AU2002222098A1 - Projection against mycobacterial infections - Google Patents
Projection against mycobacterial infectionsInfo
- Publication number
- AU2002222098A1 AU2002222098A1 AU2002222098A AU2209802A AU2002222098A1 AU 2002222098 A1 AU2002222098 A1 AU 2002222098A1 AU 2002222098 A AU2002222098 A AU 2002222098A AU 2209802 A AU2209802 A AU 2209802A AU 2002222098 A1 AU2002222098 A1 AU 2002222098A1
- Authority
- AU
- Australia
- Prior art keywords
- gene
- mycobacterial
- peptide
- sequence
- macrophage
- 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.)
- Abandoned
Links
- 208000027531 mycobacterial infectious disease Diseases 0.000 title claims description 20
- 206010062207 Mycobacterial infection Diseases 0.000 title claims description 19
- 108090000623 proteins and genes Proteins 0.000 claims description 167
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 137
- 210000004027 cell Anatomy 0.000 claims description 106
- 210000002540 macrophage Anatomy 0.000 claims description 96
- 208000015181 infectious disease Diseases 0.000 claims description 87
- 201000008827 tuberculosis Diseases 0.000 claims description 75
- 239000012634 fragment Substances 0.000 claims description 58
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 57
- 230000001105 regulatory effect Effects 0.000 claims description 56
- 238000000034 method Methods 0.000 claims description 54
- 150000007523 nucleic acids Chemical class 0.000 claims description 51
- 108020004414 DNA Proteins 0.000 claims description 50
- 230000001018 virulence Effects 0.000 claims description 47
- 230000014509 gene expression Effects 0.000 claims description 45
- 239000013598 vector Substances 0.000 claims description 41
- 230000002238 attenuated effect Effects 0.000 claims description 33
- 239000000523 sample Substances 0.000 claims description 33
- 108091026890 Coding region Proteins 0.000 claims description 30
- 102000039446 nucleic acids Human genes 0.000 claims description 29
- 108020004707 nucleic acids Proteins 0.000 claims description 29
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 27
- 239000013612 plasmid Substances 0.000 claims description 26
- 239000002773 nucleotide Substances 0.000 claims description 25
- 125000003729 nucleotide group Chemical group 0.000 claims description 25
- 102000004169 proteins and genes Human genes 0.000 claims description 24
- 108091033319 polynucleotide Proteins 0.000 claims description 22
- 102000040430 polynucleotide Human genes 0.000 claims description 22
- 239000002157 polynucleotide Substances 0.000 claims description 22
- 239000003814 drug Substances 0.000 claims description 20
- 241000186359 Mycobacterium Species 0.000 claims description 17
- 239000003112 inhibitor Substances 0.000 claims description 15
- 230000000813 microbial effect Effects 0.000 claims description 12
- 238000001943 fluorescence-activated cell sorting Methods 0.000 claims description 11
- 230000008488 polyadenylation Effects 0.000 claims description 11
- 241000186366 Mycobacterium bovis Species 0.000 claims description 10
- 241000894007 species Species 0.000 claims description 10
- 239000003242 anti bacterial agent Substances 0.000 claims description 9
- 230000000295 complement effect Effects 0.000 claims description 9
- 108700008625 Reporter Genes Proteins 0.000 claims description 8
- 230000001717 pathogenic effect Effects 0.000 claims description 8
- 230000003115 biocidal effect Effects 0.000 claims description 7
- 230000010354 integration Effects 0.000 claims description 7
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims description 5
- 241000607142 Salmonella Species 0.000 claims description 5
- 230000000692 anti-sense effect Effects 0.000 claims description 5
- 108010006025 bovine growth hormone Proteins 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 230000001939 inductive effect Effects 0.000 claims description 5
- 241000700618 Vaccinia virus Species 0.000 claims description 4
- 230000004071 biological effect Effects 0.000 claims description 4
- 210000000349 chromosome Anatomy 0.000 claims description 4
- 230000008685 targeting Effects 0.000 claims description 3
- 241000700662 Fowlpox virus Species 0.000 claims description 2
- 230000008827 biological function Effects 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 238000010188 recombinant method Methods 0.000 claims description 2
- 238000009877 rendering Methods 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims 1
- 241000894006 Bacteria Species 0.000 description 45
- 239000000427 antigen Substances 0.000 description 41
- 108091007433 antigens Proteins 0.000 description 41
- 102000036639 antigens Human genes 0.000 description 41
- 239000000047 product Substances 0.000 description 27
- 229960005486 vaccine Drugs 0.000 description 27
- 210000001744 T-lymphocyte Anatomy 0.000 description 26
- 102000004196 processed proteins & peptides Human genes 0.000 description 25
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 18
- 239000002953 phosphate buffered saline Substances 0.000 description 18
- 238000003556 assay Methods 0.000 description 17
- 239000000203 mixture Substances 0.000 description 16
- 230000001580 bacterial effect Effects 0.000 description 13
- 238000012360 testing method Methods 0.000 description 12
- 238000009396 hybridization Methods 0.000 description 11
- 239000000443 aerosol Substances 0.000 description 10
- 229940079593 drug Drugs 0.000 description 10
- 230000012010 growth Effects 0.000 description 10
- 238000003018 immunoassay Methods 0.000 description 10
- 230000004224 protection Effects 0.000 description 10
- 241000588724 Escherichia coli Species 0.000 description 9
- 244000052616 bacterial pathogen Species 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 9
- 238000009472 formulation Methods 0.000 description 9
- 230000003834 intracellular effect Effects 0.000 description 9
- 230000010076 replication Effects 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 150000001413 amino acids Chemical group 0.000 description 8
- 239000012472 biological sample Substances 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 210000004072 lung Anatomy 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 244000005700 microbiome Species 0.000 description 8
- 239000002245 particle Substances 0.000 description 8
- 244000052769 pathogen Species 0.000 description 8
- 238000003752 polymerase chain reaction Methods 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 7
- 230000000890 antigenic effect Effects 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 230000004044 response Effects 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 229940088710 antibiotic agent Drugs 0.000 description 6
- 210000000612 antigen-presenting cell Anatomy 0.000 description 6
- 210000003719 b-lymphocyte Anatomy 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 210000003527 eukaryotic cell Anatomy 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- 230000003278 mimic effect Effects 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 108020004705 Codon Proteins 0.000 description 5
- 108010041986 DNA Vaccines Proteins 0.000 description 5
- 229940021995 DNA vaccine Drugs 0.000 description 5
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 5
- 241000186362 Mycobacterium leprae Species 0.000 description 5
- 241001302239 Mycobacterium tuberculosis complex Species 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000004520 electroporation Methods 0.000 description 5
- 230000002163 immunogen Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- MDYZKJNTKZIUSK-UHFFFAOYSA-N tyloxapol Chemical compound O=C.C1CO1.CC(C)(C)CC(C)(C)C1=CC=C(O)C=C1 MDYZKJNTKZIUSK-UHFFFAOYSA-N 0.000 description 5
- 238000002255 vaccination Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 241001183012 Modified Vaccinia Ankara virus Species 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 210000001616 monocyte Anatomy 0.000 description 4
- 210000000680 phagosome Anatomy 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 230000003827 upregulation Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 101710166488 6 kDa early secretory antigenic target Proteins 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 206010061598 Immunodeficiency Diseases 0.000 description 3
- 208000032420 Latent Infection Diseases 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 241000186367 Mycobacterium avium Species 0.000 description 3
- 241000187492 Mycobacterium marinum Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 108010033276 Peptide Fragments Proteins 0.000 description 3
- 102000007079 Peptide Fragments Human genes 0.000 description 3
- 229940022005 RNA vaccine Drugs 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 102400001107 Secretory component Human genes 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000005867 T cell response Effects 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- 230000003302 anti-idiotype Effects 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- -1 for example Chemical class 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 239000012678 infectious agent Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 108700021021 mRNA Vaccine Proteins 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 239000007922 nasal spray Substances 0.000 description 3
- 229940097496 nasal spray Drugs 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 3
- 229920000053 polysorbate 80 Polymers 0.000 description 3
- 230000000241 respiratory effect Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000829 suppository Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000009885 systemic effect Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 239000003104 tissue culture media Substances 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000700199 Cavia porcellus Species 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 238000011238 DNA vaccination Methods 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 2
- 238000011510 Elispot assay Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 241001467553 Mycobacterium africanum Species 0.000 description 2
- 241000187482 Mycobacterium avium subsp. paratuberculosis Species 0.000 description 2
- 241001467552 Mycobacterium bovis BCG Species 0.000 description 2
- 241000187919 Mycobacterium microti Species 0.000 description 2
- 241000187480 Mycobacterium smegmatis Species 0.000 description 2
- 241001646725 Mycobacterium tuberculosis H37Rv Species 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 241000710960 Sindbis virus Species 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- NWMHDZMRVUOQGL-CZEIJOLGSA-N almurtide Chemical compound OC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)CO[C@@H]([C@H](O)[C@H](O)CO)[C@@H](NC(C)=O)C=O NWMHDZMRVUOQGL-CZEIJOLGSA-N 0.000 description 2
- 229960004821 amikacin Drugs 0.000 description 2
- LKCWBDHBTVXHDL-RMDFUYIESA-N amikacin Chemical compound O([C@@H]1[C@@H](N)C[C@H]([C@@H]([C@H]1O)O[C@@H]1[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O1)O)NC(=O)[C@@H](O)CCN)[C@H]1O[C@H](CN)[C@@H](O)[C@H](O)[C@H]1O LKCWBDHBTVXHDL-RMDFUYIESA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 210000000621 bronchi Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000016396 cytokine production Effects 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 235000021186 dishes Nutrition 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 230000002414 glycolytic effect Effects 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 238000001638 lipofection Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000003563 lymphoid tissue Anatomy 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 210000003097 mucus Anatomy 0.000 description 2
- 230000014207 opsonization Effects 0.000 description 2
- 230000000242 pagocytic effect Effects 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000002688 persistence Effects 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000037452 priming Effects 0.000 description 2
- 230000000750 progressive effect Effects 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 210000003705 ribosome Anatomy 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000013605 shuttle vector Substances 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 101150063569 slgA gene Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 229940031626 subunit vaccine Drugs 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000003319 supportive effect Effects 0.000 description 2
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000014621 translational initiation Effects 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 229940125575 vaccine candidate Drugs 0.000 description 2
- 230000007923 virulence factor Effects 0.000 description 2
- 239000000304 virulence factor Substances 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- YHQZWWDVLJPRIF-JLHRHDQISA-N (4R)-4-[[(2S,3R)-2-[acetyl-[(3R,4R,5S,6R)-3-amino-4-[(1R)-1-carboxyethoxy]-5-hydroxy-6-(hydroxymethyl)oxan-2-yl]amino]-3-hydroxybutanoyl]amino]-5-amino-5-oxopentanoic acid Chemical compound C(C)(=O)N([C@@H]([C@H](O)C)C(=O)N[C@H](CCC(=O)O)C(N)=O)C1[C@H](N)[C@@H](O[C@@H](C(=O)O)C)[C@H](O)[C@H](O1)CO YHQZWWDVLJPRIF-JLHRHDQISA-N 0.000 description 1
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- MIJDSYMOBYNHOT-UHFFFAOYSA-N 2-(ethylamino)ethanol Chemical compound CCNCCO MIJDSYMOBYNHOT-UHFFFAOYSA-N 0.000 description 1
- NOIRDLRUNWIUMX-UHFFFAOYSA-N 2-amino-3,7-dihydropurin-6-one;6-amino-1h-pyrimidin-2-one Chemical compound NC=1C=CNC(=O)N=1.O=C1NC(N)=NC2=C1NC=N2 NOIRDLRUNWIUMX-UHFFFAOYSA-N 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- FFKUHGONCHRHPE-UHFFFAOYSA-N 5-methyl-1h-pyrimidine-2,4-dione;7h-purin-6-amine Chemical compound CC1=CNC(=O)NC1=O.NC1=NC=NC2=C1NC=N2 FFKUHGONCHRHPE-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 101100298079 African swine fever virus (strain Badajoz 1971 Vero-adapted) pNG2 gene Proteins 0.000 description 1
- 102100035248 Alpha-(1,3)-fucosyltransferase 4 Human genes 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241000713842 Avian sarcoma virus Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- 101100327537 Caenorhabditis elegans cgp-1 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 108010039939 Cell Wall Skeleton Proteins 0.000 description 1
- 241000819038 Chichester Species 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 241000193469 Clostridium pasteurianum Species 0.000 description 1
- 108010051219 Cre recombinase Proteins 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N D-alanine Chemical compound C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- 102100024607 DNA topoisomerase 1 Human genes 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 102100024746 Dihydrofolate reductase Human genes 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108010074122 Ferredoxins Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010027992 HSP70 Heat-Shock Proteins Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001022185 Homo sapiens Alpha-(1,3)-fucosyltransferase 4 Proteins 0.000 description 1
- 101000830681 Homo sapiens DNA topoisomerase 1 Proteins 0.000 description 1
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108020003285 Isocitrate lyase Proteins 0.000 description 1
- 229930195714 L-glutamate Natural products 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-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
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 206010024229 Leprosy Diseases 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- 108700027766 Listeria monocytogenes hlyA Proteins 0.000 description 1
- 208000008771 Lymphadenopathy Diseases 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 206010027259 Meningitis tuberculous Diseases 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- 241000187474 Mycobacterium asiaticum Species 0.000 description 1
- 241000186365 Mycobacterium fortuitum Species 0.000 description 1
- 241001509451 Mycobacterium genavense Species 0.000 description 1
- 241001147828 Mycobacterium haemophilum Species 0.000 description 1
- 241000186364 Mycobacterium intracellulare Species 0.000 description 1
- 241000186363 Mycobacterium kansasii Species 0.000 description 1
- 241000908167 Mycobacterium lepraemurium Species 0.000 description 1
- 241000187493 Mycobacterium malmoense Species 0.000 description 1
- 241000187481 Mycobacterium phlei Species 0.000 description 1
- 241001147832 Mycobacterium shimoidei Species 0.000 description 1
- 241000187489 Mycobacterium simiae Species 0.000 description 1
- 241000187496 Mycobacterium szulgai Species 0.000 description 1
- 241000187917 Mycobacterium ulcerans Species 0.000 description 1
- 241000187644 Mycobacterium vaccae Species 0.000 description 1
- 241001429274 Mycobacterium virus L5 Species 0.000 description 1
- 241000187494 Mycobacterium xenopi Species 0.000 description 1
- 108700015872 N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine Proteins 0.000 description 1
- 108700020354 N-acetylmuramyl-threonyl-isoglutamine Proteins 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101710160107 Outer membrane protein A Proteins 0.000 description 1
- SZRPVOFXFLWAPX-UHFFFAOYSA-K P(=O)([O-])([O-])O.[Ca+2].[Cl-].[Rb+] Chemical compound P(=O)([O-])([O-])O.[Ca+2].[Cl-].[Rb+] SZRPVOFXFLWAPX-UHFFFAOYSA-K 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 1
- 108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 108020005067 RNA Splice Sites Proteins 0.000 description 1
- 102000004879 Racemases and epimerases Human genes 0.000 description 1
- 108090001066 Racemases and epimerases Proteins 0.000 description 1
- 208000035415 Reinfection Diseases 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 241000710961 Semliki Forest virus Species 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 230000029662 T-helper 1 type immune response Effects 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 208000022971 Tuberculous meningitis Diseases 0.000 description 1
- 108010069584 Type III Secretion Systems Proteins 0.000 description 1
- 241000607734 Yersinia <bacteria> Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000000184 acid digestion Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 208000013228 adenopathy Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000009603 aerobic growth Effects 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 210000001132 alveolar macrophage Anatomy 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004979 bone marrow derived macrophage Anatomy 0.000 description 1
- 210000003123 bronchiole Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000004520 cell wall skeleton Anatomy 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000036757 core body temperature Effects 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 108020001096 dihydrofolate reductase Proteins 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- NSYZCCDSJNWWJL-YXOIYICCSA-N erythromycin ethylsuccinate Chemical compound O1[C@H](C)C[C@H](N(C)C)[C@@H](OC(=O)CCC(=O)OCC)[C@@H]1O[C@H]1[C@@](O)(C)C[C@@H](C)C(=O)[C@H](C)[C@@H](O)[C@](C)(O)[C@@H](CC)OC(=O)[C@H](C)[C@@H](O[C@@H]2O[C@@H](C)[C@H](O)[C@](C)(OC)C2)[C@@H]1C NSYZCCDSJNWWJL-YXOIYICCSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000003176 fibrotic effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 239000012909 foetal bovine serum Substances 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 1
- 238000011554 guinea pig model Methods 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 235000011167 hydrochloric acid Nutrition 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 230000017307 interleukin-4 production Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960003350 isoniazid Drugs 0.000 description 1
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical compound NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 210000004880 lymph fluid Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 208000001223 meningeal tuberculosis Diseases 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 229960005225 mifamurtide Drugs 0.000 description 1
- JMUHBNWAORSSBD-WKYWBUFDSA-N mifamurtide Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCCCCCCCCCC)COP(O)(=O)OCCNC(=O)[C@H](C)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)OC(O)[C@@H]1NC(C)=O JMUHBNWAORSSBD-WKYWBUFDSA-N 0.000 description 1
- 108700007621 mifamurtide Proteins 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 101150006328 mpb83 gene Proteins 0.000 description 1
- 230000000420 mucociliary effect Effects 0.000 description 1
- 230000016379 mucosal immune response Effects 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000003007 myelin sheath Anatomy 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 238000001216 nucleic acid method Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 238000013492 plasmid preparation Methods 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920001515 polyalkylene glycol Polymers 0.000 description 1
- 229930001119 polyketide Natural products 0.000 description 1
- 150000003881 polyketide derivatives Chemical class 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 230000009979 protective mechanism Effects 0.000 description 1
- 238000000734 protein sequencing Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 230000037390 scarring Effects 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 229940031439 squalene Drugs 0.000 description 1
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 210000001138 tear Anatomy 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000005144 thermotropism Effects 0.000 description 1
- 230000005100 tissue tropism Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- XETCRXVKJHBPMK-MJSODCSWSA-N trehalose 6,6'-dimycolate Chemical compound C([C@@H]1[C@H]([C@H](O)[C@@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](COC(=O)C(CCCCCCCCCCC3C(C3)CCCCCCCCCCCCCCCCCC)C(O)CCCCCCCCCCCCCCCCCCCCCCCCC)O2)O)O1)O)OC(=O)C(C(O)CCCCCCCCCCCCCCCCCCCCCCCCC)CCCCCCCCCCC1CC1CCCCCCCCCCCCCCCCCC XETCRXVKJHBPMK-MJSODCSWSA-N 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 150000005691 triesters Chemical class 0.000 description 1
- 229960001005 tuberculin Drugs 0.000 description 1
- 229960002109 tuberculosis vaccine Drugs 0.000 description 1
- 230000029069 type 2 immune response Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 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
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
- A61P31/06—Antibacterial agents for tuberculosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Communicable Diseases (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oncology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Pulmonology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Description
PROTECTION AGAINST MYCOBACTERIAL INFECTIONS
The present invention relates to a method of identifying mycobacterial genes which are induced or up-regulated during mycobacterial virulence, to isolated peptide products of said genes, to inhibitors of said genes, to antibodies which bind to said peptide products, to DNA and RNA vectors encoding said products, to attenuated mycobacteria in which the activity of at least one of said genes or peptide products has been modified, to vaccines against mycobacterial infections, and to methods of detecting a mycobacterial infection.
Many microorganisms are capable of forming intracellular infections. These include: infections caused by species of Salmonella, Yersinia, Shigella, Campylobacter, Chlamydia and Mycobacteria. Some of these infections are exclusively intracellular, others contain both intracellular and extracellular components. However, it is the intracellular survival cycle of bacterial infection which is suspected as a main supportive factor for disease progression.
Generally, these microorganisms do not circulate freely in the body, for example, in the bloodstream, and are often not amenable to drug treatment regimes. Where drugs are available, this problem has been exacerbated by the development of multiple drug resistant microorganisms.
A number of factors have contributed to the problem of microbial drug resistance. One is the accumulation of mutations over time and the subsequent horizontal and vertical transfer of the mutated genes to other organisms. Thus, for a given pathogen, entire classes of antibiotics have been rendered inactive. A further factor has been the absence of a new class of antibiotics in recent years. The emergence of multiple drug-resistant pathogenic bacteria represents a serious threat to public health and new forms of therapy are urgently required.
For similar reasons, vaccine therapies have not proved effective against such intracellular microorganisms. Also, increased systemic concentration of antibiotics to improve bioavailability within cells may result in severe side effects.
Mycobacterium tuberculosis and closely related species make up a small group of mycobacteria known as the Mycobacterium tuberculosis complex (MTC) . This group comprises four species M. tuberculosis, M. microti, M. bovis and M. africanum which are the causative agent in the majority of tuberculosis (TB)
cases throughout the world.
M. tuberculosis is responsible for more than three million deaths a year worldwide. Other mycobacteria are also pathogenic in man and animals, for example M. avium subsp. paratuberculosis which causes Johne's disease in ruminants, M. bovis which causes tuberculosis in cattle, M. avium and M. mtracellulare which cause tuberculosis in immunocompromised patients (eg. AIDS patients, and bone marrow transplant patients) and M. leprae which causes leprosy in humans.
M. tuberculosis infects macrophage cells within the body, particularly alveolar macrophages in the lung. Soon after macrophage infection, most M. tuberculosis bacteria enter, persist and replicate within cellular phagosome vesicles, where the bacteria are sequestered from host defences and extracellular factors.
It is the intracellular survival and multiplication or replication of bacterial infection which is suspected as a main supportive factor for mycobacterial disease progression.
A number of drug therapy regimens have been proposed for combatting M. tuberculosis infections, and currently combination therapy including the drug isoniazid has proved most effective. However, one problem with such treatment regimes is that they are long-term, and failure to complete such treatment can promote the development of multiple drug resistant microorganisms.
A further problem is that of providing an adequate bioavailability of the drug within the cells to be treated. Whilst it is possible to increase the systemic concentration of a drug (eg. by administering a higher dosage) this may result in severe side effects caused by the increased drug concentration.
The effectiveness of vaccine prevention against M. tuberculosis has varied widely. The current M. tuberculosis vaccine, BCG, is an attenuated strain of M. bovis. It is effective against severe complications of TB in children, but it varies greatly in its effectiveness in adults particularly across ethnic groups. BCG vaccination has been used to prevent tuberculous meningitis and helps prevent the spread of M. tuberculosis to extra-pulmonary sites, but does not prevent
infection. Furthermore, it can not be administered to immunocompromised individuals, a group particularly susceptible to TB, because of the risk of systemic disease resulting from the vaccine.
The limited efficacy of BCG and the global prevalence of TB has led to an international effort to generate new, more effective vaccines. The current paradigm is that protection will be mediated by the stimulation of a Th1 immune response.
BCG vaccination in man was given orally when originally introduced, but that route was discontinued because of loss of viable BCG during gastric passage and of frequent cervical adenopathy. In experimental animal species, aerosol or intra-tracheal delivery of BCG has been achieved without adverse effects, but has varied in efficacy from superior protection than parenteral inoculation in primates, mice and guinea pigs to no apparent advantage over the subcutaneous route in other studies.
There is therefore a need for an improved and/or alternative vaccine or therapeutic agent for combatting mycobacterial infections.
This need is addressed by the present invention.
According to a first aspect, the present invention provides a method of identifying a mycobacterial nucleic acid promoter sequence which is induced or up-regulated during mycobacterial virulence, said method comprising:-
infecting a macrophage target cell with a Mycobacterium tuberculosis host cell, which host cell contains a nucleic acid construct comprising a putative mycobacterial promoter sequence operably linked to a coding sequence of a reporter gene located down-stream from the promoter;
culturing the macrophage under conditions which support mycobacterial virulence; and
identifying a promoter sequence which is induced or up-regulated during virulence by detecting expression of the reporter sequence.
M. tuberculosis is a high containment pathogen, and use of this microorganism is therefore preferably conducted under Advisory Committee for Dangerous Pathogen (ACDP) 3 conditions.
M. tuberculosis pursues a route of intracellular infection, and there are several differences in the behaviour of M. tuberculosis compared to other pathogens. Thus, nucleic acid expression profiles of other bacteria during infection, even other mycobacteria, do not accurately reflect the series of changes which occur during M. tuberculosis infection.
For example, M. tuberculosis does not use type III secretion systems to inject effector molecules to alter host cell behaviour as do Salmonella species nor does it utilise pore forming toxins such as listeriolysin to escape the phagosomal vesicle as found in L. monocytogenes infection.
Pathogenic mycobacteria appear to rely upon a repertoire of gene products which are tightly regulated in response to the interaction with a host cell, many of which have yet to be identified.
in comparison with other mycobacterial pathogens, species such as M. leprae and M. marinum appear to have a thermotropism where the infection is manifested in the periphery of the body where temperatures are lower than the typical 37°C core body temperature. M. leprae also targets the myelin sheath of the nerve cells and the disease pathology reflects this tissue tropism.
M. marinum causes a limited infection resulting in a surface lesion on the skin which can be self-limiting.
Other pathogens such as M. avium and M. mtracellulare typically cause infections in immunocompromised individuals and do not usually have the ability to cause disease in healthy subjects.
M. bovis infection is usually acquired through ingestion of contaminated milk products resulting in infection via the lymph nodes and leading to lymphoadenopathy.
Following infection, M. tuberculosis may enter a dormant state leading to a
latent infection. This latent infection may persist for decades before reactivation occurs leading to progressive active disease.
Other mycobacterial species may share in common with M. tuberculosis some components of conserved virulence mechanisms. However, with the publication of the genome sequences forM. tuberculosis and M. leprae, and the publication of other partially completed genome sequences (eg. M. smegmatis, and M. bovis), it is clear there are significant differences between the different mycobacterial pathogens that ultimately define their success as pathogens and the characteristics of the pathology of the diseases that they cause.
Thus, M. tuberculosis is a specialised bacterial pathogen typically spread by inhalation. M. tuberculosis has a complex interaction with its host, from initial contact with host macrophage onwards, which it manipulates to its own advantage leading to either progressive active disease, or a persistent latent infection or most likely a limited infection which is cleared or contained indefinitely by the host.
Mycobacterial virulence includes one or more of the events associated with infection by a mycobacterium of its natural target cell. For example, a natural target cell of M. tuberculosis is a macrophage, and virulence with respect to this bacterium and target cell would include any event involved during the sequence of events comprising phagocytosis by the macrophage or uptake via alternative pathways of the • bacterium, formation of the phagosome, multiplication of the bacterium within the phagosome, lysis of the phagosome/host cell, and spread of the lesion to a secondary site (ie. haematogenous spread). Thus, virulence conditions are culture conditions which are conducive for a mycobacterium to express at least one gene which would be normally expressed in vivo during infection of the mycobacterium's natural target cell.
In one embodiment, the putative promoter sequence is selected from the species M. phlei, M. smegmatis, M. africanum, M. caneti, M. fortuitum, M. marinum, M. ulcerans, M. tuberculosis, M. bovis, M. microti, M. avium, M. paratuberculosis, M. leprae, M. lepraemurium, M. intracellulare, M. scrofu/aceum, M. xenopi, M. genavense, M. kansasii, M. simiae, M. szulgai, M. haemophilum, M. asiaticum, M. malmoense, M. vaccae and M. shimoidei. Of
particular interest are members of the MTC, preferably M. tuberculosis or M. bovis.
In another embodiment, the reporter gene is a silent marker of gene expression, and does not require a selection pressure to be applied during the detection step. This is in contrast to other markers, for example, antibiotic resistance markers which require the presence of the antibiotic to permit selection of desirable transformants. The presence of a selection pressure may be undesirable as this pressure may itself induce or cause up-regulation of other genes, or result in the loss of transiently-expressed promoters. The induction or up-regulation of other genes, eg. growth-related genes, may result in increased growth of background mycobacteria and may cause problems in the identification of desirable transformants free of false positives.
The reporter sequences employed in the method of the present invention do not possess their own promoters, and are therefore reliant on the activity of the putative promoter sequence to effect expression thereof. Thus, by culturing mycobacterial host cells under virulence conditions, it is possible to select for those promoters which are active under virulence conditions by detecting expression of the reporter sequence.
The reporter gene is preferably a Green Fluorescent Protein (GFP). Suitable GFPs such as GFPmut 1 , 2 or 3 are described in [Cormack et al. (1996) Gene, 173, pp.33-38]. In a preferred embodiment, the GFP is GFPmut2.
In those embodiments in which the reporter sequence encodes a fluorescent protein, the desirable mycobacterial host cell transformants are capable of fluorescence by expression of the reporter sequence.
Thus, in a preferred embodiment, promoters active during virulence are detected by differential fluorescence induction methods (DFI) as, for example, described in Valdivia, R.H. and Falkow, S. (1997) [Science, vol. 277, 26 September 1997, pp. 2007-201 1 ], and desirable transformed host cells containing said promoters may be isolated by fluorescence activated cell sorting (FACS).
In use, the mycobacterial host cell infects a macrophage in vitro. Thereafter,
when recovering the mycobacteria (eg. for FACS analysis), phagosomal vesicles containing the bacteria may be recovered and the vesicles analysed (ie. sorted) whole. Alternatively, both the macrophage and phagosomal vesicles contained within are disrupted and the released bacteria are analysed by FACS.
Suitable macrophage cells include human acute-monocytic leukemic cell lines of macrophage lineage: CD14+, CD15+ (THP-I), murine bone marrow-derived macrophage cell lines, and murine BALB/c tumour derived macrophage- monocyte cell lines (eg. ATCC TIB-67, and ATCC TIB-71 ).
Example 3 of the present application describes one preferred macrophage assay of the present application. However, any one of numerous conventional macrophage infection assays may be employed in the present invention.
Suitable media for culturing mycobacteria under non-virulence conditions are described in Wayne, L.G. (1994) [in Tuberculosis: Pathogenesis, Protection, and Control published by the American Society for Microbiology, pp. 73-83]. These include Middlebrook 7H9 Medium [see Barker, L.P., et al. (1998) Molec. Microbiol., vol. 29(5), pp. 1 167-1 177, and WO00/52139 in the name of the present Applicant].
In use, an induced or up-regulated promoter is identified by detecting increased expression of the reporter sequence during macrophage infection when compared to expression of the reporter sequence under culture conditions which do not promote mycobacterial virulence. This minimises the risk of identifying mycobacterial host cells containing putative promoters which are constitutively expressed.
Suitable culture conditions which do not promote mycobacterial virulence (ie. conditions which support non-virulence) include mycobacterial culture conditions which are substantially free from macrophage.
In one embodiment, the non-virulence culture conditions are substantially non- limiting in terms of aerobic growth (eg. pH, temperature, and available nutrients) of the mycobacterial host cell culture. Thus, the mycobacterial host cell culture is preferably cultured under conventional conditions which permit a doubling time of 18-26 hours.
In use, the mycobacterial host cell culture may be cultured in batch, and is preferably harvested during mid- to late-exponential phase. Alternatively, this point in the growth phase may be mimicked under continuous culture conditions employing a steady state growth rate approximating μmax which provides a generation time of approximately 18-24 hours.
The preferred culture method employed by the present invention is that of batch fermenter culture. This method permits careful monitoring of growth culture parameters such as pH, temperature, available nutrients, and dissolved oxygen tension (DOT). In particular, temperature and DOT may be strictly controlled.
The identification of transformed host cells containing promoters which are constitutively expressed may be performed before or after identification of promoters expressed during virulence conditions. In a preferred embodiment, constitutively expressed promoters are identified after identification of induced or up-regulated promoters expressed during virulence.
Increased expression means that the signal detected as a result of expression of the reporter sequence when the mycobacterial host cell is cultured under virulence conditions is at least 1 .3 times greater than the signal detected when the host cell is cultured under non-virulence conditions. In one embodiment, the signal is at least 2 times, preferably at least 4 times greater when the host cell is cultured under virulence conditions. In another embodiment, the signal is at least 10, preferably at least 20, and particularly preferably at least 30 times greater when the host cell is cultured under virulence conditions.
Naturally, the expression profile for a given gene in a mycobacterial host cell may vary during the course of infection. For example, a gene may be induced or up-regulated most strongly during the early stages of an infection. Thus, the term "increased expression" refers to the maximum signal obtained for a given reporter gene under virulence conditions vis-a-vis non-virulence conditions.
In one embodiment, the "increased expression" concerns the maximum signal obtained within the first four days after macrophage infection, preferably within the first two days after macrophage infection.
In another embodiment, genes switched on or up-regulated at later stages of
infection are of interest, in which case a maximum signal 4 days after macrophage infection is preferred. For example, a maximum signal up to 1 1 -12 days after infection may be preferred. However, due to the invasive and destructive nature of a M. tuberculosis infection on a macrophage, a post- infection period of between 4 and 6 days is particularly preferred.
A number of methods may be employed for obtaining putative mycobacterial promoter sequences for use in the present invention. These include:- generation of a library of putative promoter sequences by use of nucleic acid digestion enzymes (see Example 1 ); and identifying nucleic acid sequence homology to known virulence or previously implicated virulence sequences in other microorganisms (see Example 2).
In application of the above homology identification method, a single promoter construct (rather than a selection of constructs generated from a library) is prepared, and a promoter-reporter unit constructed. It is then possible to assess promoter activity by direct comparison (eg. by FACS) of mycobacterial host cultured under virulence conditions versus non-virulence conditions. Since only a single promoter-reporter construct is employed, there is no need for a separate screening of the host cells.
The method of the present invention permits identification of promoters which are induced or up-regulated during virulence. Once the nucleic acid sequence and orientation of a desirable promoter sequence has been determined, the exact location can be mapped by use of convention nucleic acid homology search computer software (eg. BLASTN, or BLASTX) performed on published sequences of the mycobacterial species concerned. Accordingly, the gene or operon under the control of the identified promoter may be identified.
Thus, in a second aspect of the present invention, there is provided a method of identifying a mycobacterial gene the expression of which is induced or upregulated during mycobacterial virulence, said method comprising:-
identifying a mycobacterial promoter sequence which is induced or up- regulated during infection of a macrophage by a M. tuberculosis host cell, wherein the host cell contains a nucleic acid construct comprising said promoter sequence operably linked to a coding sequence of a
reporter gene located down-stream from the promoter;
aligning by sequence homology the nucleic acid sequence of the promoter with published nucleic acid sequence data for the same mycobacterial species; and
identifying the associated nucleic acid coding sequence under the control of said promoter.
Reference to "gene" throughout this specification embraces open reading frames (ORFs).
According to a third aspect of the present invention there is provided an isolated mycobacterial peptide or a fragment or derivative or variant thereof, wherein the peptide is encoded by a mycobacterial gene the expression of which gene is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said mycobacterial gene.
The various embodiments described for the first and second aspect of the present invention apply equally for the third and subsequent aspects of the present invention.
The terms "isolated," "substantially pure," and "substantially homogenous" are used interchangeably to describe a peptide which has been separated from components which naturally accompany it. A peptide is substantially pure when at least about 60 to 75% of a sample exhibits a single peptide sequence. A substantially pure peptide will typically comprise about 60 to 90% w/w of a protein sample, more usually about 95%, and preferably will be over about 99% pure. Peptide purity or homogeneity may be indicated by, for example, polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel. Alternatively, higher resolution may be provided by using, for example, HPLC.
A peptide is considered to be isolated when it is separated from the contaminants which accompany it in its natural state. Thus, a peptide which is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally
associated components.
The present invention provides peptides which may be purified from mycobacteria as well as from other types of cells transformed with recombinant nucleic acids encoding these peptides.
If desirable, the amino acid sequence of the proteins of the present invention may be determined by protein sequencing methods.
The terms "peptide", "oligopeptide", "polypeptide", and "protein" are used interchangeably and do not refer to a specific length of the product. These terms embrace post-translational modifications such as glycosylation, acetγlation, and phosphorylation.
The term "fragment" means a peptide having at least five, preferably at least ten, more preferably at least twenty, and most preferably at least thirty-five amino acid residues of the peptide which is the gene product of the induced or up-regulated gene in question. The fragment preferably includes at least one epitope of the gene product in question.
The term "variant" means a peptide or peptide "fragment" having at least seventy, preferably at least eighty, more preferably at least ninety percent amino acid sequence homology with the peptide which is the gene product of the induced or up-regulated peptide in question. An example of a "variant" is a peptide or peptide fragment of an induced/up-regulated gene which contains one or more analogs of an amino acid (eg. an unnatural amino acid), or a substituted linkage. The terms "homology" and "identity" are considered synonymous in this specification. In a further embodiment, a "variant" may be a mimic of the peptide or peptide fragment, which mimic reproduces at least one epitope of the peptide or peptide fragment. The mimic may be, for example, a nucleic acid mimic, preferably a DNA mimic.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences may be then compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then
calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison may be conducted, for example, by the local homology alignment algorithm of Smith and Waterman [Adv. Appl. Math. 2: 484 (1981 )], by the algorithm of Needleman & Wunsch [J. Mol. Biol. 48: 443 (1970)] by the search for similarity method of Pearson & Lipman [Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988)], by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA - Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), or by visual inspection [see Current Protocols in Molecular Biology, F.M. Ausbel et al, eds, Current Protocols, a joint venture between Greene Publishing Associates, In. And John Wiley & Sons, Inc. (1995 Supplement) Ausbubel].
Examples of algorithms suitable for determining percent sequence similarity are the BLAST and BLAST 2.0 algorithms [see Altschul (1990) J. Mol. Biol. 21 5: pp. 403-410; and "http://www.ncbi.nlm.nih.gov/" of the National Center for Biotechnology Information].
In a preferred homology comparison, the identity exists over a region of the sequences that is at least 10 amino acids, preferably at least 20 amino acids, more preferably at least 35 amino acids in length.
The term "derivative" means a protein comprising the peptide (or fragment, or variant thereof) which peptide is the gene product of the induced or upregulated gene in question. Thus, a derivative may include the peptide in question, and a further peptide sequence which may introduce one or more additional epitopes. The further peptide sequence should preferably not interfere with the basic folding and thus conformational structure of the peptide in question. Examples of a "derivative" are a fusion protein, a conjugate, and a graft. Thus, two or more peptides (or fragments, or variants) may be joined together to form a derivative. Alternatively, a peptide (or fragment, or variant) may be joined to an unrelated molecule (eg. a second, unrelated peptide). Derivatives may be chemically synthesized, but will be typically prepared by recombinant nucleic acid methods. Additional components such as lipid, and/or
polysaccharide, and/or polyketide components may be included.
All of the molecules "fragment", "variant" and "derivative" have a common antigenic cross-reactivity and/or substantially the same in vivo biological activity as the peptide product of the induced or up-regulated gene in question from which they are derived. For example, an antibody capable of binding to a fragment, variant or derivative would be also capable of binding to the gene product of the induced or up-regulated gene in question. It is a preferred feature that the fragment, variant and derivative each possess the active site of the peptide which is the induced or up-regulated peptide in question. Alternatively, all of the above embodiments of a peptide of the present invention share a common ability to induce a "recall response" of a T-lymphocyte which has been previously exposed to an antigenic component of a mycobacterial infection.
In a preferred embodiment, the peptide is selected from the group consisting of SEQ ID NO: 2; 4; 6; 8; 10; 12; 14; 1 6; 19; 21 ; 23; 25; 27; 29; 31 ; 33; 35; 38; 40; 42; 44; 46; 48; 50; 52; 55; 58; 60; 62; 64; 67; 69; 72; 75; 78; 80; 82; 85; 87; 89; 92; 94; 97; 100; 103; 105; 107; 109; 1 1 1 ; 1 13; 1 15; and 1 17.
A fourth aspect of the invention provides an inhibitor of a mycobacterial peptide, wherein the peptide is encoded by a mycobacterial gene the expression of which gene is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said mycobacterial gene, and wherein the inhibitor is capable of substantially preventing or inhibiting the mycobacterial peptide from exerting its native biological function or effect.
Inhibition of the mycobacterial peptide may be effected at the nucleic acid level (ie. DNA, or RNA), or at the peptide level.
In a further embodiment, the inhibitor may be an antibiotic capable of targeting the induced or up-regulated mycobacterial gene, or the peptide product thereof. The antibiotic is preferably specific for the gene and/or peptide product.
Inhibitors of the present invention may be prepared utilizing the sequence information provided herein. For example, this may be performed by overexpressing the peptide, purifying the peptide, and then performing X-ray
compounds are created which have similar molecular structures to all or portions of the peptide or its substrate. The compounds may be then combined with the peptide and attached thereto so as to block one or more of its biological activities.
Also included within the invention are isolated or recombinant polynucleotides that bind to the regions of the mycobacterial chromosome (eg. promoter, or coding region), or transcription products thereof, containing sequences that are associated with induction/up-regulation of a mycobacterial gene during macrophage infection by M. tuberculosis, including antisense and triplex forming polynucleotides. As used herein, the term "binding" refers to an interaction or complexation between an oligonucleotide and a target nucleotide sequence, mediated through hydrogen bonding or other molecular forces. The term "binding" more specifically refers to two types of internucleotide binding mediated through base-base hydrogen bonding. The first type of binding is "Watson-Crick-type" binding interactions in which adenine-thymine (or adenine-uracil) and guanine-cytosine base-pairs are formed through hydrogen bonding between the bases. An example of this type of binding is the binding traditionally associated with the DNA double helix and in RNA-DNA hybrids; this type of binding is normally detected by hybridization procedures.
A second type of binding is "triplex binding". In general, triplex binding refers to any type of base-base hydrogen bonding of a third polynucleotide strand with a duplex DNA (or DNA-RNA hybrid) that is already paired in a Watson-Crick manner.
In a preferred embodiment, the inhibitor may be an antisense nucleic acid sequence which is complementary to at least part of the inducible or up- regulatable gene.
The inhibitor, when in the form of a nucleic acid sequence, comprises, in use, at least 15 nucleotides, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, and most preferably at least 50 nucleotides.
In a fifth aspect there is provided an antibody which binds to a peptide encoded by a gene, or to a fragment or variant or derivative of said peptide, the expression of which gene is induced or up-regulated during infection of a
macrophage by a M. tuberculosis host cell containing said gene,
The antibody preferably has specificity for the peptide in question, and following binding thereto may initiate coating of a mycobacterium expressing said peptide. Coating of the bacterium preferably leads to opsonization thereof. This, in turn, leads to the bacterium being destroyed. It is preferred that the antibody is specific for the mycobacterium (eg. species and/or strain) which is to be targeted.
Opsonization by antibodies may influence cellular entry and spread of mycobacteria in phagocytic and non-phagocytic cells by preventing or modulating receptor-mediated entry and replication in macrophages.
The peptides, fragments, variants or derivatives of the present invention may be used to produce antibodies, including polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (eg. mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to a desired mycobacterial epitope contains antibodies to other antigens, the polyclonal antibodies may be purified by immunoaffinity chromatography.
Alternatively, general methodology for making monoclonal antibodies by hybridomas involving, for example, preparation of immortal antibody-producing cell lines by cell fusion, or other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus may be employed.
The antibody employed in this aspect of the invention may belong to any antibody isotype family, or may be a derivative or mimic thereof. Reference to antibody throughout this specification embraces recombinantly produced antibody, and any part of an antibody which is capable of binding to a mycobacterial antigen.
In one embodiment the antibody belongs to the IgG, IgM or IgA isotype families.
In a preferred embodiment, the antibody belongs to the IgA isotype family. Reference to the IgA isotype throughout this specification includes the secretory form of this antibody (ie. slgA). The secretory component (SC) of slgA may be added in vitro or in vivo. In the latter case, the use of a patient's natural SC labelling machinery may be employed.
In one embodiment, the antibody may be raised against a peptide from a member of the MTC, preferably against M. tuberculosis.
In a preferred embodiment, the antibody is capable of binding to a peptide selected from the group consisting of SEQ ID NO: 2; 4; 6; 8; 10; 12; 14; 16;
19; 21 ; 23; 25; 27; 29; 31 ; 33; 35; 38; 40; 42; 44; 46; 48; 50; 52; 55; 58;
60; 62; 64; 67; 69; 72; 75; 78; 80; 82; 85; 87; 89; 92; 94; 97; 100; 103;
105; 107; 109; 1 1 1 ; 1 13; 1 15; and 1 17 (or fragment, variant, of derivative thereof). The antibodies of the present invention are preferably employed in an isolated form.
In a further embodiment, the antigen is an exposed component of a mycobacterial bacillus. In another embodiment, the antigen is a cell surface component of a mycobacterial bacillus.
The antibody of the present invention may be polyclonal, but is preferably monoclonal.
Without being bound by any theory, it is possible that following mycobacterial infection of a macrophage, the macrophage is killed and the bacilli are released. It is at this stage that the mycobacteria are considered to be most vulnerable to antibody attack. Thus, it is possible that the antibodies of the present invention act on released bacilli following macrophage death, and thereby exert a post-infection effect.
It is possible that the passive protection aspect (ie. delivery of antibodies) of the present invention is facilitated by enhanced accessibility of the antibodies of the present invention to antigens on mycobacterial bacilli. It is possible that antibody binding may block macrophage infection by steric hindrance or disruption of its oligomeric structure. Thus, antibodies acting on mycobacterial bacilli released from killed, infected macrophages may interfere with the spread
of re-infection to fresh macrophages. This hypothesis involves a synergistic action between antibodies and cytotoxic T cells, acting early after infection, eg. yδ and NK T cells, but could later involve also CD8 and CD4 cytotoxic T cells.
According to a sixth aspect of the invention there is provided an attenuated mycobacterium in which a gene has been modified, the expression of which gene is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said mycobacterial gene, thereby rendering the mycobacterium substantially non-pathogenic.
The term "modified" refers to any genetic manipulation such as a nucleic acid or nucleic acid sequence replacement, a deletion, or an insertion which renders the mycobacterium substantially non-pathogenic or substantially incapable of macrophage infection. In one embodiment the entire inducible or up-regulatable gene may be deleted.
In one embodiment, the modification may be effected by a nucleic acid sequence encoding an anti-sense nucleic acid sequence to the induced or upregulated gene, or a transcription product thereof. Thus, by including such a nucleic acid sequence in a mycobacterium, for example in the form of a plasmid, expression of the peptide product of the induced or up-regulated gene may be reduced or substantially inhibited.
In a preferred embodiment, the gene to be modified has a wild-type coding sequence corresponding to one of the group consisting of SEQ ID NO: 3; 5; 7 9; 1 1 ; 13; 1 5; 17; 20; 22; 24; 26; 28; 30; 32; 34; 36; 39; 41 ; 43; 45; 47 49; 51 ; 53; 56; 59; 61 ; 63; 65; 68; 70; 73; 76; 79; 81 ; 83; 86; 88; 90; 93 95; 98; 101 ; 104; 106; 108; 1 10; 1 12; 1 14; 1 16; and 1 18.
It will be appreciated that the wild-type sequences may include minor variations depending on the Database employed. Reference to wild-type simply means that the sequence in question occurs in nature.
A seventh aspect of the present invention provides an attenuated microbial carrier, comprising a peptide encoded by a gene, or a fragment or variant or derivative of said peptide, the expression of which gene is induced or upregulated during infection of a macrophage by a M. tuberculosis host cell
containing said gene.
In use, the peptide (or fragment, variant or derivative) is either at least partially exposed at the surface of the carrier, or the carrier becomes degraded in vivo so that at least part of the peptide (or fragment, variant or derivative) is otherwise exposed to a host's immune system.
In a preferred embodiment, the peptide is selected from the group consisting of SEQ ID NO: 2; 4; 6; 8; 10; 12; 14; 16; 19; 21 ; 23; 25; 27; 29; 31 ; 33; 35; 38; 40; 42; 44; 46; 48; 50; 52; 55; 58; 60; 62; 64; 67; 69; 72; 75; 78; 80; 82; 85; 87; 89; 92; 94; 97; 100; 103; 105; 107; 109; 1 1 1 ; 1 13; 1 15; and 1 17.
In one embodiment, the attenuated microbial carrier is selected from the group consisting of attenuated salmonella, attenuated vaccinia virus, attenuated fowlpox virus, or attenuated M. bovis (eg. BCG strain).
According to an eighth aspect of the invention there is provided a DNA plasmid comprising a promoter, a polyadenylation signal, and a DNA sequence which corresponds to the coding sequence of a mycobacterial gene, or a fragment or variant or derivative of said DNA sequence, the expression of which gene is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said mycobacterial gene, wherein the promoter and polyadenylation signal are operably linked to the DNA sequence.
The term DNA "fragment" used in this invention will usually comprise at least about 5 codons (15 nucleotides), more usually at least about 7 to 15 codons, and preferably at least about 35 codons. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with such a sequence.
In preferred embodiments, the DNA "fragment" has a nucleotide length which is at least 50%, preferably at least 70%, and more preferably at least 80% that of the coding sequence of the corresponding induced/up-regulated gene.
The term DNA "variant" means a DNA sequence which has substantial homology or substantial similarity to the coding sequence (or a fragment
"substantially homologous" (or "substantially similar") to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95 to 98% of the nucleotide bases. Homology determination is performed as described supra for peptides.
Alternatively, a DNA "variant" is substantially homologous (or substantially similar) with the coding sequence (or a fragment thereof) of an induced/up- regulated gene when they are capable of hybridizing under selective hybridization conditions. Selectivity of hybridization exists when hybridization occurs which is substantially more selective than total lack of specificity. Typically, selective hybridization will occur when there is at least about 65% homology over a stretch of at least about 14 nucleotides, preferably at least about 70%, more preferably at least about 75%, and most preferably at least about 90%. See, Kanehisa (1984) Nuc. Acids Res. 12:203-213. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about 17 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.
Nucleic acid hybridization will be affected by such conditions as salt concentration (eg. NaCl), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions are preferably employed, and generally include temperatures in excess of 30 °C, typically in excess of 37 °C and preferably in excess of 45 °C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3.
However, the combination of parameters is much more important than the measure of any single parameter. See, eg., Wetmur and Davidson (1968) J. Mol. Biol. 31 :349-370.
The term DNA "derivative" means a DNA polynucleotide which comprises a
DNA sequence (or a fragment, or variant thereof) corresponding to the coding sequence of the induced/up-regulated gene and an additional DNA sequence which is not naturally associated with the DNA sequence corresponding to the coding sequence. The comments on peptide derivative supra also apply to DNA "derivative". A "derivative" may, for example, include two or more coding sequences of a mycobacterial operon which is induced during macrophage infection. Thus, depending on the presence or absence of a non-coding region between the coding sequences, the expression product/s of such a "derivative" may be a fusion protein, or separate peptide products encoded by the individual coding regions.
The above terms DNA "fragment", "variant", and "derivative" have in common with each other that the resulting peptide products have cross-reactive antigenic properties which are substantially the same as those of the corresponding wild-type peptide. Preferably all of the peptide products of the above DNA molecule embodiments of the present invention bind to an antibody which also binds to the wild-type peptide. Alternatively, all of the above peptide products are capable of inducing a "recall response" of a T lymphocyte which has been previously exposed to an antigenic component of a mycobacterial infection.
The promoter and polyadenylation signal are preferably selected so as to ensure that the gene is expressed in a eukaryotic cell. Strong promoters and polyadenylation signals are preferred.
In a related aspect, the present invention provides an isolated RNA molecule which is encoded by a DNA sequence of the present invention, or a fragment or variant or derivative of said DNA sequence.
An "isolated" RNA is an RNA which is substantially separated from other mycobacterial components that naturally accompany the sequences of interest, eg., ribosomes, polymerases, and other mycobacterial polynucleotides such as DNA and other chromosomal sequences.
The above RNA molecule may be introduced directly into a host cell as, for example, a component of a vaccine.
Alternatively the RNA molecule may be incorporated into an RNA vector prior to administration.
The polynucleotide sequences (DNA and RNA) of the present invention include a nucleic acid sequence which has been removed from its naturally occurring environment, and recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
The term "recombinant" as used herein intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1 ) is not associated with all or a portion of a polynucleotide with which it is associated in nature; or (2) is linked to a polynucleotide other than that to which it is linked in nature; and (3) does not occur in nature. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, eg., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions.
In embodiments of the invention the polynucleotides may encode a peptide which is induced or up-regulated during macrophage infection. A nucleic acid is said to "encode" a peptide if, in its native state or when manipulated, it can be transcribed and/or translated to produce the peptide or a fragment or variant or derivative thereof. The anti-sense strand of such a nucleic acid is also said to encode the sequence.
Also contemplated within the invention are expression vectors comprising the polynucleotide of interest. Expression vectors generally are replicable polynucleotide constructs that encode a peptide operably linked to suitable transcriptional and translational regulatory elements. Examples of regulatory elements usually included in expression vectors are promoters, enhancers, ribosomal binding sites, and transcription and translation initiation and termination sequences. These regulatory elements are operably linked to the sequence to be translated. A nucleic acid sequence is operably linked when it
is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Generally, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. The regulatory elements employed in the expression vectors containing a polynucleotide encoding a virulence factor are functional in the host cell used for expression.
The polynucleotides of the present invention may be prepared by any means known in the art. For example, large amounts of the polynucleotides may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant or other eukaryotic cell lines.
The polynucleotides of the present invention may also be produced by chemical synthesis, eg. by the phosphoramidite method or the triester method, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system recognized by the host, including the intended DNA fragment encoding the desired peptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment. Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences,
and mRNA stabilizing sequences. Secretion signals from polypeptides secreted from the host cell of choice may also be included where appropriate, thus allowing the protein to cross and/or lodge in cell membranes, and thus attain its functional topology or be secreted from the cell.
An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host, and may, when appropriate, include those naturally associated with mycobacterial genes. Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts. Useful yeast promoters include the promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others.
Appropriate non-native mammalian promoters might include the early and late promoters from SV40 or promoters derived from human cytomegalovirus, murine moloney leukemia virus, mouse mammary tumour virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. In addition, the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made.
While such expression vectors may replicate autonomously, they may less preferably replicate by being inserted into the genome of the host cell.
Expression and cloning vectors will likely contain a selectable marker, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures the growth of only those host cells which express the inserts. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, kanamycin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media, e.g. the gene encoding D-alanine racemase for Bacilli. The choice of appropriate selectable marker will depend on the host cell.
The vectors containing the nucleic acids of interest can be transcribed in vitro and the resulting RNA introduced into the host cell (e.g., by injection), or the vectors can be introduced directly into host cells by methods which vary
depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome). The cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells.
Large quantities of the nucleic acids and peptides of the present invention may be prepared by expressing the nucleic acids or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells. The most commonly used prokaryotic hosts are strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.
Mammalian or other eukaryotic host cells, such as those of yeast, filamentous fungi, plant, insect, amphibian or avian species, may also be useful for production of the proteins of the present invention. Propagation of mammalian cells in culture is per se well known. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although other cell lines may be appropriate, e.g., to provide higher expression, desirable glycosylation patterns.
Clones are selected by using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule, preferably the same DNA molecule. The transformant may be screened or, preferably, selected by any of the means well known in the art, e.g., by resistance to such antibiotics as ampicillin, tetracycline.
The polynucleotides of the invention may be inserted into the host cell by any means known in the art, including for example, transformation, transduction, and electroporation. As used herein, "recombinant host cells", "host cells", "cells", "cell lines", "cell cultures", and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transformed. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or
deliberate mutation. "Transformation", as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.
In one embodiment, a DNA plasmid or RNA vector may encode a component of the immune system which is specific to an immune response following challenge with a peptide, wherein said peptide is encoded by a mycobacterial gene which is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said mycobacterial gene.
An example of such a component is an antibody to the peptide product of an induced or up-regulated gene. Thus, in this embodiment, the nucleic acid sequence (eg. DNA plasmid or RNA vector) encodes the antibody in question.
A ninth aspect provides use of a peptide, an inhibitor, an antibody, an attenuated mycobacterium, an attenuated microbial carrier, a DNA sequence corresponding to the coding sequence of an induced or up-regulated gene or a fragment or variant or derivative of said DNA sequence, a DNA plasmid comprising said DNA sequence or said fragment or variant or derivative, an RNA sequence encoded by said DNA sequence or said fragment or variant or derivative, and/or an RNA vector comprising said RNA sequence, according to the present invention, in the manufacture of a medicament for treating or preventing a mycobacterial infection.
The term "treating" includes post-infection therapy and amelioration of a mycobacterial infection.
The term "preventing" includes reducing the severity/intensity of, or initiation of, a mycobacterial infection.
In a related aspect, there is provided a method of treating or preventing a mycobacterial infection, comprising administration of a medicament selected from the group consisting of a peptide, an inhibitor, an antibody, an attenuated mycobacterium, an attenuated microbial carrier, a DNA sequence corresponding
to the coding sequence of an induced or up-regulated gene or a fragment or variant or derivative of said DNA sequence, a DNA plasmid comprising said DNA sequence or said fragment or variant or derivative, an RNA sequence encoded by said DNA sequence or said fragment or variant or derivative, and/or an RNA vector comprising said RNA sequence, according to the present invention, to a patient.
The medicament may be administered by conventional routes, eg. intravenous, intraperitoneal, intranasal routes.
The immunogenicity of the epitopes of the peptides of the invention may be enhanced by preparing them in mammalian or yeast systems fused with or assembled with particle-forming proteins such as, for example, that associated with hepatitis B surface antigen. Vaccines may be prepared from one or more immunogenic peptides of the present invention.
Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 1 1637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1 '-2'-dipalm itoyl-sn -glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL.+ TDM + CWS) in a 2 % squalene/Tween 80 emulsion.
The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations
which are suitable for other modes of administration include suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5 % to 10 %, preferably 1 %-2 %. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10 % - 95 % of active ingredient, preferably 25 % - 70 %.
The peptides may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of 5 micrograms to 250 micrograms of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be peculiar to each subject.
The vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1 -10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reenforce the immune response, for example, at 1 -4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least
in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
In addition, the vaccine containing the immunogenic mycobacterial antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, immune globulins or cytokines, as well as antibiotics.
The outcome of administering antibody-containing compositions may depend on the efficiency of transmission of antibodies to the site of infection. In the case of a mycobacterial respiratory infection (eg. a M. tuberculosis infection), this may be facilitated by efficient transmission of antibodies to the lungs.
In one embodiment the medicament may be administered intranasally (i.n.). This mode of delivery corresponds to the route of delivery of a M. tuberculosis infection and, in the case of antibody delivery, ensures that antibodies are present at. the site of infection to combat the bacterium before it becomes intracellular and also during the period when it spreads between cells.
An intranasal composition may be administered in droplet form having approximate diameters in the range of 100-5000 μm, preferably 500-4000 μm, more preferably 1000-3000 μm. Alternatively, in terms of volume, the droplets would be in the approximate range of 0.001 -100 μl, preferably 0.1 -50 μl, more preferably 1 .0-25 μl.
Intranasal administration may be achieved by way of applying nasal droplets or via a nasal spray.
In the case of nasal droplets, the droplets may typically have a diameter of approximately 1000-3000 μm and/or a volume of 1 -25 μ\.
In the case of a nasal spray, the droplets may typically have a diameter of approximately 100-1000 μm and/or a volume of 0.001 -1 μl.
It is possible that, following i.n. delivery of antibodies, their passage to the lungs is facilitated by a reverse flow of mucosal secretions, although mucociliary action in the respiratory tract is thought to take particles within the mucus out of the lungs. The relatively long persistence in the lungs, fast
clearance from the bile and lack of transport to the saliva of some antibodies suggest the role of mucosal site specific mechanisms.
In a different embodiment, the medicament may be delivered in an aerosol formulation. The aerosol formulation may take the form of a powder, suspension or solution.
The size of aerosol particles is one factor relevant to the delivery capability of an aerosol. Thus, smaller particles may travel further down the respiratory airway towards the alveoli than would larger particles. In one embodiment, the aerosol particles have a diameter distribution to facilitate delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution may be selected to target a particular section of the respiratory airway, for example the alveoli.
The aerosol particles may be delivered by way of a nebulizer or nasal spray.
In the case of aerosol delivery of the medicament, the particles may have diameters in the approximate range of 0.1 -50 μm, preferably 1 -25 μm, more preferably 1 -5 μm.
The aerosol formulation of the medicament of the present invention may optionally contain a propellant and/or surfactant.
By controlling the size of the droplets which are to be administered to a patient to within the defined range of the present invention, it is possible to avoid/minimise inadvertent antigen delivery to the alveoli and thus avoid alveoli- associated pathological problems such as inflammation and fibrotic scarring of the lungs.
I.n. vaccination engages both T and B cell mediated effector mechanisms in nasal and bronchus associated mucosal tissues, which differ from other mucosae-associated lymphoid tissues.
The protective mechanisms invoked by the intranasal route of administration may include: the activation of T lymphocytes with preferential lung homing; upregulation of co-stimulatory molecules, eg. B7.2; and/or activation of
macrophages or secretory IgA antibodies.
Intranasal delivery of antigens may facilitate a mucosal antibody response which is favoured by a shift in the T cell response toward the Th2 phenotype which helps antibody production. A mucosal response is characterised by enhanced IgA production, and a Th2 response is characterised by enhanced IL- 4 production.
Intranasal delivery of mycobacterial antigens allows targeting of the antigens to submucosal B cells of the respiratory system. These B cells are the major local IgA-producing cells in mammals and intranasal delivery facilitates a rapid increase in IgA production by these cells against the mycobacterial antigens.
In one embodiment administration of the medicament comprising a mycobacterial antigen stimulates IgA antibody production, and the IgA antibody binds to the mycobacterial antigen. In another embodiment, a mucosal and/or Th2 immune response is stimulated.
In another embodiment monoclonal antibodies, in particular, may be used to raise anti-idiotype antibodies. Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired. These anti-idiotype antibodies may also be useful for treatment, vaccination and/or diagnosis of mycobacterial infections.
According to a tenth aspect, the peptides (including fragments, derivatives, and variants thereof) of the present invention and antibodies to them are useful in immunoassays to detect the presence of antibodies to mycobacteria or the presence of the virulence associated antigens in biological samples.
Design of the immunoassays is subject to a great deal of variation, and many formats are known in the art. The immunoassay may utilize at least one epitope derived from a peptide of the present invention. In one embodiment, the immunoassay uses a combination of such epitopes. These epitopes may be derived from the same or from different bacterial peptides, and may be in separate recombinant or natural peptides, or together in the same recombinant peptides.
An immunoassay may use, for example, a monoclonal antibody directed towards a virulence associated peptide epitope(s), a combination of monoclonal antibodies directed towards epitopes of one mycobacterial antigen, monoclonal antibodies directed towards epitopes of different mycobacterial antigens, polyclonal antibodies directed towards the same antigen, or polyclonal antibodies directed towards different antigens.
Protocols may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labelled antibody or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.
Typically, an immunoassay for an antibody(s) to a peptide, will involve selecting and preparing the test sample suspected of containing the antibodies, such as a biological sample, then incubating it with an antigenic (i.e., epitope-containing) peptide(s) under conditions that allow antigen-antibody complexes to form, and then detecting the formation of such complexes. The immunoassay may be of a standard or competitive type.
The peptide is typically bound to a solid support to facilitate separation of the sample from the peptide after incubation. Examples of solid supports that can be used are nitrocellulose (eg. in membrane or microtiter well form), polyvinyl chloride (eg. in sheets or microtiter wells), polystyrene latex (eg. in beads or microtiter plates, polyvinylidine fluoride (known as Immulon), diazotized paper, nylon membranes, activated beads, and Protein A beads. For example, Dynatech Immulon microtiter plates or 60 mm diameter polystyrene beads (Precision Plastic Ball) may be used. The solid support containing the antigenic peptide is typically washed after separating it from the test sample, and prior to detection of bound antibodies.
Complexes formed comprising antibody (or, in the case of competitive assays, the amount of competing antibody) are detected by any of a number of known techniques, depending on the format. For example, unlabeled antibodies in the
complex may be detected using a conjugate of antixenogeneic Ig complexed with a label (eg. an enzyme label).
In immunoassays where the peptides are the analyte, the test sample, typically a biological sample, is incubated with antibodies directed against the peptide under conditions that allow the formation of antigen-antibody complexes. It may be desirable to treat trie biological sample to release putative bacterial components prior to testing. Various formats can be employed. For example, a "sandwich assay" may be employed, where antibody bound to a solid support is incubated with the test sample; washed; incubated with a second, labeled antibody to the analyte, and the support is washed again. Analyte is detected by determining if the second antibody is bound to the support. In a competitive format, a test sample is usually incubated with antibody and a labeled, competing antigen is also incubated, either sequentially or simultaneously.
Also included as an embodiment of the invention is an immunoassay kit comprised of one or more peptides of the invention, or one or more antibodies to said peptides, and a buffer, packaged in suitable containers.
As used herein, a "biological sample" refers to a sample of tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumours, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components).
In a related diagnostic assay, the present invention provides nucleic acid probes for detecting a mycobacterial infection.
Using the polynucleotides of the present invention as a basis, oligomers of approximately 8 nucleotides or more can be prepared, either by excision from recombinant polynucleotides or synthetically, which hybridize with the mycobacterial sequences, and are useful in identification of mycobacteria.
The probes are a length which allows the detection of the induced or up-
regulated sequences by hybridization. While 6-8 nucleotides may be a workable length, sequences of 10-12 nucleotides are preferred, and at least about 20 nucleotides appears optimal. These probes can be prepared using routine methods, including automated oligonucleotide synthetic methods. For use as probes, complete complementarity is desirable, though it may be unnecessary as the length of the fragment is increased.
For use of such probes as diagnostics, the biological sample to be analyzed, such as blood or serum, may be treated, if desired, to extract the nucleic acids contained therein. The resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques; alternatively, the nucleic acid sample may be dot blotted without size separation. The probes are usually labeled. Suitable labels, and methods for labeling probes are known in the art, and include, for example, radioactive labels incorporated by nick translation or kinasing, biotin, fluorescent probes, and chemiluminescent probes. The nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions of suitable stringencies.
The probes may be made completely complementary to the virulence encoding polynucleotide. Therefore, usually high stringency conditions are desirable in order to prevent false positives. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, length of time, and concentration of f ormamide.
It may be desirable to use amplification techniques in hybridization assays. Such techniques are known in the art and include, for example, the polymerase chain reaction (PCR) technique.
The probes may be packaged into diagnostic kits. Diagnostic kits include the probe DNA, which may be labeled; alternatively, the probe DNA may be unlabeled and the ingredients for labeling may be included in the kit in separate containers. The kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, for example, standards, as well as instructions for conducting the test.
In a preferred embodiment, a peptide (or fragment or variant or derivative) of the present invention is used in a diagnostic assay to detect the presence of a
T-lymphocyte, which T-lymphocyte has been previously exposed to an antigenic component of a mycobacterial infection in a patient.
In more detail, a T-lymphocyte which has been previously exposed to a particular antigen will be activated on subsequent challenge by the same antigen. This activation provides a means for identifying a positive diagnosis of mycobacterial infection. In contrast, the same activation is not achieved by a T-lymphocyte which has not been previously exposed to the particular antigen.
The above "activation" of a T-lymphocyte is sometimes referred to as a "recall response" and may be measured, for example, by determining the release of interferon (eg. IFN-γ) from the activated T-lymphocyte. Thus, the presence of a mycobacterial infection in a patient may be determined by the release of a minimum concentration of interferon from a T-lymphocyte after a defined time period following in vitro challenge of the T-lymphocyte with a peptide (or fragment or variant or derivative) of the present invention.
In use, a biological sample containing T-lymphocytes is taken from a patient, and then challenged with a peptide (or fragment, variant, or derivative thereof) of the present invention.
The above T-lymphocyte diagnostic assay may include an antigen presenting cell (APC) expressing at least one major histocompatibility complex (MHC) class II molecule expressed by the patient in question. The APC may be inherently provided in the biological sample, or may be added exogenously. In one embodiment, the T-lymphocyte is a CD4 T-lymphocyte.
Example 1 - generation of putative promoter library
The routine molecular biology methods employed were as described in the standard reference protocols (Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A. and Struhl K. (1992). "Current protocols in molecular biology". John Wiley & Sons, Chichester).
The promoter library was prepared by partial restriction endonuclease digestion (Sau 3A1 ) of M. tuberculosis H37Rv chromosomal DNA. Digested DNA was size fractionated and fragments of 1 -2 kb in size recovered using the Qiaex II Gel Extraction Kit (Qiagen Ltd).
Recovered fragments were ligated into the vector pCREP8GFPTTL which had been linearised with Bgl II and dephosphorylated. The vector PCREP8GFPTTL is based upon the vector pCREP8 provided by Dr P O'Gaora, ICSM, London which is itself based upon the pNG2 replicon (Radford and Hodgson, 1991 , Plasmid 25: 149-153).
Modifications of pCREP8 to create pCREP8GFPTTL include replacement of the region encoding Cre recombinase with the gene encoding GFPmut2 (Cormack, Valdivia and Falkow. Gene. 1996, 173: 33-38). In addition transcriptional terminators from CelA (from C. thermocelum) and Ferredoxin (from C. pasteurianum) genes were inserted upstream and downstream of the GFPmut2 gene respectively. Finally translational stops were introduced in all three reading frames by insertion of a linker between the Bgl II cloning site and the ribosome binding site immediately preceding the ATG start codon of the GFPmut2 gene.
The ligation mixture was transformed into E. coli and resulting transformants banked into 96 well plates to give 23,040 individual clones. Large scale plasmid isolation was performed on pools of 480 clones using Qiagen Maxi plasmid preps (Qiagen Ltd).
Example 2 - putative promoters identified by sequence homology
Promoter regions for targeted fusions were selected by screening the annotated M. tuberculosis genome (http://www.sanger.ac.uk/Projects/M_tuberculosis/ or http://pedant.mips.biochem.mpg.de/) for secreted proteins, surface exposed
proteins and those with homology to known virulence factors or implicated previously in mycobacterial virulence using text searches.
In this way a number of candidates for targeted fusions may be isolated. Having identified a promoter, the organisation of the surrounding gene structure was examined and, where possible, a promoter was identified.
Mycobacterial promoters lack typical -35 and -10 sequences [Das Gupta SK, Bashyam MD and Tyagi AK (1993). J. Bacteriol., 175: 5186-5192].
In this instance the region immediately upstream of the gene or operon of interest was amplified by PCR using tailed primers and cloned directly into the Bgl II site of pCREP8GFPTTL. The GFPmut2 vector was used as described above using macrophage cell line J774.1 , in vitro culture grown in Middlebrook 7H9 supplemented with 10 % OADC and 0.25 % Triton WR1339 (synthetic mycobacterial culture medium), or DMEM.
Table 1 lists a number of such promoters.
as annotated in the genome sequence (www.sanger.ac.uk/Projects/M_tuberc) 'increase or decrease over DMEM control in gated population with greater than 103 fluorescence
Example 3 - Protocol for macrophage infection, harvesting, FACS analysis and sorting, and recovery of M. tuberculosis
Macrophage infection with batch culture samples of M. tuberculosis. Wells were harvested at intervals of 24hr, 48hr and 72hrs post infection.
Materials
Mouse macrophage cell line J774A.1 (obtainable from, for example, the ECACC) in 6 well plates were seeded at 5x105 cells per well.
Tissue culture medium - Dulbecco's Modified Eagle Medium supplemented with 200 mM L-glutamate,10 % foetal bovine serum (gamma-irradiated) and 10 mM HEPES.
Batch culture sample of M. tuberculosis was grown to mid-late exponential log (O.D600 0.6-0,8).
Liquid Middlebrook 7H9 medium (Difco) supplemented with 10 % OADC enrichment (Difco) and 0.25 % Triton WR1339, or organisms were cultured on solid Middlebrook 7H1 O medium (Difco) supplemented with 10 % OADC enrichment (Difco) for growth of M. tuberculosis outside of macrophage.
Procedure
1 . Macrophage were grown until confluent (4x106 cells per well).
2. Bacterial samples were prepared using the following procedure:
2-3 week old colonies were scraped from 7H10 + OADC agar plates and resuspended in 10 mis of 7H9 + OADC + Triton WR1339. A universal tube (22 mm diameter x 90 mm height) containing 10 mis of 7H9 + OADC + Triton WR1339 was inoculated with 500 μl of the bacterial suspension. Bacteria were grown on an orbital shaker (2OOrpm) at 37 °C for 7 days
500 μls of bacterial culture was inoculated into a universal tube (22 mm
diameter x 9Omm height) containing 10 mis of 7H9 + OADC + Triton WR1339 1 ml. Bacteria were grown on an orbital shaker (200 rpm) at 37
>C for 5 days (O.D600 0.6-0.8)
Bacterial samples were diluted in DMEM to a cell density of 1 x107/ml - a multiplicity of approximately 2:1 (bacteria to cells) or less is preferred.
3. Tissue culture medium was removed from each tissue culture well
4. 1 ml of bacterial suspension was added to each well and incubated at 37 °C for 3 hours in an atmosphere of 5 % CO2 in air.
5. The inoculum suspension and was removed and each well washed 4 times with 1x phosphate buffered saline (PBS) pre-warmed to 37 °C.
6. 3 ml fresh pre-warmed DMEM was added to each well.
7. Infected monolayers were incubated at 37 °C in an atmosphere of 5 % CO2 in air, for a period of 1 , 2 or 3 days.
8. Wells for each sample were harvested at appropriate time points as follows:
i) Culture medium was removed by aspiration and monolayers washed with pre-warmed PBS to remove any external bacteria.
ii) The PBS buffer was removed and replaced with 1 ml of 0.25 % Triton X-100 in water.
iii) The monolayers were Incubated for 25 mins before disruption of the monolayer with a pastette to release bacteria from the macrophage.
iv) Samples were transferred to a FACS tube and examined using a FACS scanner/sorter at Cat III collecting highly fluorescent bacteria (typically greater than 103 logs of fluorescence).
9. Bacteria were recovered by passing the sorted suspensions through a 0.2 μM filter.
10. Recovered bacteria on the filter membrane were cultured on Middlebrook 7H1 O + OADC agar plates and grown at 37 °C for 2 weeks.
1 1 . Bacteria were scraped from the filter membrane into 10 mis of 7H9 + ADC medium and grow for 5-7 days (37 °C, 200 rpm).
Notes:
i) The macrophage infection assay and sorting were repeated until the desired level of enrichment of fluorescent bacteria was attained
ii) Bacteria carrying M. tuberculosis promoters that were highly active outside of the macrophage environment were removed by culturing bacteria in Middlebrook 7H9 + ADC medium then FACS sorting to collect the non- fluorescent bacteria. (This selection procedure was carried out between passage 1 and passage 2 of the repeat macrophage infection rounds).
Example 4 - Alternative macrophage assay procedures
1 ). Triccas, J.A. et al. (1999). Microbiology 145, 2923-2930.
Procedure
Macrophage cells were seeded at 2x105 per well in 24-well plates and incubated at 37 °C in 5 % CO2 for 7 days. Macrophage monolayers were infected with bacteria at a multiplicity of infection (m.o.i) of 1 :1 . After 4 hours of infection extracellular bacteria were removed by washing 4x with PBS and incubation continued in 5 % CO2.
After 5-6 days of infection, infected macrophages were washed 3x in PBS, scraped into 1 ml PBS and analysed by FACS. To recover bacteria, sorted macrophages were centrifuged and lysed in water plus 0.1 % Tween 80. Recovered bacteria Were grown in 7H9 medium for 7days and macrophage infection and sorting repeated until the desired level of enrichment of macrophage/fluorescent bacteria was attained.
To select clones with enhanced intracellular expression the macrophage infection was performed as above, however the macrophage were then lysed with Tween 80 to release the bacteria prior to FACS sorting.
2). Kremer, L. et al. (1995). Mol Microbiol. 17, 913-922.
Procedure (macrophage infection only)
J774A.1 macrophages were seeded in eight-chamber culture slides and incubated overnight at 37 °C in 5 % CO2. Just prior to infection, the monolayer was washed with RPM 11640. Recombinant BCG was added at a m.o.i. of 1 -5 bacteria per macrophage.
After an overnight infection at 37 °C in the presence of 5 % CO2, the cells were washed 3x with RPMI to remove possible extracellular bacteria. The macrophage were then examined using a fluorescence microscope.
3). Dhandayuthapani, S. et al. (1995). Mol. Microbiol. 17, 901-912.
Procedure
Macrophages were seeded in 10O mm tissue-culture petri dishes (5x106). After overnight attachment at 37 °C in the presence of 5 % CO2 the macrophage were infected at a m.o.i of 1 bacteria per macrophage. One hour after infection, the monolayers were washed 3x with prewarmed PBS to remove non-ingested bacteria and the DMEM replaced.
The macrophages were harvested 6 days post-infection by washing with PBS and then scraped from the dish. The macrophages were collected by centrifugation and broken by 20-30 passages through a 23-gauge needle: intact cells and nuclei were pelleted and the supernatant containing bacteria collected. The bacteria were then pelleted, washed with PBS and centrifuged again. The final bacterial pellet was suspended in PBS for FACS analysis.
4). Via, L. E. et al. (1996). J. Bacteriol. 178, 3314-3321 .
Procedure
J774 macrophage monolayers were seeded at a density of 7.5x106 cells per 10Omm-diameter tissue culture petri dish. After overnight attachment at 37 °C in the presence of 5 % CO2, macrophages were infected at a m.o.i of 1 O-2O bacteria per macrophage. One hour after infection any extracellular bacteria were removed by washing 3x with prewarmed PBS, then fresh DMEM + 5%FBS added.
The medium was replaced every 3-4 days of culture. The infected macrophages were incubated for 10 mins, 3 days, 7 days and 1 1 days (FACS analysis was performed on the 1 0 min, 7 day and 1 1 day samples) before harvesting. At harvest the monolayers were washed 3x with PBS, scraped from the dish, centrifuged then homogenised in 20 mM HEPES (pH 7.2) - 25OmM sucrose. For FACS the homogenate was then subjected to three rounds of centrifugation to remove unbroken macrophage and macrophage debris, the final bacterial pellet was suspended in PBS. FACS analysis and sorting were carried out as described above in reference 3.
5). Barker L. P. et al. (1 998) . Mol. Microbiol. 29, 1 1 67-1 1 77.
Procedure
Macrophages were seeded into tissue culture dishes at a concentration of 2x1 05 cells/ml total volume. Macrophages were grown to semi-confluency before bacteria were added at a m.o.i. of 1 -5 bacteria per macrophage. After 4 hours growth at 37 °C in the presence of 5 % CO2 the medium was removed and fresh DMEM supplemented with amikacin 100 μg/ml was added to kill extracellular organisms.
After 24 hours at 32 °C the concentration of amikacin was reduced to 20 μg/ ml. At three days after infection (the point at which most phagocytic vesicles contain only one organism) the macrophages are washed in PBS, scraped from the dish and subjected to a very gentle and controlled lysis that disrupts the cell membrane without compromising the nuclear membrane.
Vesicles containing bacteria are then isolated by centrifugation, the supernatant removed and diluted with PBS for FACS analysis. Fluorescent bacterial clones
that were sorted away from non-fluorescent vesicles and vesicles containing non-fluorescent organisms were screened further using a confocal microscope.
Those clones that appeared to fluoresce intracellularly but not on 7H10 agar or in tissue culture media were passaged through macrophage again. After 3-4 days of growth the bacteria were harvested from the macrophage by lysing the macrophage with 0.1 % Triton-X in PBS for 5 min, after which the detergent was diluted with 9 volumes of PBS ready for FACS analysis.
Example 5 - elucidation of mycobacterial nucleic acid coding sequences under the transcriptional control of identified promoters
DFI allows identification of a promoter which is induced or up-regulated during infection of macrophage, a key step in the pathogenesis of tuberculosis.
Once the DNA sequence and orientation of a promoter region has been determined the exact location can be mapped by doing a DNA homology search (e.g BLASTN or BLASTX, see http://www.sanger.ac.uk/) on the published M. tuberculosis H37Rv genome to reveal the gene or operon under control of the identified promoter region.
Knockout mutants of the gene(s) under control of the identified promoter may then be prepared to see if they are essential for virulence. The fact that the genes are expressed or up-regulated during the infection means they may be essential for virulence of the organism (as demonstrated by testing a knockout mutant in a suitable model).
These data help identify a) a potential vaccine candidate which the host immune response can be targeted to; b) a gene which when inactivated sufficiently attenuates the organism that it could be considered suitable as a live vaccine; c) a target for the development of a new antibiotic.
A suitable plasmid DNA vector (Tascon RE etal, 1996 Nat. Med. 2:8 888-892; Huygen K et al, 1996 Nat. Med. 2:8 893-898) containing the DNA sequence corresponding to one or more of the genes in an identified operon may be tested as a DNA vaccine in comparison with pre-existing TB vaccines. Similarly, gene products thereof may be tested as a vaccine in a guinea pig protection model.
Example 6 - Delete one or more of the genes from M. tuberculosis in order to attenuate its virulence while retaining immunogenicity
One or more genes that are identified may be disrupted using allelic exchange. In brief, the gene of interest is cloned with 1 -2 kb of flanking DNA either side and is inactivated by deletion of part of the coding region and insertion of an antibiotic resistance marker, such as hygromycin.
The manipulated fragment is then transferred to a suitable suicide vector eg. pPR23 and is transformed into the wild-type parent strain of M. tuberculosis. Mutants are recovered by selecting for antibiotic resistant strains. Genotypic analysis (Southern Blotting with a fragment specific to the gene of interest) is performed on the selected strains to confirm that the gene has been disrupted.
The mutant strain is then studied to determine the effect of the gene disruption on the phenotype. In order to use it as a vaccine candidate it would be necessary to demonstrate attenuated virulence. This can be done using either a guinea pig or mouse model of infection. Animals are infected with the mutant strain and the progression of disease is monitored by determining the bacterial load in different organs, in particular the lung and spleen, at specific time points post infection, typically up to 16 weeks.
Comparison is made to animals infected with the wild-type strain which should have a significantly higher bacterial load in the different organs. Long-term survival studies and histopathology can also be used to assess virulence and pathogenicity.
Once attenuated virulence has been established, protection and immunogenicity studies can be performed to assess the potential of the strain as a vaccine. Suitable references for allelic exchange and preparation of TB mutants are McKinney et al., 2000 and Pelicic et al., 1997, [1 , 2].
Example 7 - Select one or more of our genes, which encode proteins that are immunogenic, and put them into BCG or an attenuated strain of M. tuberculosis to enhance its overall immunogenicity
The gene of interest is amplified from the M. tuberculosis genome by PCR. The amplified product is purified and cloned into a plasmid (pMV306) that integrates site specifically into the mycobacterial genome at the attachment site (attB) for mycobacteriophage L5 [3].
BCG is transformed with the plasmid by electroporation, which involves damaging the cell envelope with high voltage electrical pulses, resulting in uptake of the DNA. The plasmid integrates into the BCG chromosome at the attB site generating stable recombinants. Recombinants are selected and are checked by PCR or Southern blotting to ensure that the gene has been integrated. The recombinant strain is then used for protection studies.
Example 8 - Use recombinant carriers such as attenuated salmonella and the Vaccinia virus to express and present TB genes.
One of the best examples of this type of approach is the use of Modified Vaccinia virus Ankara (MVA) [4]. The gene of interest is cloned into a vaccinia virus shuttle vector, e.g. pSC1 1 . Baby Hamster Kidney (BHK) cells are then infected with wild-type MVA and are transfected with the recombinant shuttle vector. Recombinant virus is then selected using a suitable selection marker and viral plaques, selected and purified.
Recombinant virus is normally delivered as part of a prime-boost regime where animals are vaccinated initially with a DNA vaccine encoding the TB genes of interest under the control of a constitutive promoter. The immune response is boosted by administering recombinant MVA carrying the genes of interest to the animals at least 2 weeks later.
Example 9 - Sub-unit vaccines containing a single peptide/protein or a combination of proteins
To prepare sub-unit vaccines with one or more peptides or proteins it is first of all necessary to obtain a supply of protein or peptide to prepare the vaccine. Up to now, this has mainly been achieved in mycobacterial studies by purifying proteins of interest from TB culture. However, it is becoming more common to clone the gene of interest and produce a recombinant protein.
The coding sequence for the gene of interest is amplified by PCR with restriction sites inserted at the N terminus and C terminus to permit cloning in-frame into a protein expression vector such as pET-15b. The gene is inserted behind an inducible promoter such as lacZ. The vector is then transformed into E. coli which is grown in culture. The recombinant protein is over-expressed and is purified.
One of the common purification methods is to produce a recombinant protein with an N-terminal His-tag. The protein can then be purified on the basis of the affinity of the His-tag for metal ions on a Ni-NTA column after which the His-tag is cleaved. The purified protein is then administered to animals in a suitable adjuvant [5].
Example 10 - Plasmid DNA vaccines carrying one or more of the identified genes
DNA encoding a specific gene is amplified by PCR, purified and inserted into specialised vectors developed for vaccine development, such as pVAX1 . These vectors contain promoter sequences, which direct strong expression of the introduced DNA (encoding candidate antigens) in eukaryotic cells (eg. CMV or SV4O promoters), and polyadenlyation signals (eg. SV4O or bovine growth hormone) to stabilise the mRNA transcript.
The vector is transformed into E. coli and transformants are selected using a marker, such as kanamycin resistance, encoded by the plasmid. The plasmid is then recovered from transformed colonies and is sequenced to check that the gene of interest is present and encoded properly without PCR generated mutations.
Large quantities of the plasmid is then produced in E. coli and the plasmid is recovered and purified using commercially available kits (eg. Qiagen Endofree-plasmid preparation). The vaccine is then administered to animals for example by intramuscular injection in the presence or absence of an adjuvant.
Example 11 - Preparation of DNA expression vectors
DNA vaccines consist of a nucleic acid sequence of the present invention cloned into a bacterial plasmid. The plasmid vector pVAX1 is commonly used in the preparation of DNA vaccines. The vector is designed to facilitate high copy number replication in E. coli and high level transient expression of the peptide of interest in most mammalian cells (for details see manufacturers protocol for pVAXK catalog no. V26O-2O www.invitrogen.com).
The vector contains the following elements
* Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a variety of mammalian cells
* T7 promoter/priming site to allow in vitro transcription in the sense orientation and sequencing through the insert
* Bovine growth hormone (BGH) polyadenylation signal for efficient transcription termination and polyadenylation of mRNA
* anamycin resistance gene for selection in £. coli
* A multiple cloning site
* pUC origin for high-copy number replication and growth in E. coli
* BGH reverse priming site to permit sequencing through the insert
Vectors may be prepared by means of standard recombinant techniques which are known in the art, for example Sambrook et al., (1989). Key stages in preparing the vaccine are as follows:
* The gene of interest is ligated into pVAX1 via one of the multiple cloning sites
* The ligation mixture is then transformed into a competent E. coli strain (eg. TOP1 O) and LB plates containing 50 μg/ml kanamycin are used to select transformants.
* Clones are selected and may be sequenced to confirm the presence and orientation of the gene of interest.
* Once the presence of the gene has been verified, the vector can be used to transfect a mammalian cell line to check for protein expression. Methods for transfection are known in the art and include, for example, electroporation, calcium phosphate, and lipofection. * Once peptide expression has been confirmed, large quantities of the vector can be produced and purified from the appropriate cell host, e.g. E. coli.
pVAX1 does not integrate into the host chromosome. All non-essential sequences have been removed to minimise the possibility of integration. When constructing a specific vector, a leader sequence may be included to direct secretion of the encoded protein when expressed inside the eukaryotic cell.
Other examples of vectors that have been used are V1 Jns.tPA and pCMV4 (Lefevre et al., 2OOO; and Vordermeier et al, 2000).
Expression vectors may be used that integrate into the genome of the host, however, it is more common and more preferable to use a vector that does not integrate. The example provided, pVAX1 , does not integrate. Integration would lead to the generation of a genetically modified host which raises other issues.
Example 12 - RNA vaccine
As discussed on page 15 of US patent US 5,783,386, one approach is to introduce RNA directly into the host.
Thus, the vector construct (Example 1 1 ) may be used to generate RNA in vitro and the purified RNA then injected into the host. The RNA would then serve as a template for translation in the host cell. In this embodiment. In this embodiment, integration would not occur.
Another option is to use an infectious agent such as the retroviral genome carrying RNA corresponding to the gene of interest. In this embodiment, integration into the host genome will occur.
Another option is the use of RNA replicon vaccines which can be derived from virus vectors such as Sindbis virus or Semliki Forest virus. These vaccines are self-replicating and self-limiting and may be administered as either RNA or DNA which is then transcribed into RNA replicons in vivo. The vector eventually causes lysis of the transfected cells thereby reducing concerns about integration into the host genome. Protocols for RNA vaccine construction are detailed in Cheng, et al. (2001 ).
Example 13 - Diagnostic assays based on assessing T cell responses
For a diagnostic assay based on assessing T cell responses it would be sufficient to obtain a sample of blood from the patient. Mononuclear cells (monocytes, T and B lymphocytes) can be separated from the blood using density gradients such as Ficoll gradients.
Both monocytes and B-lymphocytes are both able to present antigen, although less efficiently than professional antigen presenting cells (APCs) such as dendritic cells. The latter are more localised in lymphoid tissue.
The simplest approach would be to add antigen to the separated mononuclear cells and incubate for a week and then assess the amount of proliferation. If the individual had been exposed to the antigen previously through infection, then T-cell closes specific to the antigen should be more prevalent in the sample and should respond.
It is also possible to separate the different cellular populations should it be desired to control the ratio of T cells to APCs.
Another variation of this type of assay is to measure cytokine production by the responding lymphocytes as a measure of response. The ELISPOT assay described below in Example 14 is a suitable example of this variation.
Example 14 - detection of latent mycobacteria
A major problem for the control of tuberculosis is the presence of a large reservoir of asymptomatic individuals infected with tubercle bacilli. Dormant bacilli are more resistant to front-line drugs.
The presence of latent mycobacteria-associated antigen may be detected indirectly either by detecting antigen specific antibody or T-cells in blood samples.
The following method is based on the method described in Lalvani et al. (2OO1 ) in which a secreted antigen, ESAT-6, was identified as being expressed by members of the M. tuberculosis complex but is absent from M. bovis BCG vaccine strains and most environmental mycobacteria. 60 - 80% of patients also
have a strong cellular immune response to ESAT-6. An ex-vivo ELISPOT assay was used to detect ESAT-6 specific T cells.
As applied to the present invention: A 96 well plate is coated with cytokine (eg. interferon-(, IL-2) -specific antibody. Peripheral blood monocytes are then isolated from patient whole blood and are applied to the wells. .
Antigen (ie. one of the peptides, fragments, derivatives or variants of the present invention) is added to stimulate specific T cells that may be present and the plates are incubated for 24h. The antigen stimulates cytokine production which then binds to the specific antibody.
The plates are washed leaving a footprint where antigen-specific T cells were present.
A second antibody coupled with a suitable detection system, eg. enzyme, is then added and the number of spots are enumerated after the appropriate substrate has been added.
The number of spots, each corresponding to a single antigen-specific T cell, is related to the total number of cells originally added.
The above Example also describes use of an antigen that may be used to distinguish TB infected individuals from BCG vaccinated individuals. This could be used in a more discriminative diagnostic assay.
The following Table 2 lists the preferred promoters, peptides, and corresponding DNA coding sequences of the present invention.
Table 2
References:
1 . McKinney, J.D., et al., Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase [see comments]. Nature, 2OOO. 406(6797): p. 735-8.
2. Pelicic, V., et al., Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A, 1997. 94(20): p. 10955-60.
3. Lee, M.H., et al., Site-specific integration of mycobacteriophage L5: integration- proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guerin. Proc Natl Acad Sci U S A, 1 991 . 88(8): p. 31 1 1 -5.
4. McShane, H., et al., Enhanced immunogenicity of CD4( + ) t-cell responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boost vaccination regimen for murine tuberculosis. Infect Immun, 2001 . 69(2): p. 681 -6.
5. Movahedzadeh, F., M.J. Colston, and E.O. Davis, Characterization of Mycobacterium tuberculosis LexA: recognition of a Cheo (Bacillus-type SOS) box. Microbiology, 1997. 143(Pt 3): p. 929-36.
Additional References:
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N. Y.
Lefever, P., O. Denis, L. De Wit, A. Tanghe, P. Vandenbussche, J. Content, and K. Huygen. 2OOO. Cloning of the gene encoding a 22-kilodalton cell surface antigen of Mycobacterium bovis BCG and analysis of its potential for DNA vaccination against tuberculosis. Infection and Immunity. 68:1040-1047.
Vordermeier, H. M., P. J. Cockle, A. O. Whelan, S. Rhodes, M. A. Chambers, D. Clifford, K. Huygen, R. Tascon, D. Lowrie, M. J. Colston, and R. G.
Hewinson. 2OOO. Effective DNA vaccination of cattle with the mycobacterial antigens MPB83 and MPB7O does not compromise the specificity of the comparative intradermal tuberculin skin test. Vaccine. 19:1246-1255.
Cheng, W., C. Hung, C. Chai, K. Hsu, L. He, C. Rice, M. Ling, and T. Wu. 2001 . Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of Mycobacterium tuberculosis heat shock protein 70 gene to an antigen. J. Immunol. 166:6218-6226.
Lalvani, A. et al., 2001 . Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. The Lancet 357:2017-2021 .
Claims (29)
1 . A method of identifying a mycobacterial nucleic acid promoter sequence which is induced or up-regulated during mycobacterial virulence, said method comprising:-
infecting a macrophage target cell with a Mycobacterium tuberculosis host cell, which host cell contains a nucleic acid construct comprising a putative mycobacterial promoter sequence operably linked to a coding sequence of a reporter gene located down-stream from the promoter;
culturing the macrophage under conditions which support mycobacterial virulence; and
identifying a promoter sequence which is induced or up-regulated during virulence by detecting expression of the reporter sequence.
2. A method according to Claim 1 , wherein the induced or up-regulated promoter sequence is detected by increased expression of the reporter gene under conditions which support mycobacterial virulence when compared with the corresponding level of expression when cultured under conditions which do not promote mycobacterial virulence.
3. A method according to Claim 1 or 2, wherein the putative promoter sequence is derived from M. tuberculosis or M. bovis.
4. A method according to any preceding claim, wherein the reporter sequence encodes a green fluorescence protein.
5. A method according to Claim 4, wherein a mycobacterial host cell having a promoter induced or up-regulated during mycobacterial virulence is separated from other host cells by fluorescence activated cell sorting.
6. A method of identifying a mycobacterial gene the expression of which is induced or up-regulated during mycobacterial virulence, said method comprising :- identifying a mycobacterial promoter sequence which is induced or upregulated during infection of a macrophage by a M. tuberculosis host cell, wherein the host cell contains a nucleic acid construct comprising said promoter sequence operably linked to a coding sequence of a reporter gene located down-stream from the promoter;
aligning by sequence homology the nucleic acid sequence of the promoter with published nucleic acid sequence data for the same mycobacterial species; and
identifying the associated nucleic acid coding sequence under the control of said promoter.
7. An isolated mycobacterial promoter obtainable by a method according to any of Claims 1 -5, wherein the promoter preferably has the nucleic acid sequence of SEQ ID NO: 1 ; 18; 37; 54; 57; 66; 71 ; 74; 77; 84; 91 ; 96; 99; or 102.
8. An isolated nucleic acid coding sequence obtainable by a method according to Claim 6, wherein the coding sequence preferably has the nucleic acid sequence of SEQ ID NO: 3; 5; 7; 9; 1 1 ; 13; 15; 17; 20; 22; 24; 26; 28 30; 32; 34; 36; 39; 41 ; 43; 45; 47; 49; 51 ; 53; 56; 59; 61 ; 63; 65; 68; 70 73; 76; 79; 81 ; 83; 86; 88; 90; 93; 95; 98; 101 ; 104; 106; 108; 1 10; 1 12 1 14; 1 16; or 1 18.
9. An isolated mycobacterial peptide or a fragment or derivative or variant thereof, wherein the peptide is encoded by a mycobacterial gene the expression of which gene is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said gene.
10. A pharmaceutical composition comprising a peptide, or a fragment or variant or derivative thereof, wherein the peptide is selected from the group consisting of SEQ ID NO: 2; 4; 6; 8; 10; 12; 14; 16; 19; 21 ; 23; 25; 27; 29 31 ; 33; 35; 38; 40; 42; 44; 46; 48; 50; 52; 55; 58; 60; 62; 64; 67; 69; 72 75; 78; 80; 82; 85; 87; 89; 92; 94; 97; 10O; 103; 105; 107; 109; 1 1 1 ; 1 13 1 15; and 1 17.
1 1 . An inhibitor of a mycobacterial peptide, wherein the peptide is encoded by a mycobacterial gene the expression of which gene is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said gene, and wherein the inhibitor is capable of substantially preventing or inhibiting the mycobacterial peptide from exerting its native biological function or effect.
12. An inhibitor according to Claim 1 1 , selected from the group consisting of:- an antibiotic capable of targeting the induced or up-regulated mycobacterial gene, or the gene product thereof; and an antisense or triplex-forming nucleic acid sequence which is complementary to at least part of the inducible or up- regulatable gene.
13. An antibody which binds to a peptide encoded by a gene, or to a fragment or variant or derivative of said peptide, the expression of which gene is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said gene.
14. An antibody according to Claim 13, wherein the peptide is selected from the group consisting of SEQ ID NO: 2; 4; 6; 8; 10; 12; 14; 16; 19; 21 ; 23; 25;
27; 29; 31 ; 33; 35; 38; 40; 42; 44; 46; 48; 50; 52; 55; 58; 60; 62; 64; 67; 69; 72; 75; 78; 80; 82; 85; 87; 89; 92; 94; 97; 100; 103; 105; 107; 109; 1 1 1 ; 1 13; 1 15; and 1 17.
15. An attenuated mycobacterium in which a gene has been modified, the expression of which gene is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said gene, thereby rendering the mycobacterium substantially non-pathogenic.
16. An attenuated mycobacterium according to Claim 15, wherein the gene to be modified has a wild-type coding sequence corresponding to one of the group consisting of SEQ ID NO: 3; 5; 7; 9; 1 1 ; 13; 15; 17; 20; 22; 24; 26; 28; 30; 32; 34; 36; 39; 41 ; 43; 45; 47; 49; 51 ; 53; 56; 59; 61 ; 63; 65; 68; 70; 73; 76; 79; 81 ; 83; 86; 88; 90; 93; 95; 98; 101 ; 104; 106; 108; 1 10; 1 12; 1 14; 1 16; and 1 18.
17. An attenuated microbial carrier, comprising a peptide encoded by a gene, or a fragment or variant or derivative of said peptide, the expression of which gene is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said gene.
18. An attenuated microbial carrier according to Claim 17, wherein the peptide is selected from the group consisting of SEQ ID NO: 2; 4; 6; 8; 10; 12 14; 16; 19; 21 ; 23; 25; 27; 29; 31 ; 33; 35; 38; 40; 42; 44; 46; 48; 50; 52 55; 58; 60; 62; 64; 67; 69; 72; 75; 78; 80; 82; 85; 87; 89; 92; 94; 97; 100 103; 105; 107; 109; 1 1 1 ; 1 13; 1 15; and 1 17.
19. An attenuated microbial carrier according to Claim 17 or 18, wherein the attenuated microbial carrier is attenuated salmonella, attenuated vaccinia virus, attenuated fowlpox virus, or attenuated M. bovis (eg. BCG strain).
20. A DNA plasmid comprising a promoter, a polyadenylation signal, and a DNA sequence which corresponds to the coding sequence of a mycobacterial gene, or a fragment or variant or derivative of said DNA sequence, the expression of which gene is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said gene, wherein the promoter and polyadenylation signal are operably linked to the DNA sequence.
21 . A DNA plasmid according to Claim 20, wherein the coding sequence of said gene is selected from the group consisting of SEQ ID NO: 3; 5; 7; 9; 1 1 13; 15; 17; 20; 22; 24; 26; 28; 30; 32; 34; 36; 39; 41 ; 43; 45; 47; 49; 51 53; 56; 59; 61 ; 63; 65; 68; 70; 73; 76; 79; 81 ; 83; 86; 88; 90; 93; 95; 98 101 ; 104; 106; 108; 1 10; 1 12; 1 14; 1 16; and 1 18.
22. A DNA plasmid according to Claim 20 or 21 , wherein the promoter is selected from the group consisting of:- CMV; and SV4O promoters; and the polyadenylation signal is selected from the group consisting of:- SV4O; and bovine growth hormone polyadenylation signals.
23. An isolated RNA sequence which is encoded by the coding sequence of a mycobacterial gene, or a fragment or variant or derivative of said coding sequence, the expression of which gene is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said gene.
24. An RNA vector comprising the RNA sequence of Claim 23 and an integration site for a chromosome of a host cell.
25. Use of a peptide or fragment or variant or derivative according to Claim 9 or Claim 10, an inhibitor according to Claim 1 1 or Claim 1 2, an antibody according to Claim 13 or Claim 14, an attenuated mycobacterium according to Claim 15 or Claim 16, an attenuated microbial carrier according to any of Claims 17-19, a DNA sequence corresponding to the coding sequence of a gene which is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said gene, or a fragment or variant or derivative of said DNA sequence, a DNA plasmid according to any of Claims 20-22, an RNA sequence according to Claim 23, and/or an RNA vector according to Claim 24, in the manufacture of a medicament for treating or preventing a mycobacterial infection.
26. A method of treating or preventing a mycobacterial infection, by administering to a patient a peptide or fragment or variant or derivative according to Claim 9 or Claim 10, an inhibitor according to Claim 1 1 or Claim 12, an antibody according to Claim 13 or Claim 14, an attenuated mycobacterium according to Claim 15 or Claim 16, an attenuated microbial carrier according to any of Claims 17-19, a DNA sequence corresponding to the coding sequence of a gene which is induced or up-regulated during infection of a macrophage by a,M. tuberculosis host cell containing said gene, or a fragment or variant or derivative of said DNA sequence, a DNA plasmid according to any of Claims 20-22, an RNA sequence according to Claim 23, and/or an RNA vector according to Claim 24.
27. Use of a peptide or fragment or variant or derivative according to Claim 9 or Claim 10, or an antibody according to Claim 13 or Claim 14, or a polynucleotide probe comprising at least 8 nucleotides wherein said probe binds to at least part of a gene which is induced or up-regulated during infection of a macrophage by a M. tuberculosis host cell containing said gene, in the manufacture of a diagnostic reagent for identifying a mycobacterial infection.
28. A recombinant method of preparing a mycobacterial peptide, or a fragment or derivative or a variant of said peptide, wherein the peptide is encoded by a mycobacterial gene the expression of which is induced or up- regulated during infection of a macrophage by a M. tuberculosis host cell containing said gene, comprising expressing a nucleic acid sequence corresponding to the coding sequence of said gene, or a fragment or variant or derivative of said nucleic acid sequence, in a host cell.
29. An isolated peptide, an inhibitor, an antibody, an attenuated mycobacterium, an attenuated microbial carrier, an isolated RNA molecule, an RNA vector, or a DNA plasmid substantially as hereinbefore described with reference to the Examples.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB0028966 | 2000-11-28 | ||
GBGB0028966.0A GB0028966D0 (en) | 2000-11-28 | 2000-11-28 | Protection against mycobacterial infections |
PCT/GB2001/005250 WO2002044406A2 (en) | 2000-11-28 | 2001-11-28 | Projection against mycobacterial infections |
Publications (1)
Publication Number | Publication Date |
---|---|
AU2002222098A1 true AU2002222098A1 (en) | 2002-06-11 |
Family
ID=9904013
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2002222098A Abandoned AU2002222098A1 (en) | 2000-11-28 | 2001-11-28 | Projection against mycobacterial infections |
Country Status (7)
Country | Link |
---|---|
US (1) | US20040110269A1 (en) |
EP (1) | EP1348036A2 (en) |
JP (1) | JP2004514451A (en) |
AU (1) | AU2002222098A1 (en) |
CA (1) | CA2433665A1 (en) |
GB (1) | GB0028966D0 (en) |
WO (1) | WO2002044406A2 (en) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NZ519395A (en) * | 2002-06-07 | 2004-04-30 | Agres Ltd | Techniques for identifying genes that are important for virulence in Mycobacterial vaccines |
ITRM20030411A1 (en) * | 2003-08-29 | 2005-02-28 | Consiglio Nazionale Ricerche | USE OF SPECIFIC GENE SEQUENCES OF MYCOBACTERIUM TUBERCOLOSIS IN ADDITIONAL PROTEINS FOR DIAGNOSIS AND PREVENTION OF TUBERCULAR INFECTION. |
WO2005056826A1 (en) * | 2003-12-12 | 2005-06-23 | The University Of British Columbia | Transcription-patterned biological activity screening system |
CA2738033C (en) | 2008-09-26 | 2019-11-26 | Ambrx, Inc. | Non-natural amino acid replication-dependent microorganisms and vaccines |
CN102713629B (en) | 2009-11-20 | 2016-02-24 | 俄勒冈健康科学大学 | For detecting the method for m tuberculosis infection |
ITRM20100411A1 (en) * | 2010-07-23 | 2012-01-24 | Massimo Amicosante | USE OF AMINO ACID SEQUENCES FROM MYCOBACTERIUM TUBERCULOSIS OR THEIR CORRESPONDING NUCLEIC ACIDS FOR DIAGNOSIS AND PREVENTION OF TUBERCULAR INFECTION, RELATED TO DIAGNOSTIC AND VACCINE KIT. |
KR101184566B1 (en) * | 2012-05-11 | 2012-09-20 | 케이맥(주) | Method for integrated analysis of real-time pcr and dna chip |
EP3293254B1 (en) * | 2014-03-07 | 2020-11-18 | Institute for Systems Biology | Point of care assays to detect the status of tuberculosis infection |
CN107722111B (en) * | 2017-09-28 | 2020-07-07 | 中国科学院微生物研究所 | Macrophage phagocytosis ability promoter |
US20220145366A1 (en) * | 2020-06-17 | 2022-05-12 | The Translational Genomics Research Institute | Early detection of drug-resistant mycobacterium tuberculosis |
-
2000
- 2000-11-28 GB GBGB0028966.0A patent/GB0028966D0/en not_active Ceased
-
2001
- 2001-11-28 CA CA002433665A patent/CA2433665A1/en not_active Abandoned
- 2001-11-28 EP EP01998653A patent/EP1348036A2/en not_active Withdrawn
- 2001-11-28 JP JP2002546754A patent/JP2004514451A/en active Pending
- 2001-11-28 WO PCT/GB2001/005250 patent/WO2002044406A2/en not_active Application Discontinuation
- 2001-11-28 US US10/432,934 patent/US20040110269A1/en not_active Abandoned
- 2001-11-28 AU AU2002222098A patent/AU2002222098A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
WO2002044406A3 (en) | 2003-06-26 |
JP2004514451A (en) | 2004-05-20 |
WO2002044406A2 (en) | 2002-06-06 |
EP1348036A2 (en) | 2003-10-01 |
US20040110269A1 (en) | 2004-06-10 |
GB0028966D0 (en) | 2001-01-10 |
CA2433665A1 (en) | 2002-06-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8003776B2 (en) | Mycobacterial antigens expressed during latency | |
US8017753B2 (en) | Mycobacterial antigens expressed under low oxygen tension | |
EP2571518B1 (en) | Mycobacterial antigen composition | |
AU2002314358A1 (en) | Mycobacterial antigens expressed during latency | |
US20110091881A1 (en) | Mycobacterial genes down-regulated during latency | |
RU2702194C2 (en) | Mycobacterial antigen composition | |
AU2002317940A1 (en) | Mycobacterial antigens expressed under low oxygen tension | |
US6776993B2 (en) | Tuberculosis vaccine | |
AU2002222098A1 (en) | Projection against mycobacterial infections | |
AU2002336179A1 (en) | Mycobacterial genes down-regulated during latency | |
WO2003033530A2 (en) | Mycobacterial antigens expressed under high oxygen tension | |
WO2013039069A1 (en) | Novel recombinant bcg vaccine |