IE84132B1 - OspA Lipoproteins - Google Patents
OspA Lipoproteins Download PDFInfo
- Publication number
- IE84132B1 IE84132B1 IE2000/0752A IE20000752A IE84132B1 IE 84132 B1 IE84132 B1 IE 84132B1 IE 2000/0752 A IE2000/0752 A IE 2000/0752A IE 20000752 A IE20000752 A IE 20000752A IE 84132 B1 IE84132 B1 IE 84132B1
- Authority
- IE
- Ireland
- Prior art keywords
- protein
- ospa
- seq
- salmonis
- vaccine
- Prior art date
Links
- 108090001030 Lipoproteins Proteins 0.000 title claims description 10
- 102000004895 Lipoproteins Human genes 0.000 title claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 73
- 108090000623 proteins and genes Proteins 0.000 claims description 73
- 241000192126 Piscirickettsia salmonis Species 0.000 claims description 63
- 229960005486 vaccines Drugs 0.000 claims description 44
- 230000004927 fusion Effects 0.000 claims description 33
- 230000002163 immunogen Effects 0.000 claims description 16
- 230000000240 adjuvant Effects 0.000 claims description 14
- 239000002671 adjuvant Substances 0.000 claims description 14
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 241000251468 Actinopterygii Species 0.000 claims description 10
- 244000052616 bacterial pathogens Species 0.000 claims description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- YWXYYJSYQOXTPL-SLPGGIOYSA-N Isosorbide mononitrate Chemical compound [O-][N+](=O)O[C@@H]1CO[C@@H]2[C@@H](O)CO[C@@H]21 YWXYYJSYQOXTPL-SLPGGIOYSA-N 0.000 claims 2
- 241000192127 Piscirickettsia Species 0.000 claims 1
- 210000004027 cells Anatomy 0.000 description 56
- 235000018102 proteins Nutrition 0.000 description 48
- 101700040477 ospA Proteins 0.000 description 37
- 150000001413 amino acids Chemical group 0.000 description 35
- 102000038129 antigens Human genes 0.000 description 35
- 108091007172 antigens Proteins 0.000 description 35
- 239000000427 antigen Substances 0.000 description 33
- 230000014509 gene expression Effects 0.000 description 33
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 26
- 229920001850 Nucleic acid sequence Polymers 0.000 description 25
- 150000007523 nucleic acids Chemical class 0.000 description 25
- 229920000272 Oligonucleotide Polymers 0.000 description 24
- 210000002966 Serum Anatomy 0.000 description 23
- 108090001123 antibodies Proteins 0.000 description 22
- 102000004965 antibodies Human genes 0.000 description 22
- 241000588724 Escherichia coli Species 0.000 description 21
- 210000004698 Lymphocytes Anatomy 0.000 description 19
- 108020004707 nucleic acids Proteins 0.000 description 19
- 241000283973 Oryctolagus cuniculus Species 0.000 description 16
- 241000606651 Rickettsiales Species 0.000 description 16
- 235000001014 amino acid Nutrition 0.000 description 16
- 108020001507 fusion proteins Proteins 0.000 description 16
- 102000037240 fusion proteins Human genes 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- 230000012010 growth Effects 0.000 description 14
- 241000972773 Aulopiformes Species 0.000 description 13
- 230000001580 bacterial Effects 0.000 description 13
- 235000019515 salmon Nutrition 0.000 description 13
- 101710019842 Borrelia burgdorferi ospA Proteins 0.000 description 12
- 241000277338 Oncorhynchus kisutch Species 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 12
- 230000001186 cumulative Effects 0.000 description 12
- 238000003752 polymerase chain reaction Methods 0.000 description 12
- 230000004044 response Effects 0.000 description 12
- 229940099789 OspA protein Drugs 0.000 description 11
- 238000010276 construction Methods 0.000 description 11
- 241000277331 Salmonidae Species 0.000 description 10
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 10
- 201000010099 disease Diseases 0.000 description 9
- 101710023234 Segment 5 Proteins 0.000 description 8
- 230000027455 binding Effects 0.000 description 8
- 239000002773 nucleotide Substances 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 230000000638 stimulation Effects 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 101700038420 MVP Proteins 0.000 description 7
- 238000010367 cloning Methods 0.000 description 7
- 201000009910 diseases by infectious agent Diseases 0.000 description 7
- 235000019688 fish Nutrition 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 108020004705 Codon Proteins 0.000 description 6
- 210000000987 Immune System Anatomy 0.000 description 6
- 102100013779 PPP1R14A Human genes 0.000 description 6
- 101710002147 PPP1R14A Proteins 0.000 description 6
- 230000002238 attenuated Effects 0.000 description 6
- 230000003308 immunostimulating Effects 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 108091007521 restriction endonucleases Proteins 0.000 description 6
- 239000003656 tris buffered saline Substances 0.000 description 6
- 239000012130 whole-cell lysate Substances 0.000 description 6
- 210000003000 Inclusion Bodies Anatomy 0.000 description 5
- 210000001744 T-Lymphocytes Anatomy 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000010348 incorporation Methods 0.000 description 5
- 230000001965 increased Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000001717 pathogenic Effects 0.000 description 5
- 244000052769 pathogens Species 0.000 description 5
- 210000003734 Kidney Anatomy 0.000 description 4
- 108091005503 Nucleic proteins Proteins 0.000 description 4
- 241001280377 Oncorhynchus tshawytscha Species 0.000 description 4
- 241000606701 Rickettsia Species 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 108091006028 chimera Proteins 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 230000002068 genetic Effects 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 230000003834 intracellular Effects 0.000 description 4
- 239000002609 media Substances 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- OZAIFHULBGXAKX-UHFFFAOYSA-N precursor Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000004936 stimulating Effects 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 210000001519 tissues Anatomy 0.000 description 4
- 108020004465 16S Ribosomal RNA Proteins 0.000 description 3
- 210000000612 Antigen-Presenting Cells Anatomy 0.000 description 3
- 210000003719 B-Lymphocytes Anatomy 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 3
- 229920002101 Chitin Polymers 0.000 description 3
- DJHJJVWPFGHIPH-OODMECLYSA-N Chitin Chemical compound O[C@@H]1C(NC(=O)C)[C@H](O)OC(CO)[C@H]1COC[C@H]1C(NC(C)=O)[C@@H](O)[C@H](COC[C@H]2C([C@@H](O)[C@H](O)C(CO)O2)NC(C)=O)C(CO)O1 DJHJJVWPFGHIPH-OODMECLYSA-N 0.000 description 3
- 229920001405 Coding region Polymers 0.000 description 3
- 108010067770 Endopeptidase K Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 125000000570 L-alpha-aspartyl group Chemical group [H]OC(=O)C([H])([H])[C@]([H])(N([H])[H])C(*)=O 0.000 description 3
- 241000606697 Rickettsia prowazekii Species 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000875 corresponding Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 210000002443 helper T lymphocyte Anatomy 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000001681 protective Effects 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 230000002194 synthesizing Effects 0.000 description 3
- 125000002306 tributylsilyl group Chemical group C(CCC)[Si](CCCC)(CCCC)* 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 210000004369 Blood Anatomy 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 210000001161 Embryo, Mammalian Anatomy 0.000 description 2
- 241001522878 Escherichia coli B Species 0.000 description 2
- 241000672609 Escherichia coli BL21 Species 0.000 description 2
- 241000701959 Escherichia virus Lambda Species 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 210000004185 Liver Anatomy 0.000 description 2
- 101710029070 MPN_343 Proteins 0.000 description 2
- 241000712079 Measles morbillivirus Species 0.000 description 2
- 101700080605 NUC1 Proteins 0.000 description 2
- 210000003463 Organelles Anatomy 0.000 description 2
- 241001272996 Polyphylla fullo Species 0.000 description 2
- 241000606726 Rickettsia typhi Species 0.000 description 2
- 241000277263 Salmo Species 0.000 description 2
- 230000024932 T cell mediated immunity Effects 0.000 description 2
- 108010055044 Tetanus Toxin Proteins 0.000 description 2
- 102000008579 Transposases Human genes 0.000 description 2
- 108010020764 Transposases Proteins 0.000 description 2
- 102000005626 Viral Fusion Proteins Human genes 0.000 description 2
- 108010059722 Viral Fusion Proteins Proteins 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000000295 complement Effects 0.000 description 2
- 238000010192 crystallographic characterization Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 230000000120 cytopathologic Effects 0.000 description 2
- 230000001472 cytotoxic Effects 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- -1 e.g. Chemical group 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 230000002708 enhancing Effects 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101710004978 hspX Proteins 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 239000002198 insoluble material Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000000813 microbial Effects 0.000 description 2
- 230000000051 modifying Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 229920001894 non-coding RNA Polymers 0.000 description 2
- 101700006494 nucA Proteins 0.000 description 2
- 230000036961 partial Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 230000002062 proliferating Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 229940118376 tetanus toxin Drugs 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000001052 transient Effects 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 229920000160 (ribonucleotides)n+m Polymers 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl dihydrogen phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N Ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 241000605317 Anaplasmataceae Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 229960001212 BACTERIAL VACCINES Drugs 0.000 description 1
- 238000000035 BCA protein assay Methods 0.000 description 1
- 206010006045 Boutonneuse fever Diseases 0.000 description 1
- 210000004556 Brain Anatomy 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 229960005091 Chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N Chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 210000000349 Chromosomes Anatomy 0.000 description 1
- 241000193449 Clostridium tetani Species 0.000 description 1
- 229940118765 Coxiella burnetii Drugs 0.000 description 1
- 241000606678 Coxiella burnetii Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 102000011724 DNA Repair Enzymes Human genes 0.000 description 1
- 108010076525 DNA Repair Enzymes Proteins 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 101700011961 DPOM Proteins 0.000 description 1
- 108010041525 EC 5.1.1.1 Proteins 0.000 description 1
- 241000701867 Enterobacteria phage T7 Species 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 229920002016 Extrachromosomal DNA Polymers 0.000 description 1
- 241000272184 Falconiformes Species 0.000 description 1
- 241000604959 Francisella persica Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 241000976806 Genea <ascomycete fungus> Species 0.000 description 1
- 210000002816 Gills Anatomy 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101710013836 HSPD1 Proteins 0.000 description 1
- 239000012593 Hanks’ Balanced Salt Solution Substances 0.000 description 1
- 210000002216 Heart Anatomy 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 1
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 description 1
- 108010027412 Histocompatibility Antigens Class II Proteins 0.000 description 1
- 102000018358 Immunoglobulins Human genes 0.000 description 1
- 108060003951 Immunoglobulins Proteins 0.000 description 1
- 210000000936 Intestines Anatomy 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N Kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 241000588915 Klebsiella aerogenes Species 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- 101710009221 LD Proteins 0.000 description 1
- 101710029649 MDV043 Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 229920001410 Microfiber Polymers 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 229940116542 OTHER NUTRIENTS in ATC Drugs 0.000 description 1
- 210000001672 Ovary Anatomy 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 101700036361 PCR2 Proteins 0.000 description 1
- 101700061424 POLB Proteins 0.000 description 1
- 229960005190 Phenylalanine Drugs 0.000 description 1
- 241000078275 Piscirickettsia salmonis LF-89 = ATCC VR-1361 Species 0.000 description 1
- 241000605894 Porphyromonas Species 0.000 description 1
- 229940055023 Pseudomonas aeruginosa Drugs 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 101700054624 RF1 Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000007312 Recombinant Proteins Human genes 0.000 description 1
- 108010033725 Recombinant Proteins Proteins 0.000 description 1
- 241001495396 Rickettsia japonica Species 0.000 description 1
- 229940046939 Rickettsia prowazekii Drugs 0.000 description 1
- 206010039207 Rocky mountain spotted fever Diseases 0.000 description 1
- 102100004536 SFTPB Human genes 0.000 description 1
- 101710033334 SFTPB Proteins 0.000 description 1
- 101710012186 SLC7A1 Proteins 0.000 description 1
- 241000277289 Salmo salar Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 206010039447 Salmonellosis Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 210000000952 Spleen Anatomy 0.000 description 1
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 1
- 101700056266 TRPP Proteins 0.000 description 1
- 210000004241 Th2 Cells Anatomy 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 206010061393 Typhus Diseases 0.000 description 1
- 101710015310 VI Proteins 0.000 description 1
- 229940029983 VITAMINS Drugs 0.000 description 1
- 210000003934 Vacuoles Anatomy 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 229940021016 Vitamin IV solution additives Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000001058 adult Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000890 antigenic Effects 0.000 description 1
- 229960000070 antineoplastic Monoclonal antibodies Drugs 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 125000000511 arginine group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 230000003190 augmentative Effects 0.000 description 1
- 238000002869 basic local alignment search tool Methods 0.000 description 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N benzene Substances C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000003115 biocidal Effects 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002759 chromosomal Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001351 cycling Effects 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000001086 cytosolic Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000001419 dependent Effects 0.000 description 1
- 230000001809 detectable Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000534 elicitor Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000026502 entry into host cell Effects 0.000 description 1
- 230000001747 exhibiting Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000010358 genetic engineering technique Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 239000001963 growth media Substances 0.000 description 1
- 125000003372 histidine group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 230000000984 immunochemical Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000977 initiatory Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 125000000012 isoleucine group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- 238000001738 isopycnic centrifugation Methods 0.000 description 1
- 229950003188 isovaleryl diethylamide Drugs 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 230000021633 leukocyte mediated immunity Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000000873 masking Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 108020004084 membrane receptors Proteins 0.000 description 1
- NZEDTZKNEKPBGR-UHFFFAOYSA-N methyl 3-(1-phenylethyl)imidazole-4-carboxylate;hydrochloride Chemical compound Cl.COC(=O)C1=CN=CN1C(C)C1=CC=CC=C1 NZEDTZKNEKPBGR-UHFFFAOYSA-N 0.000 description 1
- 239000003658 microfiber Substances 0.000 description 1
- 230000002438 mitochondrial Effects 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 108010045030 monoclonal antibodies Proteins 0.000 description 1
- 229960000060 monoclonal antibodies Drugs 0.000 description 1
- 102000005614 monoclonal antibodies Human genes 0.000 description 1
- 230000000877 morphologic Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 101700002291 ompT Proteins 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- 238000002888 pairwise sequence alignment Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 125000000405 phenylalanyl group Chemical group 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 229920000406 phosphotungstic acid polymer Polymers 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 108091008117 polyclonal antibodies Proteins 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 230000002516 postimmunization Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000002285 radioactive Effects 0.000 description 1
- 230000003362 replicative Effects 0.000 description 1
- 230000001718 repressive Effects 0.000 description 1
- 230000000717 retained Effects 0.000 description 1
- 102220174578 rs731236 Human genes 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 125000003616 serine group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- JUJBNYBVVQSIOU-UHFFFAOYSA-M sodium;4-[2-(4-iodophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=N1 JUJBNYBVVQSIOU-UHFFFAOYSA-M 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001225 therapeutic Effects 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 101710040460 tnpA1 Proteins 0.000 description 1
- 230000002588 toxic Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 101710023064 tpp17 Proteins 0.000 description 1
- 230000001131 transforming Effects 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamins Natural products 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/29—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Richettsiales (O)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Description
Microtek International (1998) Ltd.
OspA Lipoproteins
Field of the Invention
This invention relates generally to the field of obligate intracellular bacteria, and in
particular to agents of rickettsia type diseases, specifically Pz'scirickettsz'a salmonis in
aquatic poikilotherms. The invention also encompasses isolated genes encoding outer
surface antigens of P. salmonis and the diagnostic and therapeutic use (including in
particular the preparation of a recombinant vaccine to prevent or reduce the incidence
of infection by P. salmonis and other rickettsial diseases) of such antigens or their
homologues.
In particular aspects, this invention relates to the use of the l7 kDa outer surface
lipoprotein (OspA) of Piscirickettsia salmonis, as the basis of a recombinant vaccine.
Background of the Invention
The order Rickettsiales historically encompassed any intracellular bacterium and
taxonomy was based on only a few phenotypic characteristics (Drancourt and Raoult,
1994). More recently, 16S rRNA sequence similarity studies have helped to better
define the taxonomy of the order Rickeztsiales (Drancourt and Raoult, 1994).
Rickettsiae cause a variety of medically significant diseases in humans including
typhus fever, Rocky Mountain spotted fever, and boutonneuse fever (Pang and
Winkler, 1994; Vishwanath, et al., 1990). Rickettsiae are also agriculturally
significant, and are the aetiological agents of a variety of veterinary diseases
(Rikihisa, 1991).
The past decade has been a renaissance in the identification of rickettsial
andrickettsial-like infections as the aetiological agents of poorly understood diseases
and as emerging pathogens (Anderson, 1997; Azad, et al., 1997; Davis, et a1., 1998;
Fryer and Mauel, 1997; Stenos, et al., 1998). Inherent difficulties are associated with
rickettsials: it is very difficult to grow large quantities of rickettsiae; rickettsiae have
very slow growth rates; and rickettsiae are difficult to separate from host cell material
(Higgins, et al., 1998). Although rickettsiae lack a characterized genetic system for
genetic manipulation (Mallavia, 1991), the advent of recombinant DNA technology
has revolutionized rickettsial research. Characterization of rickettsial pathogenesis
and functional analysis of rickettsial antigens has largely relied upon antibody
inactivation studies (Li and Walker, 1998; Messick and Rikihisa, 1994; Seong, et al.,
1997). Recently major rickettsial antigens have been identified and characterized
further upon sub-cloning into Escherichia coli (Anderson, et al., 1990; Anderson, et
al., 1987; Carl, et al., 1990; Ching, er al., 1992; Ching, et al., 1996; Hahn and Chang,
1996; Musoke, et al., 1996). Successful transformation of Rickettsia typhi (Troyer, et
al., 1999) and Rickettsia prowazekii (Rachek, et al., 1998) have recently raised
exciting prospects for the future of rickettsia research.
Antibody studies of rickettsiae have shown that inactivation of specific rickettsial
surface proteins can inhibit entry into host cells and establishment of infection
(Anacker, et al., 1985; Li and Walker, 1998; Messick and Rikihisa, 1994). Failed
attempts at constructing vaccines against human rickettsial diseases have been based
on preparations of inactivated whole cells (Sumner, et al., 1995). Although these
whole cell vaccines elicit protective responses in animal models, they are only
partially effective when used in humans (Sumner, er al., 1995). Current vaccine
strategies using recombinantly expressed rickettsial proteins identified by antibody
studies have been shown to successfully elicit protective immune responses against
bacterial challenge (McDonald, et al., 1987,‘ Sumner, et al., 1995).
Piscirickettsia salmonis is the first rickettsiae to be isolated from an aquatic
poikilotherm (Fryer, et al., 1990). P. salmonis is the aetiological agent of salmonid
rickettsial septicaemia (SRS), and is an economically significant pathogen of
salmonids that is responsible for extensive mortalities in the cold water aquaculture
industry. P. salmonis, a gram-negative obligate intracellular bacterium, was first
observed in 1989 in a diseased, moribund coho sahnon from a saltwater net pen site
on the coast of Chile (Bravo and Campos, 1989). It is now known that P. sahnonis is
geographically more widespread than was initially suspected, and has recently been
observed in Ireland (Rodger and Drinan, 1993), Scotland, Norway, and on the Pacific
coast of Canada (Brocklebank, er al., 1993).
P. salmonis has been observed to infect a wide range of salmonid species and causes a
systemic infection that targets the kidney, liver, spleen, heart, brain, intestine, ovary,
and gills of salmonids (Cvitanich, et al., 1991). Pleomorphic, predominantly coccoid
bacteria that range in diameter from 0.5 to 1.5 cDm are found within cytoplasmic
vacuoles of cells from infected tissues (Bravo and Campos, 1989). While initially
difficult to culture, P. salmonis was successfully isolated from the kidney of a
diseased adult coho salmon on an immortal Chinook salmon embryo cell line (Fryer,
et al., 1990). Fryer et al. (Fryer, et al., 1992) conducted a 16S rRNA sequence
similarity study which placed P. salmonis in its own genus and species within the
order Rickettsiales. P. salmonis is most closely related to Coxiella burnetii and
Wolbachia persica with 87.5 % and 86.3 % sequence similarity respectively (Fryer, et
al., 1992). P. salmonis appears to belong within the tribe Ehrlichieae because of its
morphological characteristics (Fryer, er al., 1992).
Efficacy of antibiotic treatment of SRS is poor because of the intracellular nature of
P. salmonis, thereby making management of the disease difficult (Lannan and Fryer,
). To effectively prevent and control SRS, vaccine development is desirable.
However, vaccines prepared from whole cell bacterins of mammalian iickettsiae have
shown disappointing protection in trials (Hickman, et al., 1991)
Incorporation of highly immunogenic T lymphocyte epitopes (TCE's) into chimeric
fusion proteins is an elegant extension of the principles that underlie the
immunostimulatory effect of toxoid carrier proteins on conjugated haptens (Bixler and
Pillai, 1989). Toxoids provide TCE's that are required to elicit a strong T helper cell-
mediated immune response against haptens (Bixler and Pillai, 1989). Incorporation of
TCE's into synthetic peptide or chimeric fusion proteins can have an
immunostimulatory effect on other T cell and humoral epitopes within the peptide or
protein (Hathaway, et al., 1995; Kjerrulf, et al., 1997; O'Hern, et al., 1997; Pillai, et
al., 1995; Valmori, et al., 1992). To minimize genetic restriction of these
immunostirnulatory responses, promiscuous TCE's capable of binding major
histocompatibility complex (MHC) molecules from a variety of haplotypes are used
in chimeric vaccine constructs. Tandem repeats of TCE's can also ofien improve
immunogenicity of chimeric proteins better than single TCE's (Kjermlf, et al., 1997;
Pa1tidos,etal., 1992).
The Clostridium tetani tetanus toxin P2 (tt P2) and measles virus fusion protein
(MVF) epitopes have been established as strong TCE's that exhibit promiscuous
binding to various MHC haplotypes and are highly immunogenic in human and
murine models (Demotz, et a1., 1989; Panina-Bordignon, et al., 1989; Partidos and
Steward, 1990). Both tt P2 and MVF TCE's are MHC class II restricted and are able
to bind MHC class II molecules from a wide variety of haplotypes. Genetic restriction
of murine responses to malarial epitopes has been overcome by incorporation of the it
P2 epitope into synthetic peptide-based malarial vaccines (Valmori, et al., 1992).
Summary of the Invention
The present inventors have characterized the surface antigens of the bacterial
pathogen P. salmonis and identified and characterized an immunoreactive antigen,
namely the 17 kDA outer surface lipoprotein OspA of P. salmonis, as well as the
nucleic acid segment that encodes the OspA immunoreactive antigen. This discovery
enables the development of diagnostic techniques (including the use of hybridization
probes and primers) as well as the production of specific antigens and antibodies that
may be used in immunization techniques for inducing immunity against P. salmonis
and other rickettsial diseases. In particular, the discovery enables the development of
recombinant vaccines for SRS and other rickettsial diseases based on the 17 kDa
lipoprotein OspA.
According to "a first aspect of the invention there is provided a fish vaccine against the
bacterial pathogen Piscz'rz'ckettsz’a salmonis, comprising an immunogenic amount of a
protein of 17 kDa encoded by one of SEQ ID No: l and SEQ ID NO: 3 with or
without an adjuvant.
The nucleic acid segment of the invention may be operably linked to a recombinant
promoter and a TCE fusion partner as, for example, in SEQ ID N015.
The slow growing, rickettsia-like, piscine pathogen, P. salmonis, was grown en mass
on chinook salmon (Oncorhynchus tshawytscha) embyro cell line monolayers
(CHSE-214) to purify enough P. salmonis to allow genomic deoxyribonucleic acid
(DNA) isolation. A genomic expression library was constructed and screened with
high titre anti—P. salmonis rabbit serum identifying immunoreactive clones that
encoded a common region of P. salmonis DNA. A 4,983 bp insert was excised in E.
coli and Exo Ill/S1 deletion clones were sequenced. The insert contained 4 intact open
reading frames (OR.F) one of which encoded a homologue, ospA, of a genus specific,
rickettsia-like, outer membrane l6kDa lipoprotein antigen. OspA was recognized by
both convalescent coho salmon (0ncorhynchus kisutch) serum and rabbit antiserum to
both 10 & 20 residue peptides based on predicted protein sequence. The codon usage
of the ospA ORF was optimized for expression in E. coli by construction of a
synthetic version of the ospA gene. An N-terminal fusion partner was cloned in frame
with the ospA gene as well as tt P2 and MVF TCE's all under the control of both T7
and lambda phage promoters to direct expression into inclusion bodies as well as to
facilitate large scale expression of the protein. The various OspA ‘fusion proteins were
purified from E. coli as the insoluble inclusion body fraction of a whole cell lysate.
Suspensions of the insoluble fraction were formulated with an adjuvant and used as a
vaccine to immunize coho salmon. Vaccinates showed both an increase in anti—OspA
antibody production and increased in vitro stimulation of whole lymphocyte
populations by OspA fusion protein. Eight weeks post-vaccination, the salmon were
challenged with virulent suspensions of P. salmonis. The results indicated that the
vaccine was protective against virulent challenge and that immunogenicity and
protection were augmented by the incorporation of promiscuous TCE's into the OspA
fusion protein.
Functional presentation of antigen by salmonid MHC class I and II complexes
analogous to the role of MHC class I and II of mammals and birds has not been
confirmed in teleosts. As a result, algorithms do not exist for predicting peptide
sequences that are capable of functioning as TCE's in the salmonid immune system.
As tt P2 and MVF epitopes have been established as strong epitopes that exhibit
promiscuous binding to various MHC haplotypes, these epitopes were incorporated
onto the OspA fusion protein to elicit immunostimulatory effects. Although the
incorporation of highly immunogenic promiscuous TCE's into chimeric fusion
proteins to extend the immunostimulatory effect of toxoid carrier proteins on
conjugated haptens is not per se novel, the immunostimulating effects of TCE’s within
the salmonid immune system is novel. Furthermore, the novelty of the
immunostimulating effects of TCE's within teleosts is not dependent upon the
identification and characterization of the outer surface lipoprotein OspA of P.
Salomonis.
Sequence Listing
The nucleic and amino acid sequences listed in the accompanying sequence listing are
shown using standard letter abbreviations for nucleotide bases, and one letter code for
amino acids. Only one strand of each nucleic acid sequence is shown, but the
complementary strand is understood as included by any reference to the displayed
strand.
SEQ ID: 1 shows the ospA DNA sequence from P. salmonis
SEQ ID:2 shows the amino acid sequence of the precursor (unprocessed) protein
OspA
SEQ ID:3 shows the ospA DNA sequence, l7e2, modified for optimal codon
usage in E. coli
SEQ ID:4 shows the amino acid sequence of the modified for optimal codon
usage, in E. coli, precursor (unprocessed) protein OspA (l7E2)
SEQ ID:5 shows the DNA sequence, c17e2, of an N-terrninal fusion partner with
optimized ospA gene
SEQ ID: 6 shows the amino acid sequence of an N-terminal fusion partner with
optimized OspA (C 17E2)
SEQ lD:7 DNA sequence of the forward oligonucleotide used during pTYB1-
17kDa construction
SEQ lD:8 DNA sequence of the reverse oligonucleotide used during pTYBl—
l7kDa construction
SEQ ]D:9 oligonucleotide #1 used for construction of optimized ospA gene, l7e2
SEQ ID: 10 oligonucleotide #2 used for construction of optimized ospA gene, 17e2
SEQ ID: 11 oligonucleotide #3 used for construction of optimized ospA gene, 17e2
SEQ ID: 12 oligonucleotide #4 used for construction of optimized ospA gene, 17e2
SEQ ID: 13 oligonucleotide #5 used for construction of optimized osp/1 gene, 17e2
SEQ 1D: 14 oligonucleotide #6 used for construction of optimized 0spA gene, 17e2
SEQ ID: 15 amino acid sequence of a 10 residue synthetic polypeptide based on
residues 110-119 of OspA
SEQ 1D: 16 amino acid sequence of a 20 residue synthetic polypeptide based on
l5residues 110-129 of OspA
SEQ ID: 17 DNA sequence of the tt P2 TCE oligonucleotide
SEQ ID: 18 DNA sequence of the MVP TCE oligonucleotide
SEQ ID: 19 amino acid sequence of the tt P2 TCE
SEQ ID: 20 amino acid sequence of the MVF TCE
Brief Description of the Drawings
Figure 1. Western blot analysis of P. salmonis. Whole cell lysate and
proteinase K digest samples of P. salmonis were separated by 12% SDS-PAGE and
reacted with rabbit anti-P. salmonis polyclonal antibodies followed by
immunochemical detection. Note the immunoreactive protein migrating at 17 kDa.
The ~11 kDa antigen of P. salmonis was not susceptible to PK digestion. Molecular
weights are in kDa.
Figure 2. A. Schematic of spatial relationships of ORF's in P. salmonis clone
pB12, 4,983 bp. The Xba I and Hind III sites were used to subclone the asp/1 ORF
into pBC(+) (Example 2). B. DNA sequence of the P. salmonis ospA ORE and amino
acid sequence of the OspA protein translated from the ospA ORF. C. Pairwise
sequence alignment of the P. salmonis OspA, and the R. prowazekii 17 l
(SwissProt G1l2704). The pairwise alignment was generated using the FASTA3
algorithm. The P. salmonis 17 kDa antigen shares 41 % identity (black background)
and 62 % similarity (black box) with the 17 kDa antigen of R. prowazekii. Synthetic
peptides (SEQ ID: 15, SEQ ID: 16) representing the region from residues 110-129 of
the P. salmonis OspA antigen were used to generate rabbit polyclonal serum.
Figure 3. A. Map of pBC—17kDa, the pBC(+) plasmid encoding the subcloned
0spA ORF (Xba I/Hind HI fiagrnent of clone p1312). Cm is chloramphenicol
resistance, T7 is T7 promoter. B. Analysis of OspA expression. Whole cell lysates of
E. coli clones and P. salmonis were analyzed by SDS-PAGE (12% polyacrylamide).P.
salmonis whole cell lysate was reacted with rabbit polyclonal serum generated against
a 10 residue peptide (SEQ ID: 15) of OspA recognizing a strongly immunoreactive
product in the 17 kDa region of P. salmonis. Expression of the OspA by clone pBC-
17kDa was induced at 42°C and is visible stained by Coomassie blue. Rabbit
polyclonal serum generated against a 20 residue peptide (SEQ ID: 16) of OspA
recognized the expressed 17 kDa protein in induced pBC-17kDa samples.
Convalescent serum from coho salmon also recognized the induced 17 kDa protein in
pBC-l7l
standards are shown in kDa.
Figure 4. A. Schematic representation of the strategy employed during the
synthesis of the E. coli codon optimized ospA gene, l7e2. B. DNA sequence of the 6
overlapping oligonucleotides used. C. DNA sequence of the E. coli codon optimized
asp/1 gene, l7e2.
Figure 5. A. Amino acid sequence of the OspA protein, 17E2, expressed from
the optimized osp/1 gene, l7e2. B. DNA sequence of the N-terminal 0spA gene
fusion construct, c17e2. C. Amino acid sequence of the OspA-fusion protein, C l7E2,
containing an N-terminal fusion.
Figure 6. A. Maps of the expression vectors encoding the optimized ospA
fusion construct under the control of T7, pETC—l7E2, and lambda promoters, pKLPR—
C l7E2. Ap is ampicillin resistance, Krn is kanamycin resistance, T7 P is the T7
promoter, PLR is lambda right promoter. B. 12% polyacrylamide SDS—PAGE
analysis of Cl7E2 expression. Samples from the lambda promoter expression
represent the insoluble fraction (i.f.) of whole cells lysates. Whole cell (w.c.) samples
from T7 expression are loaded along with a sample of the insoluble fraction Note the
abundant expression of the OspA-fusion product at 28.5 kDa in the induced samples.
Molecular weight standards are shown in kDa.
Figure 7. Map of pTYBl-l7kDa. An ospA-fusion construct encoding a C-
terminal fusion partner was placed under the control of T7 promoter. "The C—te1minal
fusion partner contained a self-cleaving spacer region and chitin binding domain.
Figure 8. A. A diagram illustrating the cloning strategy employed to create the
OspA fusion protein constructs encoding promiscuous TCE's. 17E2 is the synthetic
ospA gene that was created using codons optimized for E. coli high level expression.
tt P2 and MVF are the DNA sequences (SEQ ID: 19, SEQ ID:20) encoding the tetanus
toxin and measles virus fusion protein T cell epitopes (SEQ ID:l7, SEQ lD:18). B. (a)
Sequences of the tt P2 and (b) MVF oligonucleotides (SEQ lD:l7, SEQ ID:l8) used
to incorporate the (c) tt P2 and (d) MVF TCE's (SEQ ID:l9, SEQ lD:20) into the
OspA fusion protein constructs. Bold nucleotides indicate the TCE coding region of
the oligonucleotides.
Figure 9. Antibody titres of coho salmon groups against OspA-fusion protein
candidate vaccines. Salmon were immunized with either C l7E2, CT 17E2, CM
l7E2, or CMTl7E2. Antibody titres were defined as the maximum serum dilution
that resulted in a signal corresponding to 3 times the background obtained with the
diluent vaccinated serum group at a dilution of l 2320.
Figure 10. Proliferative lymphocyte responses of vaccinated Atlantic salmon (Salmo
salar). The highest lymphocyte stimulation occurred in salmon that were vaccinated
with an OspA fusion protein containing two promiscuous TCE's (CMTl7E2).
Figure 11. Vaccine trial of OspA fiision protein constructs containing promiscuous
TCE's in an outbred population of coho salmon. Adjuvant-injected salmon
experienced a cumulative mortality of 85.5% when challenged with P. salmonis by 1]?
injection. Cl7E2 vaccinated salmon reached a cumulative mortality of 59.6%. CT
17E2 vaccinated salmon experienced 35.6% cumulative mortality. CMl7E2 and the
CMl7E2 + CTl7E2 groups experienced 20 and 18.6% cumulative mortality,
respectively. The CMTl7E2 vaccinated group experienced only 14.5% cumulative
mortality. RPS values of Cl7E2, CTl7E2, CMl7E2, CMl7E2 + CTl7E2, and
CMTI 7E2 were 30.2, 58.4, 76.6, and 83.0%, respectively.
Detailed Description of the Invention
. Definitions
Epitope: An epitope refers to an immunologically active region of an immunogen
(most often a protein, but sometimes also a polysaccharide or lipid) that binds to
specific membrane receptors for antigen on lymphocytes or to secreted antibodies. To
generate an immune response to a foreign antigen, lymphocytes and antibodies
recognize these specific regions (epitopes) of the antigen rather than the entire
molecule.
B cell epitope: The region (epitope) of an immunogen which is recognized by B cells
when it binds to their membrane bound antibody. The B cells which recognize that
particular region then proliferate and secrete antibody molecules which are specific
for that region of the immuno gen. B cell epitopes tend to be highly accessible regions
on the exposed surface of the immunogen. Stimulation of the immune system by B
cell epitopes results in "humoral" immunity.
T cell epitope: The region (epitope) of an immunogen which is recognized by a
receptor on T cells alter being processed and presented on the surface of an antigen
presenting cell (APC) in the context of a major histocompatability complex (MHC)
class 1 or II molecule. T cells can be split into two distinct groups, T helper cells (Th)
and T cytotoxic cells (TC). T helper cells recognize epitopes bound to MHC class It
molecules whereas T cytotoxic cells recognize epitopes bound to MHC class I
molecules. T helper cells can be further subdivided into two classes, Th1 and Th2, Tm
being responsible for stimulation of cell-mediated immunity and Th2 cells stimulating
the humoral arm of the immune system. When a given T cell recognizes the epitope-
MHC complex at the surface of the APC it becomes stimulated and proliferates,
leading to the production of a large number of T cells with receptors specific for the
stimulating epitope. Stimulation of the immune system by T cell epitopes normally
results in "cell—rnediated" immunity.
Attenuated Bacterial Vaccine: This refers to bacterial strains which have lost their
pathogenicity while retaining their capacity for transient growth Within an inoculated
host. Because of their capacity for transient growth, such vaccines provide prolonged
immune-system exposure to the individual epitopes on the attenuated organisms,
resulting in increased immunogenicity and memory—cell production, which sometimes
eliminates the need for repeated booster injections. The ability of many attenuated
vaccines to replicate within host cells makes them very suitable to induce a cell-
mediated immunity. Typically, bacterial strains are made attenuated by introducing
multiple defined gene mutations into the chromosome thereby impairing growth in
vivo.
Recombinant Vector Vaccine: This refers to the introduction of genes (or pieces of
genes) encoding major antigens (or epitopes) from especially virulent pathogens into
attenuated viruses or bacteria. The attenuated organism serves as a vector, replicating
within the host and expressing the gene product of the pathogen.
Sequence Identity: Identity between two nucleic acid sequences, or two amino acid
sequences is expressed in terms of the level of identical residues shared between the
sequences. Sequence identity is typically expressed in terms of percentage identity;
the higher the percentage, the more similar the two sequences are.
Sequence Similarity: Similarity between two amino acid sequences is expressed in
terms of the level of sequence conservation, including shared identical residues and
those residues which differ but which share a similar size, polarity, charge or
hydrophobicity. Sequence similarity is typically expressed in terms of percentage
similarity; the higher the percentage, the more similar the two sequences are.
Recombinant: A recombinant nucleic acid is one that has a sequence that is not
normally occurring or has a sequence that is made by an artificial combination of two
otherwise separated segments of sequence. This artificial combination is often
accomplished by chemical synthesis or, more commonly, by the artificial
manipulation of isolated segments of nucleic acids, e.g., by genetic engineering
techniques.
Oligonucleotide (oligo) : A linear polymer sequence of up to approximately 100
nucleotide bases in length.
Probes and primers: Nucleic acid probes and primers may readily be prepared based
on the amino acid and DNA sequence provided by this invention. A probe comprises
an isolated nucleic acid attached to a detectable label or reporter molecule. Typical
labels include radioactive isotopes, ligands, chemilmninescent agents, and enzymes.
Methods for labeling and guidance in the choice of labels appropriate for various
purposes are discussed, e.g., in Sambrook et al.
Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or
more in length. Primers may be annealed to a complementary target DNA strand, and
then extended along the target DNA strand by a DNA polymerase enzyme. Primer
pairs can be used for amplification of a nucleic acid sequence, e. g., by the polymerase
chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
Methods for preparing and using probes and primers are described, for example, in
Sambrook, 1989, Ausubel, 1987, and Innis, 1990. PCR primer pairs can be derived
from a known sequence, for example, by using computer programs intended for that
purpose such as DNAStar Lasergene software. One of skill in the art will appreciate
that the specificity of a particular probe or primer increases with its length. Thus, for
example, a primer comprising 20 consecutive nucleotides will anneal to a target with
a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order
to obtain greater specificity, probes and primers may be selected that comprise 20, 25,
, 35, 40, 50 or more consecutive nucleotides.
Isolated: An "isolated" biological component (such as nucleic acid or protein or
organelle) has been substantially separated or purified away from other biological
components in the cell of the organism in which the component naturally occurs, i.e.,
other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins
purified by standard purification methods. The term also embraces nucleic acids and
proteins prepared by recombinant expression in a host cell as well as chemically
synthesized nucleic acids. An "isolated" bacterial strain or colony is purified away
from other colonies and yields a pure culture without any contaminants upon plating
on selective media.
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a
transformed host cell. A vector may include nucleic acid sequences that permit it to
replicate in a host cell, such as an origin of replication. A vector may also include one
or more selectable marker genes and other genetic elements known in the art. A
"temperature-sensitive" vector is one which replicates normally at a low growth
temperature (i.e., 28°C) and will not replicate at a higher growth temperature (i.e.,
°C) due to mutations at or near the origin of replication. An "imperfectly
segregating" vector is one which is not stably inherited by new daughter cells at the
time of cell division in the absence of selection pressure due to mutations within the
vector sequence.
Host Cell: Refers to those cells capable of growth in culture and capable of
expressing OspA protein and/or OspA fusion protein. The host cells of the present
invention encompass cells in in vizro culture and include prokaryotic and eukaryotic,
including insect cells. A host cell strain may be chosen which modulates the
expression of the inserted sequences, or modifies and processes the gene product in
the specific fashion desired. Expression from certain promoters can be elevated in the
presence of certain inducers (i.e. temperature, small inducer molecules such as B-
galactosides for controlling expression of T7 or lac promoters or variants thereof).
The preferred host cell for the cloning and expression of the OspA protein and OspA-
filSlOI1 protein is a prokaryotic cell. An example of a prokaryotic cell useful for
cloning and expression of the OspA protein of the present invention is E. coli BL21.
Cell Culture: a) Refers to the growth of eukaryotic (non-bacterial) cells in a complex
culture medium generally consisting of vitamins, buffers, salts, animal serum, and
other nutrients. (b) Refers to the growth of P. salmonis on CHSE—2l4 and any other
cell line that sustains P. salmonis growth.
Fusion Partner: Any DNA sequence cloned in frame to the 5‘ or 3' end of an ORF
that results in transcription and translation of amino acid sequence added to the N— or
C-terrninus of the original protein.
Fusion Protein: The term fiision protein used herein refers to the joining together of
at least two proteins, an OspA protein and a second protein. In some embodiments of
the present invention, the second protein may be fused or joined to a third protein. In
the present invention, examples of second proteins include any polypeptide that
facilitates the following: expression, secretion, purification, condensation,
precipitation, or any property which facilitates concentration or purification.
Variant: Any molecule having amino acid substitutions, deletions, and/or insertions
provided that the final construct possesses the desired ability of OspA. Amino acid
substitutions in OspA may be made on a basis of similarity in polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the
residues involved. Also included within the definition of variant are those proteins
having additional amino acids at one or more of the C-terminal, N—terminal, and
within the naturally occurring OspA sequence as long as the variant protein retains the
desired capability to elicit an immune response against P. rickettsia and hence to
function effectively as a vaccine against same.
The substitutions which in general are expected to produce the greatest changes in
protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or
threonyl, is substituted for (or by) a to hydrophobic residue, e.g., leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any
other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or
histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or
aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted
for (or by) one not having a side chain, e.g., glycine. Variant proteins having one or
more of these more substantial changes may also be employed in the invention,
provided that immunogenicity of OspA is retained. Covalent modification such as
lipidation is also included as the protein is known to be lipidated in vivo.
More extensive amino acid changes may also be engineered into variant OspA. As
noted above however, these variants will typically be characterized by possession of
at least 40% sequence identity counted over the full length alignment with the amino
acid sequence of their respective naturally occurring sequences using the alignment
programs described herein. In addition, these variant OspA proteins would retain
immunogenicity.
Confinnation that OspA has immunogenic activity may be achieved using the
immunological and protection experiments described herein. Following confirmation
that OspA has the desired immunogenic effect, a nucleic acid molecule encoding
OspA may be readily produced using standard molecular biology techniques. Where
appropriate, the selection of the open reading frame will take into account codon
usage bias of the bacterial or eukaryotic species in which OspA is to be expressed.
Inclusion body: Intracellularly confined, insoluble, protein-containing particles of
bacterial cells comprised of either homologous or heterologous proteins. These
particles are the reservoirs and consequence of overproduction of bacterial
recombinant proteins. Inclusion bodies can be purified or semi-purified and used
directly as protein antigens or can be solubilized by various procedures and used as
soluble protein antigen preparations.
Alignment programs: Methods for aligning sequences for comparison purposes are
well known in the art. Various programs and alignment algorithms are described in
Smith and Waterman (1981), Needleman and Wunsch (1970), Pearson and Lipman
(1988), Higgins and Sharp (1988, 1989), Corpet et al. (1988), Huang et al. (1992),
Pearson et a1. (1994). Altschul et al. (1990) presents a detailed consideration of
sequence alignment methods.
The National Centre of Biotechnology Information (NCBI) Basic Local Alignment
Search Tool (BLAST; Altschul er al., 1990) is available from several sources,
including the NCBI (Bethesda, MD) and on the Internet, for use in connection with
the sequence analysis programs BLASTP, BLASTN, BLASTX, TBLASTN,
TBLASTX. BLAST can be accessed at Altschul et al. 1997 Nucleic Acid Research
2523389-3402.
For comparisons of amino acid sequences of greater than 30 amino acids, the
"BLAST 2 Sequences" function in the BLAST program is employed using the
BLASTP program with the default BLOSUM62 matrix set to default parameters,
(open gap 11, extension gap 1 penalties). When aligning short peptides (fewer than 30
amino acids), the alignment should be performed using the "Blast 2 Sequences"
function employing the BLASTP program with the PAM3O matrix set to default
parameters (open gap 9, extension gap 1 penalties). Proteins having even greater
similarity to the reference sequences will show increasing percentage identities when
assessed by this method, such as at least 45 %, at least 50 %, at least 60 %, at least 70
%, at least 75 %, at least 80 %, at least 85 %, at least 90 %, or at least 95 % sequence
identity.
Promoter: A region of DNA to which either RNA polymerase or any other enhancer
protein binds before initiating transcription of the DNA code into the RNA gene
product. For example; lambda, phage T7, lac, tac, srpP, trpP, or araB etc. promoter
DNA. A promoter region therefore determines the efficiency of the RNA gene
product.
Fragments: Those parts of either the DNA encoding a gene for a protein, a TCE, or a
fusion partner and those parts of the protein, TCE, or fusion partner itself
II. Selection and Creation of Nucleic Acid Sequences Encoding the 17 kDa OspA
Protein
21. Growth & Purification of P. salmonis
P. salmonis strains were routinely passaged on Chinook salmon embryo cell line
CHSE-214 (ATCC CRL—l681) at 17°C in Eagle's minimal essential media (MEM)
with Earle's salts supplemented with 10% newborn calf serum. Type strain P.
salmonis LF-89 was obtained from the American Type Culture Collection (ATCC
VR-1361) and is herein referred to as P. salmonis.
A protocol for purifying P. salmonis was developed by combining and modifying the
protocols of Tamura et al (Tamura, er al., 1982) and Weiss et al (Weiss, et al., 1975).
A 6,320 cmz NuncIM- cell factory was seeded with cell line CHSE-214 and infected
with 450 ml of cell culture supernatant from fully lysed CHSE—2l4 monolayers
infected with P. salmonis. Infection was allowed to continue 14-17 days until
cytopathic effects obliterated the entire monolayer. Upon destruction of the
monolayers cell culture supematants were collected and centrifiiged at 10,000 x g for
min at 4°C. Pellets were resuspended in MEM and homogenized in a 15 ml
Dounce tissue homogenizer. The homogenized suspension was centrifuged at 200 x g
for 10 min at 4°C to pellet large host cell debris. The supernatant was filtered twice
through glass microfibre and centrifuged at 17,600 x g for 15 min at 4°C. Pellets were
resuspended in TS—buffer (33 mM Tris-HCI, 0.25 M sucrose; pH 7.4). Samples were
loaded onto Percollm gradients with a final concentration of 40% and centrifuged in a
fixed angle rotor (type JA-14) at 20,000 x g for 60 min at 4°C in a Beekman—“—‘ J2-21
centrifuge. Bands were collected by aspiration, diluted with phosphate buffered
saline, pH 7.4 (Sambrook, et al., 1989) and centrifuged at 20,000 x g for 10 min at
4°C. Pellets were washed twice with phosphate buffer solution (PBS). Contents of the
bands were negative stained with 0.5 phosphotungstic acid and analyzed by
transmission electron microscopy on a Phillips EM 300 at an accelerating voltage of
75 kV.
b. Demonstration of Immunoreactive Molecules
In order to characterize the antigenic profile of P. salmonis, western blot analysis was
canied out using anti-P. salmonis rabbit serum (Fig. 1). Proteinase K digestion was
used to determine if any observed antigens may have been carbohydrate. Six P.
salmonis immunoreactive antigens were observed at relative molecular weights of 65,
60, 54, 51, 17, and 11 kDa (Fig. 1). Proteinase K digestion destroyed all
immunoreactive antigens except the 11 kDa antigen (Fig. 1).
c. Purification of Genomic DNA & Construction of Library
P. salmonis was purified by density gradient centrifugation as previously described
(Kuzyk, er al., 1996) from 12,000 cm2 of CHSE—214 cells exhibiting fiall cytopathic
effect 14 days after infection with P. salmonis. A single step DNA isolation solution
was used to obtain genomic DNA from the purified P. salmonis. Genomic DNA was
further purified by equilibrium centrifugation using a CsCl-ethjdium bromide gradient
to yield 250
DNA was partially digested using serially diluted Ec0R I. Digests containing an
average fragment size of 10 kb were chosen for creation of a P. salmonis gene
expression library using a lambda ZAP II cloning kit.
d. Immunological Screening of Library
Approximately 10,000 plaques of P. salmonis lambda expression library were
screened per round with a desired density of 1,000 plaques per 80 mm petri dish.
Plaques were lifted in duplicate using 80 mm nitrocellulose discs impregnated with 10
mM isopropyl-B-D-thiogalactoside (IPTG). Screening followed the protocol of
Sambrook et al. (1989) using anti-P. salmonis rabbit serum. Imrnunoreactive plaques
were picked and rescreened until pure cultures were obtained. Lambda clones were
then amplified and the pBluescript phagemid excised into E. coli.
Screening of the P. salmonis expression library with high titre anti-P. salmonis rabbit
serum identified several strongly immunoreactive plaques. These plaques were picked
and rescreened until pure and were confirmed to contain inserts. Initial attempts to
excise the clones into E. coli from the lambda clones were unsuccessful which
suggested the clones may encode products toxic to E. coli. Restriction fragment length
analysis using frequently cutting enzymes suggested that all clones contained a
common region of DNA. The clones contained a 5 kb insert (Example 1).
Genomic DNA from all the lambda clones, P. salmonis, CHSE—214, and vector
plasmid DNA was analyzed by DNA dot blotting using insert DNA from one clone
(Clone pBl2) as the probe. Hybridization revealed that the pBl2 insert was of P.
salmonis origin. The pBl2 insert also hybridized with all other immunoreactive
lambda clone samples indicating that all the inserts encoded an overlapping fragment
of P. salmonis DNA.
e. DNA Sequence Analysis of Clone pBl2
DNA sequence analysis of clone pBl2 (Example 1) identified 4 complete ORF's
within the 4,983 bp insert and 1 partial ORF (Example 1). The predicted amino acid
sequences of these ORF's was subjected to homology searches using alignment
programs (eg. BLAST2 and FASTA3). No significant matches were found when
searching for DNA sequence homology to the pBl2 insert. The 499 bp ‘alr ORF
(Example 1) was predicted to encode a 176 residue (res.) protein fused to the N-
terminus of LacZ. The predicted molecular weight (m.w.) of the LacZ-'Alr fusion is
22.2 kDa. The predicted 'Alr ORF amino acid sequence shares 44 % identity and 63
% similarity with C—terrninal portions of known alanine racemase enzymes from
Klebsiella aerogenes (GenBank AAC38l40), Salmonella typlzimurium (GenBank
A295l9), and E. coli (GenBank BA.‘/X36048).
A 732 bp ORF (bax; Example 1) was predicted to encode a 243 res., 27.6 kDa protein.
Both FASTA3 and BLAST2 only identified low scoring similarity (33% identical,
49% similar) between the central 187 amino acid region of the bax ORF and a 274
res. uncharacterized, hypothetical protein in E. coli K12 (BAX; GenBank
AABl8547).
A 1368 bp ORF (radA; Example 1) was predicted to encode a 456 res., 49.4 kDa
protein. A high degree of amino acid homology was found over the entire length of
the radA ORF and RadA DNA repair enzymes fiom a variety of bacteria. P. salmonis
RadA is most homologous to RadA of Pseudomonas aeruginosa (SwissProt P96963)
with 62% identity and 77% similarity. P. salmonis RadA also exhibits 59 % identity
and 75 % similarity to E. coli RadA (SwissProt P24554).
A 486 bp ORF (ospA; Example 1), immediately following raa'A, was predicted to
encode a 162 res., 17.7 kDa protein with amino acids 21-162 having substantial
sequence similarity with the mature chain of the rickettsial 17 kDa genus common
antigen. The predicted 17 kDa antigen was up to 41% identical and 62% similar to the
17 kDa protein antigens of R. prowazekiz’ (SwissProt G112704), Rickettsia japonica
(SwissProt Q52764), Rickettsia rz'ckettsz‘z' (SwissProt PO5372), and Rickettsia typhi
(SwissProt P22882). The 17 kDa protein of rickettsiae is translated as a precursor
protein containing a 20 amino acid signal peptide. During processing the signal
peptide is removed and the N-terminal cysteine residue is lipid-modified to form the
mature protein. The first 21 amino acids of the P. salmonis OspA protein are
predicted to be a signal peptide and contain a bacterial lipidation pattern as well.
The final 717 bp ORF (mp/1; Example 1) was predicted to encode a 239 res., 27.7
kDa protein. This ORF is flanked by a perfect 288 bp direct repeat. Amino acid
similarity searches returned strong matches between the tnpA ORF and a variety of
transposases. The closest match was a transposase (GenBank U83995) in a
Porphyromonas gz’ngz'valz's insertion element, ISl95, with 47% identity and 65
similarity (Lewis and Macrina, 1998).
f. Identification of the ospA ORF as the 16 kDa Antigen
Rabbit antibodies raised against lO—mer and 20—mer synthetic peptides of this region
reacted with an immunoreactive product in P. salmonis around the 16kDa predicted
mass of the ospA ORF product (Example 2). Expression of the 16kDa antigen was
induced in clone pBC—16kDa and was recognized by rabbit serum against the
synthetic peptides (Example 2). Serum from coho salmon fry that had survived a
challenge with P. salmonis also recognized the induced l6kDa product (Example 2).
These data confirm that the ospA ORF encodes the immunoreactive l6kDa OspA
anti gen.
g. Optimization of the ospA ORF for E. coli Expression
The coding sequence of ospA was optimized using codons used frequently by E. coli
(Example 3). Six overlapping oligonucleotides representing the optimized ospA gene
were synthesized using standard phosphoamidite method. The gene was assembled
using 2 successive PCR reactions with the oligonucleotides and the full length
product was cloned into an appropriate cloning vector. DNA sequence of the
optimized ospA gene was verified by sequence analysis using an automated
sequencer. Production of the OspA protein from the optimized ospA gene was
confirmed upon subcloning the optimized ospA gene to the pET21(+) (Novagene)
expression vector and inducing expression using the T7 promoter (Example 3).
h. Description of the Fusion Protein Constructs
The level of OspA production from the optimized ospA gene was still relatively low.
It is well known to persons skilled in the art that filSlOI1 paitners can aid in increasing
the level of production of proteins. We constructed both N- and C—terminal fusions
(Examples 4 & 5) with the 0SpA gene. In our examples we show that some fusions
resulted in increased production of the OspA-fusion with the N-terminal fusion
partner being more favourable than the C-terminal fusion partner. It is possible that
presence of a signal peptide on the N-terminus of OspA may hamper high level
production of OspA. Therefore, the N-terminal fusion partner may increase OspA
production by masking the signal peptide. Similar increases in OspA production may
be obtained from deletion of the region of the ospA gene that encodes the signal
peptide.
TCE's tt P2 (SEQ ID 17) and MVF (SEQ ID 18) were synthesized as oligonucleotides
using codons optimized for high level expression in E. coli. The epitope coding
regions of the MVF and tt oligonucleotides were flanked by BamH I, Nde I and Vsp I,
Hind Ill restriction endonuclease sites and primer binding sites for subsequent PCR
amplification and subcloning. The MVF and tt P2 oligonucleotides were converted to
double stranded DNA and amplified by PCR using standard conditions (Giovannoni,
1991) and cloned into pBC—V using BamH I and Hind III restriction endonuclease
sites to create pBC-MVF and pBC—ttP2. Vector pBC-V is a variant of pBC KS(+) that
lacks Vsp I restriction endonuclease sites at 925 and 984 bp. pBC KS(+) was digested
with Vsp I, single stranded ends were filled in using Klenow fragment, and blunt end
ligation was performed to create pBC-V.
The BamH I and Vsp I fragments of pBC—MVF and pBC—ttP2 were separately
subcloned into the BamH I and Nde I sites of pET-C17B2 (Fig. 8). This subcloning
step placed the TCE's in frame between 0spA and the N-terminal fusion partner to
create pET—CMl7E2 and pET-CT17E2 (Fig. 8). Ligation of the Vsp I and Nde I
cohesive ends destroyed the respective restriction sites while an Nde I site was
encoded in the 5'-terminal region of the TCE insert to allow subsequent ligation of
inserts in frame and upstream of the TCE using BamH I and Nde I (Fig. 8).
A third construct encoding both TCE's was created by subcloning the BamH I and
Vspl fragment of pBC-MVF into the BamH I and Nde 1 sites of pET-CT17E2 to
create pET—CMT17E2 (Fig. 8).
EXAMPLES
The following examples are included to demonstrate preferred embodiments of the
invention, and it will be appreciated by those skilled in the art, in light of this
disclosure, that many changes can be made in the specific embodiments disclosed
without departing from the scope of the invention.
1. Sequence Analysis of P. salmonis Insert Producing Immunoreactive Material
A directional deletion library of P. salmonis clone pBl2 was constructed to facilitate
sequence analysis. Exo III and S1 nuclease were used to construct double—stranded
nested deletions in the direction of IacZ. Restriction endonucleases EcoR I and Sac I
were used to generate opposing overhangs protecting the vector from Exo III
digestion. Upon ligation and screening, 32 deletion clones were selected that
represented the entire insert and differed in size by 100-500 bp.
Double stranded plasmid DNA samples were sequenced using a combination of dye
primer and dye termination. Sequencing reactions were analyzed using an automated
DNA sequencer. Sequence data were assembled and analyzed using commercially
available computer software packages.
DNA sequencing of pB12 Exo III/SI nuclease deletion clones revealed that the insert
was 4,983 bp. Coding predictions identified 4 intact ORF's and 1 partial ORF creating
a fusion in frame with LacZ (Fig. 2). The predicted ORF's were subjected to BLAST2
(Altschul, et al., 1997) and FASTA3 (Pearson, 1998) analysis to determine if any
similar sequences were known (Fig. 2).
. Identification of the ospA ORF as the Source of OspA
Residues 110-129 of the 17 kDa antigen encoded by the predicted 0spA ORF were
predicted to be a B cell epitope by the Jameson-Wolf method (Jameson and Wolfl
1988). Antibodies were generated in New Zealand white rabbits against 10 and 20
amino acid synthetic peptides (SEQ ID: 15; SEQ 1D:l6) representing amino acids
-129 of the predicted OspA amino acid sequence (SEQ 1D:2). Peptides were
glutaraldehyde conjugated to for 1 h at 4°C in a 10 ml reaction volume with 500
CDg/ml keyhole limpet hemocyanin and 1 % glutaraldehyde. For the primary
immunization, rabbits received 250 CDg of conjugated peptide mixed 1:1 with Freund's
complete adjuvant. Each rabbit was boosted three times at 2 week intervals with 250
CDg of conjugated peptide per boost mixed 1:1 with Freund‘s incomplete adjuvant.
Table 2: Synthetic polypeptides used to generate polyclonal rabbit antibodies against
OspA.
Peptide Sequence
mer Pro—Val-Arg-Thr-Tyr-Gln-Arg-Tyr-Asn-Lys
(SEQ H3215)
mer Pro-Val-Arg-"lhr-TynGln-Arg-'l‘yr—Asn-Lys-Gln-Glu-Arg-Arg-Gln-Gln-Tyr-Cys—Arg-Glu
(SEQ ID:16)
The 17 kDa antigen ospA ORF was subcloned into pBC(+) under control of the T7
promoter. The Xba I/Hind III fragment of clone pB12 was ligated with Xba I/Hind III
digested pBC(+) to generate clone pBC-17kDa. Induction of the T7 promoter by
shifting growth temperature to 42°C resulted in expression of a 17 kDa protein
observed by Coomassiem staining of whole cell lysates of induced clone pBC-17 kDa
SDS-PAGE samples (Fig. 3). Western blot analysis of whole cell lysates of P.
salmonis and pBC-17 kDa with rabbit antibodies generated against synthetic peptides
of OspA reacted with a 17 kDa protein in both P. salmonis and the induced sample of
pBC-l7kDa confirming the ospA ORF as the source of then translated OspA protein
(Fig. 3).
. Synthesis & Cloning of Optimized ospA GeneA nucleic acid molecule was
designed to encode the OspA protein precursor (OspA including signal peptide). This
nucleic acid was constructed by PCR using 6 overlapping oligonucleotides (SEQ
ID:9, SEQ lD:l0, SEQ ID: 11, SEQ lD:12, SEQ ID:13, and SEQ lD:l4). Synthesis of
ospA gene was done by three subsequent PCR using the six synthetic overlapping
oligonucleotides (Fig. 4A & Fig. 4B). PCR—l involved overlapping oligonucleotides
SEQ ID: 11, SEQ ID: 12 (0.05 pmol/(D1 each) and SEQ lD:l0, SEQ ID: 13 (0.25
pmol/CD1 each). Product of PCR-l (1 CD1) was used as a template in PCR-2 using
oligonucleotides SEQ ID:9 and SEQ lD:l4 as primers (0.25 pmol/CD1). Both PCR
were performed using Taq I polymerase (Boehringer), supplied buffer and
deoxynucleotide triphosphates (dNTP) (Amersham Pharmacia). Temperature cycling
was as follows: 4
PCR-1 & 2: 92°C 30 sec., 55°C 30 sec., 72°C 30 sec., 1 cycle
°C 30 sec., 70°C 30 sec., 72°C 30 sec., 29 cycles
Product of PCR2 (Fig. 4C) was cloned into plasmid vector pBC(+) as a BamH I —
Hind III fragment resulting to pBC—l7E2. DNA sequence of the insert was verified by
DNA sequencing using methods known to those skilled in the art. The DNA fragment
of pBCKS-l7E2 carrying optimized 0spA gene was than cloned to pET2l(+) as a Nde
I — Hind III DNA fragment resulting to pET-17E2.
. Expression of Optimized OspA Antigen With N-Terminal Fusion Partner
A. Expression using T7 promoter system
DNA fragment of pBCKS-l7E2 carrying optimized asp/1 gene was cloned, using
methods known to one skilled in the art, to pETC (Microtek International) resulting to
pETC-17E2 as a BamHI-HindIII fragment carrying ospA fused to a desired fusion
partner under control of T7 promoter (Fig. 5, Fig. 6A).
Strain E. coli BL2l [E. coli B, F', ompT, hsdS (rs', ms'), gal, dcm] (Pharmacia) canied
the recombinant expression plasmid pETC—17E2 and helper plasmid pGPl-2 (Tabor
and Richardson, 1985). Expression experiment was performed in 4 L flask. During
the growth phase, the culture was grown in Tenific Broth (TFB) with agitation (~ 300
RPM) at 28—30°C to late log phase. Then cells were diluted with an equal volume of
fresh TFB media and growth continued at 42°C 3-6 hours. Product was accumulated
inside cells as insoluble aggregates of protein. Cells from 1 ml of culture were
sedimented in a microcentrifuge, washed with water, resuspended in 1 ml of water
and disrupted by sonication. Insoluble material was sedimented, washed with water
and analyzed by 15 % SDS-PAGE as is known to one skilled in the art (Fig. 6B).
B. Expression usinglarnbda promoter system
DNA fragment of pETC-l7E2 carrying fused optimized ospA gene was subcloned,
using methods known to one skilled in art, to pKLPR—8 (Microtek International 1998
Ltd.) resulting in pKLPR-Cl7E2 as a Xba I - Kpn I fragment carrying the ospA fiision
under control of phage lambda promoter. Plasmid also carries repressor gene C1875
of the lambda promoter (Fig. 5).
Strain E. coli BL2l [E. coli B, F’, 0mpT, hsdS (rs", ms"), gal, dcm] (Pharmacia)
carried the recombinant expression plasmid pKLPR-C17E2 (Fig. 6A). During the
growth phase, the culture was grown in TFB with agitation (300 RPM) at 28—3O °C to
late log phase. Then cells were diluted with an equal volume of fresh TFB media and
growth continued at 42°C 3-6 hours. Product was accumulated inside cells as
insoluble aggregates of protein. Cells fiom 1 ml of culture were sedirnented in a
microcentrifuge, washed with water, resuspended in 1 ml of water and disrupted by
sonication. Insoluble material was sedimented, washed with water and analyzed by 15
% SDS-PAGE as is known to one skilled in the art (Fig. 6B).
. Expression of Optimized OspA Antigen With C-Terminal Fusion Partner
The P. salmonis 0spA ORF was subcloned into the Impact CN Expression System
(New England Biolabs) to add a C—termina1 fusion partner containing a self-cleaving
spacer region and chitin binding domain to aid in purification and antibody generation
of OspA (Fig. 7).
The 0spA ORF was PCR amplified from clone pBl2 using custom primers (Table 3)
designed to incorporate Nde I and Sap I restriction enzyme cleavage sites onto the 5'
and 3' ends of the 0spA ORF. The OspA PCR product was digested with Nde I and Sap
I restriction enzymes and ligated with the pTYB 1 vector (NEB) of the Impact CN
system digested with Nde I and Sap I to create the OspA fusion construct, pTYB1-
l7kDa (Fig. 7). Positive clones were identified by screening Kpn I and Nde I digests
of plasmid preps from potential positive clones by agarose gel electrophoresis.
Positive clones were confirmed to contain the osp/1 ORF in frame with the chitin
binding domain by DNA sequence analysis.
Table 3: Oligonucleotide primers used during construction of pTYBl-17kDa. Bold
nucleotides are not homologous to the template ospA ORF.
Primer Sequence
Forward (SEQ lD:7) 5'- GAG AGA ACA TAT GAA CAG AGG ATG rrr GCA AGG — 3'
. Salmonid Antibody Response to OspA-fusion Vaccine
Coho salmon antibody response to the OspA with N-terminal fusion partner Vaccine
candidate (Example 4) was assayed by enzyme linked immunosorbant assay (ELISA).
Coho salmon fry (125 per group; ~15 g mean weight) were each injected
intraperitoneally (TP) 0.2 ml of a formalin inactivated (1 ml/L) adjuvanated
(MicrogenTM) vaccine (5:1 vaccine:adjuvant) containing 50 (Pg of total protein
purified as the insoluble fraction from E. coli BL21 expressing the osp/I fusion
construct pET—Cl7E2 (Example 4). A control group of fish received 0.2 ml of
adjuvant diluted with saline 5:1. A second control group was comprised of non-
vaccinated salmon.
Four weeks post-immunization, 5 fish from each group were bled from the
caudalvein, kept on ice, blood was pooled for each group and serum was collected
bycentrifiigation of pooled blood at 5,000 rpm for 20 min in a clinical
centrifuge.ELISA plates were coated with 10 g of C l7E2 protein in 100 (D1 of
coating buffer (Tris buffered saline (TBS), pH 7.5, 0.5% Tween-TM-20). Plates were
covered with parafilm and incubated at 4°C overnight. Coating solution was removed
and wells were blocked with 200 (D1 of TBS with 3 % bovine serum. Plates were
washed 3 times with -TBS. Fish serum from each group was serially diluted in -TBS
with 3 % bovine serum and added to wells. Plates were then incubated at 15°C for 1 h
and then washed 3 times with Second antibody, a mixture of 2 monoclonal
antibodies (mAb) against salmon immunoglobulin, IPAZC7 (dil. 1/ 100) and Beecrofi
Reverse (SEQ IDi8) 5'- GCC ATA AGC TCT TCC GCA TTT TTC TGT TGA AAT GAC TTG C -
(dil. 1/500), were diluted in TBS with 3% bovine serum, added to plates and
incubated at room temperature for l h. Plates were washed 3 times with TBS-Third
antibody, alkaline phosphatase conjugated goat anti-mouse IgG1 (dil. 1/2000), was
added to plates and incubated at room temperature for 1 h. Plates were washed 3
times. The ELISA was developed with 100 CD1 of 1 mg/ml paranitrophenyl phosphate
in alkaline phosphatase buffer and incubated at room temperature overnight and
absorbance at 405 nm was measured spectrophotometrically.
Antibody titres were defined as the maximum serum dilution that resulted in a signal
corresponding to 3 times the background obtained with the diluent vaccinated serum
group at a dilution of 1:320. Background serum was pooled from coho salmon
vaccinated with adjuvant alone at each time point. The results indicate that all OspA
fusion protein constructs are capable of elicitingan antibody response in immunized
coho higher than the response obtained with adjuvant alone. The highest antibody
responses were found in coho salmon immunized with OspA fusion proteins
containing promiscuous TCE's (Fig. 9).
7. Salmonid Lymphocyte Response to OspA-fusion Vaccine
The lymphocyte response to the OspA-fiision protein vaccine constructs was
measured using the lymphocyte proliferation assay.
Isolation of lymphocytes. Atlantic salmon that had been vaccinated 4 weeks prior
with 0.2 ml of each OspA fusion protein vaccine were euthanized with an overdose of
marinil and their head kidneys were aseptically harvested and immediately placed in 5
ml of cold MEM-10 (10% fetal bovine serum; Life Technologies) on ice. All
subsequent manipulations were conducted on ice. Cells were dissociated by repeated
passage through a 5 ml syringe. The tissue suspension was placed in a 15 ml tube and
7 ml of additional MEM—l0 were added. Tissue fragments were allowed to settle out
of solution for 10 min. Cells suspended in the media were collected and layered on 4
ml of 51 % PercollT—M (10 ml 10H HBSS, 51 ml Percollm-, made up to 100 ml with
H20). The step gradient was centrifuged for 30 min at 400 x g, 4°C. Lymphocytes
were collected from the MEM-10/Percoll-TM interface. Lymphocytes were centrifuged
and washed once in MEM—l0 and resuspended in 1 ml MEM-10. The numbers of
viable cells was determined using Trypan blue (0.4%; Sigma) staining. Cells were
diluted to a final concentration of 5 x 106 cells/ml with MEM-10.
Lymphocyte proliferation assay. Isolated lymphocytes were added to 96 well cell
culture plates with 5 x 105 cells/well (100 (D1 vol.). OspA fi.lSl0I1 protein Cl7E2 was
added as a stimulating antigen (2 CDg/well) and cells were incubated for 6 days at
17°C. Lymphocyte proliferation was determined spectrophotometiically using WSTI
(4-[3-(4-iodophenyl)(4-nitrophenyl)~2H—5-tetrazolio]-l,3-benzene disulfonate) cell
proliferation reagent (Roche Molecular Biochemicals). WST-1 allows colorimetric
quantification of cell proliferation based on cleavage of WST-l by mitochondrial
dehydrogenases in viable cells. WST-l (10 CD1) was added to each well and plates
were incubated at 17°C until sufficient colour development prior to absorbance
measurement at 450 nm with a reference wavelength of 630 nm. Bacterial
lipopolysaccharide (LPS) (100 CDg/ml) and conconavalin A (ConA) (50 CDg/ml) were
used as B and T lymphocyte mitogens for positive controls.
The degree of lymphocyte stimulation was detennined by calculating the stimulation
index for each sample of lymphocytes exposed to antigen (Fig. 10). Stimulation index
was calculated by dividing the average absorbance of lymphocyte samples presented
with stimulating antigen by the average absorbance of lymphocytes presented with no
antigen (Fig. 10). The results indicate the addition of promiscuous TCE’s to the OspA
fusion protein candidate enhance the proliferative lymphocyte responses of salmon
vaccinated with the TCE—encoding vaccines against OspA (Fig. 10).
8. Protection of Immunized Salmonids Against P. salmonis Challenge
OspA fusion proteins were purified as inclusion bodies from E. coli BL2l and protein
concentrations were determined using the BCA protein assay (Pierce). The relative
percentages of the OspA fusion proteins within each preparation were determined by
SDS—PAGE analysis and quantification of the fusion protein bands using a Gel
Documentation system and AlphaEase software. Each protein sample was fixed by
the addition of formalin (1 ml/L) and incubation with shaking at 15°C for 24 hr. Each
protein solution was added aseptically to diluent (oil in water adjuvant) to obtain a
final target protein concentration of 250 mg/L.
Coho salmon (~15 g) were anaesthetized (1 ppm metomidate hydrochloride), fin
clipped for group identification, and intraperitoneally injected with 0.2 ml of vaccine
with 60 fish per group. There were 6 groups in total: C17E2, CT17E2, CMl7E2,
CMT17E2, CM l7E2 plus CT l7E2 (1:1), and an adjuvant control. Salmon were held
for 8 weeks in freshwater at 85°C post-vaccination.
All vaccinated coho were anaesthetized (1 ppm Marinil) and IP injected with 0.1 ml
of P. salmonis infected CHSE-214 cell culture supernatant ( ~106 T C1D5o/ml).
Salmon were maintained in freshwater at 13°C post—challenge and mortalities were
logged. External and internal observations along with PCR of kidney and central liver
sections using P. salmonis 16S rRNA primers (Giovannoni, 1991; Marshall, et al.,
) were performed for confirmation of mortality.
RPS is calculated to generate a numerical value representing the level of protection
elicited by a vaccine. In general, RPS is calculated as a ratio of the cumulative
mortality of a test group to the cumulative mortality of an unvaccinated group. RPS =
[1 — (% mortality of test group - % mortality of control group)] x 100%.
Mortalities in the TCE OspA construct vaccinated groups began 7-10 days afier the
control group (Fig. 11). Cumulative mortality reached 85.5% in the control group
(Fig. 11). The C17E2 vaccinated group reached 59.6% cumulative mortality, 30.2%
RPS (Fig. 11). The CT17E2 vaccinated group reached a cumulative mortality of
.6%, 58.4% RPS (Fig. 11). CMl7E2 vaccinated salmon reached 20.0%, 76.6 RPS
(Fig. 11). Salmon vaccinated with a 1:1 mixture of CMl7E2 and CT17E2 reached
.6% cumulative mortality giving a 78.2% RPS (Fig. 11). The lowest mortality was
observed in the CMT17E2 vaccinated group, with only 14.5 cumulative mortality and
an 83.0% RPS (Fig. 11).
The results indicate that adjuvant controls (0) had severe mortalities (> 80 %) and the
CMT17E2 Vaccinates (H) were significantly protected with only 14.5% mortality
(Fig. 11).
REFERENCES
Altschul, et al. (1990). J. Mol. Biol. 215:403-410.
Altschul, et al. (1997). Nucleic Acids Res. 2513389-402.
Anacker, et al. (1985). J. Infect. Dz's. 15121052-60.
Anderson (1997). Rickettsial Infection and Immunity. Plenum Press, New York
NY. ppl-11.
Anderson, et al. (1990). Infect. Immun. 58:2760—9.
Anderson, et al. (1987). J. Bacteriol. 169:2385—90.
Azad, et al. (1997). Emerg. Infect. Dis. 3:319-27.
Bixler, et al. (1989). Contrib. Microbiol. Immunol. 10:18-47.
Bravo, et al. (1989). FHS/AFSNewsl. 17:3.
Brocklebank, et al. (1993). Can. Vet. J. 34:745-8.
Carl, et al. (1990). Proc. Natl. Acad. Sci. U. S. A. 87:8237-41.
Ching, et al. (1992). Mol. Immunol. 29:95-105.
Ching, et al. (1996). Infect. Immun. 64:1413-9.
Corpet, et al. (1988). Nuc. Acids Res. 16:10881-10890.
Cvitanich, et al. (1991). J. Fish Dis. 14:121—45.
Davis, et al. (1998). Curr. Microbial. 36:80-4.
Demotz, et al. (1989). J. Immunol. 142:394-402.
Drancourt, et al. (1994). FEMS Microbiol. Rev. 13:13-24.
Fryer, et al. (1990). Fish Pathol. 25:107-14.
Fryer, et al. (1992). Int. J. Syst. Bacteriol. 421120-6.
Fryer, et al. (1997). Emerg. Infect. Dis. 3:137-44.
Giovannoni (1991). Nucleic Acid Techniques in Bacterial Systematics. John Wiley
and Sons, New York. pp177-201.
Hahn, et al. (1996). Microbiol. Immunol. 40:233—6.
Hathaway, et al. (1995). Vaccine 13:1495-500.
Hickman, et al. (1991). Microb. Pathog. 11:19-31.
Higgins, et al. (1998). J. Clin. Microbiol. 36:1793-4.
Huang, et al. (1992). Comp. Appl. Biosci. 8:155-165.
Jameson, et al. (1988). Comput. App]. Biosci. 4:181—6.
Kj€1Tll1f, et al. (1997). Mol. Immunol. 34:599-608.
Kuzyk, et al. (1996). Infect. Immun. 6415205-10.
Lannan, et al. (1993). Fish. Res. 171115-21.
Lewis, et al. (1998). Infect. Immun. 66:3035-42.
Li, et al. (1998). Microb. Pathog. 242289-98.
Mallavia (1991). Eur. J. Epidemiol. 7:213—21.
Marshall, et al. (1998). Appl. Environ. Microbiol. 64:3066—9.
McDonald, et al. (1987). Science 235:83-5.
Messick, et al. (1994). Infect. Immun. 62:3156-61.
Musoke, et al. (1996). Br. Vet. J. 152:621-39.
Needleman, et al. (J 970). J. Mol. Biol. 48:443.
O'Hem, et al. (1997). Vaccine 1521761-6.
Olivier, et al. (1985). Dev. Comp. Immunol. 9:419-32.
Pang, et al. (1994). J Bacteriol. l76:923-6.
Panina-Bordignon, et al. (1989). Eur. J. Immunol. 19:2237—42.
Partidos, et al. (1992). Eur. J. Immunol. 22:2675—80.
Partidos, et al. (1990). J. Gen. Virol. 7122099-105.
Pearson, et al. (1988). Proc. Natl. Acad. Sci. 85:2444
Pearson, et al. (1994). Meth. Mol. Biol. 242307-33 1.
Pearson (1998). J. Mol. Biol. 276171-84.
Pillai, et al. (1995). Infect. Immun. 6321535-40.
Rachek, et al. (1998). J. Bacterial. 18022118-24.
Rikihisa (1991). Clin. Microbiol. Rev. 4:286-308.
Rodger, et al. (1993). J. Fish Dis. 162361-9.
Sambrook, et al. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring
Harbour Laboratory Press, Cold Spring Harbour, NY.
Seong, et al. (1997). Vaccine 15:l74l—7.
Smith, et al. (1981). Adv. Appl. Math. 2:482.
Stenos, et al. (1998). Int. J. Syst. Bacterial. 48 Pt 4:l399—404.
Sumner, et al. (1995). Vaccine 13:29-35.
Tabor, et al. (J 985). Proc. Natl. Acad. Sci. U. S. A. 82:1074—8.
Tarnura, et al. (1982). Microbial. Immunol. 262321-8.
Troyer, et al. (1999). Infect. Immun. 67:3308-l l.
Valrnori, et al. (1992). J. Immunal. 1491717-21.
Vishwanath, et al. (1990). Infect. Immun. 582646-53.
Weiss, et al. (1975). Appl. Microbiol. 30:456—63.
Claims (18)
1. A fish vaccine against the bacterial pathogen Piscirickettsia salmonis, comprising an immunogenic amount of a protein of 17 kDa encoded by one of SEQ ID NO: 1 and SEQ ID N023, with or without an adjuvant.
2. The vaccine of claim 1, wherein said protein is post-translationally modified into a lipoprotein.
3. The vaccine of claim 1 or 2, wherein said protein is fused to at least one other protein or protein fragment either at the N or C terminus or both.
4. The fish vaccine as claimed in claim 3, comprising an immunogenic amount of a protein of substantially 17 kDa fused with an N—terminal fusion partner, as encoded by SEQ ID NO: 5.
5. A fish vaccine against the bacterial pathogen Piscirickettsia sa/monis comprising an immunogenic amount of a protein comprising an amino acid sequence of one of SEQ ID NO: 2, and SEQ ID NO: 4 or sequence variants thereof retaining the same immunogenicity, with or without an adjuvant.
6. The vaccine of claim 5. wherein said protein is post-translationally modified into a lipoprotein.
7. The vaccine of claim 5 or 6, wherein said protein is fused to at least one other protein or protein fragment either at the N or C terminus or both.
8. The vaccine of claim 7, wherein said protein is fused with an N-terminal fusion partner as encoded by SEQ ID NO: 6.
9. A use of an immunogenic amount of a protein of substantially 17 kDa encoded by one of SEQ ID NO: 1 and SEQ ID NO: 3 in the manufacture of a vaccine for the treatment of Piscirickettsia salmonis in a fish.
10. The use of claim 9, wherein said protein is post-translationally modified into a lipoprotein.
11. The use of claim 9 or 10, wherein said protein is fused to at least one other protein or protein fragment either at the N or C terminus or both.
12. The use as claimed in claim 11, wherein the protein is fused to an N—terminal fusion partner, as encoded by SEQ ID NO: 5.
13. A use of an immunogenic amount of a protein comprising the amino acid sequence of one of SEQ ID NO: 2 or SEQ ID NO: 4 or sequence variants thereof retaining the same immunogenicity, in the manufacture of a vaccine for the treatment of P/scirickettsi'a sa/monis in a fish.
14. The use of claim 13, wherein said protein is post-translationally modified into a lipoprotein.
15. The use of claim 13 or 14, wherein said protein is fused to at least one other protein or protein fragment either at the N or C terminus or both.
16. The use of claim 15, wherein said protein is fused to an N-terminal fusion partner comprising the amino acid sequence of SEQ ID NO: 6.
17. A fish vaccine substantially as described herein, with reference to the accompanying drawings.
18. A use, as defined in any one of Claims 10 to 16, with reference to the accompanying drawings.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CACANADA17/09/19992,281,913 | |||
CA002281913A CA2281913A1 (en) | 1999-09-17 | 1999-09-17 | A vaccine against piscirickettsia salmonis based on a recombinant 17 kd protein |
Publications (2)
Publication Number | Publication Date |
---|---|
IE20000752A1 IE20000752A1 (en) | 2002-12-11 |
IE84132B1 true IE84132B1 (en) | 2006-02-08 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US4888170A (en) | Vaccines obtained from antigenic gene products of recombinant genes | |
Abdullah et al. | Cloning, nucleotide sequence, and expression of the Pasteurella haemolytica A1 glycoprotease gene | |
Vodkin et al. | A heat shock operon in Coxiella burnetti produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli | |
CA1338705C (en) | Vaccines obtained from antigenic gene products of recombinant genes | |
JP4301415B2 (en) | Lyme disease combination composition and use thereof | |
JP5566684B2 (en) | Recombinant toxin A / toxin B vaccine against Clostridium difficile | |
JP2002541808A (en) | Recombinant toxin A protein carrier for polysaccharide conjugate vaccine | |
JPH09500538A (en) | High level expression, purification and regeneration of Neisseria meningitidis outer membrane group B porin protein | |
JP2008212160A (en) | Microbial protein, microorganism producing the same and their use for vaccine and for detection of tuberculosis | |
EP0068693A2 (en) | Production of foot and mouth disease vaccine from microbially expressed antigens | |
JP2002511492A (en) | Proboscis | |
US20050002946A1 (en) | Vaccines and agents for inducing immunity in fish against rickettsial diseases, and associated preventative therapy | |
EP3062816A1 (en) | Attenuated pasteurella multocida vaccines & methods of making & use thereof | |
JP2001504329A (en) | Nucleic acid and amino acid sequences related to Helicobacter pylori and vaccine compositions thereof | |
JPH10234388A (en) | Gonococcus pi protein and production of its vaccine | |
KR100510906B1 (en) | Attenuated live bacteria of Actino Bacillus pluronomymoniae | |
CA2719041C (en) | A method for identifying polypeptides which comprise a cross-reactive antigenic determinant | |
JP2001510992A (en) | Nucleic acid and amino acid sequences related to Helicobacter pylori and vaccine compositions thereof | |
FI104496B (en) | Overexpression systems for cholera B subunit expression by foreign promoters and leader peptides | |
RU2129611C1 (en) | Polypeptide, recombinant polynucleotide, vaccine | |
GB2356632A (en) | OspA lipoproteins | |
IE84132B1 (en) | OspA Lipoproteins | |
EP0087735A2 (en) | Recombinant DNA, microorganism transformed therewith and their use | |
Canales et al. | Anaplasma marginale major surface protein 1a directs cell surface display of tick BM95 immunogenic peptides on Escherichia coli | |
IE20000752A1 (en) | Immunoreactive 17 kDa outer surface lipoprotein (OspA) of Piscirickettsia salmonis, vaccine for inducing immunity against P. Salmonis and other rickettsial diseases |