EP1220924A1 - Chlamydia antigene und entsprechende gene und ihre verwendungen - Google Patents
Chlamydia antigene und entsprechende gene und ihre verwendungenInfo
- Publication number
- EP1220924A1 EP1220924A1 EP00962124A EP00962124A EP1220924A1 EP 1220924 A1 EP1220924 A1 EP 1220924A1 EP 00962124 A EP00962124 A EP 00962124A EP 00962124 A EP00962124 A EP 00962124A EP 1220924 A1 EP1220924 A1 EP 1220924A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- polypeptide
- nucleic acid
- chlamydia
- vaccine
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 241000606161 Chlamydia Species 0.000 title claims abstract description 52
- 239000012634 fragment Substances 0.000 title claims description 41
- 239000000427 antigen Substances 0.000 title description 31
- 108091007433 antigens Proteins 0.000 title description 31
- 102000036639 antigens Human genes 0.000 title description 31
- 241001647372 Chlamydia pneumoniae Species 0.000 claims abstract description 68
- 238000000034 method Methods 0.000 claims abstract description 58
- 239000013598 vector Substances 0.000 claims abstract description 51
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 36
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 28
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 28
- 239000002773 nucleotide Substances 0.000 claims abstract description 24
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 24
- 102000023805 ATP:ADP antiporter activity proteins Human genes 0.000 claims abstract description 19
- 108040000931 ATP:ADP antiporter activity proteins Proteins 0.000 claims abstract description 19
- 230000003053 immunization Effects 0.000 claims abstract description 12
- 230000000694 effects Effects 0.000 claims abstract description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 216
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 206
- 229920001184 polypeptide Polymers 0.000 claims description 202
- 229960005486 vaccine Drugs 0.000 claims description 65
- 239000002671 adjuvant Substances 0.000 claims description 28
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 28
- 208000007190 Chlamydia Infections Diseases 0.000 claims description 22
- 150000001413 amino acids Chemical class 0.000 claims description 20
- 230000004927 fusion Effects 0.000 claims description 19
- 241000124008 Mammalia Species 0.000 claims description 17
- 230000028993 immune response Effects 0.000 claims description 17
- 239000008194 pharmaceutical composition Substances 0.000 claims description 14
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 10
- 239000003937 drug carrier Substances 0.000 claims description 8
- 230000002163 immunogen Effects 0.000 claims description 6
- 230000000692 anti-sense effect Effects 0.000 claims description 5
- 108020001507 fusion proteins Proteins 0.000 claims description 5
- 102000037865 fusion proteins Human genes 0.000 claims description 5
- 230000005847 immunogenicity Effects 0.000 claims description 5
- 230000000295 complement effect Effects 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 2
- 239000013613 expression plasmid Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000009007 Diagnostic Kit Methods 0.000 claims 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims 1
- 210000001124 body fluid Anatomy 0.000 claims 1
- 239000010839 body fluid Substances 0.000 claims 1
- 239000002853 nucleic acid probe Substances 0.000 claims 1
- 208000015181 infectious disease Diseases 0.000 abstract description 48
- 108090000623 proteins and genes Proteins 0.000 abstract description 41
- -1 DNA Chemical class 0.000 abstract description 13
- 238000002649 immunization Methods 0.000 abstract description 11
- 230000004048 modification Effects 0.000 abstract description 8
- 238000012986 modification Methods 0.000 abstract description 8
- 201000010099 disease Diseases 0.000 abstract description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 7
- 102000040430 polynucleotide Human genes 0.000 description 75
- 108091033319 polynucleotide Proteins 0.000 description 75
- 239000002157 polynucleotide Substances 0.000 description 75
- 108020004414 DNA Proteins 0.000 description 52
- 239000000203 mixture Substances 0.000 description 33
- 239000000523 sample Substances 0.000 description 28
- 210000004027 cell Anatomy 0.000 description 27
- 235000001014 amino acid Nutrition 0.000 description 21
- 102000004169 proteins and genes Human genes 0.000 description 21
- 235000018102 proteins Nutrition 0.000 description 20
- 229940024606 amino acid Drugs 0.000 description 18
- 239000013612 plasmid Substances 0.000 description 18
- 239000013615 primer Substances 0.000 description 18
- 239000000047 product Substances 0.000 description 16
- 102000053602 DNA Human genes 0.000 description 15
- 239000003153 chemical reaction reagent Substances 0.000 description 15
- 238000003752 polymerase chain reaction Methods 0.000 description 14
- 230000001225 therapeutic effect Effects 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 208000006673 asthma Diseases 0.000 description 12
- 238000012217 deletion Methods 0.000 description 12
- 230000037430 deletion Effects 0.000 description 12
- 238000009396 hybridization Methods 0.000 description 12
- 230000036039 immunity Effects 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 12
- 230000000069 prophylactic effect Effects 0.000 description 12
- 241000606153 Chlamydia trachomatis Species 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 11
- 239000002502 liposome Substances 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 10
- 241001647378 Chlamydia psittaci Species 0.000 description 10
- 206010061041 Chlamydial infection Diseases 0.000 description 10
- 239000012472 biological sample Substances 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 150000001875 compounds Chemical class 0.000 description 10
- 239000003814 drug Substances 0.000 description 10
- 230000001681 protective effect Effects 0.000 description 10
- 238000011282 treatment Methods 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 9
- 238000006467 substitution reaction Methods 0.000 description 9
- 108091026890 Coding region Proteins 0.000 description 8
- 208000035473 Communicable disease Diseases 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 206010035664 Pneumonia Diseases 0.000 description 8
- 210000003719 b-lymphocyte Anatomy 0.000 description 8
- 239000003085 diluting agent Substances 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 241000498849 Chlamydiales Species 0.000 description 7
- 108010049048 Cholera Toxin Proteins 0.000 description 7
- 102000009016 Cholera Toxin Human genes 0.000 description 7
- 210000001744 T-lymphocyte Anatomy 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 210000004962 mammalian cell Anatomy 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 238000010561 standard procedure Methods 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 6
- 101710164702 Major outer membrane protein Proteins 0.000 description 6
- 230000008827 biological function Effects 0.000 description 6
- 125000002091 cationic group Chemical group 0.000 description 6
- 238000002405 diagnostic procedure Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 238000007918 intramuscular administration Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 230000036961 partial effect Effects 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 238000007920 subcutaneous administration Methods 0.000 description 6
- 239000013603 viral vector Substances 0.000 description 6
- 241001674218 Chlamydia pecorum Species 0.000 description 5
- 241000701022 Cytomegalovirus Species 0.000 description 5
- 102000013462 Interleukin-12 Human genes 0.000 description 5
- 108010065805 Interleukin-12 Proteins 0.000 description 5
- 102000000588 Interleukin-2 Human genes 0.000 description 5
- 108010002350 Interleukin-2 Proteins 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 239000012620 biological material Substances 0.000 description 5
- 206010006451 bronchitis Diseases 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 229940117681 interleukin-12 Drugs 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 5
- 210000004379 membrane Anatomy 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 201000001320 Atherosclerosis Diseases 0.000 description 4
- 241001185363 Chlamydiae Species 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 241001529936 Murinae Species 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000001476 gene delivery Methods 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 238000001990 intravenous administration Methods 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- 108040007629 peroxidase activity proteins Proteins 0.000 description 4
- 102000013415 peroxidase activity proteins Human genes 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 230000002685 pulmonary effect Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000003053 toxin Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 108700012359 toxins Proteins 0.000 description 4
- 239000003981 vehicle Substances 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 208000035086 Chlamydophila Infections Diseases 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108010081690 Pertussis Toxin Proteins 0.000 description 3
- 206010057190 Respiratory tract infections Diseases 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N 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 3
- 241000607626 Vibrio cholerae Species 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 125000000129 anionic group Chemical group 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 230000027645 antigenic variation Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 201000000902 chlamydia Diseases 0.000 description 3
- 208000012538 chlamydia trachomatis infectious disease Diseases 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000009260 cross reactivity Effects 0.000 description 3
- 101150028842 ctxA gene Proteins 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- 230000029226 lipidation Effects 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 239000011859 microparticle Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 238000007911 parenteral administration Methods 0.000 description 3
- 229920000136 polysorbate Polymers 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 210000001635 urinary tract Anatomy 0.000 description 3
- 229940118696 vibrio cholerae Drugs 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- LEBVLXFERQHONN-UHFFFAOYSA-N 1-butyl-N-(2,6-dimethylphenyl)piperidine-2-carboxamide Chemical compound CCCCN1CCCCC1C(=O)NC1=C(C)C=CC=C1C LEBVLXFERQHONN-UHFFFAOYSA-N 0.000 description 2
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 208000003322 Coinfection Diseases 0.000 description 2
- 206010011224 Cough Diseases 0.000 description 2
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 241000700721 Hepatitis B virus Species 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 208000032376 Lung infection Diseases 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 208000035415 Reinfection Diseases 0.000 description 2
- 208000037656 Respiratory Sounds Diseases 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 241000607762 Shigella flexneri Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 241000256251 Spodoptera frugiperda Species 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 241000700618 Vaccinia virus Species 0.000 description 2
- 206010047924 Wheezing Diseases 0.000 description 2
- HIHOWBSBBDRPDW-PTHRTHQKSA-N [(3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-yl] n-[2-(dimethylamino)ethyl]carbamate Chemical compound C1C=C2C[C@@H](OC(=O)NCCN(C)C)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HIHOWBSBBDRPDW-PTHRTHQKSA-N 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000009798 acute exacerbation Effects 0.000 description 2
- 230000000240 adjuvant effect Effects 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000002238 attenuated effect Effects 0.000 description 2
- 229960004099 azithromycin Drugs 0.000 description 2
- MQTOSJVFKKJCRP-BICOPXKESA-N azithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)N(C)C[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 MQTOSJVFKKJCRP-BICOPXKESA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229960003150 bupivacaine Drugs 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 210000004351 coronary vessel Anatomy 0.000 description 2
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- MXHRCPNRJAMMIM-UHFFFAOYSA-N desoxyuridine Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000000521 hyperimmunizing effect Effects 0.000 description 2
- 210000004201 immune sera Anatomy 0.000 description 2
- 229940042743 immune sera Drugs 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 229940126578 oral vaccine Drugs 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000002987 primer (paints) Substances 0.000 description 2
- 230000037452 priming Effects 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 150000003431 steroids Chemical class 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 2
- 229910052721 tungsten Inorganic materials 0.000 description 2
- 239000010937 tungsten Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- RKDVKSZUMVYZHH-UHFFFAOYSA-N 1,4-dioxane-2,5-dione Chemical compound O=C1COC(=O)CO1 RKDVKSZUMVYZHH-UHFFFAOYSA-N 0.000 description 1
- MXHRCPNRJAMMIM-SHYZEUOFSA-N 2'-deoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-SHYZEUOFSA-N 0.000 description 1
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical group OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- 229940117976 5-hydroxylysine Drugs 0.000 description 1
- 206010000211 Abortions and stillbirth Diseases 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 241000238421 Arthropoda Species 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 208000037260 Atherosclerotic Plaque Diseases 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 231100000699 Bacterial toxin Toxicity 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 206010006458 Bronchitis chronic Diseases 0.000 description 1
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical class [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 1
- 241000178270 Canarypox virus Species 0.000 description 1
- 241000701931 Canine parvovirus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000014882 Carotid artery disease Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 206010009137 Chronic sinusitis Diseases 0.000 description 1
- 241000193163 Clostridioides difficile Species 0.000 description 1
- 108091033380 Coding strand Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 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
- 102100036912 Desmin Human genes 0.000 description 1
- 108010044052 Desmin Proteins 0.000 description 1
- 206010013952 Dysphonia Diseases 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000701832 Enterobacteria phage T3 Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 108010088729 HLA-A*02:01 antigen Proteins 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 101500016539 Histophilus somni (strain 2336) Protein p76 IgBP Proteins 0.000 description 1
- 208000010473 Hoarseness Diseases 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 241000235058 Komagataella pastoris 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
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-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
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 206010024971 Lower respiratory tract infections Diseases 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 241000202934 Mycoplasma pneumoniae Species 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 206010068319 Oropharyngeal pain Diseases 0.000 description 1
- 108700006640 OspA Proteins 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 201000007100 Pharyngitis Diseases 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010013381 Porins Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 101710194807 Protective antigen Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 238000011579 SCID mouse model Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241001222774 Salmonella enterica subsp. enterica serovar Minnesota Species 0.000 description 1
- 241000710961 Semliki Forest virus Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 241000287219 Serinus canaria Species 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 101710084578 Short neurotoxin 1 Proteins 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241000194026 Streptococcus gordonii Species 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 101710182532 Toxin a Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 206010046306 Upper respiratory tract infection Diseases 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108010051583 Ventricular Myosins Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- PTTGRYBBCYZPSL-UHFFFAOYSA-H [Al+3].[Al+3].OOP([O-])([O-])=O.OOP([O-])([O-])=O.OOP([O-])([O-])=O Chemical compound [Al+3].[Al+3].OOP([O-])([O-])=O.OOP([O-])([O-])=O.OOP([O-])([O-])=O PTTGRYBBCYZPSL-UHFFFAOYSA-H 0.000 description 1
- 208000024434 abnormal chest sound Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000012387 aerosolization Methods 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical class [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 229940047712 aluminum hydroxyphosphate Drugs 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229940069428 antacid Drugs 0.000 description 1
- 239000003159 antacid agent Substances 0.000 description 1
- 230000001458 anti-acid effect Effects 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000002555 auscultation Methods 0.000 description 1
- 239000000688 bacterial toxin Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000002051 biphasic effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000010083 bronchial hyperresponsiveness Effects 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 1
- 229960003669 carbenicillin Drugs 0.000 description 1
- 210000001715 carotid artery Anatomy 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 150000001841 cholesterols Chemical class 0.000 description 1
- 208000007451 chronic bronchitis Diseases 0.000 description 1
- 208000027157 chronic rhinosinusitis Diseases 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- YSMODUONRAFBET-UHFFFAOYSA-N delta-DL-hydroxylysine Natural products NCC(O)CCC(N)C(O)=O YSMODUONRAFBET-UHFFFAOYSA-N 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 210000005045 desmin Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- UMGXUWVIJIQANV-UHFFFAOYSA-M didecyl(dimethyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCC[N+](C)(C)CCCCCCCCCC UMGXUWVIJIQANV-UHFFFAOYSA-M 0.000 description 1
- PSLWZOIUBRXAQW-UHFFFAOYSA-M dimethyl(dioctadecyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC PSLWZOIUBRXAQW-UHFFFAOYSA-M 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 239000001177 diphosphate Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical group [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006872 enzymatic polymerization reaction Methods 0.000 description 1
- 238000009585 enzyme analysis Methods 0.000 description 1
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical compound NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 244000052637 human pathogen Species 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000010324 immunological assay Methods 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 244000000056 intracellular parasite Species 0.000 description 1
- 229940065638 intron a Drugs 0.000 description 1
- 238000007852 inverse PCR Methods 0.000 description 1
- 101150027374 irgA gene Proteins 0.000 description 1
- 239000012948 isocyanate Substances 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 208000030500 lower respiratory tract disease Diseases 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000016379 mucosal immune response Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 229940127249 oral antibiotic Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000008024 pharmaceutical diluent Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 1
- 229920002627 poly(phosphazenes) Polymers 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 102000007739 porin activity proteins Human genes 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 210000001747 pupil Anatomy 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000011555 rabbit model Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 201000009890 sinusitis Diseases 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- MNQYNQBOVCBZIQ-JQOFMKNESA-A sucralfate Chemical compound O[Al](O)OS(=O)(=O)O[C@@H]1[C@@H](OS(=O)(=O)O[Al](O)O)[C@H](OS(=O)(=O)O[Al](O)O)[C@@H](COS(=O)(=O)O[Al](O)O)O[C@H]1O[C@@]1(COS(=O)(=O)O[Al](O)O)[C@@H](OS(=O)(=O)O[Al](O)O)[C@H](OS(=O)(=O)O[Al](O)O)[C@@H](OS(=O)(=O)O[Al](O)O)O1 MNQYNQBOVCBZIQ-JQOFMKNESA-A 0.000 description 1
- 229960004291 sucralfate Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 229940040944 tetracyclines Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- IOTZOAHTTSZFQY-SEYXRHQNSA-N trimethyl-[3-[(z)-octadec-9-enoxy]propyl]azanium Chemical compound CCCCCCCC\C=C/CCCCCCCCOCCC[N+](C)(C)C IOTZOAHTTSZFQY-SEYXRHQNSA-N 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- 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
- C07K14/295—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Chlamydiales (O)
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to the Chlamydia Npt2cp (ADP/ATP translocase) antigen and corresponding DNA molecules, which can be used to prevent and treat Chlamydia infection in mammals, such as humans.
- Chlamydia Npt2cp ADP/ATP translocase
- Chlamydiae are prokaryotes . They exhibit morphologic and structural similarities to gram-negative bacteria including a trilaminar outer membrane, which contains lipopolysaccharide and several membrane proteins that are structurally and functionally analogous to proteins found in E coli . They are obligate intra-cellular parasites with a unique biphasic life cycle consisting of a metabolically inactive but infectious extracellular stage and a replicating but non- infectious intracellular stage. The replicative stage of the life-cycle takes place within a membrane-bound inclusion which sequesters the bacteria away from the cytoplasm of the infected host cell . C.
- C. pneumoniae is a common human pathogen, originally described as the T AR strain of Chlamydia psi ttaci but subsequently recognised to be a new species.
- C. pneumoniae is antigenically, genetically and morphologically distinct from other chlamydia species (C. trachomati s , C. pecorum and C. psi ttaci ) . It shows 10% or less DNA sequence homology with either of C. trachomatis or C. psi ttaci .
- C. pneumoniae is a common cause of community acquired pneumonia, only less frequent than Streptococcus pneumoniae and Mycoplasma pneumoniae (Grayston et al .
- C. pneumoniae infection usually presents as an acute respiratory disease (i.e., cough, sore throat, hoarseness, and fever; abnormal chest sounds on auscultation) .
- cough i.e., cough, sore throat, hoarseness, and fever; abnormal chest sounds on auscultation
- the cough persists for 2 to 6 weeks, and recovery is slow.
- upper respiratory tract infection is followed by bronchitis or pneumonia.
- subsequent co- infection with pneumococcus has been noted in about half of these pneumonia patients, particularly in the infirm and the elderly.
- C. pneumoniae infection is also linked to diseases other than respiratory infections.
- C. pneumoniae appears to spread slowly through a population (case-to-case interval averaging 30 days) because infected persons are inefficient transmitters of the organism. Susceptibility to C. pneumoniae is universal. Reinfections occur during adulthood, following the primary infection as a child.
- C. trachomatis infection does not confer cross- immunity to C. pneumoniae . Infections are easily treated with oral antibiotics, tetracycline or erythromycin (2 g/d, for at least 10 to 14 d) . A recently developed drug, azithromycin, is highly effective as a single-dose therapy against chlamydial infections . In most instances, C. pneumoniae infection is often mild and without complications, and up to 90% of infections are subacute or unrecognized.
- pneumoniae infection usually happens between the ages of 5 and 20 y. In the USA, for example, there are estimated to be 30,000 cases of childhood pneumonia each year caused by C. pneumoniae . Infections may cluster among groups of children or young adults ⁇ e . g. , school pupils or military conscripts) .
- C. pneumoniae causes 10 to 25% of community-acquired lower respiratory tract infections (as reported from Sweden, Italy, Finland, and the USA) . During an epidemic, C. pneumonia infection may account for 50 to 60% of the cases of pneumonia. During these periods, also, more episodes of mixed infections with S . pneumoniae have been reported.
- mice which have recovered from a lung infection with C. trachomatis are protected from infertility induced by a subsequent vaginal challenge (Pal et al . (1996) Infection and Immunity .64 : 5341) .
- sheep immunized with inactivated C. psi ttaci were protected from subsequent chlamydial -induced abortions and stillbirths (Jones et al . (1995) Vaccine 13:715) .
- C. pneumoniae serotypes and the defining antigens, is not yet possible.
- the entire genome sequence of C. pneumoniae strain CWL-029 is now known (http : //chlamydia-www.berkeley . edu: 4231/) and as further sequences become available a better understanding of antigenic variation may be gained.
- the present invention provides purified and isolated polynucleotide molecules that encode the Chlamydi a polypeptides designated Npt2cp (ADP/ATP translocase) (SEQ ID No: 1) which can be used in methods to prevent, treat, and diagnose Chlamydia infection.
- the polynucleotide molecules are DNA that encode the polypeptide of SEQ ID No: 2.
- Another form of the invention provides polypeptides corresponding to the isolated DNA molecules.
- the amino acid sequence of the corresponding encoded polypeptide is shown as SEQ ID No : 2.
- the invention having provided the polynucleotide sequences encoding the Chlamydia Npt2cp (ADP/ATP translocase) protein, also provides polynucleotides encoding fragments derived from such a polypeptide. Moreover, the invention is understood to provide mutants and derivatives of such polypeptides and fragments derived therefrom, which result from the addition, deletion, or substitution of non-essential amino acids as described herein. Those skilled in the art would also readily understand that the invention, having provided the polynucleotide sequences encoding Chlamydia polypeptides, further provides monospecific antibodies that specifically bind to such polypeptides.
- the present invention has wide application and includes expression cassettes, vectors, and cells transformed or transfected with the polynucleotides of the invention. Accordingly, the present invention further provides (i) a method for producing a polypeptide of the invention in a recombinant host system and related expression cassettes, vectors, and transformed or transfected cells; (ii) a vaccine, or a live vaccine vector such as a pox virus, Salmonella typhimurium, or Vibrio cholerae vector, containing a polynucleotide of the invention, such vaccines and vaccine vectors being useful for, e . g.
- RNA or DNA molecule of the invention either in a naked form or formulated with a delivery vehicle, a polypeptide or combination of polypeptides, or a monospecific antibody of the invention, and related pharmaceutical compositions
- a method for diagnosing the presence of Chlamydia in a biological sample which can involve the use of a DNA or RNA molecule, a monospecific antibody, or a polypeptide of the invention
- a method for purifying a polypeptide of the invention by antibody-based affinity chromatography a method for purifying a polypeptide of the invention by antibody-based affinity chromatography .
- Figure 1 shows the nucleotide sequence of the Npt2cp (ADP/ATP translocase) gene (SEQ ID No: 1) and the deduced amino acid sequence of the Npt2cp (ADP/ATP translocase) from Chlamydia pneumoniae (SEQ ID No: 2) .
- FIG. 2 shows the restriction enzyme analysis of the C. pneumoniae Npt2cp (ADP/ATP translocase) gene.
- Figure 3 shows the construction and elements of plasmid pCABk663.
- Figure 4 illustrates protection against C. pneumoniae infection by pCABk663 following DNA immunization.
- An open reading frame (ORF) encoding the chlamydial Npt2cp (ADP/ATP translocase) protein has been identified from the C. pneumoniae genome. The gene encoding this protein has been inserted into an expression plasmid and shown to confer immune protection against chlamydial infection. Accordingly, this Npt2cp (ADP/ATP translocase) protein and related polypeptides can be used to prevent and treat Chlamydia infection.
- isolated polynucleotides which encode Chlamydia polypeptides, whose amino acid sequences are shown in SEQ ID No: 2.
- isolated polynucleotide is defined as a polynucleotide removed from the environment in which it naturally occurs. For example, a naturally-occurring DNA molecule present in the genome of a living bacteria or as part of a gene bank is not isolated, but the same molecule separated from the remaining part of the bacterial genome, as a result of, e . g. , a cloning event (amplification), is isolated.
- an isolated DNA molecule is free from DNA regions (e.g., coding regions) with which it is immediately contiguous at the 5' or 3 ' end, in the naturally occurring genome.
- isolated polynucleotides may be part of a vector or a composition and still be defined as isolated in that such a vector or composition is not part of the natural environment of such polynucleotide.
- the polynucleotide of the invention is either RNA or DNA (cDNA, genomic DNA, or synthetic DNA) , or modifications, variants, homologs or fragments thereof.
- the DNA is either double-stranded or single-stranded, and, if single-stranded, is either the coding strand or the non-coding (anti -sense) strand.
- Any one of the sequences that encode the polypeptides of the invention as shown in SEQ ID No: 1 is (a) a coding sequence, (b) a ribonucleotide sequence derived from transcription of (a) , or (c) a coding sequence which uses the redundancy or degeneracy of the genetic code to encode the same polypeptides.
- polypeptide or “protein” is meant any chain of amino acids, regardless of length or post-translational modification (e . g. , glycosylation or phosphorylation) . Both terms are used interchangeably in the present application.
- amino acid sequences are provided which are homologous to SEQ ID No: 2.
- homologous amino acid sequence is any polypeptide which is encoded, in whole or in part, by a nucleic acid sequence which hybridizes at 25-35°C below critical melting temperature (Tm) , to any portion of the nucleic acid sequence of SEQ ID No : 1.
- a homologous amino acid sequence is one that differs from an amino acid sequence shown in SEQ ID No: 2 by one or more conservative amino acid substitutions.
- Such a sequence also encompass serotypic variants (defined below) as well as sequences containing deletions or insertions which retain inherent characteristics of the polypeptide such as immunogenicity .
- such a sequence is at least 75%, more preferably 80%, and most preferably 90% identical to SEQ ID No: 2.
- homologous amino acid sequences include sequences that are identical or substantially identical to SEQ ID No: 2.
- amino acid sequence substantially identical is meant a sequence that is at least 90%, preferably 95%, more preferably 97%, and most preferably 99% identical to an amino acid sequence of reference and that preferably differs from the sequence of reference by a majority of conservative amino acid substitutions .
- amino acids having uncharged polar side chains such as asparagine, glutamine, serine, threonine, and tyrosine
- amino acids having basic side chains such as lysine, arginine, and histidine
- amino acids having acidic side chains such as aspartic acid and glutamic acid
- amino acids having nonpolar side chains such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.
- sequence analysis software such as Sequence Analysis Software Package of the Genetics
- a homologous sequence is one that is at least 45%, more preferably 60%, and most preferably 85% identical to the coding sequence of SEQ ID No: 1.
- polypeptides having a sequence homologous to SEQ ID No: 2 include naturally-occurring allelic variants, as well as mutants or any other non-naturally occurring variants that retain the inherent characteristics of the polypeptide of SEQ ID No: 2.
- an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does not alter the biological function of the polypeptide.
- biological function is meant the function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells.
- biological function of a porin is to allow the entry into cells of compounds present in the extracellular medium.
- Biological function is distinct from antigenic property.
- a polypeptide can have more than one biological function.
- Allelic variants are very common in nature.
- a bacterial species such as C. pneumoniae
- C. pneumoniae is usually represented by a variety of strains that differ from each other by minor allelic variations.
- a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence (and polynucleotide sequence) that is not identical in each of the strains.
- an immune response directed generally against many allelic variants has been demonstrated.
- cross-strain antibody binding plus neutralization of infectivity occurs despite amino acid sequence variation of MOMP from strain to strain, indicating that the MOMP, when used as an immunogen, is tolerant of amino acid variations.
- Polynucleotides encoding homologous polypeptides or allelic variants are retrieved by polymerase chain reaction (PCR) amplification of genomic bacterial DNA extracted by conventional methods. This involves the use of synthetic oligonucleotide primers matching upstream and downstream of the 5' and 3' ends of the encoding domain. Suitable primers are designed according to the nucleotide sequence information provided in SEQ ID No : 1. The procedure is as follows: a primer is selected which consists of 10 to 40, preferably 15 to 25 nucleotides.
- a standard PCR reaction contains typically 0.5 to 5 Units of Taq DNA polymerase per 100 ⁇ L, 20 to 200 ⁇ M deoxynucleotide each, preferably at equivalent concentrations, 0.5 to 2.5 mM magnesium over the total deoxynucleotide concentration, 10 ⁇ to 10° " target molecules, and about 20 pmol of each primer. About 25 to 50 PCR cycles are performed, with an annealing temperature 15°C to 5°C below the true Tm of the primers.
- a more stringent annealing temperature improves discrimination against incorrectly annealed primers and reduces incorportion of incorrect nucleotides at the 3' end of primers.
- a denaturation temperature of 95°C to 97°C is typical, although higher temperatures may be appropriate for dematuration of G+C- rich targets. The number of cycles performed depends on the starting concentration of target molecules, though typically more than 40 cycles is not recommended as non-specific background products tend to accumulate.
- An alternative method for retrieving polynucleotides encoding homologous polypeptides or allelic variants is by hybridization screening of a DNA or RNA library. Hybridization procedures are well-known m the art and are described in Ausubel et al . , (Ausubel et al . , Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994), Silhavy et al . (Silhavy et al . Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, 1984), and Davis et al . (Davis et al . A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, 1980)).
- stringent conditions are achieved for both pre-hyb ⁇ dizmg and hybridizing incubations (I) within 4-16 hours at 42°C, m 6 x SSC containing 50% formamide, or (ii) within 4-16 hours at 65°C in an aqueous 6 x SSC solution (1 M NaCl , 0.1 M sodium citrate (pH 7.0)) .
- hybridization experiments are performed at a temperature from 60 to 68°C, e.g. 65°C.
- stringent hybridization conditions can be achieved m 6xSSC, preferably m 2xSSC or lxSSC, more preferably m 0.5xSSc, 0.3xSSC or 0. lxSSC (m the absence of formamide).
- lxSSC contains 0.15 M NaCl and 0.015 M sodium citrate.
- Useful homologs and fragments thereof that do not occur naturally are designed using known methods for identifying regions of an antigen that are likely to tolerate amino acid sequence changes and/or deletions. As an example, homologous polypeptides from different species are compared; conserved sequences are identified. The more divergent sequences are the most likely to tolerate sequence changes. Homology among sequences may be analyzed using, as an example, the BLAST homology searching algorithm of Altschul et al . , Nucleic Acids Res.; 25:3389-3402 (1997).
- sequences are modified such that they become more reactive to T- and/or B-cells, based on computer-assisted analysis of probable T- or B-cell epitopes
- Yet another alternative is to mutate a particular amino acid residue or sequence within the polypeptide in vi tro, then screen the mutant polypeptides for their ability to prevent or treat Chlamydia infection according to the method outlined below.
- a person skilled in the art will readily understand that by following the screening process of this invention, it will be determined without undue experimentation whether a particular homolog of SEQ ID No. 2 may be useful in the prevention or treatment of Chlamydia infection.
- the screening procedure comprises the steps: immunizing an animal, preferably mouse, with the test homolog or fragment ; inoculating the immunized animal with Chlamydia; and selecting those homologs or fragments which confer protection against Chlamydia.
- polypeptide derivatives are provided that are partial sequences of SEQ ID No. 2, partial sequences of polypeptide sequences homologous to SEQ ID No. 2, polypeptides derived from full- length polypeptides by internal deletion, and fusion proteins. It is an accepted practice in the field of immunology to use fragments and variants of protein immunogens as vaccines, as all that is required to induce an immune response to a protein is a small ⁇ e . g.
- polypeptide fragments preferably are at least 12 amino acids in length.
- polypeptide fragments are at least 20 amino acids, preferably at least 50 amino acids, more preferably at least 75 amino acids, and most preferably at least 100 amino acids in length.
- Polynucleotides of 30 to 600 nucleotides encoding partial sequences of sequences homologous to SEQ ID No: 2 are retrieved by PCR amplification using the parameters outlined above and using primers matching the sequences upstream and downstream of the 5 ' and 3 ' ends of the fragment to be amplified.
- the template polynucleotide for such amplification is either the full length polynucleotide homologous to SEQ ID No: 1, or a polynucleotide contained in a mixture of polynucleotides such as a DNA or RNA library.
- screening hybridization is carried out under conditions described above and using the formula for calculating Tm.
- the calculated Tm is corrected by subtracting (600/polynucleotide size in base pairs) and the stringency conditions are defined by a hybridization temperature that is 5 to 10°C below Tm.
- Tm 4 x (G+C) + 2 (A+T) .
- Epitopes which induce a protective T cell -dependent immune response are present throughout the length of the polypeptide. However, some epitopes may be masked by secondary and tertiary structures of the polypeptide. To reveal such masked epitopes large internal deletions are created which remove much of the original protein structure and exposes the masked epitopes. Such internal deletions sometimes effect the additional advantage of removing immunodominant regions of high variability among strains.
- Polynucleotides encoding polypeptide fragments and polypeptides having large internal deletions are constructed using standard methods (Ausubel et al . , Current Protocols in
- a fusion polypeptide is one that contains a polypeptide or a polypeptide derivative of the invention fused at the N- or C-terminal end to any other polypeptide (hereinafter referred to as a peptide tail) .
- a simple way to obtain such a fusion polypeptide is by translation of an in- frame fusion of the polynucleotide sequences, i.e., a hybrid gene.
- the hybrid gene encoding the fusion polypeptide is inserted into an expression vector which is used to transform or transfect a host cell.
- the polynucleotide sequence encoding the polypeptide or polypeptide derivative is inserted into an expression vector in which the polynucleotide encoding the peptide tail is already present.
- vectors and instructions for their use are commercially available, e . g. the pMal-c2 or pMal-p2 system from New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen.
- a fusion polypeptide is one where the polypeptide or homolog or fragment of the invention is fused to a polypeptide having adjuvant activity, such as subunit B of either cholera toxin or E. coli heat- labile toxin.
- a polypeptide having adjuvant activity such as subunit B of either cholera toxin or E. coli heat- labile toxin.
- Another advantageous fusion is one where the polypeptide, homolog or fragment is fused to a strong T-cell epitope or B-cell epitope.
- Such an epitope may be one known in the art (e . g. the Hepatitis B virus core antigen, D.R. Millich et al . , "Antibody production to the nucleocapsid and envelope of the Hepatitis B virus primed by a single synthetic T cell site", Nature. 1987.
- a fusion polypeptide comprising T- or B-cell epitopes from SEQ ID No : 2 or its homolog or fragment, wherein the epitopes are derived from multiple variants of said polypeptide or homolog or fragment, each variant differing from another in the location and sequence of its epitope within the polypeptide.
- Such a fusion is effective in the prevention and treatment of Chlamydia infection since it optimizes the T- and B-cell response to the overall polypeptide, homolog or fragment.
- the polypeptide of the invention is fused to the N- , or preferably, to the C-terminal end of the polypeptide having adjuvant activity or T- or B-cell epitope.
- a polypeptide fragment of the invention is inserted internally within the amino acid sequence of the polypeptide having adjuvant activity.
- the T- or B-cell epitope may also be inserted internally within the amino acid sequence of the polypeptide of the invention.
- the polynucleotides of the invention also encode hybrid precursor polypeptides containing heterologous signal peptides, which mature into polypeptides of the invention.
- heterologous signal peptide is meant a signal peptide that is not found in naturally-occurring precursors of polypeptides of the invention.
- Polynucleotide molecules according to the invention including RNA, DNA, or modifications or combinations thereof, have various applications.
- a DNA molecule is used, for example, (i) in a process for producing the encoded polypeptide in a recombinant host system, (ii) in the construction of vaccine vectors such as poxviruses, which are further used in methods and compositions for preventing and/or treating Chlamydia infection, (iii) as a vaccine agent (as well as an RNA molecule) , in a naked form or formulated with a delivery vehicle and, (iv) in the construction of attenuated Chlamydia strains that can over-express a polynucleotide of the invention or express it in a non-toxic, mutated form.
- vaccine vectors such as poxviruses
- a second aspect of the invention encompasses (i) an expression cassette containing a DNA molecule of the invention placed under the control of the elements required for expression, in particular under the control of an appropriate promoter; (ii) an expression vector containing an expression cassette of the invention; (iii) a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, as well as (iv) a process for producing a polypeptide or polypeptide derivative encoded by a polynucleotide of the invention, which involves culturing a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, under conditions that allow expression of the DNA molecule of the invention and, recovering the encoded polypeptide or polypeptide derivative from the cell culture.
- a recombinant expression system is selected from procaryotic and eucaryotic hosts.
- Eucaryotic hosts include yeast cells (e . g. , Saccharomyces cerevisiae or Pichia pastoris) , mammalian cells ( e . g. , C0S1, NIH3T3, or JEG3 cells), arthropods cells ( e . g. , Spodoptera frugiperda (SF9) cells), and plant cells.
- a preferred expression system is a procaryotic host such as E. coli .
- Bacterial and eucaryotic cells are available from a number of different sources including commercial sources to those skilled in the art, e . g.
- the American Type Culture Collection (ATCC; Rockville, Maryland) .
- ATCC American Type Culture Collection
- Commercial sources of cells used for recombinant protein expression also provide instructions for usage of the cells.
- the choice of the expression system depends on the features desired for the expressed polypeptide. For example, it may be useful to produce a polypeptide of the invention in a particular lipidated form or any other form.
- the host In selecting a vector, the host must be chosen that is compatible with the vector which is to exist and possibly replicate in it. Considerations are made with respect to the vector copy number, the ability to control the copy number, expression of other proteins such as antibiotic resistance.
- an expression control sequence a number of variables are considered. Among the important variable are the relative strength of the sequence (e . g. the ability to drive expression under various conditions) , the ability to control the sequence's function, compatibility between the polynucleotide to be expressed and the control sequence ( e . g. secondary structures are considered to avoid hairpin structures which prevent efficient transcription) .
- unicellular hosts are selected which are compatible with the selected vector, tolerant of any possible toxic effects of the expressed product, able to secrete the expressed product efficiently if such is desired, to be able to express the product in the desired conformation, to be easily scaled up, and to which ease of purification of the final product.
- an expression cassette includes a promoter that is functional in the selected host system and can be constitutive or inducible; a ribosome binding site; a start codon (ATG) if necessary; a region encoding a signal peptide, e . g. , a lipidation signal peptide; a DNA molecule of the invention; a stop codon; and optionally a 3 ' terminal region (translation and/or transcription terminator) .
- the signal peptide encoding region is adjacent to the polynucleotide of the invention and placed in proper reading frame.
- the signal peptide-encoding region is homologous or heterologous to the DNA molecule encoding the mature polypeptide and is compatible with the secretion apparatus of the host used for expression.
- the open reading frame constituted by the DNA molecule of the invention, solely or together with the signal peptide, is placed under the control of the promoter so that transcription and translation occur in the host system.
- Promoters and signal peptide encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coli (as described in U.S. Patent No.
- the expression cassette is typically part of an expression vector, which is selected for its ability to replicate in the chosen expression system.
- Expression vectors e . g. , plasmids or viral vectors
- plasmids or viral vectors can be chosen, for example, from those described in Pouwels et al . (Cloning Vectors: A Laboratory Manual 1985, Supp . 1987). Suitable expression vectors can be purchased from various commercial sources. Methods for transforming/transfecting host cells with expression vectors are well-known in the art and depend on the host system selected as described in Ausubel et al . , (Ausubel et al . , Current Protocols in Molecular Biology, John Wiley & Sons Inc. , 1994) .
- a recombinant polypeptide of the invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane.
- the polypeptide is recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation of the recombinant cell culture.
- the recombinant polypeptide is purified by antibody-based affinity purification or by other well-known methods that can be readily adapted by a person skilled in the art, such as fusion of the polynucleotide encoding the polypeptide or its derivative to a small affinity binding domain.
- a polynucleotide of the invention can also be useful as a vaccine. There are two major routes, either using a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e . g. , inserted into a plasmid. Therapeutic or prophylactic efficacy of a polynucleotide of the invention is evaluated as described below.
- a third aspect of the invention provides (i) a vaccine vector such as a poxvirus, containing a DNA molecule of the invention, placed under the control of elements required for expression; (ii) a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier; specifically (iii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector of the invention; (iv) a method for inducing an immune response against Chlamydia in a mammal ( e . g. , a human; alternatively, the method can be used in veterinary applications for treating or preventing Chlamydia infection of animals, e . g.
- a vaccine vector such as a poxvirus, containing a DNA molecule of the invention, placed under the control of elements required for expression
- a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier
- a pharmaceutical composition containing a
- the third aspect of the invention encompasses the use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.
- a vaccine vector expresses one or several polypeptides or derivatives of the invention.
- the vaccine vector may express additionally a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12), that enhances the immune response (adjuvant effect) .
- IL-2 interleukin-2
- IL-12 interleukin-12
- each of the components to be expressed is placed under the control of elements required for expression in a mammalian cell.
- a composition comprising several vaccine vectors, each of them capable of expressing a polypeptide or derivative of the invention.
- a composition may also comprise a vaccine vector capable of expressing an additional Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof; optionally together with or a cytokine such as IL-2 or IL-12.
- Vaccination methods for treating or preventing infection in a mammal comprises use of a vaccine vector of the invention to be administered by any conventional route, particularly to a mucosal (e . g. , ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e . g. , subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.
- a mucosal e . g. , ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract
- parenteral e . g. , subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal
- Treatment may be effected in a single dose or repeated at intervals.
- Live vaccine vectors available in the art include viral vectors such as adenoviruses and poxviruses as well as bacterial vectors, e . g. , Shigella, Salmonella , Vibrio cholerae, Lactobacillus , Bacille bilie de Calmette-Guerin (BCG) , and Streptococcus .
- viral vectors such as adenoviruses and poxviruses as well as bacterial vectors, e . g. , Shigella, Salmonella , Vibrio cholerae, Lactobacillus , Bacille bilie de Calmette-Guerin (BCG) , and Streptococcus .
- BCG Bacille bilie de Calmette-Guerin
- Streptococcus An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a DNA molecule of the invention, are described in U.S. Patent No
- Poxvirus vectors include vaccinia and canary pox virus, described in U.S. Patent No. 4,722,848 and U.S. Patent No. 5,364,773, respectively. (Also see, e . g. , Tartaglia et al . , Virology (1992) 188:217) for a description of a vaccinia virus vector and Taylor et al , Vaccine (1995) 13:539 for a reference of a canary pox.) Poxvirus vectors capable of expressing a polynucleotide of the invention are obtained by homologous recombination as described in Kieny et al .
- the dose of vaccine viral vector for therapeutic or prophylactic use, can be of from about lxlO 4 to about lxlO 11 , advantageously from about lxlO 7 to about lxlO 10 , preferably of from about lxlO 7 to about lxlO 9 plaque-forming units per kilogram.
- viral vectors are administered parenterally; for example, in 3 doses, 4 weeks apart. It is preferable to avoid adding a chemical adjuvant to a composition containing a viral vector of the invention and thereby minimizing the immune response to the viral vector itself .
- Non-toxicogenic ViJbrio cholerae mutant strains that are useful as a live oral vaccine are known.
- Mekalanos et al . Nature (1983) 306:551 and U.S. Patent No. 4,882,278 describe strains which have a substantial amount of the coding sequence of each of the two ctxA alleles deleted so that no functional cholerae toxin is produced.
- WO 92/11354 describes a strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations.
- WO 94/01533 describes a deletion mutant lacking functional ctxA and attRSl DNA sequences.
- An effective vaccine dose of a Vibrio cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a DNA molecule of the invention contains about lxlO 5 to about lxlO 9 , preferably about 1x10 s to about lxlO 8 , viable bacteria in a volume appropriate for the selected route of administration.
- Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
- Attenuated Salmonella typhimurium strains genetically engineered for recombinant expression of heterologous antigens or not, and their use as oral vaccines are described in Nakayama et al . (Bio/Technology (1988) 6:693) and WO 92/11361.
- Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
- the polynucleotide of the invention is inserted into the bacterial genome or remains in a free state as part of a plasmid.
- composition comprising a vaccine bacterial vector of the present invention may further contain an adjuvant.
- adjuvants are known to those skilled in the art. Preferred adjuvants are selected as provided below.
- a fourth aspect of the invention provides (i) a composition of matter comprising a polynucleotide of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a polynucleotide of the invention; (iii) a method for inducing an immune response against Chlamydia in a mammal by administration of an immunogenically effective amount of a polynucleotide of the invention to elicit a protective immune response to Chlamydia ; and particularly, (iv) a method for preventing and/or treating a Chlamydia ( e . g. , C.
- a Chlamydia e . g. , C.
- the fourth aspect of the invention encompasses the use of a polynucleotide of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.
- a preferred use includes the use of a DNA molecule placed under conditions for expression in a mammalian cell, especially in a plasmid that is unable to replicate in mammalian cells and to substantially integrate in a mammalian genome.
- polynucleotides of the invention include their administration to a mammal as a vaccine, for therapeutic or prophylactic purposes.
- Such polynucleotides are used in the form of DNA as part of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome.
- a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell.
- the promoter functions either ubiquitously or tissue- specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (described in U.S. Patent No.
- CMV Cytomegalovirus
- tissue-specific promoter is the desmin promoter which drives expression in muscle cells (Li et al . , Gene (1989) 78:243, Li & Paulin, J. Biol. Chem. (1991) 266:6562 and Li & Paulin, J. Biol. Chem. (1993) 268:10403).
- Use of promoters is well-known to those skilled in the art. Useful vectors are described in numerous publications, specifically WO 94/21797 and Hartikka et al . , Human Gene Therapy (1996) 7:1205.
- Polynucleotides of the invention which are used as vaccines encode either a precursor or a mature form of the corresponding polypeptide.
- the signal peptide is either homologous or heterologous.
- a eucaryotic leader sequence such as the leader sequence of the tissue-type plasminogen factor (tPA) is preferred.
- a composition of the invention contains one or several polynucleotides with optionally at least one additional polynucleotide encoding another Chlamydia antigen such as urease subunit A, B, or both, or a fragment, derivative, mutant, or analog thereof.
- the composition may also contain an additional polynucleotide encoding a cytokine, such as interleukin-2 (IL-2) or interleukin- 12 (IL-12) so that the immune response is enhanced.
- additional polynucleotides are placed under appropriate control for expression.
- DNA molecules of the invention and/or additional DNA molecules to be included in the same composition are present in the same plasmid.
- a polynucleotide of the invention is formulated according to various methods outlined below.
- One method utililizes the polynucleotide in a naked form, free of any delivery vehicles.
- a polynucleotide is simply diluted in a physiologically acceptable solution such as sterile saline or sterile buffered saline, with or without a carrier.
- the carrier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e . g. , a solution containing 20% sucrose.
- An alternative method utilizes the polynucleotide in association with agents that assist in cellular uptake.
- agents are (i) chemicals that modify cellular permeability, such as bupivacaine (see, e . g. , WO 94/16737), (ii) liposomes for encapsulation of the polynucleotide, or (iii) cationic lipids or silica, gold, or tungsten microparticles which associate themselves with the polynucleotides .
- chemicals that modify cellular permeability such as bupivacaine (see, e . g. , WO 94/16737)
- liposomes for encapsulation of the polynucleotide or
- cationic lipids or silica gold, or tungsten microparticles which associate themselves with the polynucleotides .
- Cationic lipids are also known m the art and are commonly used for gene delivery.
- Such lipids include LipofectinTM also known as DOTMA (N- [1- (2 , 3 -dioleyloxy) propyl] - N,N,N-t ⁇ methylammon ⁇ um chloride), DOTAP (1 , 2-b ⁇ s (oleyloxy) -3- (trimethylammonio) propane) , DDAB (dimethyldioctadecylammonium bromide) , DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Choi (3 beta- (N- (N 1 ,N' - dimethyl ammomethane) -carbamoyl) cholesterol) .
- DC-Choi 3 beta- (N- (N 1 ,N' - dimethyl ammomethane) -carbamoyl) cholesterol
- Cationic lipids for gene delivery are preferably used m association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamme) , as described m WO 90/11092 as an example.
- DOPE dioleyl phosphatidylethanolamme
- Formulation containing cationic liposomes may optionally contain other transfection-facilitatmg compounds.
- transfection-facilitatmg compounds A number of them are described m WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/02397. They include spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane- permeabiliz g compounds such as GALA, Gramicidme S, and cationic bile salts (see, for example, WO 93/19768) . Gold or tungsten microparticles are used for gene delivery, as described m WO 91/00359, WO 93/17706, and Tang et al .
- microparticle-coated polynucleotide is injected via mtradermal or mtraepidermal routes using a needleless injection device ("gene gun"), such as those described in U.S. Patent No. 4,945,050, U.S. Patent No. 5,015,580, and WO 94/24263.
- a needleless injection device such as those described in U.S. Patent No. 4,945,050, U.S. Patent No. 5,015,580, and WO 94/24263.
- the amount of DNA to be used in a vaccine recipient depends, e . g . , on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the mammal intended for administration ( e . g. , the weight, age, and general health of the mammal), the mode of administration, and the type of formulation.
- a therapeutically or prophylactically effective dose from about 1 ⁇ g to about 1 mg, preferably, from about 10 ⁇ g to about 800 ⁇ g and, more preferably, from about 25 ⁇ g to about 250 ⁇ g, can be administered to human adults.
- the administration can be achieved in a single dose or repeated at intervals.
- the route of administration is any conventional route used in the vaccine field.
- a polynucleotide of the invention is administered via a mucosal surface, e . g. , an ocular, intranasal , pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface; or via a parenteral route, e . g. , by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route.
- the choice of administration route depends on the formulation that is selected.
- a polynucleotide formulated in association with bupivacaine is advantageously administered into muscles.
- the formulation can be advantageously injected via intravenous, intranasal (aerosolization) , intramuscular, intradermal, and subcutaneous routes.
- a polynucleotide in a naked form can advantageously be administered via the intramuscular, intradermal, or sub- cutaneous routes.
- such a composition can also contain an adjuvant.
- a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable such as, e . g. , QS21, which is described in U.S. Patent No. 5,057,546.
- sequence information provided in the present application enables the design of specific nucleotide probes and primers that are used for diagnostic purposes.
- a fifth aspect of the invention provides a nucleotide probe or primer having a sequence found in or derived by degeneracy of the genetic code from a sequence shown in SEQ ID No : 1.
- probe refers to DNA (preferably single stranded) or RNA molecules (or modifications or combinations thereof) that hybridize under the stringent conditions, as defined above, to nucleic acid molecules having SEQ ID No : 1 or to sequences homologous to SEQ ID No:l, or to its complementary or anti-sense sequence.
- probes are significantly shorter than full-length sequences. Such probes contain from about 5 to about 100, preferably from about 10 to about 80, nucleotides.
- probes have sequences that are at least 75%, preferably at least 85%, more preferably 95% homologous to a portion of SEQ ID No : 1 or that are complementary to such sequences.
- Probes may contain modified bases such as inosine, methyl- 5 -deoxycytidine, deoxyuridine, dimethylamino-5- deoxyuridine, or diamino-2, 6-purine.
- Sugar or phosphate residues may also be modified or substituted.
- a deoxyribose residue may be replaced by a polyamide (Nielsen et al .
- phosphate residues may be replaced by ester groups such as diphosphate, alkyl, arylphosphonate and phosphorothioate esters.
- ester groups such as diphosphate, alkyl, arylphosphonate and phosphorothioate esters.
- 2 ' -hydroxyl group on ribonucleotides may be modified by including such groups as alkyl groups .
- Probes of the invention are used in diagnostic tests, as capture or detection probes. Such capture probes are conventionally immobilized on a solid support, directly or indirectly, by covalent means or by passive adsorption.
- a detection probe is labelled by a detection marker selected from: radioactive isotopes, enzymes such as peroxidase, alkaline phosphatase, and enzymes able to hydrolyze a chromogenic, fluorogenic, or luminescent substrate, compounds that are chromogenic, fluorogenic, or luminescent, nucleotide base analogs, and biotm.
- Probes of the invention are used m any conventional hybridization technique, such as dot blot (Maniatis et al . , Molecular Clonmg: A Laboratory Manual (1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) , Southern blot (Southern, J. Mol. Biol. (1975) 98:503), northern blot (identical to Southern blot with the exception that RNA is used as a target) , or the sandwich technique (Dunn et al . , Cell (1977) 12:23).
- the latter technique involves the use of a specific capture probe and/or a specific detection probe with nucleotide sequences that at least partially differ from each other.
- a primer is a probe of usually about 10 to about 40 nucleotides that is used to initiate enzymatic polymerization of DNA an amplification process (e . g. , PCR), in an elongation process, or m a reverse transcription method. Primers used m diagnostic methods involving PCR are labeled by methods known m the art . As described herein, the invention also encompasses
- a reagent comprising a probe of the invention for detecting and/or identifying the presence of Chlamydia m a biological material;
- a method for detectmg and/or identifying the presence of Chlamydia m a biological material which (a) a sample is recovered or derived from the biological material, (b) DNA or RNA is extracted from the material and denatured, and (c) exposed to a probe of the invention, for example, a capture, detection probe or both, under stringent hybridization conditions, such that hybridization is detected; and (in) a method for detecting and/or identifying the presence of Chlamydia in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA is extracted therefrom, (c) the extracted DNA is primed with at least one, and preferably two, primers of the invention and amplified by polymerase chain reaction, and (d) the amplified DNA fragment is produced.
- a sixth aspect of the invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide of the invention.
- substantially purified polypeptide as used herein is defined as a polypeptide that is separated from the environment in which it naturally occurs and/or that is free of the majority of the polypeptides that are present in the environment in which it was synthesized.
- a substantially purified polypeptide is free from cytoplasmic polypeptides.
- the polypeptides of the invention may be purified from a natural source, i.e., a Chlamydia strain, or produced by recombinant means .
- Consistent with the sixth aspect of the invention are polypeptides, homologs or fragments which are modified or treated to enhance their immunogenicity in the target animal, in whom the polypeptide, homolog or fragments are intended to confer protection against Chlamydia.
- modifications or treatments include: ammo acid substitutions with an amino acid derivative such as 3 -methyhistidine, 4-hydroxyproline, 5- hydroxylysine etc., modifications or deletions which are carried out after preparation of the polypeptide, homolog or fragment, such as the modification of free amino, carboxyl or hydroxyl side groups of the amino acids.
- Identification of homologous polypeptides or polypeptide derivatives encoded by polynucleotides of the invention which have specific antigenicity is achieved by screening for cross-reactivity with an antiserum raised against the polypeptide of reference having an amino acid sequence of SEQ ID No : 1.
- the procedure is as follows: a monospecific hyperimmune antiserum is raised against a purified reference polypeptide, a fusion polypeptide (for example, an expression product of MBP, GST, or His-tag systems, the description and instructions for use of which are contained in Invitrogen product manuals for pcDNA3.
- l/Myc-His (+) A, B, and C and for the Xpress Tm System Protein Purification) or a synthetic peptide predicted to be antigenic.
- an antiserum is raised against a fusion polypeptide, two different fusion systems are employed. Specific antigenicity can be determined according to a number of methods, including Western blot (Towbin et al . , Proc. Natl. Acad. Sci. USA (1979) 76:4350), dot blot, and ELISA, as described below.
- the product to be screened is submitted to SDS-Page electrophoresis as described by Laemmli (Nature (1970) 227:680).
- SDS-Page electrophoresis as described by Laemmli (Nature (1970) 227:680).
- the material is further incubated with the monospecific hyperimmune antiserum diluted in the range of dilutions from about 1:5 to about 1:5000, preferably from about 1:100 to about 1:500. Specific antigenicity is shown once a band corresponding to the product exhibits reactivity at any of the dilutions in the above range.
- the product to be screened is preferably used as the coating antigen.
- a purified preparation is preferred, although a whole cell extract can also be used. Briefly, about 100 ⁇ l of a preparation at about 10 ⁇ g protein/ml are distributed into wells of a 96-well polycarbonate ELISA plate. The plate is incubated for 2 hours at 37 °C then overnight at 4°C. The plate is washed with phosphate buffer saline (PBS) containing 0.05% Tween 20 (PBS/Tween buffer) . The wells are saturated with 250 ⁇ l PBS containing 1% bovine serum albumin (BSA) to prevent non- specific antibody binding.
- PBS phosphate buffer saline
- BSA bovine serum albumin
- the plate After 1 hour incubation at 37°C, the plate is washed with PBS/Tween buffer. The antiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA. 100 ⁇ l of dilutions are added per well . The plate is incubated for 90 minutes at 37°C, washed and evaluated according to standard procedures. For example, a goat anti -rabbit peroxidase conjugate is added to the wells when specific antibodies were raised in rabbits. Incubation is carried out for 90 minutes at 37°C and the plate is washed. The reaction is developed with the appropriate substrate and the reaction is measured by colorimetry (absorbance measured spectrophotometrically) .
- a purified product is preferred, although a whole cell extract can also be used.
- a solution of the product at about 100 ⁇ g/ml is serially two- fold diluted in 50 mM Tris-HCl (pH 7.5) .
- 100 ⁇ l of each dilution are applied to a nitrocellulose membrane 0.45 ⁇ m set in a 96- well dot blot apparatus (Biorad) .
- the buffer is removed by applying vacuum to the system.
- Wells are washed by addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried.
- the membrane is saturated in blocking buffer (50 mM Tris-HCl (pH 7.5) 0.15 M NaCl, 10 g/L skim milk) and incubated with an antiserum dilution from about 1:50 to about 1:5000, preferably about 1:500.
- the reaction is revealed according to standard procedures. For example, a goat anti-rabbit peroxidase conjugate is added to the wells when rabbit antibodies are used. Incubation is carried out 90 minutes at 37°C and the blot is washed. The reaction is developed with the appropriate substrate and stopped. The reaction is measured visually by the appearance of a colored spot, e . g. , by colorimetry.
- a positive reaction is shown once a colored spot is associated with a dilution of at least about 1:5, preferably of at least about 1:500.
- Therapeutic or prophylactic efficacy of a polypeptide or derivative of the invention can be evaluated as described below.
- a seventh aspect of the invention provides (i) a composition of matter comprising a polypeptide of the invention together with a diluent or carrier; specifically (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention; (iii) a method for inducing an immune response against Chlamydia in a mammal, by administering to the mammal an immunogenically effective amount of a polypeptide of the invention to elicit a protective immune response to Chlamydia ; and particularly, (iv) a method for preventing and/or treating a Chlamydia ( e . g. , C. trachomatis . C.
- a Chlamydia e . g. , C. trachomatis . C.
- the seventh aspect of the invention encompasses the use of a polypeptide of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.
- the immunogenic compositions of the invention are administered by conventional routes known the vaccine field, in particular to a mucosal (e . g. , ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral ⁇ e . g. , subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.
- a mucosal e . g. , ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract
- the choice of administration route depends upon a number of parameters, such as the adjuvant associated with the polypeptide. If a mucosal adjuvant is used, the intranasal or oral route is preferred.
- lipid formulation or an aluminum compound is used, the parenteral route is preferred with the sub-cutaneous or intramuscular route being most preferred.
- the choice also depends upon the nature of the vaccine agent. For example, a polypeptide of the invention fused to CTB or LTB is best administered to a mucosal surface .
- composition of the invention contains one or several polypeptides or derivatives of the invention.
- the composition optionally contains at least one additional Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof.
- a composition of the invention a polypeptide or derivative thereof is formulated into or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response.
- liposomes preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response.
- Adjuvants other than liposomes and the like are also used and are known m the art.
- Adjuvants may protect the antigen from rapid dispersal by sequestering it m a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system.
- An appropriate selection can conventionally be made by those skilled m the art, for example, from those described below (under the eleventh aspect of the invention) .
- Treatment is achieved in a single dose or repeated as necessary at intervals, as can be determined readily by one skilled in the art. For example, a priming dose is followed by three booster doses at weekly or monthly intervals.
- An appropriate dose depends on various parameters including the recipient ( e . g. , adult or infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect ( e . g. , protection and/or treatment), as can be determined by one skilled in the art.
- a vaccine antigen of the invention is administered by a mucosal route in an amount from about 10 ⁇ g to about 500 mg, preferably from about 1 mg to about 200 mg .
- the dose usually does not exceed about 1 mg, preferably about 100 ⁇ g-
- polynucleotides and polypeptides of the invention may be used sequentially as part of a multistep immunization process.
- a mammal is initially primed with a vaccine vector of the invention such as a pox virus, e . g. , via the parenteral route, and then boosted twice with the polypeptide encoded by the vaccine vector, e . g. , via the mucosal route.
- liposomes associated with a polypeptide or derivative of the invention is also used for priming, with boosting being carried out mucosally using a soluble polypeptide or derivative of the invention in combination with a mucosal adjuvant (e.g., LT) .
- a mucosal adjuvant e.g., LT
- a polypeptide derivative of the invention is also used in accordance with the seventh aspect as a diagnostic reagent for detecting the presence of anti - Chlamydia antibodies, e . g. , in a blood sample.
- Such polypeptides are about 5 to about 80, preferably about 10 to about 50 amino acids in length. They are either labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below.
- a polypeptide or polypeptide derivative is produced and purified using known laboratory techniques.
- the polypeptide or polypeptide derivative may be produced as a fusion protein containing a fused tail that facilitates purification.
- the fusion product is used to immunize a small mammal, e . g. , a mouse or a rabbit, in order to raise antibodies against the polypeptide or polypeptide derivative (monospecific antibodies) .
- an eighth aspect of the invention provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention.
- Monospecific antibody an antibody that is capable of reacting with a unique naturally-occurring Chlamydia polypeptide.
- An antibody of the invention is either polyclonal or monoclonal.
- Monospecific antibodies may be recombinant, e . g. , chimeric (e . g. , constituted by a variable region of murine origin associated with a human constant region) , humanized (a human immunoglobulin constant backbone together with hypervariable region of animal, e . g. , murine, origin), and/or single chain.
- Both polyclonal and monospecific antibodies may also be in the form of immunoglobulin fragments, e . g.
- the antibodies of the invention are of any isotype, e . g. , IgG or IgA, and polyclonal antibodies are of a single isotype or a mixture of isotypes.
- Antibodies against the polypeptides, homologs or fragments of the present invention are generated by immunization of a mammal with a composition comprising said polypeptide, homolog or fragment.
- Such antibodies may be polyclonal or monoclonal. Methods to produce polyclonal or monoclonal antibodies are well known in the art. For a review, see “Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Eds. E. Harlow and D. Lane (1988), and D.E. Yelton et al . , 1981. Ann. Rev. Biochem. 50:657-680.
- For monoclonal antibodies see Kohler & Milstein (1975) Nature 256:495-497.
- the antibodies of the invention which are raised to a polypeptide or polypeptide derivative of the invention, are produced and identified using standard immunological assays, e . g. , Western blot analysis, dot blot assay, or ELISA (see, e . g. , Coligan et al . , Current Protocols in Immunology (1994) John Wiley & Sons, Inc., New York, NY) .
- the antibodies are used in diagnostic methods to detect the presence of a Chlamydia antigen in a sample, such as a biological sample.
- the antibodies are also used in affinity chromatography for purifying a polypeptide or polypeptide derivative of the invention. As is discussed further below, such antibodies may be used in prophylactic and therapeutic passive immunization methods .
- a ninth aspect of the invention provides (i) a reagent for detecting the presence of Chlamydia in a biological sample that contains an antibody, polypeptide, or polypeptide derivative of the invention; and (ii) a diagnostic method for detecting the presence of Chlamydia in a biological sample, by contacting the biological sample with an antibody, a polypeptide, or a polypeptide derivative of the invention, such that an immune complex is formed, and by detecting such complex to indicate the presence of Chlamydia in the sample or the organism from which the sample is derived.
- the immune complex is formed between a component of the sample and the antibody, polypeptide, or polypeptide derivative, whichever is used, and that any unbound material is removed prior to detecting the complex.
- a polypeptide reagent is useful for detecting the presence of anti - Chlamydia antibodies in a sample, e . g. , a blood sample, while an antibody of the invention is used for screening a sample, such as a gastric extract or biopsy, for the presence of Chlamydia polypeptides.
- the reagent i.e., the antibody, polypeptide, or polypeptide derivative of the invention
- a solid support such as a tube, a bead, or any other conventional support used in the field. Immobilization is achieved using direct or indirect means. Direct means include passive adsorption (non-covalent binding) or covalent binding between the support and the reagent. By “indirect means” is meant that an anti-reagent compound that interacts with a reagent is first attached to the solid support.
- an antibody that binds to it can serve as an anti-reagent, provided that it binds to an epitope that is not involved in the recognition of antibodies in biological samples.
- Indirect means may also employ a ligand-receptor system, for example, where a molecule such as a vitamin is grafted onto the polypeptide reagent and the corresponding receptor immobilized on the solid phase. This is illustrated by the biotin-streptavidin system.
- a peptide tail is added chemically or by genetic engineering to the reagent and the grafted or fused product immobilized by passive adsorption or covalent linkage of the peptide tail.
- Such diagnostic agents may be included in a kit which also comprises instructions for use.
- the reagent is labeled with a detection means which allows for the detection of the reagent when it is bound to its target .
- the detection means may be a fluorescent agent such as fluorescein isocyanate or fluorescein isothiocyanate, or an enzyme such as horse radish peroxidase or luciferase or alkaline phosphatase, or a radioactive element such as 125 I or 51 Cr.
- a tenth aspect of the invention provides a process for purifying, from a biological sample, a polypeptide or polypeptide derivative of the invention, which involves carrying out antibody-based affinity chromatography with the biological sample, wherein the antibody is a monospecific antibody of the invention.
- the antibody is either polyclonal or monospecific, and preferably is of the IgG type.
- Purified IgGs is prepared from an antiserum using standard methods (see, e . g. , Coligan et al . , Current Protocols in Immunology (1994) John Wiley & Sons, Inc., New York, NY.) .
- Conventional chromatography supports, as well as standard methods for grafting antibodies, are described in, e . g. , Antibodies: A Laboratory Manual, D. Lane, E. Harlow, Eds. (1988) and outlined below.
- a biological sample such as an C. pneumoniae extract preferably in a buffer solution
- a chromatography material preferably equilibrated with the buffer used to dilute the biological sample so that the polypeptide or polypeptide derivative of the invention (i.e., the antigen) is allowed to adsorb onto the material .
- the chromatography material such as a gel or a resin coupled to an antibody of the invention, is in either a batch form or a column.
- the unbound components are washed off and the antigen is then eluted with an appropriate elution buffer, such as a glycine buffer or a buffer containing a chaotropic agent, e . g.
- An eleventh aspect of the invention provides (i) a composition of matter comprising a monospecific antibody of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a monospecific antibody of the invention, and (iii) a method for treating or preventing a Chlamydia (e . g. , C. trachomatis, C. psi ttaci , C. pneumoniae or C. pecorum) infection, by administering a therapeutic or prophylactic amount of a monospecific antibody of the invention to an infected individual .
- a Chlamydia e . g. , C. trachomatis, C. psi ttaci , C. pneumoniae or C. pecorum
- the eleventh aspect of the invention encompasses the use of a monospecific antibody of the invention in the preparation of a medicament for treating or preventing Chlamydia infection.
- the monospecific antibody is either polyclonal or monoclonal, preferably of the IgA isotype (predominantly).
- the antibody is administered to a mucosal surface of a mammal, e . g. , the gastric mucosa, e . g. , orally or intragastrically, advantageously, in the presence of a bicarbonate buffer.
- systemic administration not requiring a bicarbonate buffer, is carried out.
- a monospecific antibody of the invention is administered as a single active component or as a mixture with at least one monospecific antibody specific for a different Chlamydia polypeptide.
- the amount of antibody and the particular regimen used are readily determined by one skilled in the art. For example, daily administration of about 100 to 1,000 mg of antibodies over one week, or three doses per day of about 100 to 1,000 mg of antibodies over two or three days, are effective regimens for most purposes .
- Therapeutic or prophylactic efficacy are evaluated using standard methods in the art, e . g. , by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e . g. , the C. pneumoniae mouse model.
- the C. pneumoniae strain of the model may be replaced with another Chlamydia strain.
- the efficacy of DNA molecules and polypeptides from C. pneumoniae is preferably evaluated in a mouse model using C. pneumoniae strain.
- Protection is determined by comparing the degree of Chlamydia infection to that of a control group. Protection is shown when infection is reduced by comparison to the control group.
- Such an evaluation is made for polynucleotides, vaccine vectors, polypeptides and derivatives thereof, as well as antibodies of the invention.
- Adjuvants useful in any of the vaccine compositions described above are as follows.
- Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate.
- the antigen is precipitated with, or adsorbed onto, the aluminum compound according to standard protocols.
- Other adjuvants such as RIBI (ImmunoChem, Hamilton, MT) , are used in parenteral administration.
- Adjuvants for mucosal administration include bacterial toxins, e . g. , the cholera toxin (CT) , the E. coli heat -labile toxin (LT) , the Clostridium difficile toxin A and the pertussis toxin (PT) , or combinations, subunits, toxoids, or mutants thereof such as a purified preparation of native cholera toxin subunit B (CTB) . Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity.
- CT cholera toxin
- LT heat -labile toxin
- PT pertussis toxin
- CTB native cholera toxin subunit B
- a mutant having reduced toxicity is used. Suitable mutants are described, e . g. , in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/06627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant) . Additional LT mutants that are used in the methods and compositions of the invention include, e . g. , Ser-63-Lys, Ala-69Gly, Glu-110-Asp, and Glu-112- Asp mutants. Other adjuvants, such as a bacterial monophosphoryl lipid A (MPLA) of, e . g. , E. coli , Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri ; saponins, or polylactide glycolide (PLGA) microspheres, is also be used in mucosal administration.
- MPLA bacterial
- Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/02415) , DC-chol (3 b- (N- (N' ,N' -dimethyl aminomethane) -carbamoyl) cholesterol; U.S. Patent No. 5,283,185 and WO 96/14831) and QS-21 (WO 88/09336) .
- composition of the invention containing a polynucleotide, a polypeptide, a polypeptide derivative, or an antibody of the invention, is manufactured in a conventional manner.
- a pharmaceutically acceptable diluent or carrier e . g. , water or a saline solution such as phosphate buffer saline.
- a diluent or carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers or diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington ' s Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF.
- the invention also includes methods in which Chlamydia infection are treated by oral administration of a Chlamydia polypeptide of the invention and a mucosal adjuvant, in combination with an antibiotic, an antacid, sucralfate, or a combination thereof.
- antibiotics including, e . g. , macrolides, tetracyclines, and derivatives thereof (specific examples of antibiotics that can be used include azithromycin or doxicyclin or immunomodulators such as cytokines or steroids) .
- compounds containing more than one of the above-listed components coupled together, are used.
- compositions for carrying out these methods i.e., compositions containing a Chlamydia antigen (or antigens) of the invention, an adjuvant, and one or more of the above-listed compounds, in a pharmaceutically acceptable carrier or diluent.
- the 60kDa cysteine rich membrane protein contains a sequence cross-reactive with the murine alpha-myosin heavy chain epitope M7A-alpha, an epitope conserved in humans (Bachmaier et al . , Science (1999) 283:1335) .
- This cross-reactivity is proposed to contribute to the development of cardiovascular disease, so it may be beneficial to remove this epitope, and any other epitopes cross-reactive with human antigens, from the protein if it is to be used as a vaccine.
- a further embodiment of the present invention includes the modification of the coding sequence, for example, by deletion or substitution of the nucleotides encoding the epitope from polynucleotides encoding the protein, as to improve the efficacy and safety of the protein as a vaccine.
- a similar approach may be appropriate for any protective antigen found to have unwanted homologies or cross-reactivities with human antigens.
- Amounts of the above- listed compounds used the methods and compositions of the invention are readily determined by one skilled m the art.
- Treatment/immunization schedules are also known and readily designed by one skilled m the art.
- the non-vaccme components can be administered on days 1-14, and the vaccine antigen + adjuvant can be administered on days 7, 14, 21, and 28.
- This example illustrates the preparation of a plasmid vector pCABk663 containing the Npt2cp (ADP/ATP translocase) gene.
- the Npt2cp (ADP/ATP translocase) gene was amplified from Chlamydia pneumoniae genomic DNA by polymerase chain reaction (PCR) using a 5' primer (5' ATAAGAATGCGGCCGCCACCATGCAGTCATCAGAAGTGAAAC 3 ' ; SEQ ID No : 3 ) and a 3' primer (5' GCGCCGGATCCCCAGAACAGCCATTTCTTCTTG 3 ' ; SEQ ID No : 3
- the 5' primer contains a Not I restriction site, a ribosome binding site, an initiation codon and a sequence at the 5' end of the Npt2cp (ADP/ATP translocase) coding sequence.
- the 3' primer includes the sequence encoding the C- terminal sequence of the Npt2cp (ADP/ATP translocase) and a Bam HI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in- frame fusion with the Histidine tag.
- the PCR fragment was purified using QIAquickTM PCR purification kit (Qiagen) and then digested with Not I and Bam HI and cloned into the pCA-Myc-His eukaryotic expression vector describe in Example 2 ( Figure 3) with transcription under control of the human CMV promoter .
- This example illustrates the preparation of the eukaryotic expression vector pCA/Myc-His.
- Plasmid pcDNA3.1 ( - ) Myc-His C (Invitrogen) was restricted with Spe I and Bam HI to remove the CMV promoter and the remaining vector fragment was isolated.
- the CMV promoter and intron A from plasmid VR-1012 (Vical) was isolated on a Spe I / Bam HI fragment .
- the fragments were ligated together to produce plasmid pCA/Myc-His.
- the Not I/Bam HI restricted PCR fragment containing the Npt2cp (ADP/ATP translocase) gene was ligated into the Not I and Bam HI restricted plasmid pCA/Myc- His to produce plasmid pCABk663 ( Figure 3) .
- the resulting plasmid, pCABk663, was transfered by electroporation into E. coli XL-1 blue (Stratagene) which was grown in LB broth containing 50 ⁇ g/ml of carbenicillin.
- the plasmid was isolated by Endo Free Plasmid Giga KitTM (Qiagen) large scale DNA purification system. DNA concentration was determined by absorbance at 260 nm and the plasmid was verified after gel electrophoresis and Ethidium bromide staining and comparison to molecular weight standards. The 5' and 3' ends of the gene were verified by sequencing using a LiCor model 4000 L DNA sequencer and IRD-800 labelled primers.
- Example 3 Example 3 :
- mice are susceptible to intranasal infection with different isolates of C. pneumoniae .
- Strain AR-39 (Grayston, 1989) was used in Balb/c mice as a challenge infection model to examine the capacity of chlamydia gene products delivered as naked DNA to elicit a protective response against a sublethal C. pneumoniae lung infection.
- Protective immunity is defined as an accelerated clearance of pulmonary infection.
- mice Groups of 7 to 9 week old male Balb/c mice (6 to 10 per group) were immunized intramuscularly (i.m.) plus intranasally (i.n.) with plasmid DNA containing the coding sequence of C. pneumoniae Npt2cp (ADP/ATP translocase) as described in Examples 1 and 2. Saline or the plasmid vector lacking an inserted chlamydial gene was given to groups of control animals. For i.m. immunization, alternate left and right quadriceps were injected with lOO ⁇ g of DNA in 50 ⁇ l of PBS on three occasions at 0 , 3 and 6 weeks. For i.n.
- mice aspirated 50 ⁇ l of PBS containing 50 ⁇ g DNA on three occasions at 0 , 3 and 6 weeks.
- immunized mice were inoculated i.n. with 5 x 10 5 IFU of C. pneumoniae, strain AR39 in lOO ⁇ l of SPG buffer to test their ability to limit the growth of a sublethal C. pneumoniae challenge.
- Lungs were taken from mice at day 9 post-challenge and immediately homogenised in SPG buffer (7.5% sucrose, 5mM glutamate, 12.5mM phosphate pH 7.5) .
- the homogenate was stored frozen at -70°C until assay. Dilutions of the homogenate were assayed for the presence of infectious chlamydia by inoculation onto monolayers of susceptible cells.
- the inoculum was centrifuged onto the cells at 3000rpm for 1 hour, then the cells were incubated for three days at 35°C in the presence of l ⁇ g/ml cycloheximide .
- Figure 4 and Table 1 show that mice immunized i.n. and i.m. with pCABk663 had chlamydial lung titers less than 36,000 in 5 of 6 cases at day 9 (mean 32,766) whereas the range of values for control mice sham immunized with saline was 13,600-458,100 IFU/lung (mean 107,641) at day 9.
- DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCAI190, failed to protect, with lung titers in immunised mice similar (mean 78,633 IFU/lung) to those obtained for saline-immunized control mice.
- the construct pCAI190 is identical to pCABk663 except that the nucleotide sequence encoding the Npt2cp (ADP/ATP translocase) is replaced with a C. pneumoniae nucleotide sequence encoding a hypothetical protein based on the open reading frame.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15432699P | 1999-09-17 | 1999-09-17 | |
US154326P | 1999-09-17 | ||
PCT/CA2000/001087 WO2001021803A1 (en) | 1999-09-17 | 2000-09-15 | Chlamydia antigens and corresponding dna fragments and uses thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1220924A1 true EP1220924A1 (de) | 2002-07-10 |
Family
ID=22550905
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP00962124A Withdrawn EP1220924A1 (de) | 1999-09-17 | 2000-09-15 | Chlamydia antigene und entsprechende gene und ihre verwendungen |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1220924A1 (de) |
AU (1) | AU7398400A (de) |
WO (1) | WO2001021803A1 (de) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB0709373D0 (en) * | 2007-05-16 | 2007-06-27 | Thrombosis Res Inst | BCG-based anti-atheroma vaccine |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK1032674T3 (da) * | 1997-11-21 | 2007-04-10 | Serono Genetics Inst Sa | Chlamydia pneumoniae genomisk sekvens og polypeptider, fragmenter deraf og anvendelser deraf, især til diagnosen, forebyggelsen og behandlingen af infektion |
-
2000
- 2000-09-15 AU AU73984/00A patent/AU7398400A/en not_active Abandoned
- 2000-09-15 EP EP00962124A patent/EP1220924A1/de not_active Withdrawn
- 2000-09-15 WO PCT/CA2000/001087 patent/WO2001021803A1/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO0121803A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO2001021803A1 (en) | 2001-03-29 |
WO2001021803B1 (en) | 2001-05-10 |
AU7398400A (en) | 2001-04-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7662391B2 (en) | Chlamydia outer membrane protein (OMP) and vaccine uses of the protein | |
US7736873B2 (en) | Chlamydia polypeptides and corresponding DNA fragments and uses thereof | |
WO2001021810A1 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
AU4388500A (en) | Chlamydia antigens and corresponding DNA fragments and uses thereof | |
WO2001046224A2 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
US7629327B2 (en) | Chlamydia 60 Kda CRMP antigens and vaccine uses | |
US7335370B2 (en) | Chlamydia antigens and corresponding DNA fragments and uses thereof | |
WO2001046225A2 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
WO2001046226A2 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
EP1140999A1 (de) | Chlamydia antigene, entsprechende dna fragmente und ihre verwendungen | |
WO2000039157A1 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
WO2001036455A2 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
WO2001021803A1 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
WO2001002575A1 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
WO2001021805A1 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
WO2001021802A1 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
WO2001021806A1 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
WO2000053764A1 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof | |
WO2001036457A2 (en) | Chlamydia antigens and corresponding dna fragments and uses thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20020415 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
AX | Request for extension of the european patent |
Free format text: AL PAYMENT 20020415;LT PAYMENT 20020415;LV PAYMENT 20020415;MK PAYMENT 20020415;RO PAYMENT 20020415;SI PAYMENT 20020415 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20040401 |