,7) (GST-PorA) combined with a modified heat-labile enterotoxin of Escherichia coli as an adjuvant and later orally challenged with C31 strain or three heterologous strains: 48 (O:19), 75 (O:3), and 111 (O:1,44). The vaccine produced robust antibody responses against both antigens in serum and secretion. Since strain C31 was a poor colonizer, homologous protection could not be studied. The protective efficacies of heterologous strains were 43% (for strain 48, P < 0.001), 29% (for strain 75, P < 0.005), and 42% (for strain 111, P < 0.001) for the 9-day period compared to control mice given phosphate-buffered saline. Thus, PorA provided appreciable protection against colonization with heterologous serotypes (Abimiku et al., 1989).
  • Related Vaccine(s): C. jejuni PorA protein vaccine
1. IgA
2. IgG
  • Gene Name : IgG
  • Sequence Strain (Species/Organism) : Mus musculus
  • NCBI Gene ID : 16059
  • Genbank Accession : AF010213
  • Taxonomy ID : 10090
  • Chromosome No : 12
  • Gene Starting Position : 881411
  • Gene Ending Position : 914690
  • Gene Strand (Orientation) : +
  • Protein Name : immunoglobulin heavy chain (V7183 family)
  • Protein Note : Also known as IgG; IgH; VI24H; VH7183; B9-scFv; IgVH1(VSG)
  • DNA Sequence : Show Sequence
    >gi|94393741:881411-914690 Mus musculus strain 129/SvJ chromosome 12 unlocalized genomic contig, MGSCv37 alternate locus group 129/SvJ
    CATGGACTTTGGGCTCAGCTGTGTTTTCCTTGTCCTCATTTTAAGAGGTAATTTGTAGAAATAAGATCCT
    GCCAGTATTGTGTACAGGAGAAATAGAAAAATTTTTCTTTCCTCTATTTTGTTTTGTTTTGTTAGTGACA
    GTTTACAAATAAGCATTCTCTGTTGTGAGGTGCCCAGTGTGAGGTGAAGATGGTGGAGTCTGTGGGAGGC
    TTAGTGCAGCCTGTAGGGTCATTGAAACCCTCCTGTGCAGCCTCTGGATTCATTCTCACTGACTACTGAA
    TGACCTGGATCCTTCAGGCTTCAAAGAAAAGGATGGAGAGGGTGACAATAAAATTTTTCCCACTCACACC
    CACTTGCTGGCCCTGGCGTTCCCCTGTAATGAGGCACATAAAGTTTGCAAAACCAAGGGGCCTCTCTTCT
    CAATGATGGCTGACTAGGACATCTTCTGATACATATGCAGCTAGAGACATGAGCTCCCGGGATACTGGTT
    ACTTCATATTGTTGTTCCCCCACAACAGTTGCAGACCCTTTTATTTCTGTGGGTACTTTCACTAGCTCCT
    CCATTGGGGGCCCTGTGTTCCATCCAATAGCTGACTGTGAGCATCCACTTCTGTAGTTGCTAGGCCCCGG
    CATAGCCTCACAAGAGACAGCTATATCAGGGTCCTTTCAGCAAAATCTTGCTGGTGTATGCAATGGTGTC
    AGAGTTTGGAGTCTGATTATGGGATGGATCCCCAGGTATGGCAAACTCTAGATGGTCCATCCTTTCCCAA
    CAATAATTTTTAATGGTGGAGATAGCACCTACTATCCAGACACAATGAAGGGCCAATTCACTATCTCTAG
    AGATGATGCCAAAAACACACTTTACCTGAAAATAAACAGTCTGAGGTCTGAGTACACAGCCATGTATACA
    TATATTCCTAAGCTTGAGTAAGTGGACTTAAACTGATTCCATCACAACTTGCATGAGATGGATATTCCCC
    AGTGTTAAGACCTGTCACCATCACTGTCAATCAGAAGACAAAGTTTATGCACAACAAAACAAAAAAACCA
    AAAGCAGAGGCCTCCAATTACAAGTAATAGACCCAGACCCACAGTCTCTGAAAACTGACTGTACAGTTGG
    ATCCAGTCTTTTACTTCTTTTCCCTGGATTTTATATTACTGAGGAAATGAGGAAAGCTCTACAATATCTG
    TTCTCCATAGTGCTCAACACCTCCAAGCACAGGTTACCCATATTCATGCCTGCCTTCTGCTACACTTCTT
    GTCTTGTAGACTACTTCAACCTATTTTGTACTCCAGTTAATGAAACTCAAGTCTAGCAGCCTGTCACTGT
    TTATTCTAAAGTATTATGAACAGGTGACCTCCCATCCTTCCCCAACGCAATAATCATATTTAGGAATTTG
    AGGTTTTATGAGATATGATCTCAGGGTAGAGAGAGAAAGCAAACTACATAGAAATATAGACTGACATAAA
    TCAAGACTTGCATAAGCTAGTCCCCAAGTTCCATGTCCCTAAGTGGCAAGGACTATCTTCTGAGCCTAAT
    GAGATGAGATCCAAATCAAACCTCCTGGGTCTTTTTAGATAAACATGTGAGATCAATAGACTAAATGCTT
    TGGCTGGGCTTCCTTGCAATCCAATTCCCAAACAAAAATGGATCTGGCTCCACAGACACCACAAGAATAG
    TCTTAAATAGTTCTTTAAGTAGAATGTCTGATCACTACGAGCCCAATTCCATCCTAAATACTCTTCTGGA
    TTATACATAAATAAAAATTGAACATAGGGCATGGGGCACTGATCTCCCTGTGCTACATGAATGGGGGCTC
    ATTTACTAAATGTTCCCATTTTTCTTTCTAGCGCTGCACAGTGCAAATCCTACAACTTCCTGTTTATCTA
    CAATGTGAACTCCAAACAGTACAAGAAAAAACGTTCCTTATGTTCCTCTCCAGGTTCTCCAACAAGCACA
    GAGCAACCTTCTGTCACCAGGACGGAAAACTGGAAACCTTGCTCACTGCTTCCTTTTATTCCCTCGGGAA
    CCTCCCCCAATGCAAAGCAGCCCTCAGGCAGAGGATAAAAGCTCACACAAAGACGAGAAGCCCCATCCTC
    TTCTCATAGAGCATCCATCAGAGCATGGCTGTCCTGGGGCTGCTTCTCTGCCTGGTGACTTTCCCAAGCT
    GTAAGTGTTTCAGGGTTTCAGAAGAGGGACTAAGACATGTCAGCTAGAAGATGTGTGACTAATGGTGATG
    TTGCTTGTCCCCAGGTGTCCTGTCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCA
    CAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACTAGCTATGGTGTACACTGGGTTCGCC
    AGCCTCCAGGAAAGGGTCTGGAGTGGCTGGTAGTGATATGGAGTGATGGAAGCACAACCTATAATTCAGC
    TCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTC
    CAAACTGATGACACAGCCATGTACTACTGTGCCAGAAACACAGTGTGGGAAGTCCAATGTGAGCCTGCAC
    AAATACTTCTCTGCAGGGATGATCACAACCAGCAGGGGGCGCTGATGACCCAAAGGGACTTCCCAGGATC
    TCTTCTGGAATCTAGGGAGTTCTGGCCTGTGTCTATCAGCATGTGTTTCAATGTTAGAGTTGTGAGTTTT
    CCTTCCAGCAACAGAGATATTTTAGAGCCCACTTTTCATGGTCATTCTACTAAATTTGTTCACATAGTGG
    AAAGATTTGTTAAATGATTCTATAGTCTAATAGGGTCAAACAAAAACAAATAATTGAGTTCATATCTACC
    AAAAAAAAAAAAATTCCCTCAAAGTGGACAACTGTGTAGGTGAGGAAAACCAGGGGGATTTGGGAATAGA
    TTATTTCTCTCCCGACTGTGGTGTTAGTCACCTTTCAGCCATTTACTGTATTTAATTCATGTTTAAAGTT
    AATTGTATTCATCTTCCCTAACATGAAGTTTTCAAACACATGTCCACAATTTAATGATGACACAGTTATC
    ATTGTGAATGATAACTCATAAGATCTCTCTCCATATATGAAACACACGATATTCTGTTATTAGCTATAGC
    CAACAAAGTTACATATCATTATTATTACTGAAATAATTCTTCCCATCTAACTGAATAGTTTTCTCACTCG
    ACACTGCCTATGCTGCGATCAGCCTGCTCTAATTTTACCTTTGCTCAGAGCACTGCTTTCTGTTTCATTT
    GATAAGATGGTGCCTGGTTGTCACTGAAAATGTGTCCTCCAGTTTCACAGTGATTAATGTGATTTTCAGT
    ACATTGAGAACAGAGCCACAGCAAAAATGAGATGGAATGTCATCATTTTTACGCCAGGTGCTGCAGAAAT
    AGCTAGGTGGTTAAGAGAGAATATTGAACTTTGAGGGGTTCAGATTAAATTCCTGATACCCACAATGTGC
    ACCTCATAACCACATGTACATATAGAAAACTCAGTATAAATGTGGCCTCCGTGAGCACCACACTACTCCC
    ACAAACACATATACACATAATTAAAAGTAAAGATTTTAGAAAAATGGCTAATCTAATGATAGGAAGTAGT
    CAAAAAGAGAGTTCTTTTGTCATGTACATATGTGTAGCAGACTTAAATGTTAAACATTCAAGAATACATT
    CCTCGATCCACATTAAAATTTTGCAAAGAGTAGCACAGACGGTGGCAATTGCTAAACTTATATCTAGAAA
    CACAATTATGTGTGGTGATATTTAATTACACATTTATACCAGGACATATGACAATATGGAAACCAAACAT
    GTTGTATCCACATGCTCTAAGGAAAACTAAATGGAGTGTGATAAAACCAAAGAAAATGTGAATATGAAAT
    ATTTTTCCAACTCTGCATCTTAAAACGGTTTCTTTCATGTGTCATATCTGCTATGAGGACTTTCTTCTGC
    CCATGTCCAACTCCAGAGCATGCCACAGCAGGAAGACCTACAGGTATTACTTCTCTGCACCCAGGAAAAC
    CACCTCTGTCCTGACCCTGCAGCTCTCAGAAGAGCCCAGACCTTCATTCTCAGGCCCTCATCCAGTAATC
    AGCACTGAATACAGAGCACTCACCATGGACTTTGGGCTCAGCTTGGTTTTCCTTGTCCTTATTTTAAAAG
    GTAATTCATAGAGATAAGATTCTATCTGTTTTGTGTACATGAGAAACAGAAAAATTGTATTGTTTCTCTA
    TTTTGTTTTGTTTTGTTAGTGACAGTTTCTGACTCAAGATTCTCTGTTTGAAGGTACCCAGTGTGAGGTG
    AAGCTGGTGAAGTCTAAGGGGAGGCATAGTGCAGCCTAGAAGGTCCATGATACTCTACTGTGCAGCCTCG
    GATTCACTGTAAGTGACGACTGGTTTGTCCGTGTTTGCCAGGCTCCAAAGAAGGGGCTGCAGTGGGGGAT
    GGGAATAATTTTTCATGGTTGTGGTAGCCCCTCTTATGCAGACACCTTGAAGAAGTGGGTTGGACGTAAC
    ATATTCAGAATCAATATTTAAAGATTCTAATCCTTGAAGATATCACTTTTGACCAAGTATATATGAACCA
    TGTTACTGAGGTTTATGGAGGTTTGAGTATGTTAGGTCCATGGATAGGGAAAATATTAGGGGATTTTAGG
    AGTAACTGAGGCTTGTTGTAGGAAGGACATCACTGTAGGGGTAGGCTCCGTGTTCCTATCTTCAAGCTCT
    ACCCAGTGCAGAATAGAGCCCTCTTCTGCCGGCAAGTGGAAAGAGTTTCTCTTCCTGGCTGCCTTCAATC
    CAAGTTGTAGAATATCAAATCTCCTAACACTATGACTGCGAGCATGCTGACATGCGTCCCACCATAAAAA
    TAGACTGAACCTCTGAAGCTTTAAGCCAGCCTCTAGTAACTGTATTCTTTAATAAGACTGAACTTGGCTA
    TGGTGTCTTTTCACAGTAAAATGGAAACATAGACAAGGTTCTAACTCTCTGTACTAGAGGACCATCTCTA
    TGTCCTTGGTGTTGTATGTAAATTATTGAACAAATACTATAGACAAGTTGTATGGCCAAAAGTCCATCCC
    CTTCAACAATCTACTATCTGGAACCATATAGATTAGATTCCTTTCCTGCACTCATTTCCCTTACTTGCTG
    AGACATTTTGAAGACATGCTCAGAATCCCTGAACTTCCTGCTGAAAAAAAAATACCCCTCCAATTTGAAG
    ACAGTTCTCTTCCAAAATTCATCATAAATACTGATCCACATGTCCAGGCATATCATGTAGTTTTAAAAAA
    CAAACCAATCAATGCTAAAGTGGGTAAGTGCCTACATCATTGGTTTATGTCTGTTCATAACCTGGTATAT
    AGCTAGAGACCAGGACTGCATGTTTCTCCTAGGCCACACCTCTCCCACAAATGCTGGCTCTGCCTCTCCA
    GAATTCCAAGATCATGGATCTCTGGGTCCCCAAGGAACAATAAGTGTGCACTCTTGCCAGCCTGTACACT
    ACTGCACTAGCTTCTCCCCCTGGAGCTTGGTCATTGCTCAATCTTCCCTTTCCAAAGCCTGGCCAAATTC
    CTCCTACCTTTTGTTCTTTCAGTCAGGCCACCTAAGTCATCTTGAATGAAAAACAACACAAAATCTGAAC
    TTAAAATCAACAAATAATACAAGTGCTGCGCACAAAATCAATCATTCTAAACTCATCAACTATTTTATGA
    TGGAAATCTTCCAACATACAAGCTACCACCAGGTCTAAGAATTACTCATCTACATGTTGCTTCCTCTCAC
    TCACAGCCTAACCATAAAAGGGCTGTTTCTTCCTCCCATGTCCCCTCTTTTGCCTTCAGAAGCAGAAGCT
    CCACCTCCTCCTCTTTGCCCAGCAATTGGCTTCTTGTCGTCTTTATTATCATATTAATTACTTAGGGGAA
    AATCCCGTGTAGTGGCTATTCCTGGTTGTCAACTTGACAATATTTGGAATGAACTACAATCCGGAATTGG
    AAGGCTCACCAGTGACCCTTATCTGGAGGCTTGGAGATCCTTATCTGGATCTTGGTTTGAAGATCTTGAG
    CCATAGGGGCTATGGATTCCAGAAGATTGAATCTCCGAGTTTAAGGAACACACCTTTAATCTGGGCTACG
    CCTTTCATCTGGGATTAAAGGTGTGGCGGAACACACCTTTAATCTGGACTACACCTTCTGCTGGAGACAA
    TATAAGGACATTGAAAGAAGGGAGTCTAGCTCTTGCTCTTGCTCCTTCGCCTGCTTGCTGCGTGAGACTG
    AGTAACTGCTAGATCCTTGGACTTCCATTCACAGCTGCGACTGAACAATTGTTGGGAATTGGGCTGCCGA
    CTCTAAGTCATCAATAAATTCCTTTACTATCTAGAGACTATCCATAGTTCTGTGACTCTAGAGAACCCTG
    ACTAATACAGAAGTTGGTACCAGGAGTGGTTCTAGAGTAACAGAAGTACAAGGATGAATCTTTTAAAATA
    CTGGAATTGGCTTGTTGATCCACCAGCACTTTCAACTATTGAAACCTCTCCAGATTCTCTCCCTCCTGGG
    AGCTCAGAGAATTTTGAAGACCCATGGTTGAAACTATATTCCGAACTTAAAGAAGCAAATGCCCTTGATT
    TTCTTAATGAATTAGGTGATTCAGTGCACAAAGCTTTCTACAAGATGGGGAAAAAATTGGAAAATGATTT
    TACTGGCTGGCTGCTCTTAGTATCTGTGGAAAAAATGATGAATGAAAGGAAGGAGTTGTGTGATAAAATC
    GAAAGGCTCCAGACACAAGTAAACGATCTAAAAGTTGCTAAGTGTGTCCTTGAGGAGAATCTTCTCTCTT
    GTAGCAATAGAGCTCAAGTTGCAGAAAATCAAACAGAAACTCTCATTGTAAGGTTGGCTGAACTACAGCG
    AAAATTCAAGTCTCAGCCTCAGAGTGTGTCAACAGTTAAAGTAAGGGCTCTAATTGGCAAAGAATGGGAT
    CCTACAACATGGGACGGGGATGTGTGGGAAGACCATGTTGAAGCTGAGAATTTTGAATCTTCAGATTCTC
    AAGGGTTTGCCCCACCTGAGGAAGTAGTACCCTCAGCCCCACCCCTTGAAATAATGCCTTCCCCACATGA
    GGAAATTAATTTTGCAGAGTCTGATAAACCAGCAATGATTTTCACTACTGATGTTTCTCAAGGCCCACCA
    ATAGTTTCTTCTAGACCTGTAACCAGACTCAAAGCAAAACAGGCTCCTAGAGGGGAGGTAGAAAGTGTAG
    TCCATGAGGAAATTCGCTACACTACTAAGGAGCTTAATGAGTTTGCTAATTCATTCAAGCAGAAACCTGG
    TGAATATGTGTGGGAATGGATTTTAAGGGTGTGGGATAAGGGTGGAAGGAACATAAGACTAGAGCAGGCT
    GAGTTTATTGACATGGGTCCTCTGAGTAGAGATTCTAGGTTTAATACGGAAGCTCGCATAATTAAAAAAG
    GTGTCAAAAGTTTGTTTGAATGGTTGGCTGAGGTGTTTATCAAAAGATGGCCTACTGGAAATGACTTGGA
    GATGCCTGATATTCCGTGGCTTAGTGTTGATGAAGGGATTTTAAGACTTAGGGAAATTGCAATGCTAGAG
    TGGATATATTGTGTAAAGCATAATTGTCCACAATGGGAAGGTCCAGAAGATATGCCTTTCACTAGCTCTA
    TAAGACGCAAATTGGTGAGAGGGGCACCAGCACATTTGAAGGGTTTTGTTCTTTCCCTTTTCCTTGTACC
    AGATCTTAGCATTGGAGATGCTTCTGCTCAATTAGATGAATTAAATTCACTGGGTTTAGTTGGATTCCGA
    GGTAACAAGGGCCAGGTGGCAGCATTGAATCGCCGGAGACAAGGTGATTCTAGTTATTATAATGGACAGC
    GTAGACAAAAGAATGTTTATAATAACATACCCAGCAATGGTCAGCACAGGAGAGGTGAAATTTATAATGG
    CATGACTCGGTTGGACCTTTGGTACTGGCTAACCAATCATGGTGTTTCCAGGAATGAAATACATAGGAAG
    CCTACTGCATATTTGTTTGATCTGTATAAGCAGAAAAATTCTCAAACAAATGAAAGAAAGGCTACATTAG
    ATCATGGTAAACAGCAATCTCGGCCAGTGAATCAATTTCCAGACTTGAGACAGTTTGCAGATCCAGAACC
    CCTTGAATGAAGGGGTGGCCAGGTTCCGCTGAGGAAGGATCTTGATAAGACACCCAAAGGTTTTGCTGTT
    ACCCTTTCTCCAGTTCTTCCCCAGAGGGACCTAAGGCCTTTTACAAGGGTAACTGTACACTGGGGAAAAG
    GAAATAATCAGACTTTTCAGGGTCTGCTGGATACTGGTTCTGAGTTGACACTGATTCCAGGGGATCCCAA
    GAAACATTGTGGCCCTCCAGTTAAAGTAGAGGCTTATGGAGGGCAGGTGATTAATGGAGTTTTGACTGAT
    GTCCGACTCACAATAGGTCCAGTAGGTCCCCAGACACATCCTGTGGTGATTTCCCCAGTTCCAGAATGTA
    TAATTGGGATAGATATACTCAGAAATTGGCAGAATTCTCATATTGGTTCCCTGAACTGTAGAGTGAGGGC
    TATTATGGTTGGAAAGGCCAAATGGAAACCTTTAGAGTTGCCTCTGCCAAAGAAAATAGTGAATCAAAAA
    CAGTATCGTATTCCTGGAGGCATTGCAGAAATTACTGCCACTATCAAGGACTTGAAAGATGCAGGGGTGG
    TGGTTCCCACCACATCTCCGTTTAACTCTCCTATCTGGCCAGTGCAGAAAACAGATGGATCATGGAGAAT
    GACAGTTGATTATCGAAAACTAAATCAGGTAGTAACTCCAATTGCAGCTGCTGTACCAGATGTAGTTTCA
    TTACTTGAGCAAATTAACACATCTCCTGGCACCTGGTATGCGGCTATTGATCTGGCAAATGCCTTCTTCT
    CAGTACCTGTCCATAAGGACCACCAGAAGCAATTTGCTTTCAGTTGGCAAGGCCAACAGTATACTTTCAC
    AGTTTTGCCTCAAGGATATATTAACTCTCCTGCCCTGTGTCATAATTTAGTTAGAAGGGATCTTGATCGT
    TTGGATCTTCCACAAAATATCACATTGGTGCACTATATTGATGACATTATGCTGATTGGACCAAGTGAGC
    AGGAAGTAGCAACCACTTTGGACTCATTGGTAACACATATGCGTATCAGAGGATGGGAAATAAATCCAAC
    CAAAATTCAAGGACCATCTACCTCAGTGAAATTCTTAGGAGTCCAGTGGTGTGGAGCATGCAGAGATATT
    CCTTCTAAGGTGAAAGATAAGTTATTGCACCTGGCCCCTCCTACAACCAAGAAAGAAGCACAACGTTTAG
    TGGGTCTATTTGGATTCTGGAGACAACACATCCCTCACTTGGGTGTGTTACTTAGGCCTATTTACCAAGT
    GACTCGGAAAGCTGCTAGCTTTGTGTGGGGCCTGGAACAGGAGAAGGCCCTTCAACAGGTCCAGGCTGCT
    GTGCAGGCTGCACTACCACTTGGACCATATGACCCAGCAGACCCGATGGTACTTGAGGTGTCTGTGGCTG
    ATAGAGATGCTGTTTGGAGCCTCTGGCAGGCCCCTGTAGGTGAATCACAGAAAAGACCTTTGGGATTTTG
    GAGCAAAGCTCTACCATCATCTGCAGACAACTATTCTCCCTTTGAAAAACAGCTCTTGGCCTGCTATTGG
    GCCTTAGTGGAAACTGAACGTTTGACAATAGGACACCAAGTTACTATGCGACCTGAACTACCCATCATGA
    GCTGGGTACTATCAGACCCTGCAAGTCATAAAGTGGGACGCGCACAGCAGCAGTCTGTTATCAAATGGAA
    GTGGTATATACGTGATCGGGCCAGAGCAGGTCCTGAAGGCACAAGCAAGTTACATGAAGAAGTTGCTCAA
    ATGCCTATGGTTTCTACTCCTGTTACACTGCCATCTGCTGCCAAACATGTGCCTATAGCCTCATGGGGTG
    TTCCCTATGATCGACTGACCGAAGAGGAAAAGACTAGAGCCTGGTTTACTGATGGCTCTGCACGTTATGC
    AGGCACCACCCAGAAGTGGACAGACAGCTGCAGCATTACAACCCCTTTCTGGGACAACCCTGAAAGACAC
    AGGTGAAGGGAAATCTTCACAGTGGGCAGAACTTCGGGCAGTACACATGGTATTACAGTTTGTTTGCAAG
    AAGAAATGGCCAGATGTACGATTATTCACTGACTCATGGGCTGTAGCCAATGGATTGGCTGGATGGTCAG
    GGACTTGGAAAGATCACAATTGGAAAATTGGTGAGAAAGACATCTGGGGAAGAAGTATGTGGATAGATCT
    CTCCAAATGGGCAAAGGATGTGAAGATATTTGTGTCCCATGTAAATGCTCACCAAAAGGTGACTTCAGCT
    GAGGAGGAGTTCAATAATCAAGTGGATAAGATGACCCGTTCTGTGGACAGTCAGCCTCTCTCCCCAGCCA
    TCCCTGTCATTGCTCAATGGGCACATGAACAAAGTGGCCATGGTGGTCGAGATGGAGGTTATGCTTGGGC
    TCAGCAACACGGGCTTCCACTCACCAAGGCTGACCTGGCTACAGCTGCTGCTGATTGCCAGATCTGCCAA
    CAGCAGAAACCAACACTGAGTCCCAGATATGGCACCATTCCTCGAGGTGACCAGCCAGCAACCTGGTGGC
    AGGTTGACTACATTGGACCACTTCCTTCGTGGAAAGGACAGCGTTTTGTTCTTACTGGAGTAGATACTTA
    TTCTGGTTATGGATTTGCCTTTCCTGTACGTAATGCCTCTGCTAAAACCACCATTAACGGACTGACAGAA
    TGCCTTATCTATCGTCATGGTATTCCACACAGTATTGCTTCTGACCAAGGAACTCATTTCACAGCCAGAG
    AAGTACGACAGTGGGCCCACGATCATGGAATTCACTGGTCTTACCACATTCCCCATCATCCTGAAGCAGC
    TGGTCTGATAGAAAGATGGAATGGCCTTCTGAAGACGCAGTTACAGCGCCAATTAGGTGGTAACAGCTTG
    GAAGGCTGGGGTAGAGTTCTTCAGAAGGCAGTATATGCTTTGAATCAGCGCTCGATATATAGTACAGTTT
    CACCCATAGCCAGGATTCATGGGTCCAGGAATCAAGGGGTGGAAAAAGGAATAGTTCCACTTACTATCAC
    TCCTAGTGACCCTCTAGGAAAATTTTTGCTTCCTGTCCCCATAACTCTAGGTTCTGCTGGCTTAGAAGTT
    TTGGCTCCAGAGAGGGGAGTGCTCCTACCAGGAGCTACAACAAACATTCCATTGAACTGGAAGCTCAGAC
    TTCCCCCTGGTCATTTTGGGCTTCTAATGCCCTTAAACCAACAGGCTAAAAAAGGAGTAACAGTGTTAGG
    AGGGGTGATAGATCCAGATTACCATGGGGAAATTGGATTACCTCTTCACAATGGTGGTAAGCAAGATTAT
    GTCTGGAGTGTAGGAGATCCCTTAGGGCGTCTCTTAGTACTACCATGTCCTGTGATTAAAGTCAATGGGA
    AACTACAACAGCCTAATCCAAGCAGGATGACAAAGGACACAGACCCATCAGGAATGAAGGTATGGGTCAA
    TCCTCCAGGAAAAGAGCCAAGACCTGCTGAGGTGCTGACTGAAGGAGAAGGAAATATAGAATGGGTAGTA
    GAGGAAGGTAGTTATAAATACCAATTAAGGCCACGTAACCAGTTGCAGAAACGAGGATTATAAAGTAATA
    TGAATGCCCATTGTAAATTTACTAATGCGTTTGCGATTGTACGAGGGATAGTTATATCATGTTAGGCGTA
    TTTACAAACTTGTTATTGTTTTATGTGAACATGAGATATTATTTGTGTCAAGTTGACAAGGGGTGGATTG
    TAGTGGCTATTCCTGGTTGTCAACTTGACAATATTTGGAATGAACTACAATCCGGAATTGGAAGGCTCAC
    CAGTGACCCTTATCTGGAGGCTTGGAGATCCTTATCTGGATCTTGGTTTGAAGATCTTGAGCCATAGTGG
    CTATGGATTCCAGAAGATTGAATCTCCGAGTTTAAGGAACACACCTTTAATCTGGGCTACGCCTTTCATC
    TGGGATTAAAGGTGTGGCGGAACACACCTTTAATCTGGACTACACCTTCTGCTGGAGACAATATAAGGAC
    ATTGAAAGAAGGGAGTCTAGCTCTTGCTCTTGCTCCTTCGCCTGCTTGCTGCGTGAGACTGAGTAACTAC
    TAGATCCTTGGACTTCCATTCACAGCTGCGACTGAACAATTGTTGGGAATTGGGCTGCCGACTCTAAGTC
    ATCAATAAATTCCTTTACTATCTAGAGACTATCCATAGTTCTGTGACTCTAGAGAACCCTGACTAATACA
    TCCCTACATACAGGAAACTTGATGTATAAGTAAAGAAAATAGTAAATCTAAAAATTTCCTTATCAAATAC
    ATGCAAGAAATCCAAAATACCATGAAAAGACCTGATCTAAAAATAGTAGGAACTGAAGGTGAAGTGTCCC
    AGCTCTGAAAACTAGAAAATGTTTTCAATATTTTTCCTAACAGTGAAGACATGGATCTAATGAGGCCACC
    TCTTATAGCCATGCAAGACTGACAGTGGAGGGATAAGGACACCAATCCACCCACAAAACTTTTGACCCTA
    AATCAGTTTTATCTAAAAGCAATGCAGGGGCACAAATGGAGCAGAGACTGGAGGAATGGTCAAACAATAA
    CCTGTCATATCTGAGACCCACCTAATTGGCATACACCAGTCACTGACATTATTAATAATGTTCCATTATG
    CTTACAGGCATTTCTCTAGCATTACTATTCTTGGAGAGACTTCACACAGCATCTTATTGAAACAGATGCA
    GACACCCAGAACCAAACATTGGATGGAGATTGGGATCCTTAAAGAAGAGTTGGGAGGATAATTGAGAGAC
    CTCAAAGGAATAGCAACCCCATAGAAAGACACGAACAGGGTCAGTAAACCTGGACCCATGGGGTATCTCA
    GAGACTGATCACCAACCAAAAATCACAGAGGGCCTGGATTGATTCCCCTGGCACACATGTAGCAGAGGTC
    TGCCTTGTCCTTCAGTAGGTGAAGATGAGCCTAAGGCTTCAGAGACTTGATATATCAGCATACAGCAACA
    CCCAAGTGTGTCCCACTATCTCAGAGGTGAAGGGGTGGAGATGGGCAGAAGGGTCCTGATAGGGGACAAC
    TGCAAAGAGGCAATATTTGGGACAGAAAGAAAGGAAGAGAGAGGAAGATAAAGAGAGAGATGAAGGAAGG
    AAACAAACAAACCAAGATGAAGAGAGAGAAAAGGGGGAAAATAGCCTTGAATGGTTCAGGGATTCAAAGG
    GACATACCTAAGCATGGTAAAACAATGCACATAATGGCAGCAAGTAGCTAACACTGAATTAAATAGAAAG
    ACACATAAAGCAGTTCTACTAAAGTAAGAGACAGAGTTGCCCAATCTCACCCTATTTATTCAATAATAAT
    GTACTTGAAGTTCTAGCTAGGGCTGTAAGACAACTCAAGGAGATCGGAGGATACAAATTAGAAAGAAAAA
    TTCAAAGAATTGTAATTGGTAGATGATAGGATAATATACATAAATGACCTCAAAATTCTTCCAGAGAACT
    CTGAAAGCTGATAAATACCTTCAGCAAAGTGGCTAGAAACAAAATTACTTCATAGAATTCAGGAGCTATC
    CTTTATAGAAAAGTTAAAGTGGCTGAGAAAGAAATTAGGAAAATCACATTCTTTGCAATAACCAAAAATA
    ATATGAATTAGCTTGGTGTGACTCTAATCTTGCAAGTGAAAGATGTGTGTGACAAGAGCTTCAGGACCCT
    GAAGAAAGAAATCAAAGAACTCAAAGATGCAAAGTACTCTAATGCTCATGGATAGGCAGAATTAACATAA
    TGAAAATGCCAAATTTAACAATTCAATCTACAGATTCAGTGGAATTCCCATTAAATATCCAACCCAATTT
    ATTACATTCTTGAAAAAGCAAATCTCAACTTCATATAGAAAAACAAGAAATTGAGGGTAGCTAACAAAAT
    CCTGAACAATGAAAACACTTCAGGAGAATTCACCATCCCCTACCTCAAGCTGTATTTTAGAGCAATAGTT
    ATTAAAACTGCATGGTATTTGTATAGAAACAGATATGATGATCAATGTAATTGAATTGAATACACTGACA
    TAAAACCACACTGTTATGGACACTTGATTTTTGACAAAGAACCCAATAATCATAATAAAAATCATAATAA
    GAATGCATCCCCCAACAAATGGTCCTGATCTAAATGCAAATAGATTCATATCTATCATCCTGCACGAAAT
    TCAAATCAAAGTGAATTGCAGACTTCGACCTAATACTGGATTAACTAAATCTAAATGAACAAAAAGTAGA
    GAATAGTTTTGAACTCATTGATGCAGGATTCAATTTCCTGAACAGAACCACAATGGCTCAGGCTCTAGGA
    TCATAAATTGGTAGATAGGATCTCATGAAACTGAAAAACTTCTGTAAGGCAAAGGCAATAAAACAAAATG
    GCAACCTACAGATTGGAAAAAAAATAATCTTCAGTATCCCTAAATCCCATTGAAGGCCAATATGCAAAGT
    ATATAAAGAACTCAAGATGTTATCCTCCACAAAACCAAATAAATCAATTAAAAATGAACTACAGAGCTAA
    CAAGAGAATTCTCAACAGAGGAATCACCAATGACTGAGAATCACTTAAAGAAACATTCAATATTTGTAGT
    CACCAGAGAATTGCAAATCAAAACTACCCTGAGATTCTACTTTATACCAATCATAATGGTCAAGATAAAA
    ATTCAAATGACAGCGCATGTTGGTGAGGATGTGTATACTTTTGCATTGCTGGTGAGGAAAACAATCTGGC
    AGCTCCTCAGAAAATTGTAAATAATTCTACCTGAAGATCCAGCCATACCACTCCTGTGCATATACATAAA
    ATGTGCTCCACCATACCACTAGGAGATATGTGCCACTATGCCCATAGCAGCCTTATTTGTTAATAGCCAA
    AAGATGGATACAACCCTGATGTGCCTCAAACAAAGATTGTATATAGAAAATATGGGTTCCCTTACACAAT
    GGTATTCTACTCAGCAATTAAAACGTGAGAACATCATAAAGTTTTCAGGCAAACTGATCAAATGAAAAAG
    TATAATCTTGAGTGAGGTAACAGAACCAAAATGACCTGCATGGGATGTACTCACTGGTAAGTGAATATTA
    GACTAAAAGTATAGAATATCAATGATGAAAGCCACAGACCATAAAGAGTTTAATAAGATGGAAAGCTCAA
    GTACGGATTCTTCAATCCAATATAGAAAGAGGGACAAAATAATCAGGGGAGGCAGAGGGAGAGAGAGACA
    TTTGGTGGAAATGGGAGAAGAAGGGAGAAAGGCAGAAAGGATCAGGCGTTGGGGAACACTGGAAAGCAGC
    CCAGGGGTCCTGGTAAATGGATTAAATATTCAGCTGAATGGAGGTGGGAGGCAGGGGGAAGCTCTGCAAA
    GTCCAAGAGACCTGGGATGTGAGAGGCTCCCCAGATGATAATCTTAGCCTTCATGCCTAACAGTTGTAGA
    TAAACCCTGAAGAGATCACTTCCAATAGATGCAGAGGGCCCTAAGTGGTTGGATGGAGTCCCCCAACTTA
    CCTCAAAATTGTCAATACTCATAGAAATTAGGACAATGAAAATCAAAACACCCTGAGATTGTATCTTATG
    TCTGTCACAATGGTTAAGACCAAAACCTCAAGTGATGGCTTGTGCTGGCAAAGATTCAGAATAGTAAAAG
    TCTCCCCATGGCTTGTGGGAATGCAAACTCTTACAACCTCTTTGGAAGTATATTTGATTGTTACCTGGTA
    TAAAATGGCAAAAAAAAAAAATGGTTGAAGGGGGACTAAAAAAGGAAGAAAGGGGAGAACTATGGGATGC
    GAAACAAGAAAGTTTGTTGCAAAAGAAATATGTTTCCACTGCAAACCCTGAGTCTCAGACAGAAGGGGAC
    CTGGAATTCTTCAGATACAAAGAATCTCTAAACCCTGAGGACATTCTATCACAAATAAGTAAAATTCAGA
    AAATTCTGAGTGCTCCCATCACGGAGATGAATCTGCTATGAACAGCTCATAGGTGTGACCCTCTACAAAA
    GCCATATTATTGAAAAGCCACATTGTGCCCAGACTTTGGAAAGACTGAGCTCATATCCTGAAATACAGTT
    ATGTGTGGTTCTATTTAATTACACATTTACACTAAGAAAACATGGCAGTATGGGAATGAAGCTTGTTCTG
    TACACATTAACAGAGGGAAACTAAACAAAGTATGGTGAATCCCTAACCAAAAGTAAAAAAAAAAAAAGAA
    AGAAAAGAAAATAAAAGTGAAACTACAATATGTTTCAAATGCTGTAACTGAAATCTGGTTTTTTGATGCT
    TATATCTGGTATCATCAGTGACTTCAGATTTAGTCCAACTCCAGAGCATGGTATAGCAGAAAGACATGCA
    AATAAGCCTTCTCTCTGCCCATGAAAAACACCTCGGCCCTGACCCTGCAGCTCTGACAGAGGAGGCCAGT
    CCTGGATTCGATTCCCAGTTCCTCACATTCAGTGATCAGCACTGAACACGGACCCCTCACCATGAACTTG
    GGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTAATTTATTGAGAAGAGATGACATCTGTTGTA
    TGCTCATGAGACAGAAAAATTGTTTGTTTTGTTAGTGACAGTTTTCCAACCAGTATTCTCTGTTTGCAGG
    TGTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTC
    TCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGA
    GGCTGGAGTGGGTCGCATACATTAGTAATGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCG
    ATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCCGTCTGAAGTCTGAGGAC
    ACAGCCATGTATTACTGTGCAAGACACACAATGAGGAAATGTTACTGTGAGCTCAAACTAAAACCTCCTG
    CAGAGCACCCAGGACCAGCAGGGGGCGCAGAGAGCACATGGAGTTCTGATTCACAGAAGAGTTACAGCCT
    GTACAATTAGACCCAATCTTCAACAAACCGTCAAAATATTCGATCCAAAATTGTTCCTGTCTAAAAGTAA
    TTCAAGGACAAAATGGACCAGAGACTGAAGAAATGGCTGACCTGTGACCCTCCCAACTTTGGATCTATCT
    CATAGGCAGGTACCAAACCTTGACATTTGTCACTGACACTGTATTGTGCTTGCAGACAGGAGCATAGCAT
    GGCTGACCTCTAAGAGGCTCTGCAAGCACCTGAATGAGACAGATGTAAATAGAGTGTACAACTGGATTAA
    GTTTGAAGACTGCAATGGAAGAGTTAGAGGAAGGAATGAAAAATCTGAAGGGGATCACAACCCCAGAAGA
    AGACCAACAGTGCCAATTAAACTGGATGCCTGGGAACTCCCAGAGACTAAGCCACAAACCAAGAATCATG
    CAGGCTGGTCTGAGAATCCTGGCACCCAAGTCTCAAGGAACTGCCTTTTCTTGGCTCAGATGGGAGAAGA
    TGAGCCTAAAGCTTTAGACTTGACGCCCCAGGGATGGGCAATACAAAGTTGAGTTTCTTGGGAAAGTGAA
    GAGGATGGTGGGAGGAGGAATAAATCTGGGAAGTGGGACTGGTTGGGGACAACATATTGGTCCTAAATTT
    ATGAAATAATTATCTAATTGATAAAAAGAGTCCTTGGGGTAATGGAGGGCTAGACTCCTCTGTGCCTAGT
    TTGTAACTCTACAGGGATCCTCTTTAAAAGAATAGGGTGCACATAGAGTATATGTGTGTGACACTAATAC
    AGGGGTAAGTGTTTCTGTAAGAACTTTATGAATAAACTTTATTAAAACATCAAAAAGTATAGGTTGTAGC
    AACCCTTGACCTGTACGAATGTTTATAAAACTTTCTATTTTCCTTAATTATATCTGTTTTGCATTTGTTT
    ATTTATTTATTTACTTACTTACTTATTTATGATTTCACTGTGTCTTTCTGTAGTCCTGTATGTGTTTATT
    TGTCTCTTTATTGCTCTGTTTCTGTTTCTTTCTCTCTGTCTAGTATTGTATCTCTCTGTGTCTCTATGTC
    GCCGATTGTGTCTGTGTGTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCATATTGAAA
    TGAGATCTCTTCTTCTATCAGTGTACATAATGACTTTGAAATAAAGTCCATCACTGGGCTTGGATATCAC
    TATCTTGTTAGAATGGTTGCCCAAAGTGTTCCAACCATCACACTGTTTCTACATATAATAGATGGGTTTA
    TTGATTTACTACCAAAACTACCCCATCTTAAGTTATGGATATTAAACACAGATCCTCATGGTGTGTTAAG
    AACTTGAAGCACAGAGCTATATGTACAGCCCACTAAAAATGACTTAAATATAAGATAAACTCTGGCTTGA
    ACAAAACACAGAGCATCACACGAGGCAGAAGTGAATCTCTATGAGGACCAGGATCACATCCCAAAGAACC
    AAAGAACACTTGAGACTGTGGAACCCTAGAGTCAGGGCTGACACTCAGTAAGATTTACTTCATAGAGCAT
    GATGTGGGATTCAAGAAGAAATCAGGCAGGGCCTGTGACCCATGAACCAGATGACTTCCAGTCTCTCCAT
    CATCCTCACACAAACCATAGGCAACAGCTCAGCACTGACTCACCTGTGATGCTGGTGACAAAATGTGTGC
    AAAGCCTCAGAACTGAGACGTGAGGCCAATGTTGCTACCGACACTCAGTGAGCTTCAATTTGCTGCAAAC
    TGCTCCAAAACAACTCAAAGGGTAACTTCTTCTTTTTCTATTTGTACTTGTGATGCCTTGGTAGATATAG
    TTCCCCAATAATTTAATAGGACAAATCATTTTTCCTTTTAACACATTAATCCCCTTTCCTTCCCCCAAGA
    TTCTAAAATTTTTCTTTAACACTGATGTCTGAGAGATATGGCATAATAGATGTTTGTCTTATTCTGAGTT
    GTTCCTTATCAAAAGAAATCAGCTGTGGCACTTTGAACCTAAATATAACAATATTTCACAAAGGCATTCT
    GGGAATATATAGATGGTATTGCTACAAAAGAATAGGAGGATCTATGGCCATACCACCCTTAATGAACCAG
    ATACTATCTAAAAGATTAGGACAGCTGGGCATGTTGGCACAGGCCTTTAATCCCAGCACTAAGGAGGCAG
    AGGCAGGCAGATTTCTGATTCGAGGCCAGACTGGACTACAAAGTGAGTTCCAAGACAACCAGGGCTATAC
    AGAGAAACCCTGTCTCGAAAAAACAAAACAAAACAAAACAAGAGTAGGAGGGTATTAGATGCGAGGTGAT
    GATGGCCATTCAATCAAACCAGAATGTGACCTGAGAAAAGGTCTAAGTTTGTCAGCTTGTCCTGGAGATG
    TGTTGCCTCTGATAAATGCAGAGCTGGAACTGCAATACATTGGATTCCTGTGAGTTACGGAGAAATAAAA
    GGAATAGCCTCTGTTTCCTTTGGTTCATCAAGTTGGCAGTGAGCTAGAAGATGATACTGGGAACCATCAC
    ATGTACTTCTACAGAGTTATTCTACATTCTACAAGTCAGTCTTGCTTGCTTTCGTTTCTGTGAGGAGCTA
    AATACAGAATGTTTTCAATGTCTCATAATGTTCTGTGCACACCCATGGGTGCCTAGCCAAGGATAGAAAC
    TTGTATCTGATAAGGAAAGAGGATGCTGCAGTGGCCACACTCTGAGATCACCTCCTGCCAAATTATACTA
    GAGTGTTTTACATAGTCTTGAAAACACTAGGTCAAATATGTTGTCATCCAGATATGAAAACTCTGTGCAA
    CAATTCAGAAATACAGTGGTCAGGACAACTGGTATGTAACACTTTAGTTCCAGCCCACATTAATCTCACA
    TCTTTTCTGTGGTTTCAATAAAGAGCACACATGTTTGGAACACCATCTCCACTTTGTATGCTTATCCTTA
    TGCATCAAACTTTTCCTGTAATTTCCTACCCCGCACAGGTTCCTGTCTTGATCTCTTTTTCTAATGAACA
    GCAATACAGAAGTGTTGGCCAAATGAACACTTTCACCCCAAGTTGCTTTGCCATTGTGTTTCATCACAGC
    ATTAGTAACTCCCAGTAAGGCTACTGGTTACCCTGCATCTTCTATGTAGATCTTTTGATGAAGTTCCAGG
    CTTGGGGTCTTTATCTGACTGTTATCCTGGTTAATTCATGTCCACCAATAGAATATTGTTCTCATTTTCA
    ATATCATATGAAGTTACCTGTTTATAGGATCTTCCTAAAGCAGAAAACTATATAAAATGTCTGCTTACTT
    TAAGAAAAGTATGTGTCCTTGGAGGTAAATGGAGGAAAGGGTGGAGGATGTTGAGTAATGGAAGACTGGC
    TAATACAGGAAGGTCACAGAATTGAATGATTTTAAAAATTTTAACTCCTCATCAATGAGGTACTTCAAGT
    TGAGGCAGGAGTCCTTGATGCACAGGTGACCCAAAGGCCCCAGCCTCTGTTTCTTGGTCTGCTCCAGTCT
    CCCGGCTCAGCACAGTTCACTGAGTGACAATCTTTAAGATAGTCTTTCAGGATACAGTTATCACAGTTTT
    CAAACTCTCTTCATTGAAACAATATAATGAGTCAGAGTCCAGTGTGCCATTAATAGGCCCTATGCAATAG
    GATTTTACTTCATCTGTGGAAAGAAACCACCATGGCCTCATCAAGGCCTCATTTGATGATTAGTGTAGAA
    AGCGTCCCCAGAGCTTTGCTGGGATGAAAACCCTGGCAAGATGTTTAGGGCACTCTCCATCTGACAGGAC
    AATTCTATCAAGGGTTGCTTCCATATAGAGAGAAATCTCCACATCTGCAGTATTTCCCGATTTATCAACA
    GTTCTTTACCCCGTGTCATATGCATAAAATCTTTTCTCTGTAGAATTTTCAGTACTAAAAACTAACAAGG
    ATGTTGGTCCTGATATCAGACATAATAAATAAATAAACATCAAATTATACCCTAAACATGTGGAGATGGT
    TTTCTGTTATGAATTTATTGGCTCTCTAAAAAGTGTTAATTAAGCCAGGCGTGATGGCACATGCCTTTAA
    TCCCAGCACTTGTGAGGCAGAGGCAGGCGAATTTCTGAGTTTGAGGCCAAACTGATCTACAGAGTGATTT
    CCAGGACAGCCAGGGTTACACAAAAAAACCCTGTCTCCAAAAAGTAACAAAAAGAAAGTGTTAATTAACA
    TCTACACTTTAAGATAATTTGGTGGCAAAGGTTAATAGTCTGAAATACTAGATATGAAAGTATGACACCT
    AAATTGCATGCAGAGAATAAAGCTGAATTCCAATGAGATTCATGCCTTGTGAATCCATTCCAATCTGATC
    TCAATCTTCAAACATGTTTGTGCATAATTTACATCTTAATCAGTTGCTGGAAATTAAAAGGACATAAGCT
    AAACTTAAAACTCTTTCTATTTAATGTGTCAGAATAGAATATGTGCACTGAAGTTTTCATGTGCATAGGA
    TATCATTGTTACAATGATGGTTCAAATACGCACCAGTAGCACAGGTGATGCTTGACATGAAAAAAACTGT
    GACCAGAGCCCAGACGTTGATGTTGGCTGAAAGAGCCTAGTGTCTCACATTTGATCAAGCAAAGTGTTAT
    TCAGAGAAGCAGTGCCAGATTTGGGATCCTTCTTAATGGATCAAGAACATGAGGAAGATTTGGAAGCCAG
    ATTGATGAGATTACTAGAATCCTGGATCTGAACACACAACACACAATAGTAAGTTGAACTTTTCCACGCA
    ATGCTAAAGATAAAGCATCCAGTATCATGTTATACAGGACCAAGGTGAGGATCAGTCTGTGGGTGAAGTA
    CAATGAACTGGAAATTGTTTGGATTTTAAATGTTGGTTTTTAAATTATATATAAAATATATATATATATA
    TATATATATACATATATATATATATATATATATATATATATATATCGGAACCAACATTTAAAATTTGGAT
    TTTAAATGTTGGTTTTTAAATTATATATAAATTATATATATATATATATATATATATATATATATATATA
    TATATATATATATATCGGAACCAACATTTAAATTCCTAATTCTTAAGGACTTCACTTTTGACCAAAAATA
    TTTGAAATGGGTTATTTAGGTCTGTGGTGGTTTGAATATGCATAGCCTATAGGAAGTGGAAGTATGGCCA
    TTTTGTAGTATGTGTAGCCTTGATGGAGGAAGTACATCACTATATGGTGTTGTGAGAGTTGAGATCCTAT
    GTTCAAGTTCTGCCAAAAACAGAAGAGAGTCCCCTCCTGGATATTTGAAACAGATGCAGAGGCCCAAAGC
    CAAACATTGGGTATATCTCAGGGTTTCTTATGGAAAAGTTGGCTGAAGGATTGAAGGCCTTAAAGGGGAT
    AGGAACTTCACAGGAAAACCAACAGATTCATCTAACCTAGACTCTAGGGGTCTTTCAAAGGTTGAGTCAC
    CATACAAAAAGGACACAATGGCTAGACCTAGGTCCCTGATACATATGTAGCAAATGTGCAGCTTAGACTC
    CATGTGGATTCCCCAACGACTAGAGCAGGTATTGTCCCTGAAGTTATTGCTTGTCTGTAGAATACATTTC
    CCAACATAATTGTCTTGTCTGGCCTCAGTAGGAGAGTATGTTCCAATCCTGGAGAGACTTGATATGCCAG
    GATTGAGAGATTAAGGGTCTCCCACCATCTCAAAGTTCATGGGTATGACAGGGGAGGGGCAGTGCAGGTT
    TTCCCCATCTCTGATTTCCAGCATTTGCACCCCCAATATTGAGGGGGAGGTCTAGCAATTGGAGGGGGGA
    CAAACAGTATGAGAAAGCTAACTTTAGTGGTGTTGTCACAGTGTGGGGTATTGTGGGTGGGAGTGGAGAT
    GAATCCTCCTCCTCCTCAGGAGGCTGTAGCAGGTGCTTGCCCTCTTTTGGGACAGTAGAGCTGTGGTCAG
    TACTGGCCTGTTCCTGACAAATTTATTCTGTGCTGTGACCATTTATTCAAGGGAGGAAGATTTAAGACTT
    GCTATGATAACCTGACGAATCTGGACAAACAAGAGACAGGAGGTAACACCTGAGTTACTCTTTTGCTTGC
    TAGTACAGAAGAGCACTATAATCCACAAATTGGGGTCCCACATATTCTTGAGTGGGATCCAAGGTGCCAT
    AATTTCAGCCACATTCCAAAGTCCCTCAAGGTCTGACAGTTTAGTCTTTAGGCTTCTCCTAACCAAATCA
    TATTTGGAGTTCTTTAACATAAAAGGGTAGTTTCCCCATATCACTGCCAGCAAAACAGGTTGAATTTGGA
    CAGACAAGCAAAACTATCACAAACAAGACAGACTATGTGTGCGATCCAAATGTCTTGTGATTAGAGGGGA
    AGTTCTGCAGCTTATTAGGCTGTGTCCTTATGCCCACAAATGTCTTCAGATAGGAAAGGGCCATTGGGTG
    CCAATCCTTAGTTCTTCTGTTTCCTGGGCTGTCAGTTTTTATGTGGAGAAGACATGAACCAAATGCAAAG
    TCATGTATCCTACAAGAACTGACATACACAGACAAACAAGGAATAACAATGAGCTCTCTCATTCACCTAA
    GGGGGTCTTATAACATAGAACAGAAAGGGGCCAACTTAGCAAGAATTGACCAAAAGGTGGTCACCTAGAC
    CACACCACAATGCAGGAAGGATATCAATGAAGGAAGAACAAAGGAAAGGCCAAATCAAGTTTGTTGGCAC
    ATGACATAAAACAGTGCTCAAAAAACAATAGATGCGAGCAAAATGAAGCATTGGAATCATATCGGGGTAC
    ATAGGGGTCACTCTTACCCCTGTTCAAGTGCCAGATGTTGTTTTAGCCTTAGGCTCCCTGAGTTTGTCTT
    TAGGGGAAATGGGTAAGAATACACAAAAATAATGTCACAGAATCTTAGAGGCAGATGAGAAGTCAAAGCA
    GACTCATTTATTGCTATAGAAAAGAATGCCTTTATACCTTCTGTCCATAACACCTCTGCCCATATATGCT
    CATCTCTGTGAGGCAATGGACTACCTGCAGACAGGCTGGTATCCAGTCAAGCTGAGGTCTGAACCACAAT
    GGTCGTAACTCACCTAAAGGACGTGGTAGGCATGACGCGATAGGCGTTCCCTCATGCTCCTGGAACTCCG
    GCCCCTGCCTAAGGTACCACCTCCCACAGCCCCCACAAGAGAAGCATGGTCAGTAGTCATGTAAGCAATG
    GCCCAAGCTTCTGACCTTCAGCCTAAACTCCTCCCCAGTTACCTAGCAACAGTGAAGACCATAAAATGGG
    ATGTTAGGCCCCCACCTCACTCTCTTACTCCTTTGTTCCTTTACCTCTCACTTCTCTCAATTCTCTCTCC
    TCTTCCCTTTCTTTGTCTTCTTCTTTCCTTGTCTCTCTGTCTTTCTCTTTCTCTCTAGCCTTCTCTGCCT
    CTCTCTCTCTCTCTGTCTCTCTGTCTCTCTCTTCTCTCTTTCCCTTGAATTTCTTTTTTTCTTTTTTCTT
    TTTTTGATGAGCACAGTATAAATAGTTTATTGCATTGTTTCTGAATGTGGCTGTATTCTCTATTGTTCTC
    TTTCTTATTTTTTTTCTTTTTTTGGTTGTTTTTGTTTTTATAGTCTCGCACAGAATTCTACATCTACCAA
    ATCCCCATTGTGTACTTTAGTTTTGTCTTGTATTCAAAGGTTCTTTCAGAAAGAATCTTAAAGATGAAAT
    TTGCCTTCCAACTTTTCAAAAATACATATAAAGATAGATGTTACATAGTTCACGGAATAGACACATATAC
    GGAGATGCAGCAATATCTCCTTTGTGCCTCTTGTATATTCATGTTATTTCCTCAACTAAAAATACATAGC
    CACAAAGACATACAATGAGCACTCCACTTAGGAAAATATGGCCATATTTTTTTACATTTTTATGATTTTA
    TGATTAGGTATATTCTTCATTTACATTTCCAATGCTACCCCAAAAGTCCCCCAACCCTTCCCTCACTCCC
    CTTCCCCACTCCCACTTGTTGGCCCTGGAGTTCCCCTGTACTGAGGCATATAAAGTTTGCAAGACCAATG
    GGCCTCTCTTTCCACTGATGGCCCACAAGGTCATCTTCTGATACATATGCAGCTAGATACATATACATAC
    ACGAGCTCCAGGGGGTACTGGTTAGTTCATATTGTTGTTCCACCTATAGGGTTGCAGATCCCTTCAGCTC
    CTTGGGTACTTTCTCTAGCTCCTCCATTGAGGGCCCTGTGATCCATCCAATAGCTGACTGTGAGCATCCA
    CTTCTGTGTTTGCTAGGCCCCATTATAGCCTTGCAAGACAGCTATATCAGGGTCCTTTCAGCAAAATCTT
    GCTAGTGTGTGCAATGGTGTCAGCATTTGGGGGCTGATTATGGCATGGATCCCCGGATATGGCAGTCTCT
    AGATGGTCCATCCTTTCATCTCAGCTCCAATCTTTGTCTCTGTAACTCTTTCCATGGGTGTTTTGTTCCC
    AATTCTAAGAAGGAGCAAAGTGTCCACACATTTGTATTCATTACTCTTGAGTTTCATGTGTTTCCCTTGC
    ATTTCTATAATAAACCATAAGGAGTCTCTGCTCTACCAAGACCCGCTGCACACTCTGGTCAGTGTTGGGA
    ACTTTTCCCCTATTCCCTCTCTCCTATAACTCCGGGGCTACAGGGTGTCTCCTTTGGGTCCCGGTTGGGA
    GCTGTCTCTTCTCAACCCCCTGACTCATGGGTCAGAGGCCTAAATCTCCACCCAAGGCTGTGTGAAAAGC
    ACCGGGTGGTCTCCCCATATCTCCCTGTCCAGAGCACAGGAACTCTGGCCGGACATGGCATATTTTTCCT
    CCCCAACTTCTTCCCCGGCCTCCTCAGGGCTGTCCCTTCATTTTGTTCCCCACACATCTCCACATGGCTG
    CCACCATCCAATTGGCTGCTGAGGTCACACACTCTGTCTTTGTTCTTCTCTGAGTCTCATGCAGGTTTTG
    TGTTAGTAAATGCAAAAGTGCCTGCTGTGCATGCATGAACCATTACAGGCTGCTAGGCAGAACATGCTGA
    CTTGCTCCTGGATTATAAATCCATCACTGTGATAGCAGGACATATCCAGAAGACATTATTTAACAACACT
    CCTCTTCGTGCTCAACCTCTCATGGTCTTTCTACCTCCTCTTTCTTAGTGTTCTCTGAGCCTACATGAAA
    TGTGAACAAACCTGAAAAAAATCTCTGTGTCAAATACATAGTGTGGATTGGGAAGGAAAAACCCTGCACT
    TGTGTGTGGGCAGGGAAATGCTGGTGCTTTAACAGGACAAGTGCGGTGTGGCTAGAGAAAGTGATGAGAA
    GAAGGGAAGCATGAGAACTGGGTGGGTGGATCCCATGTCTACTGAAAATGCTATGTTGATGTCAAGATAT
    TGTGTCTTGTTACAAAAATTCATTGAGTACCTAGGTTATTCTCAGTATATAAAAGGGGAGGAATGAAACT
    AGAATTAAACACACACACACACACACACACACACACACACAAACACACACACACGAGTAGAGAAAAGAAA
    TAGTCATGAGTTGCCTAGTGAATGAAGAGCGGAAAATGGTGCAGTTAATAGGGATCATAGAAAGAACTGG
    GGGATGCACACGAATGGGTTTCCTGTAATTATGTCTTCATAGTAATCTCTGCTCAATAATCAGACAGTGA
    CAATGTATGCCTCATTTACAAGCCCTGATGGCCTCTCAGAGGAAAGCATCTCTCTGAAATGAATAAAGTT
    CAGAAATGTCCTAAGTGTCCCTGTCACAGGAAAGCAGTGTTGGTAACGTTTCCAGGAAGCTCAGTCTTTG
    TCAAATATCCACAACAAGAAGAAGCCATGTCTAGACAGACAACAGACTGGGAAAGACTGAGCTCATGTAT
    GGAAACACAACTATGTGTCATGCTTTTATTTTCACATTATACTGAGGGGATATGACATTATAGAAACACA
    GTTTGTTCTTTGCATATGCTGAGAGAGGAACTAGATGAAGTGTGCTAATTCTTAGCAATAAAAAGATATA
    CAAAGTGGTACTTTACTGTTTCAATTTTTGTAACTGGCATCATTGTCTTTGATATTTTATATCTTCCTTG
    AGCACTGCCTTCTCTCAAGTGTCCAACTTCAGAGTATGCTATAGCAGGAAGACTACCAAATAAGATTAAT
    TTTTTTGTACCCATGAAAAAATTATGTGCCCTGACCCCTGTTCTCTGAAAGAGGAGCCAAGTTCTGGATT
    CCCAGATCCTCATATTCATTGATCAGCAGTGAACACAGATCATTCACCATGGACATGGGGCTCATCTGGA
    TTCTCCTTATTGTTTTTAAAAAAGGATTTCATTGGGAAAAGCTGCCTCATATTTCTATGACCAGGAGAGG
    AAGATACAGCTAGAGACACAAGCCCCACCATGTAAAATCTGTATGGTACTCTCGTTTTCATTTTACAACA
    TTTTGCTTTTGCATTGCAAAGGAGCAATAAGGGTTCAGCAATCAGCACTGTAACTGTACTTTTAAAATTT
    CCACCTCTACCCTCTGTGTCCTCTATCTAGTGATAGAAAGTGAAATAAGCACATAAAGCATGGGACAAGA
    AGAACACACAGAGGAGCAGAGACTGGCTACAAGTGGCAGCCAGCATGGAGCTGGACAGAATTTAAAAAGA
    TAAAGAGAAATGTCATGGCATGAAGCAGTGACTTTTTCTGTTCACAATGCCTGCAAAAACTTTATGGAGC
    CTACTACTGCTGAGATATGCAGGTTTGGTTCCAGAGAGGATTTTCTGTTTTATTTAATTTGCTTTTAAAT
    TTTTCTTCTTTGTATAGTCTGTATTTTTAAAAAGGGGTTGAATGAATACCTAAGCAGTTGGAAAATTTGT
    GAGTAGAAACCGAGGGCCTGAGAAACAAATAAATCAAAATTTTGAAATGGCAAAAGAAAGGGTTCATTCT
    ATTTTCTCTTTTCAGAAAAGGTTGCAAAGTTGGATGTGCAAATGGAGATGTTATGGGTGGACTTGGAACA
    TCTTGGGAAGGAAGGAACCCAGGAAAAGAAAGAATGAAATGGCTCAGCCTCAATGACTGAGCAAGAGAGT
    TAGAGTCACCCACAGGAGAAAAGAAGAAAATAAACAGCACTGGGGTGAATTTGGGTCAGAGAAAACCTTA
    AAATGCCAGAGGACAATGTGATGAAAGAGACCATTTGAAAGAAAGGGTTAACCTCATTCCATCAGTCATG
    CCAGAGTTAGAGAATGGGCAGTCTGGAGTTACAAGATCACGATTAGCCTTCCCAGTATGTATACAGCATA
    TTCCTGATAATGAGATGAAGCAAAGTTTGACAAGGTAAGATTGTATCCTATAGTAATATATAGATCAAAG
    GCATGTTGCAGTAAATCTCCAACAGAAATAAGCCCGGGCAATGAAAACACAACTCAATTAATATGAATAC
    GTGCTGTGCACCTAGACTGGGCAGATCTACCACTCCACTACCATATGAGAGATCCCTTATAACTTGTGGT
    TTCTCCAGGCCAGCTGCTTCTGCTCTACTTTCCTTCCTCCTCCTCCTATGTCATCCTCTCCCTCACTCTT
    TCTCTCCCAAAACTTTCAGCTGCACCTTCCCCTCTTCATCCCCCAATCAGTGGCTCTAGCCTTTATTTAT
    AAATTAAGGTAGGAAGAAGGTTTATAGGAAATTACCTGAGTGTTGACGTGTTGATAACCCCACACAAGAG
    AACAAAATTAATATCAAATATGATTAGCTCCAGGGTTATATGCAACAAAGGCATGATAAAGTGGTCATGA
    TTTCATATGAGATGTACTGGACTCATGTGAAAAAACGTTTAAATACATGGGCTTACTCAAAATATAGACT
    CATACCCCCAAGATGGGAAGAGACTGGTAACATTTGTACTGGAGGCTGGTCTGCTGTGGCAGCCATTAAC
    ACAGTGGAGGGAAGAGCCTCAAAGAGTGAAATAGAGGAAGGGGAACAAATACAGTAAACCACCAGTTACT
    ACTTAAAGGGCAGTGTTCTGATGTCCTAGGGAGGGCACTCCAAAATACCAATGGCCTTATCCTTAAATTT
    ATTGTTTAATGAGCCTACACTGATCTCAGTGGCCCACTACTAAGGAGAAATGACAGGCACTTGAACAGCT
    GATATGAGAACAGCTGGAGGCACAGTATACAGAAGAGTTTACAAGCCTATGGAACTCCCCTGTATTTTTT
    TATGATGAGGAAATCTGAAAAATGTAAAATGTTGAAAGATTTAAGAGCAGTTATTAGAGTAGATCAACCA
    ATGAGTCTCTTGCAGCCTGGAATTCTTTTATCTTGTTTGTAACAACAATCAGGCTCCATAAATTATAAAC
    TATAAAAATTTACTTTATAAATTATAAATATATATATATGTGTGTCTGTAGATAGATAGATAGATAAATA
    GATAGATAGATAGATAGATAGATAGATAGATATATGGATTTGATGGTTGCAGCAAATTCTAATTGGTTCC
    AACAACCCTACTCACATAATACATTCATACAGGTTTGATAGATGGAGCTAAGTTTTAAATAAATTATTTT
    TCTCCCATGGCTTATATATACACCACCAAAATTCTCAAAAATTAAAATGTGATTGTGTTGTATTTTTCTT
    TTACTGAATGACTATAAAAAGTTACAACATTCTCCATAAATTTACATGAAAAAATATTATGTAGTGCAGG
    TAAAGAAAACAAATTGACCCAAGAATGGTGTATATTCATTACTAAGCAACTTTTTAGATTCACAAAGTGT
    GGGTAAACAAGGTAATTTTTTCAATCAGTTTTTTTTAACTGGCAGGCAGCAATTCAGAGTTACAATGAGA
    AGATTAATAATTTTATTGTGTATTTTAAAATAAATCTTACAAAAATATTAATAGAATCACAAATTTATAC
    CTTTGTATAAAAACAATCAGTCATTTCTACTTTAAGAAACAGAACTCACATCTACTCATCAATCATTTTA
    TTAAGTCATATTACAAAGCTGAGTGCTAACATGGGTATAAGAAAACCATAACCTTATTCACCAGCCCAGC
    GTCAAAAAGAAAAAAACCAGTCATATCAGCTGCTGCTTCAAGAGTTCTTGTTCCTTGACATTAACAAAAT
    CCCTAGCTTAACTATTAAATTTTTTTTCAAAACTTCTAATTGATCCCTTAGATAAATGTTTGTGCTAACC
    ATCGGGACACATCCCATGAGTTCTGAAGCAGTGTGTTGTTCTTCATGCATGGCCCTTTTGTAGAGCTGTG
    AGTGTAGGGGAAGAGGGGGGAGAGAGAGCCCGTGTCCAGCCAGAGATCCTGTGCTCTGGGCAGGCAGACA
    CGGGAGGACAACGGAACACTTTTCACTCGGCCTTTGGTGGGCATCTGGCTGTGTGAAGTTACTGACCCCA
    CATGGTGGGGGATGGACAAGGGGCAGCCCCTGGTACCAGGAGCCCCAGGGCTACACTCTCGGCCCCAGAT
    ATACAAGAAGAGGGCAGAGGGAGAGAGGCTCCCACACAGGCGAGAGTCCTTAGTCTGGTCTGTGGCTGGA
    GCAGGGGAATTCCTTCTGATTGGAGATTAGGCACAGCTGATTAGGGGAAAGCCTACCCCATCATCCAAGC
    ACAATGGACTTTGAGGAACAGAGCCAGTCTAAGCTTTTATAGCTTTATGGTAGAAAGGCAGGGAGAAAGG
    AGAGAATGTGGAGAGAGACAGAGAGAGACTGGCCATGGCCAAGAGGAGGGAAGGGGGAAGAGAGAGAAAC
    AGGAAAAGCTAGAAAGTAAGATAAGAGAAAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGCAAAGTG
    GGGCCAAGCAGCCCCTTTTCTAGTGAGCTTGTCATCTCACAGTTGCTAGGTAACTGGGGAGGAGTTTAGT
    CTGAAGGTCAGAAGCTTGGGACCTTGTGTATGTGACTACTAACCACAGCTTCTCCTGTCGGGACTGTGGG
    AGCAGTAACTTTGACAGGAGACAGGGATCCAGGAGACATGAGGGAACACCTTCTGTCCCACATAGGTGGA
    ATCGGTTGTACCAGGGTTCAGAACTCAACTCCACTGGAGACCAGCCTATGTGTGCATAGCCCATTGCCCT
    ACAGCCTTTGACATTCTACAGAGTTCTGAGACTGGTAGAGATACAAAATAGCTAGGATTACACAGAGAAA
    CACTGGCTTGACAATAAAACTTCCAAAACAAATATAAATGAATAAATACACAAATTGGAGCAAGTGAACT
    TGCACTAAATCCATCAAAACTTGCACAGGATGGCTGTCTCCATAATGTTCAGATCTACTTCTATTAATGT
    CATTAAGAAGACAAAGATTATGCACAAAAAGAAACAAAACAAGAAACCCAAAAGGTAAAGGCTCCAATTG
    TAAGTAATAGACCCACAGTCTCTGAGGCCTGACTGTGCAGGTCCTCACCTTTGGTAATCAGCACTGAATG
    CAGACCACTCCTCATGGACTTTGGGCTCAGGTTTGTTTTCCTTGTCCTTATTTTAAAAGGTAATTCATAG
    AAATGAGATCCTGCCAGTATTGTGTACATGAGAAATAGAAAAATTGGTTTTCTTTGCTCTATTTTGTTTT
    GTTTGGTTAATGACAGTTTCCAAATCAGCATTCTTTGCTTTGAGGTGCCCAGTGTGAGGTGAAGCTGGTA
    GGGTCAGGGCAGCCTGGAGGGTCCCTGAAACACTCCTGTGCAGCCTCTGTAGTCACTGTGAGTGACTACT
    GAATGACCTGGGTCCTTCAGGCTCTAAAGAAGGGGCTGGAGAGGGTGGAAATAATTTTTAATGGTGGAGG
    TAGCACCTATTATCCAGACACCATGAAGGGCTGATTCACCATCTACAGAGATGATGCCAGAAACACACTT
    TACCTGAAAATAAACAGTCTGAGGTCTGAGTACACAGCCATGTGTGTGTGTGTGTGTGTGTGTGTGTGTG
    TGTGTGTGTGTGTGTGTGTGTGTGAGTGTGTATTCATATATATATATATAGTGTGTATGTATATTCATAT
    ATATATGTATATTCATAAGGTTGAGTAAATGGACTTAAACTGATTCCATCACAACTTCCATGGGATGGAT
    ATTTCCCAGTGTTAAGACCTGTCAACATCACTGTCATTCAGGAGACAATGATTATGCACAACAAAACAAG
    AAACCCAAAAGCAGAGGACTCCAATTACAATAGACCCAGACCCACAGTCTCTGAAGATTGACTGTACAGT
    TCAACCCAGCCCTGTACTTCTCTTCCCTAGAATTTACATTACTGAGTAACTGAGGAAAGCTCTACAATAT
    CTGTTCTCTATAGTGGTCAACACCTCCAAACACAGGTTACCCATATTCATGCCTGCCTTCTGCTACACTT
    CTTGCCATAATGTAGACTACTTCAGCCTATTTTGTACTCCAGTTAACGAAACTCAAGACTAGCTGCATGT
    TAGTCCTTATTCTGAAATATTATGAACAGGTGACCTCCCATCATTCACCAATGCAATAATCATATTTAGG
    AATATGAGGTTTTATGAGATATAAGCACAAGGGAGAGAAAGTAAGAAAACTACATAGATATATAGACTGA
    CATAAATCAAGACTTGCATGAGCTAGTGCCCAAGTACCATGTCCCTAAGTGGCAAGGAGTATCTTTTGAG
    CCTAGTGAGATGAGGTACAAATCGGACTCTTACATCTTTTTAGATAAACATGTGAGATCAATGGACTAAA
    GGCGTGAGCTGGGCTTCCTTGCAATCCATTTTCCATACAAGATAACAATAGATCTGGCTCCACAGACACG
    ATGAGAATAGTCTTAATTAGTTCTTTAAGTAGAATGACTGATCACTAAGAGCCCAATTCCATACTAAATA
    CTCTTCTGGATTATGCACAGATAAAAATTGCACATAGGGCATGGGGCACTGATCTCCCTGCACTACATGA
    ATGGGGGCTCATTTACTAAATGTTCCCATGTTTCTTCCTAGTGCTGCACAGAGCAAATCCTACAACTTCC
    TGCTTGTCTACAATGTAAACTCCAGACAGTACCAGAAAATCATTCCTTATGTTCCTCTCCAGGTGCTTCA
    ACAAGCACAGTGCAAATTTCTGTCACCCTG
  • Molecule Role : Vaximmutor
  • Additional Molecule Role : Vaximmutor
  • Related Vaccine(s): C. jejuni DNA vaccine pcDNA3.1(+)-cadF , C. jejuni DNA vaccine pcDNA3.1(+)-peblA , C. jejuni FspA1 protein vaccine , C. jejuni MBP-FlaA protein vaccine
IV. Vaccine Information
1. Avirulent Salmonella vaccine strain carrying C. jejuni cjaA gene
a. Vaccine Ontology ID:
VO_0004121
b. Type:
Recombinant vector vaccine
c. Antigen
Three C jejuni genes [cjaA ( cj0982c ) , cjaC ( cj0734c ) and cjaD ( cj0113 )] encoding highly immunogenic proteins which are conserved among different Campylobacter serotypes have been introduced into avirulent Salmonella enterica sv. Typhimurium (chi 4550 and chi 3987) strains of two different serotypes (UK-1 and SR) (Wyszynska et al., 2004). C. jejuni 72Dz/92 cjaA gene encodes a highly immunogenic protein which is conserved among different Campylobacter serotypes (Wyszynska et al., 2004). The surface antigen CjaA of Campylobacter jejuni is supported by the presence of an upstream gene with significant homology to ATP binding proteins (Martin et al., 1999).
d. Gene Engineering of CjaA from C. jejuni RM 1221
  • Type: Recombinant vector construction
  • Description: pUWA10A plasmid carrying CjaA was ligated into the pYA3341 plasmid and then transformed into E. coli χ6097 competent cells. It was later introduced by electroporation into two S. enterica sv. Typhimurium (χ3987 and χ4550) strains (Wyszynska et al., 2004).
  • Detailed Gene Information: Click here.
e. Gene Engineering of CjaA from C. jejuni
  • Type: Recombinant protein preparation
  • Description: pUWM80 carrying cjacC was cut and ligated into pYA3341. The resulting plasmid was tranformed into E. coli χ6097 competent cells, and later introduced by electroporation into two S. enterica sv. Typhimurium (χ3987 and χ4550) strains (Wyszynska et al., 2004).
  • Detailed Gene Information: Click here.
f. Adjuvant: E. coli heat-labile toxin, LT
g. Preparation
Three gene libraries of Campylobacter jejuni 72Dz/92 DNA were prepared using lambda gt11, pSupercos and pWSK129 cloning vectors. Screening of the libraries revealed several immunoreactive clones (Pawelec et al., 1997).
h. Virulence
The vaccine vector is avirulent Salmonella enterica sv. Typhimurium (chi 4550 and chi 3987) strains of two different serotypes (UK-1 and SR) (Wyszynska et al., 2004).
i. Description
The constitutive expression of all analysed genes as measured by Western immunoblot technique was independent of the particular host strain. Specific rabbit anti-rCjaA antibody reacted not only with CjaA but also with other solute-binding proteins, components of the ABC transport system (CjaC protein), chosen as the protective antigen for animal experiments. Chickens orally immunized with Salmonella expressing Campylobacter cjaA gene developed serum IgG and mucosal IgA antibody responses against Campylobacter membrane proteins and Salmonella OMPs. Protection experiments show that chicken immunization with avirulent Salmonella carrying Campylobacter cjaA gene greatly reduces ability of heterologous wild type C. jejuni strain to colonize the bird cecum (Wyszynska et al., 2004).
j. Chicken Response
  • Host Strain: Commercial broiler chickens (Gallus gallus)
  • Vaccination Protocol: Commercial broiler chickens were obtained from the local hatchery on the day of hatch. Briefly, chickens deprived of food and water for 4 h were immunized orally with 100 μl of 109 CFU/ml of Salmonella χ3987 carrying pUWM251 (cjaA). Booster doses were administrated 2 weeks after primary immunization. Following vaccination, chickens were observed for the development of diarrhoea and other potential adverse side effects (Wyszynska et al., 2004).
  • Persistence: There was high anti-Campylobacter IgG titer present at the first time point (week 0), dropping at weeks 2 and 4. A moderate increase of Campylobacter specific IgG level was observed at week 6 followed by significant increase of anti-Campylobacter IgG level at week 8. Serum IgG titers to Campylobacter whole cell lysates followed a similar pattern, as those measured with membrane antigens, with respect to time, although the increase of anti-Campylobacter IgG titer at week 8 was not so significant. The results suggest that high IgG titer observed at the 0 week reflects maternally derived immunity while increasing serum IgG titers at 6 and 8 weeks is a consequence of vaccination (Wyszynska et al., 2004).
  • Immune Response: Mucosal anti-Salmonella and anti-Campylobacter IgA antibodies were present in samples taken from chickens inoculated with Salmonella/pUWM251 at every tested time point. In contrast to serum IgG, the kinetics of IgA responses to both antigens were similar. In intestinal secretions the level of the antibodies dropped at week 2 and then peaked 2 weeks after the booster. During the next 2 weeks, IgA titers decreased and maintained at almost the same level during the remainder of the experiment. Since IgA antibodies directed towards both antigens were present in the intestinal fluids of 1-day-old birds, similarly to IgG antibodies, IgA present at 0 week were maternally derived. In intestinal secretions, taken from different parts of chicken gut (rectum and jejunum), levels of IgA antibodies to Campylobacter and Salmonella antigens also peaked 2 weeks after the secondary immunization. Intestinal IgA titers to Campylobacter whole cell lysates followed the similar patterns as those measured with membrane antigens (Wyszynska et al., 2004).
  • Side Effects: No adverse side effects were observed (Wyszynska et al., 2004).
  • Challenge Protocol: Two weeks after booster, all chicks received a dose containing approximately 2×109 CFU/ml of the wild type C. jejuni/pUOA18 strain in 0.1 ml PBS with gelatine. Campylobacter colonization was confirmed on days 3, 6, 9 and 12 after the challenge. In each instance, four chicks were euthanized, the intestine was excised and the C. jejuni present in chicken cecal contents were enumerated by plating (Wyszynska et al., 2004).
  • Efficacy: Protection experiment showed that chicken immunization with avirulent Salmonella carrying Campylobacter cjaA gene greatly reduced the ability of heterologous wild type C jejuni strain to colonize the bird cecum (Wyszynska et al., 2004).
  • Description: It is well documented that poultry and poultry products are the major source of human campylobacteriosis and salmonellosis. The avirulent Salmonella vaccine strains expressing Campylobacter antigen can be potentially used as a bivalent chicken vaccine (Wyszynska et al., 2004).
2. C. jejuni DNA vaccine pcDNA3.1(+)-cadF
a. Vaccine Ontology ID:
VO_0011545
b. Type:
DNA vaccine
c. Status:
Research
d. Antigen
C. jejuni fibronectin binding protein cadF
e. Gene Engineering of CadF
  • Type: DNA vaccine construction
  • Description: The chitosan-DNA vaccines was prepared by embedding pcDNA3.1(+)-cadF with chitosan (Zheng et al., 2007).
  • Detailed Gene Information: Click here.
f. Vector:
pcDNA3.1(+) (Zheng et al., 2007)
g. Immunization Route
Intranasal
h. Mouse Response
  • Host Strain: BALB/c
  • Vaccination Protocol: BALB/c mice were intranasally immunized in a four-dose primary series (7 d intervals) at doses of 60 microg chitosan-DNA vaccines each time (Zheng et al., 2007).
  • Challenge Protocol: Mice were attacked repeatedly through intragastric administration of C. jejuni HS:19 at the 8th week after the immunization and protective efficacy was determined by detecting the degrees of protection afforded against C. jejuni invaded (Zheng et al., 2007).
  • Efficacy: The mice immunized with chitosan-DNA vaccines have generated high levels of IgA and IgG from the sera and IgA from the intestinal secretions and the P/N value went up to 20.58, 30.13 and 6.87 respectively. Moreover the chitosan-DNA vaccines induced strongest level of protection in BALB/c mice against challenge with C. jejuni HS:19 strain and the protective efficacies was 93.70. The results of this study indicate that the chitosan-DNA vaccines could induce significant protective immunity against C. jejuni challenge in the mice model (Zheng et al., 2007).
3. C. jejuni DNA vaccine pcDNA3.1(+)-peblA
a. Vaccine Ontology ID:
VO_0011488
b. Type:
DNA vaccine
c. Status:
Research
d. Antigen
C. jejuni bifunctional adhesin/ABC transporter aspartate/glutamate-binding protein
e. Gene Engineering of Peb1A
  • Type: DNA vaccine construction
  • Description: The chitosan-DNA vaccine was prepared by embedding pcDNA3.1(+)-peblA with chitosan (Zheng et al., 2007).
  • Detailed Gene Information: Click here.
f. Vector:
pcDNA3.1(+) (Zheng et al., 2007)
g. Immunization Route
Intranasal
h. Mouse Response
  • Host Strain: BALB/c
  • Vaccination Protocol: BALB/c mice were intranasally immunized in a four-dose primary series (7 d intervals) at doses of 60 microg chitosan-DNA vaccines each time (Zheng et al., 2007).
  • Immune Response: The mice immunized with chitosan-DNA vaccines have generated high levels of IgA and IgG from the sera and IgA from the intestinal secretions and the P/N value went up to 20.58, 30.13 and 6.87, respectively (Zheng et al., 2007).
  • Challenge Protocol: Mice were attacked repeatedly through intragastric administration of C. jejuni HS:19 at the 8th week after the immunization and protective efficacy was determined by detecting the degrees of protection afforded against C. jejuni invaded (Zheng et al., 2007).
  • Efficacy: The chitosan-DNA vaccines induced strongest level of protection in BALB/c mice against challenge with C. jejuni HS:19 strain and the protective efficacies was 93.70 (Zheng et al., 2007).
4. C. jejuni FlaC protein vaccine
a. Vaccine Ontology ID:
VO_0011495
b. Type:
Subunit vaccine
c. Status:
Research
d. Antigen
C. jejuni flagellin subunit protein FlaC
e. Gene Engineering of FlaC
  • Type: Recombinant protein preparation
  • Description: The flaC gene from C. jejuni 81-176 was expressed in Escherichia coli as hexahistidine-tagged proteins in pET-19b. Strains of BL21(DE3) containing each clone were grown in Luria broth containing 100 μg/ml ampicillin and proteins were purified by nickel chromatography under native conditions (Baqar et al., 2008).
  • Detailed Gene Information: Click here.
f. Adjuvant: LTR192G
g. Immunization Route
Intranasal
h. Mouse Response
  • Host Strain: BALB/c
  • Vaccination Protocol: Mice were lightly anesthetized with isoflurane, and 30 μl of phosphate-buffered saline (PBS) alone or PBS containing 5 μg (7 to 10 mice per group) or 25 or 100 μg (10 to 18 mice per group) of protein was applied a drop (5 to 6 μl) at a time to the external nares. A total of three vaccinations for each dose level of each protein were delivered at 2-week intervals. To determine efficacy, animals receiving 100 μg of each protein alone or with 1 μg of LTR192G as the adjuvant were challenged with homologous or heterologous strains of C. jejuni. Following vaccination, animals were observed for two consecutive days for the development of vaccine-associated side effects (Baqar et al., 2008).
  • Challenge Protocol: Twenty-eight days following the last vaccination, mice were intranasally challenged with 3 × 109 CFU of C. jejuni 81-176 or CG8486 (Baqar et al., 2008).
  • Efficacy: FlaC provided an 18% protection against disease from C. jejuni 81-176 (Baqar et al., 2008).
5. C. jejuni FspA1 protein vaccine
a. Vaccine Ontology ID:
VO_0011485
b. Type:
Subunit vaccine
c. Status:
Research
d. Antigen
C. jejuni flagellum-secreted protein FspA1
e. Gene Engineering of FspA1
  • Type: Recombinant protein preparation
  • Description: The fspA1 gene from C. jejuni 81-176 was expressed in Escherichia coli as hexahistidine-tagged proteins in pET-19b. Strains of BL21(DE3) containing each clone were grown in Luria broth containing 100 μg/ml ampicillin and proteins were purified by nickel chromatography under native conditions (Baqar et al., 2008).
  • Detailed Gene Information: Click here.
f. Adjuvant: LTR192G
g. Immunization Route
Intranasal
h. Mouse Response
  • Host Strain: BALB/c
  • Vaccination Protocol: Mice were lightly anesthetized with isoflurane, and 30 μl of phosphate-buffered saline (PBS) alone or PBS containing 5 μg (7 to 10 mice per group) or 25 or 100 μg (10 to 18 mice per group) of protein was applied a drop (5 to 6 μl) at a time to the external nares. A total of three vaccinations for each dose level of each protein were delivered at 2-week intervals. To determine efficacy, animals receiving 100 μg of each protein alone or with 1 μg of LTR192G as the adjuvant were challenged with homologous or heterologous strains of C. jejuni. Following vaccination, animals were observed for two consecutive days for the development of vaccine-associated side effects (Baqar et al., 2008).
  • Challenge Protocol: Twenty-eight days following the last vaccination, mice were intranasally challenged with 3 × 109 CFU of C. jejuni 81-176 or CG8486 (Baqar et al., 2008).
  • Efficacy: Immunization with FspA1 resulted in 57.8% protection without adjuvant or 63.8% protection with adjuvant against homologous challenge with 81-176 (Baqar et al., 2008).
  • Host Gene Response of IgA
    • Gene Response: FspA1 induced significantly high levels of fecal IgA in mice 7 days after last vaccination. These results are compared to PBS vaccinated mice (Baqar et al., 2008).
    • Detailed Gene Information: Click here.
  • Host Gene Response of IgG
    • Gene Response: FspA1 induced significantly high levels of serum immunoglobulin G (IgG) in mice 18-20 days after 3rd vaccination. These results are compared to PBS vaccinated mice (Baqar et al., 2008).
    • Detailed Gene Information: Click here.
6. C. jejuni FspA2 protein vaccine
a. Vaccine Ontology ID:
VO_0011487
b. Type:
Subunit vaccine
c. Status:
Research
d. Antigen
C. jejuni flagellum-secreted protein FspA2
e. Gene Engineering of FspA2
  • Type: Recombinant protein preparation
  • Description: The fspA2 gene from C. jejuni CG8486 were expressed in Escherichia coli as hexahistidine-tagged proteins in pET-19b. Strains of BL21(DE3) containing each clone were grown in Luria broth containing 100 μg/ml ampicillin and proteins were purified by nickel chromatography under native conditions (Baqar et al., 2008).
  • Detailed Gene Information: Click here.
f. Adjuvant: LTR192G
g. Immunization Route
Intranasal
h. Mouse Response
  • Host Strain: BALB/c
  • Vaccination Protocol: Mice were lightly anesthetized with isoflurane, and 30 μl of phosphate-buffered saline (PBS) alone or PBS containing 5 μg (7 to 10 mice per group) or 25 or 100 μg (10 to 18 mice per group) of protein was applied a drop (5 to 6 μl) at a time to the external nares. A total of three vaccinations for each dose level of each protein were delivered at 2-week intervals. To determine efficacy, animals receiving 100 μg of each protein alone or with 1 μg of LTR192G as the adjuvant were challenged with homologous or heterologous strains of C. jejuni. Following vaccination, animals were observed for two consecutive days for the development of vaccine-associated side effects (Baqar et al., 2008).
  • Challenge Protocol: Twenty-eight days following the last vaccination, mice were intranasally challenged with 3 × 109 CFU of C. jejuni 81-176 or CG8486 (Baqar et al., 2008).
  • Efficacy: Immunization with FspA2 provided 38.4% (without adjuvant) or 47.2% (with adjuvant) protection against disease from homologous challenge with CG8486 (Baqar et al., 2008).
7. C. jejuni MBP-FlaA protein vaccine
a. Vaccine Ontology ID:
VO_0011493
b. Type:
Subunit vaccine
c. Status:
Research
d. Antigen
C. jejuni structural flagella protein flaA and maltose-binding protein MBP
e. Gene Engineering of FlaA from C. jejuni 81-176
  • Type: Recombinant protein preparation
  • Description: Purification schemes were essentially as recommended by NEB. DH5α containing the flagellin-MBP fusion was grown overnight in 10 ml of rich medium (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 2 g of glucose/liter) supplemented with 100 μg of ampicillin per ml and used to inoculate a fresh 1-liter culture of the same medium. This culture was grown with shaking at 37°C to an optical density at 600 nm of 0.5, and IPTG (isopropyl-β-d-thiogalactoside; Gibco, Gaithersburg, Md.) was added to a final concentration of 0.3 mM (Lee et al., 1999).
  • Detailed Gene Information: Click here.
f. Adjuvant: LTR192G
g. Immunization Route
Intranasal
h. Mouse Response
  • Host Strain: BALB/c
  • Vaccination Protocol: Mice were anesthetized with methoxyflurane (Metofane; Pitman-Moore, Mundelein, Ill.) and immunized intranasally with 30 to 35 μl of fusion protein by using a micropipette. The doses used were 0, 3, 6, 12, 25, or 50 μg of fusion protein in phosphate-buffered saline (PBS), either alone or in combination with 5 μg of the genetically modified heat-labile enterotoxin of E. coli, designated LTR192G as an adjuvant. A second dose was administered 8 days after the first vaccination (Lee et al., 1999).
  • Challenge Protocol: Mice were intranasally challenged with 2 × 109 C. jejuni bacteria/mouse 26 days after the second vaccination, and the animals were monitored for sickness and death for 5 days (Lee et al., 1999).
  • Efficacy: The protective efficacies of a 50 microgram of MBP-FlaA plus LT(R192G) dose against disease symptoms and intestinal colonization were 81.1 and 84%, respectively. When mice which had been immunized with 50 microgram of MBP-FlaA plus LT(R192G) intranasally were challenged orally with 8 x 1010, 8 x 109, or 8 x 108 cells of strain 81-176, the protective efficacies against intestinal colonization at 7 days postinfection were 71.4, 71.4, and 100%, respectively (Lee et al., 1999).
  • Host Gene Response of IgA
    • Gene Response: Stimulation of FlaA-specific intestinal secretory IgA (sIgA) responses required immunization with higher doses of MBP-FlaA (>/=25 microgram) or coadministration of lower doses with the adjuvant. IgA titers were significant 7 days after immunization as compared to PBS-vaccinated mice (Lee et al., 1999).
    • Detailed Gene Information: Click here.
  • Host Gene Response of IgG
    • Gene Response: The full range of MBP-FlaA doses were effective in eliciting significant antigen-specific serum immunoglobulin G (IgG) responses, and these responses were enhanced by adjuvant use, except in the highest dosing group. The results were compared to PBS vaccinated mice 7 days after immunization (Lee et al., 1999).
    • Detailed Gene Information: Click here.
8. C. jejuni PorA protein vaccine
a. Vaccine Ontology ID:
VO_0004203
b. Type:
Subunit vaccine
c. Status:
Research
d. Antigen
Recombinant PorA protein
e. Gene Engineering of PorA
  • Type: Recombinant protein preparation
  • Description:
  • Detailed Gene Information: Click here.
f. Adjuvant: EtxB vaccine adjuvant
g. Immunization Route
orally
h. Mouse Response
  • Host Strain: BALB/c
  • Vaccination Protocol: BALB/c mice were orally vaccinated twice at a weekly interval with the purified GST-PorA fusion protein combined with a modified heat-labile enterotoxin of Escherichia coli toxin, LT R192G, as an adjuvant. Mice immunized with LT R192G or PBS (pH 7.2) alone were included as controls. Vaccination was done by using a stainless steel, curved-ball-tip feeding needle (20 gauge, 1.5-in. long; Popper and Sons, Inc., New Hyde Park, NY). Just prior to vaccination, gastric acidity was neutralized with two doses (0.5 ml each) of a 5% sodium bicarbonate (pH 8.5) solution given as an oral gavage at an interval of 15 min. An amount of 300 µg of the GST-PorA fusion protein mixed with 5 µg of LT R192G in a total volume of 300 µl in PBS (pH 7.2) was given to each of nine animals. Another group of mice was each fed with 5 µg of LT R192G and yet another group each fed with 1x PBS (pH 7.2) (Abimiku et al., 1989; Islam et al., 2010)
  • Challenge Protocol: Mice were challenged with bacterial culture 3 weeks after the second vaccine dose, immediately after tail vein blood collection. A 48-h bacterial culture grown on campylobacter agar was suspended in PBS (pH 7.2) to a concentration of 2 x 109 CFU per ml, and 0.5 ml of the suspension was orally fed to the mice immediately after neutralization of the gastric acidity (Islam et al., 2010).
  • Efficacy: The vaccine produced robust antibody responses against both antigens in serum and secretion. Since strain C31 was a poor colonizer, homologous protection could not be studied. The protective efficacies of heterologous strains were 43% (for strain 48, P < 0.001), 29% (for strain 75, P < 0.005), and 42% (for strain 111, P < 0.001) for the 9-day period compared to control mice given phosphate-buffered saline (Islam et al., 2010).
9. deltaphoP/Q S. typhimurium strains expressing PEB1-ss
a. Vaccine Ontology ID:
VO_0004124
b. Type:
Recombinant vector vaccine
c. Antigen
Campylobacter PEB1 is an adherence factor that is highly immunogenic in humans (Sizemore et al., 2006).
d. Preparation
Three live, attenuated deltaphoP/Q Salmonella enteric serovar Typhimurium strains expressing PEB1 minus its signal sequence (PEB1-ss) from three different plasmids were prepared. The three plasmids include a pBR-based asd plasmid, an arabinose-based runaway plasmid, which each expressed PEB1-ss in the bacterial cytosol, and a PEB1::HlyA fusion plasmid that directs secretion of PEB1-ss into the extracellular milieu (Sizemore et al., 2006).
e. Virulence
PEB1 is an adherence factor that is highly immunogenic in humans (Sizemore et al., 2006).
f. Description
PEB1, the major cell-binding factor of Campylobacter jejuni, is a homolog of the binding component in Gram-negative nutrient transport systems (Prokhorova et al., 2006).
g. Mouse Response
  • Host Strain: Female, BALB/c Mouse Specific Pathogen Free (MSP) 7–9 weeks of age from Taconic
  • Vaccination Protocol: Mice that had been acclimated for 5 days were vaccinated with freshly prepared inocula on days 1 and 12 of the experiment by pipet feeding. The target concentration for this experiment was 1–2.5E8 in 50 μl (Sizemore et al., 2006).
  • Persistence: MGN4735 was able to colonize the intestine to a high level over the entire test period of 9 days post-inoculation while those mice that survived challenge with 81-176 cleared the infection (Sizemore et al., 2006).
  • Immune Response: Serum IgG responses specific for PEB1-ss were induced by pBR-derived and runaway plasmids, with 100 and 90% seroconversion, respectively, at a 1:500 dilution of anti-sera as measured by Western blot analysis, while the PEB1-ss::HlyA fusion plasmid induced serum IgG in only 20% of the mice (Sizemore et al., 2006).
  • Side Effects: no adverse side effects were observed
  • Challenge Protocol: Challenge of vaccinated BALB/c mice was based on the model developed by Baqar et al. (Baqar et al., 1995b). C. jejuni strain 81-176 served as the challenge strain in the oral model (Sizemore et al., 2006).
  • Efficacy: Although significant levels of anti-PEB serum IgG were induced, no protection against oral Campylobacter jejuni challenge was observed (Sizemore et al., 2006).
  • Description: Three live attenuated ΔphoP/Q Salmonella enteric serovar Typhimurium strains expressing PEB1 minus its signal sequence (PEB1-ss) from three different plasmids: a pBR-based asd plasmid, an arabinose-based runaway plasmid, which each expressed PEB1-ss in the bacterial cytosol, and a PEB1::HlyA fusion plasmid that directs secretion of PEB1-ss into the extracellular milieu are described above (Sizemore et al., 2006).
10. Inactivated C. jejuni whole-cell (CWC)
a. Vaccine Ontology ID:
VO_0004100
b. Type:
Inactivated or "killed" vaccine
c. Antigen
inactivated whole-cell Campylobacter jejuni (CWC)
d. Adjuvant: E. coli heat-labile toxin, LT
  • VO ID: VO_0001322
  • Description: 25 mg of a mucosal adjuvant, the heat-labile enterotoxin of Escherichia coli (LT) has been used (Baqar et al., 1995b).
e. Preparation
The CWC vaccine is prepared from the 81-176 strain of C. jejuni and consists of a 1:1 mixture of heat (60 C, 60 min)- and formalin (0.02 M)-inactivated Campylobacter whole cells. Each vaccination group receives oral doses of CWC vaccine containing bacterial cells alone or in combination with 25 mg of LT. Just prior to vaccination, gastric acidity is neutralized by 2 doses of a 5% NaHCO3 solution (pH 8.5) with a 15-min interval (Baqar et al., 1995b).
f. Virulence
Volunteers given the CWC vaccine have similar IgA-ASC and sIgA responses and in vitro induction of IFN to Campylobacter antigens (Walker, 2005).
g. Description
Whole-cell vaccine formulations deserve further evaluation as candidate vaccines and the potential value of mucosal adjuvants, like LT, in enteric vaccine development are not in doubt (Baqar et al., 1995).
h. Mouse Response
  • Host Strain: BALB/c
  • Vaccination Protocol: Mice were orally immunized in a three-dose primary series with CWC vaccine particles alone or in combination with a mucosal adjuvant, the heat-labile enterotoxin of Escherichia coli (LT) (Baqar et al., 1995b).
  • Persistence: Ninety percent of control animals remain colonized for 9 days post-challenge. A similar colonization pattern is observed in mice vaccinated with LT alone. Clearance of challenge organisms from mice receiving CWC vaccine alone appear to be dependent on the amount of vaccine administered. Although 80% of the mice receiving the highest vaccine dose cleared the infection within 3 days of challenge, the remaining 20% were colonized for the remainder of the study period (Baqar et al., 1995b).
  • Immune Response: Campylobacter-specific intestinal IgA responses are dependent on the use of LT, whereas IgA and IgG responses in serum are not. Intestinal lavage fluid collected from sham-immunized animals at 7 d post-vaccination have no detectable levels of Campylobacter- or LT-specific secretory IgA (sIgA) or IgG. When CWC is administered over a 10,000-fold range of doses, only 20% of the mice immunized with the low or intermediate doses mount a significant Campylobacter-specific sIgA response. In contrast, administering the same doses with LT resulted in a substantial enhancement of the sIgA response to vaccine-associated Campylobacter antigens in all vaccination groups (Baqar et al., 1995b).
  • Side Effects: Both the CWC and CWC-LT vaccine formulations are well tolerated, with no weight loss or signs consistent with toxicity in immunized animals. Histopathological examination of the intestinal mucosa and other organ systems following vaccination fails to detect evidence of tissue damage or severe inflammation (Baqar et al., 1995b).
  • Challenge Protocol: At ~4 wks post-vaccination, mice were challenged orally with C. jejuni, and duration of colonization was determined by the monitoring of fecal shedding. Efficacy was determined by measuring the degree of protection afforded against intestinal colonization and systemic dissemination of challenge organisms (Baqar et al., 1995b).
  • Efficacy: Colonization resistance was induced over a broad range of vaccine doses when LT was included. However, only the highest dose of CWC alone gave comparable levels of protection. Both formulations provided equivalent protection against systemic spread of challenge organisms. These results indicate that both whole-cell vaccine formulations deserve further evaluation as candidate vaccines and also highlight the potential value of mucosal adjuvants, like LT, in enteric vaccine development (Baqar et al., 1995b).
  • Description: Inactivated Campylobacter whole-cell vaccines (CWC) must be given orally in large doses to be effective. Drawbacks could be overcome by the coadministration of LT (Baqar et al., 1995b).
i. Ferret Response
  • Host Strain: Mustela putorius furo
  • Vaccination Protocol: Food was withheld prior to challenge. The vaccine dose was administered orally via a pediatric nasogastric tube. After 60 min, animals were administered paragoric i.p. to slow peristalsis. Drinking water was supplemented with tetracycline for three days prior to rechallenge to decrease the competing intestinal microflora (Burr et al., 2005).
  • Persistence: An oral regimen delivered on days 0, 3, 5, and 7 provided enhanced protection following oral homologous challenge delivered at Days 0 and 14. The percent protection observed was 100% in previously infected ferrets, 50% in a two-dose CWC group, and 89% in a four-dose CWC group. Protection was afforded through vaccination with sufficient inactivated whole cell vaccine, but protection was obtained regardless of the inclusion of the adjuvant LTR192G (Burr et al., 2005).
  • Immune Response: Antibodies generated during natural infection in ferrets by either strain 81-176 or strain VC167 appeared to react more strongly to glycosylated flagellins isolated from Campylobacter spp . than to unglycosylated , recombinant flagellins isolated from E coli . (Lee et al., 1999)
  • Side Effects: Following infection, animals were monitored three times daily for signs of diarrhea, dehydration, appetite and water consumption. Rectal swabs from each ferret were cultured for C. jejuni by direct plating on C. jejuni selective agar. At the appropriate time, blood samples were obtained by bleeding the animals from the jugular vein while under light anesthesia using acepromazine-ketamine. Collected sera were assayed for specific anti-Campylobacter immunoglobulin levels. At the conclusion of each experiment, animals were lightly anaesthetized with acepromazine-ketamine then euthanized by intracardiac injection of sodium pentobarbital (Burr et al., 2005).
  • Challenge Protocol: Food was withheld prior to challenge. Animals received ketamine plus acepromazine i.m. Following sedation, the vaccine or challenge dose was administered orally. Animals being fed live organisms were administered paragoric i.p. For secondary challenge the procedure was otherwise identical, except that NaHCO3 was delivered prior to challenge. In addition, drinking water was supplemented with tetracycline prior to rechallenge. All challenge doses were monitored by plate counts (Burr et al., 2005).
  • Efficacy: Ferrets show some conservation of protection against disease induced by Campylobacter. Upon rechallenge, homologous protection was 67-84%. Heterologous challenge resulted in 67-80% protection. These data indicate that the strain chosen for use in humans protects against disease caused by two of the major serotypes responsible for human disease (Burr et al., 2005).
  • Description: The strong IgG responses seen upon rechallenge of vaccinated or previously challenged animals may not be protective in themselves but could indicate the magnitude of other unmeasured antibody responses following immunization. Whatever the exact nature of the protective immune responses associated with the CWC vaccine, it is clear that the vaccine offers protection which may be relatively conserved among clinically important serotypes of Campylobacter (Burr et al., 2005).
j. Chicken Response
  • Vaccination Protocol: A hybrid protein was administered as a vaccine to chickens either orally or i.m. Alimentary secretions were collected , and specific antibodies were assayed by western blot analyses (Khoury et al., 1995).
  • Persistence: Seventy-two percent of the birds vaccinated orally with 1000 micrograms protein showed detectable antibodies against C jejuni flagellin in the excreta . None of the control birds produced detectable antibody to this antigen (Khoury et al., 1995).
  • Immune Response: Chickens orally immunized developed serum IgG and mucosal IgA responses against Campylobacter membrane proteins, as measured by an ELISA test. Protection experiments show that chicken immunization greatly reduces the ability of heterologous wild type C jejuni strain to colonize the bird cecum (Wyszynska et al., 2004).
  • Side Effects: No adverse side effects have been associated with this vaccine in chickens.
  • Challenge Protocol: For trials to demonstrate clearance of Campylobacter, groups of chickens were vaccinated with the hybrid protein at 2 and 4 wk of age and challenged at 3 wk with an excess of Cjejuni. The number of birds that remained colonized at 5 wk of age was significantly lower among the vaccinated birds than among controls (Khoury et al., 1995).
  • Efficacy: The nature of the antibody response to MOMP during in vivo infection is not well understood. Antibody responses elicited in Campylobacter-colonized chickens demonstrates that proteoliposomes restore reactivity to rabbit antibodies. Sera from naturally or experimentally infected chickens reacts weakly, suggesting that the chicken antibody response is predominantly directed against conformational epitopes. These observations provide direct evidence for conformation-dependent humoral responses induced by Campylobacter infection, demonstrate that C jejuni is immunogenic in its natural host and suggest that proteoliposomes may be potentially used for the evaluation of vaccines (Huang et al., 2007).
  • Description: This is a conserved immunodominant protein in this organism and a promising vaccine candidate because the protein is expressed in multiple Campylobacter isolates from both chicken and human hosts (Martin et al., 1999).
11. MBP fused on Campylobacter FlaA (MBP-FlaA)
a. Vaccine Ontology ID:
VO_0004101
b. Type:
Conjugate vaccine
c. Antigen
recombinant protein comprising the maltose-binding protein (MBP) of E. coli fused to amino acids 5 to 337 of the FlaA flagellin of Campylobacter coli VC167 (Lee et al., 1999)
d. Gene Engineering of FlaA from C. jejuni NCTC 11168
  • Type: flagellin protein
  • Description:
  • Detailed Gene Information: Click here.
e. Adjuvant: LTR192G
  • VO ID: VO_0001321
  • Description: immunization occurs with the mutant E. coli heat-labile enterotoxin (LT( R192G)) as a mucosal adjuvant (Lee et al., 1999)
f. Preparation
Part of the flaA gene ( 780 base pairs ) was cloned in plasmid pBEB downstream and in frame with the LT-B to allow expression of a hybrid protein . Transformed E coli chi 6097 expressed the hybrid protein ( 43 kdaltons ) in inclusion bodies at mid log phase . The inclusion bodies were isolated , and the identity of the protein was verified by western blot . (Khoury et al., 1995)
g. Virulence
The full range of MBP-FlaA doses were effective in eliciting antigen-specific serum IgG responses, and these responses were enhanced by adjuvant use. Stimulation of FlaA-specific intestinal secretory IgA (sIgA) responses required immunization with higher doses of MBP-FlaA or coadministration of lower doses with the adjuvant (Lee et al., 1999).
h. Description
A mucosal vaccine was used in an effort to elicit serum IgG and intestinal secretory IgA. A genetic hybrid of the Campylobacter jejuni flaA gene with LT-B of Escherichia coli and assessment of the efficacy of the hybrid protein has been developed an oral chicken vaccine (Wilkinson et al., 2003).
i. Mouse Response
  • Host Strain: BALB/c
  • Vaccination Protocol: Mice were immunized intranasally with two doses of 3 to 50 mg of MBP-FlaA, given 8 days apart, with or without 5 mg of the mutant E. coli heat-labile enterotoxin (LTR192G) as a mucosal adjuvant (Lee et al., 1999).
  • Persistence: The results showed that, when challenged with bacteria, there was a reduction in colonization as early as 3 days after infection and that no campylobacter organisms could be detected in stools by 7 days post-feeding (Lee et al., 1999).
  • Immune Response: Stimulation of FlaA-specific intestinal secretory IgA (sIgA) responses required immunization with higher doses of MBP-FlaA or co-administration of lower doses with the adjuvant (Lee et al., 1999).
  • Side Effects: Animals were monitored for sickness and death for 5 days and only minimal adverse side effects were encountered (Lee et al., 1999).
  • Challenge Protocol: Mice were intranasally challenged after the second vaccination. Fecal excretion of C. jejuni was monitored daily for 10-14 days after challenge by culturing fecal homogenates. Putative colonies were confirmed by morphology and oxidase reactions. Mice were challenged orally with 0.5 ml of various doses of C. jejuni. Fecal excretion was monitored as described above for 7-9 days (Lee et al., 1999).
  • Efficacy: The full range of MBP-FlaA doses were effective in eliciting antigen-specific serum IgG responses, and these responses were enhanced by adjuvant use, except in the highest dosing group. When vaccinated mice were challenged intranasally 26 days after immunization, the best protection was seen in animals given 50 mg of MBP-FlaA plus LTR192G. The protective efficacies of this dose against disease symptoms and intestinal colonization were 81.1 and 84%, respectively. When mice which had been immunized intranasally were challenged orally with 8 x 1010, 8 x 109, or 8 x 108 cells of strain 81-176, the protective efficacies against intestinal colonization at 7 days postinfection were 71.4, 71.4, and 100%, respectively (Lee et al., 1999).
  • Description: It is interesting that antibodies generated during natural infection by either strain 81-176 or strain VC167 appear to react more strongly to glycosylated flagellins isolated from Campylobacter spp. than to unglycosylated, recombinant flagellins isolated from E. coli. Immunization with the recombinant fusion protein lacking post-translational 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. Further evaluation of this recombinant flagellin is ongoing as a vaccine in a ferret diarrheal disease model (Lee et al., 1999).
V. References
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