AU2007234526B2 - Latent human tuberculosis model, diagnostic antigens, and methods of use - Google Patents
Latent human tuberculosis model, diagnostic antigens, and methods of use Download PDFInfo
- Publication number
- AU2007234526B2 AU2007234526B2 AU2007234526A AU2007234526A AU2007234526B2 AU 2007234526 B2 AU2007234526 B2 AU 2007234526B2 AU 2007234526 A AU2007234526 A AU 2007234526A AU 2007234526 A AU2007234526 A AU 2007234526A AU 2007234526 B2 AU2007234526 B2 AU 2007234526B2
- Authority
- AU
- Australia
- Prior art keywords
- tuberculosis
- granuloma
- vitro
- mycobacteria
- protein
- 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.)
- Expired
Links
- 238000000034 method Methods 0.000 title claims description 124
- 201000008827 tuberculosis Diseases 0.000 title claims description 89
- 239000000427 antigen Substances 0.000 title description 68
- 108091007433 antigens Proteins 0.000 title description 68
- 102000036639 antigens Human genes 0.000 title description 68
- 108090000623 proteins and genes Proteins 0.000 claims description 132
- 206010018691 Granuloma Diseases 0.000 claims description 126
- 238000000338 in vitro Methods 0.000 claims description 95
- 210000002540 macrophage Anatomy 0.000 claims description 40
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 31
- 102000004127 Cytokines Human genes 0.000 claims description 24
- 108090000695 Cytokines Proteins 0.000 claims description 24
- 239000003814 drug Substances 0.000 claims description 24
- 229940079593 drug Drugs 0.000 claims description 21
- 210000002950 fibroblast Anatomy 0.000 claims description 19
- 238000004519 manufacturing process Methods 0.000 claims description 18
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 14
- 238000012216 screening Methods 0.000 claims description 13
- 229940000406 drug candidate Drugs 0.000 claims description 9
- 241000186359 Mycobacterium Species 0.000 claims description 8
- 210000001616 monocyte Anatomy 0.000 claims description 8
- 230000035772 mutation Effects 0.000 claims description 8
- 230000017074 necrotic cell death Effects 0.000 claims description 7
- 230000007420 reactivation Effects 0.000 claims description 7
- 108010002350 Interleukin-2 Proteins 0.000 claims description 6
- 101100490139 Mus musculus Acer3 gene Proteins 0.000 claims description 6
- 230000002829 reductive effect Effects 0.000 claims description 6
- -1 30 IFN-y Proteins 0.000 claims description 5
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 5
- 101150114893 oxyR gene Proteins 0.000 claims description 5
- 101150067544 sigF gene Proteins 0.000 claims description 5
- 229960002109 tuberculosis vaccine Drugs 0.000 claims description 5
- 102100040247 Tumor necrosis factor Human genes 0.000 claims description 4
- 230000002365 anti-tubercular Effects 0.000 claims description 4
- 230000001225 therapeutic effect Effects 0.000 claims description 4
- 101100297439 Dictyostelium discoideum phg1b gene Proteins 0.000 claims description 3
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 101150003531 sigC gene Proteins 0.000 claims description 3
- 101150077142 sigH gene Proteins 0.000 claims description 3
- 230000035899 viability Effects 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 description 110
- 235000018102 proteins Nutrition 0.000 description 99
- 101710095306 Alpha-crystallin Proteins 0.000 description 61
- 210000004027 cell Anatomy 0.000 description 54
- 150000007523 nucleic acids Chemical class 0.000 description 50
- 108090000765 processed proteins & peptides Proteins 0.000 description 42
- 150000001413 amino acids Chemical group 0.000 description 41
- 235000001014 amino acid Nutrition 0.000 description 36
- 229940024606 amino acid Drugs 0.000 description 34
- 108020004707 nucleic acids Proteins 0.000 description 34
- 102000039446 nucleic acids Human genes 0.000 description 34
- 238000001514 detection method Methods 0.000 description 32
- 102000004196 processed proteins & peptides Human genes 0.000 description 28
- 239000012634 fragment Substances 0.000 description 26
- 206010065048 Latent tuberculosis Diseases 0.000 description 23
- 239000000203 mixture Substances 0.000 description 23
- 230000014509 gene expression Effects 0.000 description 22
- 208000015181 infectious disease Diseases 0.000 description 22
- 229920001184 polypeptide Polymers 0.000 description 22
- 239000000523 sample Substances 0.000 description 21
- 229960005486 vaccine Drugs 0.000 description 21
- 230000009870 specific binding Effects 0.000 description 19
- 238000009739 binding Methods 0.000 description 18
- 238000002360 preparation method Methods 0.000 description 18
- 239000013615 primer Substances 0.000 description 18
- 241000304886 Bacilli Species 0.000 description 17
- 150000001875 compounds Chemical class 0.000 description 17
- 230000002163 immunogen Effects 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 16
- 230000027455 binding Effects 0.000 description 16
- 230000015572 biosynthetic process Effects 0.000 description 16
- 210000004072 lung Anatomy 0.000 description 16
- 108020004999 messenger RNA Proteins 0.000 description 16
- 230000003321 amplification Effects 0.000 description 15
- 230000001900 immune effect Effects 0.000 description 15
- 238000003199 nucleic acid amplification method Methods 0.000 description 15
- 210000001519 tissue Anatomy 0.000 description 15
- 238000001262 western blot Methods 0.000 description 15
- 238000003556 assay Methods 0.000 description 14
- 241001465754 Metazoa Species 0.000 description 13
- 239000012472 biological sample Substances 0.000 description 13
- 230000028993 immune response Effects 0.000 description 13
- 230000003308 immunostimulating effect Effects 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 12
- 238000000746 purification Methods 0.000 description 12
- 238000002965 ELISA Methods 0.000 description 11
- 239000002671 adjuvant Substances 0.000 description 11
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 11
- 201000010099 disease Diseases 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 description 10
- 229920002684 Sepharose Polymers 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 10
- 230000004927 fusion Effects 0.000 description 10
- 230000012010 growth Effects 0.000 description 10
- 238000011534 incubation Methods 0.000 description 10
- 239000012528 membrane Substances 0.000 description 10
- 238000003757 reverse transcription PCR Methods 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 238000007792 addition Methods 0.000 description 9
- 239000011324 bead Substances 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 238000003018 immunoassay Methods 0.000 description 9
- 238000003752 polymerase chain reaction Methods 0.000 description 9
- 241000700199 Cavia porcellus Species 0.000 description 8
- 241000283973 Oryctolagus cuniculus Species 0.000 description 8
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- 239000011230 binding agent Substances 0.000 description 8
- 210000004899 c-terminal region Anatomy 0.000 description 8
- 238000009472 formulation Methods 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 125000005647 linker group Chemical group 0.000 description 8
- 229920000642 polymer Polymers 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- 206010062207 Mycobacterial infection Diseases 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 230000016396 cytokine production Effects 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 230000003053 immunization Effects 0.000 description 7
- 239000006166 lysate Substances 0.000 description 7
- 208000027531 mycobacterial infectious disease Diseases 0.000 description 7
- 239000008188 pellet Substances 0.000 description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 7
- 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 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 6
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 6
- 230000000890 antigenic effect Effects 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 108020001507 fusion proteins Proteins 0.000 description 6
- 102000037865 fusion proteins Human genes 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 238000002649 immunization Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 230000004936 stimulating effect Effects 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 5
- 102000000588 Interleukin-2 Human genes 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- 206010036790 Productive cough Diseases 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- 230000016784 immunoglobulin production Effects 0.000 description 5
- 208000033353 latent tuberculosis infection Diseases 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 239000004005 microsphere Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 208000024794 sputum Diseases 0.000 description 5
- 210000003802 sputum Anatomy 0.000 description 5
- 230000009466 transformation Effects 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 4
- 239000003155 DNA primer Substances 0.000 description 4
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 239000000443 aerosol Substances 0.000 description 4
- 239000012491 analyte Substances 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 239000012228 culture supernatant Substances 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 239000000539 dimer Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 230000033687 granuloma formation Effects 0.000 description 4
- 238000010324 immunological assay Methods 0.000 description 4
- 238000001114 immunoprecipitation Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 230000000670 limiting effect Effects 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000004224 protection Effects 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 229960001005 tuberculin Drugs 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- 241000701022 Cytomegalovirus Species 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 208000032420 Latent Infection Diseases 0.000 description 3
- 101100038261 Methanococcus vannielii (strain ATCC 35089 / DSM 1224 / JCM 13029 / OCM 148 / SB) rpo2C gene Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108091005461 Nucleic proteins Proteins 0.000 description 3
- 102000057297 Pepsin A Human genes 0.000 description 3
- 108090000284 Pepsin A Proteins 0.000 description 3
- 108010004729 Phycoerythrin Proteins 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 230000009603 aerobic growth Effects 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 239000013060 biological fluid Substances 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000005059 dormancy Effects 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000005538 encapsulation Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 230000036571 hydration Effects 0.000 description 3
- 238000006703 hydration reaction Methods 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 238000011532 immunohistochemical staining Methods 0.000 description 3
- 238000012423 maintenance Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 230000001338 necrotic effect Effects 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 230000003362 replicative effect Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 101150085857 rpo2 gene Proteins 0.000 description 3
- 101150090202 rpoB gene Proteins 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 150000003573 thiols Chemical class 0.000 description 3
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108010042708 Acetylmuramyl-Alanyl-Isoglutamine Proteins 0.000 description 2
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 2
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 2
- 206010011224 Cough Diseases 0.000 description 2
- 108020001019 DNA Primers Proteins 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 108010036684 Glycine Dehydrogenase Proteins 0.000 description 2
- 102100033495 Glycine dehydrogenase (decarboxylating), mitochondrial Human genes 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 2
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 241000276498 Pollachius virens Species 0.000 description 2
- 108700043532 RpoB Proteins 0.000 description 2
- 241000700584 Simplexvirus Species 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 150000001350 alkyl halides Chemical class 0.000 description 2
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 229940037003 alum Drugs 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 238000002869 basic local alignment search tool Methods 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000013270 controlled release Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007123 defense Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 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 2
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- AEUTYOVWOVBAKS-UWVGGRQHSA-N ethambutol Chemical compound CC[C@@H](CO)NCCN[C@@H](CC)CO AEUTYOVWOVBAKS-UWVGGRQHSA-N 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000009851 immunogenic response Effects 0.000 description 2
- 238000010874 in vitro model Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000007834 ligase chain reaction Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- BSOQXXWZTUDTEL-ZUYCGGNHSA-N muramyl dipeptide Chemical compound OC(=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)O[C@@H](O)[C@@H]1NC(C)=O BSOQXXWZTUDTEL-ZUYCGGNHSA-N 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 239000002853 nucleic acid probe Substances 0.000 description 2
- 229940023146 nucleic acid vaccine Drugs 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 2
- 229940033663 thimerosal Drugs 0.000 description 2
- 150000003568 thioethers Chemical class 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 229940125575 vaccine candidate Drugs 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 239000012130 whole-cell lysate Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- QRXMUCSWCMTJGU-UHFFFAOYSA-L (5-bromo-4-chloro-1h-indol-3-yl) phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP([O-])(=O)[O-])=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-L 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
- ZORQXIQZAOLNGE-UHFFFAOYSA-N 1,1-difluorocyclohexane Chemical compound FC1(F)CCCCC1 ZORQXIQZAOLNGE-UHFFFAOYSA-N 0.000 description 1
- 150000003923 2,5-pyrrolediones Chemical class 0.000 description 1
- FMYBFLOWKQRBST-UHFFFAOYSA-N 2-[bis(carboxymethyl)amino]acetic acid;nickel Chemical compound [Ni].OC(=O)CN(CC(O)=O)CC(O)=O FMYBFLOWKQRBST-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- HLXHCNWEVQNNKA-UHFFFAOYSA-N 5-methoxy-2,3-dihydro-1h-inden-2-amine Chemical compound COC1=CC=C2CC(N)CC2=C1 HLXHCNWEVQNNKA-UHFFFAOYSA-N 0.000 description 1
- 101150109930 ACR gene Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 108010083590 Apoproteins Proteins 0.000 description 1
- 102000006410 Apoproteins Human genes 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000557626 Corvus corax Species 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 238000011238 DNA vaccination Methods 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
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 1
- 108091092566 Extrachromosomal DNA Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 208000000616 Hemoptysis Diseases 0.000 description 1
- GVGLGOZIDCSQPN-PVHGPHFFSA-N Heroin Chemical compound O([C@H]1[C@H](C=C[C@H]23)OC(C)=O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4OC(C)=O GVGLGOZIDCSQPN-PVHGPHFFSA-N 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000934372 Homo sapiens Macrosialin Proteins 0.000 description 1
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 1
- 208000008454 Hyperhidrosis Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 229930194542 Keto Natural products 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 102100025136 Macrosialin Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000187480 Mycobacterium smegmatis Species 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 102000004213 Neuropilin-2 Human genes 0.000 description 1
- 108090000770 Neuropilin-2 Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920001710 Polyorthoester Polymers 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000710799 Rubella virus Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 201000005010 Streptococcus pneumonia Diseases 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 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
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 241000223997 Toxoplasma gondii Species 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- UZQJVUCHXGYFLQ-AYDHOLPZSA-N [(2s,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-4-[(2r,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-6-(hydroxymethyl)-4-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-6-(hy Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2[C@@]1(C=O)C)C)(C)CC(O)[C@]1(CCC(CC14)(C)C)C(=O)O[C@H]1[C@@H]([C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O[C@H]4[C@@H]([C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)[C@H](O)[C@@H](CO)O4)O)[C@H](O)[C@@H](CO)O3)O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UZQJVUCHXGYFLQ-AYDHOLPZSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 244000000022 airborne pathogen Species 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000003282 alkyl amino group Chemical group 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 102000007362 alpha-Crystallins Human genes 0.000 description 1
- 108010007908 alpha-Crystallins Proteins 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 208000022531 anorexia Diseases 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 230000010310 bacterial transformation Effects 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 210000000621 bronchi Anatomy 0.000 description 1
- 238000013276 bronchoscopy Methods 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 244000309464 bull Species 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
- 238000011088 calibration curve Methods 0.000 description 1
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000005081 chemiluminescent agent Substances 0.000 description 1
- 239000002975 chemoattractant Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012504 chromatography matrix Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229940028617 conventional vaccine Drugs 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 210000000852 deltoid muscle Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229960002069 diamorphine Drugs 0.000 description 1
- 208000013219 diaphoresis Diseases 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 150000002019 disulfides Chemical class 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
- 239000002552 dosage form Substances 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 239000008151 electrolyte solution Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000005712 elicitor Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 229960000285 ethambutol Drugs 0.000 description 1
- 125000001033 ether group Chemical group 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000005182 global health Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000011544 gradient gel Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000007236 host immunity Effects 0.000 description 1
- 101150007644 hspX gene Proteins 0.000 description 1
- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 238000010820 immunofluorescence microscopy Methods 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000002480 immunoprotective effect Effects 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 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
- 238000011901 isothermal amplification Methods 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- MJGFBOZCAJSGQW-UHFFFAOYSA-N mercury sodium Chemical class [Na].[Hg] MJGFBOZCAJSGQW-UHFFFAOYSA-N 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 229960000282 metronidazole Drugs 0.000 description 1
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000012514 monoclonal antibody product Substances 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 1
- 238000007837 multiplex assay Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 201000001119 neuropathy Diseases 0.000 description 1
- 230000007823 neuropathy Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- JPXMTWWFLBLUCD-UHFFFAOYSA-N nitro blue tetrazolium(2+) Chemical compound COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 JPXMTWWFLBLUCD-UHFFFAOYSA-N 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 230000000422 nocturnal effect Effects 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 210000004043 pneumocyte Anatomy 0.000 description 1
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 230000027317 positive regulation of immune response Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000003725 proteoliposome Substances 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000011537 solubilization buffer Substances 0.000 description 1
- 239000001593 sorbitan monooleate Substances 0.000 description 1
- 235000011069 sorbitan monooleate Nutrition 0.000 description 1
- 229940035049 sorbitan monooleate Drugs 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell 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
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000583 toxicological profile Toxicity 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 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
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 208000016261 weight loss Diseases 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant: THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES, CENTERS FOR DISEASE CONTROL AND PREVENTION Invention Title: LATENT HUMAN TUBERCULOSIS MODEL, DIAGNOSTIC ANTIGENS, AND METHODS OF USE The following statement is a full description of this invention, including the best method for performing it known to us: - la LA TENT HUMAN TUBERCULOSIS MODEL, DIAGNOSTIC ANTIGENS, AND METHODS OF USE The entire disclosure in the complete specification of our Australian Patent 5 Application No. 2002237764 is by this cross-reference incorporated into the present specification. FIELD OF THE DISCLOSURE The present disclosure relates to the field of mycobacterial latency, and in 10 particular relates to an in vitro granuloma model for the study of mycobacteria and for the development of tuberculosis drug and vaccine candidates, and to the detection of latent mycobacterial infection using immunoassays. BACKGROUND 15 All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art 20 publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. Approximately every ten seconds, a person dies of tuberculosis somewhere in the world. Tuberculosis is the world's number one killer among infectious diseases and 25 the leading cause of death among women of reproductive age. Although developing countries bear the greatest burden of disease, the United States is greatly affected by tuberculosis, reporting 16,377 cases in 2000. The infectious agent causing almost all cases of tuberculosis is Mycobacterium tuberculosis (M. tuberculosis). M. tuberculosis is easily spread between individuals 30 through the air. A single cough by an infected individual can generate as many as 3000 infected droplet nuclei, while less than 10 bacilli may initiate a pulmonary infection in a N \MleIb ,\CasesPatem,\49000-49999\P49867 AU I\Spccis\P49867.AU I Specification 2007-11-16 doc 16/11/07 - lb susceptible individual. Because simply inhaling an airborne pathogen may infect individuals, tuberculosis outbreaks are difficult to contain and require isolating the infected individuals in negative air pressure rooms. Although it was believed that tuberculosis would eventually be eliminated after 5 the development of antibiotics in the 1950s, in 1999, tuberculosis was labeled as a global health emergency by the World Health Organisation. One of the major reasons for the perseverance of tuberculosis is the evolution of multi-drug resistant strains. Multi-drug resistant strains have evolved in part due to infected patients' poor compliance with drug therapy, which lasts for a period of at least six months. One 10 multi-drug resistant strain, strain W, has evolved resistance to all first-line drugs (isoniazid, rifampin, ethambutol, and pyrazidine), as well as one second-line drug (kanamycin). It is therefore evident that tuberculosis continues to be a serious health threat to individuals worldwide. Initial infection with M. tuberculosis only rarely leads to immediate disease 15 because the infection is typically controlled by the host's immune system. Among people infected with M tuberculosis, approximately 5% manifest the disease within a few years after infection. Upon initial N :McIboume\Cases\Paten\4900-49999\P49867 AU. I\Spccis\P49867.AU I Specification 2007-11-16 doc 16/11/07 -2 infection, the mycobacteria enter unactivated macrophages and multiply therein. Following a rapid growth phase, infected macrophages and their bacilliary cargo are surrounded and walled off by newly recruited activated macrophages. This walling off of the infected macrophages results in the characteristic granuloma. The granuloma is a compact, organized collection of activated 5 macrophages, including epithelioid and multinucleated giant cells, surrounded by T lymphocytes, and later by fibroblasts and collagen, which aggregate around the macrophage core. Mycobacterial dormancy results in a disease stage termed latent tuberculosis. An individual with latent tuberculosis may later develop a case of reactivated tuberculosis, and in fact, the majority of the tuberculosis cases reported in the United States are the result of reactivation of a mycobacterial 10 infection and not an initial infection. (Am. Rev. Respir. Dis. 146:1623-1633, 1992). Reactivation of the M tuberculosis bacilli usually occurs in the apex of the lung where large numbers of tubercle bacilli cause necrosis of the small bronchi of the lung. The characteristic bloodstained sputum of tuberculosis results from the erosion of small blood vessels during this necrotic process. Approximately one-third of the population worldwide has been estimated to be latently 15 infected with M tuberculosis. (Sudre et al., Bull. W.H.O. 70:149-159, 1992). Currently, the tuberculin skin test is the only available diagnostic for those infected with M. tuberculosis. Unfortunately, no currently available test can specifically identify latently infected individuals. The tuberculin test is only capable of identifying all individuals either exposed to the pathogen or vaccinated against the pathogen. Due to the high number of latently infected individuals and the risk 20 of reactivation of tuberculosis in those individuals, diagnostics and therapeutics targeted to latent tuberculosis need to be developed. In addition, the development of an in vitro granuloma model for the study of mycobacteria and for the development of tuberculosis drug and vaccine candidates would be desirable. 25 SUMMARY OF THE DISCLOSURE An in vitro model for tuberculosis latency is described in certain embodiments of this disclosure. In particular, an in vitro granuloma model and methods for using the model are provided. In some embodiments, the in vitro granuloma model contains human peripheral blood mononuclear cells, autologous macrophages and mycobacteria. In some embodiments these components are 30 combined in a low-attachment container. In specific examples, the in vitro granuloma model further contains fibroblasts, for example, human lung fibroblasts. Further embodiments are methods for using the in vitro granuloma model to screen new or known compounds for their effects on granuloma, for instance to screen candidate tuberculosis drugs, to identify candidate tuberculosis vaccines, and to analyze and characterize the process of granuloma 35 formation and granuloma necrosis. Also provided herein are immunological methods for detecting latent tuberculosis infections. Such methods are based on detecting specific bacterial antigens (or antibodies against these antigens) that are present in a subject with tuberculosis only (or predominately) during latent infection. By way of example, one such latency-specific antigen is alpha-crystallin (Acr).
-3 Further embodiments include an immunological assay for detection of latent tuberculosis in a subject, which assay involves contacting a biological sample from the subject, wherein the sample is suspected of containing a first latency-specific binding partner (LSBP) (such as a latency-specific antigen or an antibody thereto), with a second (corresponding) LSBP, and detecting binding between 5 the first LSBP and the corresponding LSBP. Binding between the first and second LSBPs is indicative of latent tuberculosis in the subject. Thus, in one example where the first LSBP is a M tuberculosis latency-specific antigen (for instance, Acr or an immunogenic fragment thereof), the corresponding LSBP may be an antibody that is capable of binding to that antigen. Where the first LSBP is an antibody, the corresponding LSBP (to make a specific binding pair) is an antigen. 10 Also provided are kits for the detection of latent tuberculosis in a subject, which kits include at least one LSBP (e.g., a latency-specific antigen or antibody thereto) and instructions for carrying out an immunological assay to detect binding of the LSBP to a cognate LSBP found in a biological sample. Also provided are kits comprising one or more elements of an in vitro granuloma model, for 15 instance cell culture media and, optionally, low-attachment containers and/or instructions for growing in vitro granulomas. Further embodiments provide methods for eliciting an immune response in a subject by administering to the subject an immune stimulatory amount of a M tuberculosis latency-specific antigen (e.g., Acr), or immunogenic fragment thereof. Compositions containing such 20 immunostimulatory molecules, and kits for their administration, are also provided. The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures and sequence listing. 25 BRIEF DESCRIPTION OF THE FIGURES FIG. I is a series of micrographs of aerosol-infected guinea pig lung granuloma tissue. FIG. IA shows tissue that was stained with hematoxylin and eosin (H & E). FIG. IB shows tissue that was stained with acid-fast stain; representative mycobacterium are indicated by the arrows. FIG. IC shows tissue that was subjected to immunohistochemical staining using a polyclonal antibody against 30 the Acr protein; the arrows indicate two stained mycobacteria. FIG. 2 is a ribonuclease protection assay (RPA) blot. Lanes 1-5 are various negative and positive controls, as indicated. Lanes 6-9 represent hybridizations to mRNA froin mycobacteria grown 5 or 7 days either aerobically or in the anoxic chamber. Acr mRNA was observed at all four time points while rpoB mRNA was only observed in aerobically grown cultures. 35 FIG. 3 is a RPA blot. Lanes 1 and 2 represent hybridizations to mRNA from mycobacteria extracted after 7- or 12-day incubations in the in vitro granuloma model. Both acr and rpoB mRNA were observed at both time points, likely indicating that aerobic bacilli were present in the granuloma. Lanes 3 and 4 are positive controls; acr mRNA and acr and rpoB DNA, respectively.
-4 FIG. 4 is a pair of agarose gels, showing the results of RT-PCR. Acr transcript is clearly induced with longer infection in the in vivo model (lanes 2, 3, and 4), and is even more strongly induced in anoxic culture (lane 5). Key: Lane 1, Uninfected Guinea Pig Lung; lane 2, MTB-Infected Guinea Pig Lung - 3 5 weeks P.1; lane 3, MTB-Infected Guinea Pig Lung - 5 weeks P.I.; lane 4, MTB-Infected Guinea Pig Lung - 10 weeks P.I.; lane 5, MTB-7 Day Anoxic Culture. FIG. 5 is a schematic drawing of an example anoxic growth vessel. FIG. 6 is a pair of Western blots demonstrating the production of the N-terminal construct Acr-N-FLAG in transformant 3. The blot in FIG. 6A was probed with anti-Acr polyclonal 10 immunoglobin. The blot in FIG. 6B was probed with anti-FLAG epitope imnimunoglobin. FIG. 7 is a pair of Western blots of whole cell lysates demonstrating the production of the C terminal construct Acr-C-FLAG in two Mycobacteriun smegmatis transformed strains versus non transformed M. smegnatis and M tuberculosis controls. The blot in FIG. 7A was probed with anti FLAG immunoglobin. The blot in FIG. 7B was probed with anti-Acr immunoglobin. 15 - FIG. 8 shows five strips cut from a Western blot of whole cell lysates; the arrows indicate the location of recombinant Acr protein. The strips were developed with the indicated primary antibodies. In addition to the control antibody, this blot demonstrates the presence of tyrosine phosphorylation in the final strip. FIG. 9 shows two Western blots from culture supernatants of M. tuberculosis bacilli grown 20 under various conditions. FIG. 9A was probed using rabbit anti-Acr antibody; FIG. 9B is a control blot. Protein is detected in 7 day and 12 month anoxic cultures and in vitro granuloma. Lower molecular weight variants are observed in the 12-month and in vitro granuloma supernatants. Key: Lane 1, molecular weight marker; lane 2, 3 day aerobic growth; lane 3, 7 day aerobic growth; lane 4, 7 day anoxic growth; lane 5, blank; lane 6, 7 day react.; lane 7, 12 months react.; lane 25 8, control cells; lane 9, 7 day in vitro granulomas; lane 10, M tuberculosis lysate. . FIG. 10 is a two-dimensional gel electrophoresis analysis of a sample taken from M tuberculosis grown under anoxic conditions. Acr protein is indicated by the circle. FIG. 11 is a Coomassie stained SDS-PAGE gel of culture supernatants from M tuberculosis bacilli cultured under a variety of conditions. 30 Key: Lane 1, MW marker; lane 2, 5-day aerobic (logarithmic); lane 3, anoxic 12 months; lane 4, anoxic 7 days; lane 5, 30-hour aerobic reactivated (logarithmic); lane 6, MW marker. FIG. 12 is a graph showing the growth (measured by optical density at 580 nm) of M tuberculosis under anoxic and aerobic growth conditions. 35 SEQUENCE LISTING The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the -5 complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing: SEQ ID NOs: 1 and 2 are the sequences of primers used to generate the N terminal FLAG-Acr fusion. 5 SEQ ID NOs: 3 and 4 are the sequences of primers used to generate the C terminal Acr-FLAG fusion. DETAILED DESCRIPTION 10 I. Abbreviations Acr alpha ((x) crystalline ELISA enzyme-linked immunosorbent assay HS human serum 15 LSA latency-specific antigen LSBP latency-specific binding partner PBMCs peripheral blood mononuclear cells RPA ribonuclease protection assay RT-PCR reverse-transcription polymerase chain reaction 20 hm. Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); 25 Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitate review of the various embodiments of the disclosure, the 30 following explanations of specific terms are provided: In the claims which follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the 35 presence or addition of further features in various embodiments of the invention. The terms o i "an, and "the" as used herein are defined to mean one or more and include the plural unless the context is inappropriate. H:\cintae\Keep\speci\2002237764.doc 11/08/04 - 5a The term "antibody" refers to a protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the 5 myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. The basic immunoglobulin (antibody) structural unit is generally a tetramer. 10 Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N Terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer, respectively, to these light and heavy 15 chains. H:\cintae\Keep\speci\2002237764.doc 11/08/04 -6 As used herein, the term antibodies includes intact immunoglobulins as well as a number of well-characterized fragments produced by digestion with various peptidases, or genetically engineered "artificial" antibodies. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab which itself is a light chain joined to 5 VH -CH I by a disulfide bond. The F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)' 2 dimer into an Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y., 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, it will be appreciated that Fab' fragments may be synthesized de novo 10 either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Antibodies for use in the methods and devices of this disclosure can be monoclonal or polyclonal. Merely by way of example, monoclonal antibodies can be prepared from murine 15 bybridomas according to the classical method of Kohler and Milstein (Nature 256:495-497, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). The term "antigen" refers to a molecule, or fragment thereof, which can induce an immune response in a mammal. The term includes immunogens and regions responsible for antigenicity or 20 antigenic determinants. A chemical or biochemical structure, determinant, antigen or portion thereof that is capable of inducing the formation of an antibody can be referred to as being "antigenic." "Antigenic determinant" refers to a region of a specified protein that is recognized by an antibody. When referring to macrophages, the term "autologous" refers to macrophages that are derived from the same individual as the peripheral blood mononuclear cells. Alternatively, a 25 macrophage cell line such as, but not limited to the THP-1 macrophage cell line is used as the macrophage component of the granuloma model. In one embodiment of the present disclosure, the beginning concentration of the autologous macrophages is between approximately 5 X 104 and 1 X 10', per two-milliliter sample, and optionally at a beginning concentration of approximately I X 10'. A "biological sample" is a sample of bodily fluid or tissue used for laboratory testing or 30 examination. As used herein, biological samples include all clinical samples useful for detection of microbial infection in subjects. Tissue samples may be taken from the oropharyngeal tract, for instance from lung or bronchial tissue. Samples can be taken by biopsy (such as during a bronchoscopy) or during autopsy examination, as appropriate. Biological fluids include blood, derivatives, and fractions of blood such 35 as serum, urine, semen and fluids of the oropharyngeal tract, such as sputum. Examples of specimens for use with the current disclosure for the detection of latent M tuberculosis include conventional clinical samples, for instance blood or blood-fractions (e.g., serum), urine, bronchoalveolar lavage (BAL), sputum, and induced sputum samples. Techniques for acquisition of such samples are well known in the art. Blood and blood fractions can be prepared in -7 traditional ways. Oropharyngeal tract fluids can be acquired through conventional techniques, including sputum induction, bronchoalveolar lavage (BAL), and oral washing. Obtaining a sample from oral washing involves having the subject gargle with an amount normal saline for about 10-30 seconds and then expectorate the wash into a sample cup. 5 The "condition" or "conditions" under which a DNA strand is synthesized include the presence of nucleotides, cations, and appropriate buffering agents in amounts and at temperatures such that the nucleic acid molecule and a DNA primer will anneal and oligonucleotides will be incorporated into a synthesized DNA strand. As used herein, the terms "detecting" or "detection" refers to quantitatively or qualitatively 10 determining the presence of a biomolecule under investigation. "Epitope tags" are short stretches of amino acids to which a specific antibody can be raised, which in some embodiments allow one to specifically identify and track a protein tagged with the epitope. Detection of the tagged molecule can be achieved using a number of different techniques. Examples of such techniques include: immunohistochemistry, immunoprecipitation, flow cytometry, 15 immunofluorescence microscopy, ELISA, immunoblotting ("westem"), and affinity chromatography. Examples of epitope tags include FLAG, T7, HA (hemagglutinin) and myc. As used herein, the term "granuloma" refers to a compact, organized collection of activated macrophages, including epithelioid and multinucleated giant cells, surrounded by T lymphocytes, fibroblasts and collagen. It is to be understood, however, that the term "in vitro granuloma" is not 20 limited to a collection of cells as described above. The term "in vitro granuloma" refers to a collection or aggregate of cells containing at least human peripheral blood mononuclear cells and autologous macrophages, wherein the collection or aggregate of cells mimics the granuloma as described above. Whether the in vitro granuloma mimics the granuloma as described above, and as found in vivo, is determined by methods known to those skilled in the art, such as microscopic 25 examination of the cell aggregates, phenotypic analysis of cells within the aggregates, via FACS (fluorescence activated cell sorter) analysis for example, and analysis of cytokine production by the cells within the aggregates. As used herein, the term "human peripheral blood mononuclear cells" (PBMCs) includes, but is not limited to, monocytes, B lymphocytes, and T lymphocytes. The human PBMCs included in 30 examples of the in vitro granuloma model can be monocytes and T lymphocytes. Optionally, in certain embodiments the in vitro granuloma model contains monocytes at a beginning concentration of between approximately 5 X 104 and 1 X 106, per two milliliter sample, and T lymphocytes at a beginning concentration of between approximately I X 105 and I X 106. In some embodiments, the in vitro granuloma model contains monocytes at a beginning concentration of approximately I X 106 35 and T lymphocytes at a beginning concer.tration of approximately 1 X 106. It is to be understood that the term "beginning concentration" refers to the concentration of material as it is added to a low attachment container. In some embodiments, the in vitro granuloma model contains fibroblasts, such as human lung fibroblasts. Optionally, the fibroblasts can be added at a beginning concentration of between -8 approximately 1 X 105 and I X 106, for instance, at a beginning concentration of approximately 5 X 10'. A "low attachment container" is a container whose surface inhibits or reduces the attachment of cells in culture. In some embodiments, the low attachment container is a low 5 attachment tissue culture dish such as, but not limited to, the COSTARTM Ultra Low Attachment Surface or Clusters (Costar Corp., Cambridge, MA) used in accordance with the manufacturer's recommended procedures. Optionally, the low attachment container can have a surface composed of a covalently bound hydrogel layer that is hydrophilic and neutrally charged, so that it inhibits (for instance, by 5%, 10%, 20%, 40%, 50% or more compared to a non-coated container) the attachment 10 and activation of macrophages and neutrophils. Because proteins and other biomolecules passively adsorb to surfaces through hydrophobic and ionic interactions, a hydrogel surface naturally inhibits non-specific immobilization via these forces, thus inhibiting subsequent cell attachment. Optionally, the surface of the low attachment container may be rehydrated at a temperature consistent with the application or cell growth requirements of the cells described above and the rehydration media 15 aspirated or decanted prior to the addition of cells and fresh media. Alternatively, the cells may be added directly to the rehydration media. "In vitro amplification" refers to techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen. An example of in vitro amplification is the polymerase chain reaction (PCR), in which a biological sample collected from a subject is contacted with a pair of 20 oligonucleotide primers, under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify copies of the nucleic acid. Other examples of in vitro amplification techniques include strand displacement amplification (see U.S. Patent No. 5,744,311); transcription-free isothermal amplification (see U.S. Patent No. 25 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889); and NASBATM RNA transcription-free amplification (see U.S. Patent No. 6,025,134). The product of in vitro amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, 30 oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques. An "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and/or organelles. Nucleic acids and proteins that have been "isolated" 35 include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. As with the term purified, isolated is a relative term. A "label" is any molecule or composition that is detectable by, for instance, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Examples of -9 labels, including radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, enzymes, colloidal gold particles, colored latex particles, and epitope tags, have been disclosed previously and are known to those of ordinary skill (see, for instance, U.S. Patents No. 4,275,149; 4,313,734; 4,373,932; and 4,954,452). 5 The attachment of a compound (e.g., an antibody) to a label can be through covalent bonds, adsorption processes, hydrophobic and/or electrostatic bonds, as in chelates and the like, or combinations of these bonds and interactions and/or may involve a linking group. "Latent tuberculosis" refers to a stage in the M tuberculosis infection where the bacilli remain viable but are slowly replicating or non-replicating, may be encapsulated in localized lesions 10 within the lung, and do not cause active necrotic disease. The latent stage may exist for the remainder of a host's life, or the infection may reactivate during, for instance, a period of decreased host immunity or in response to other stressors. Though latent M tuberculosis infections have not previously been able to be specifically identified, they are within the group of individuals that possess a positive tuberculin skin test but do not possess the characteristic symptoms of active disease. 15 A "latency specific antigen" is an antigen that is expressed at higher levels (or exclusively) by a M tuberculosis bacterium in its dormant or stationary rather than its active or logarithmic phase of growth. Latency specific antigens (LSAs) can be identified, for instance, by comparing the protein expression found in in vitro cultured M tuberculosis grown under standard aerobic/logarithmic conditions with bacilli grown under conditions that mimic latency (e.g., in a latency model). 20 A "linking group" is a chemical arm between two compounds, for instance a compound and a label (e.g., an antibody and a label). To accomplish the requisite chemical structure of the linkage, each of the reactants must contain a reactive group. Representative combinations of such groups are amino with carboxyl to form amide linkages; carboxy with hydroxy to form ester linkages; amino with alkyl halides to form alkylamino linkages; thiols with thiols to form disulfides; or thiols with 25 maleimides or alkylhalides to form thioethers. Hydroxyl, carboxyl, amino and other functionalities, where not present in the native compound, may be introduced by known methods. Likewise, a wide variety of linking groups may be employed. The structure of the linkage should be a stable covalent linkage formed to attach two compounds to each other (e.g., the label to the antibody). In some cases the linking group may be designed to be either hydrophilic or 30 hydrophobic in order to enhance a desired characteristic, for instance a binding characteristic of a modified ligand and its cognate receptor. The covalent linkages should be stable relative to the solution conditions to which linked compounds are subjected. Examples of linking groups will be from 1-20 carbons and 0-10 heteroatoms (NH, 0, S) and may be branched or straight chain. Without limiting the foregoing, it should be obvious that only 35 combinations of atoms that are chemically compatible comprise the linking group. For example, amide, ester, thioether, thiol ester, keto, hydroxyl, carboxyl, and ether groups in combinations with carbon-carbon bonds are particular examples of chemically compatible linking groups. The term "mycobacteria" as used herein includes, but is not limited to, M. tuberculosis. Any mycobacteria that form granulomas can be used in the compositions and methods provided herein.
-10 Exemplary mycobacteria include M avium, M bovis, M marinum, M ulcerans, M smegmatis, and M haemophilum. Optionally, the beginning concentration of mycobacteria in the in vitro granuloma model is between approximately 1 X 10' and I X 105 cfu/2 ml sample. A first nucleic acid sequence is "operably linked" with a second nucleic acid sequence when 5 the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. "Peptides," "polypeptides," and "oligopeptides" are chains of amino acids (typically L 10 amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. The terminal amino acid at one end of the chain (i.e., the amino terminal) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminal) has a free carboxyl group. As such, the term "amino terminus" 15 (abbreviated N-terminus) refers to the free alpha-amino group on the amino acid at the amino terminal end of the peptide, or to the alpha-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. The term "carboxy terminus" (abbreviated C-terminus) refers to the free carboxyl group on the amino acid at the carboxy terminal end of a peptide, or to the carboxyl group of an amino acid at any other location within the peptide. 20 Typically, the amino acids making up a peptide are numbered in order, starting at the amino terminus and increasing in the direction toward the carboxy terminus of the peptide. Thus, when one amino acid is said to "follow" another, that amino acid is positioned closer to the carboxy terminal end of the peptide than the preceding amino acid. As used herein, the term "primer" or "DNA primer" means an oligonucleotide that anneals 25 to a nucleic acid molecule in a particular orientation to allow for the synthesis of a nascent DNA strand. As used herein, the phrase "primer pair" refers to two primers, one having a forward designation and the other having a reverse designation (relative to their respective orientations when annealed to a double-stranded DNA molecule that consists of a sense and antisense sequence). Under 30 in vitro amplification conditions, the forward primer anneals to and primes amplification of the sense sequence and the reverse primer anneals to and primes amplification of the antisense sequence. Primers can be selected for use in an amplification reaction on the basis, for instance, of having minimal complementarity with other primers in the reaction (to minimize the formation of primer dimers) and having T, values with a range of reaction temperatures appropriate for the amplification 35 method, such as PCR. In addition, primers can be selected to anneal with specific regions of a DNA or RNA template such that the resulting DNA amplification product of specific size, for instance from 100 to 5000 base pairs in length, for instance around 300 base pairs in length or longer. By "probe" is meant a nucleic acid sequence that can be used for selective hybridization with complementary nucleic acid sequences for their detection. The probe varies in length, for - 11 instance from about 5 to 100 nucleotides, or from about 10 to 50 nucleotides, or about 18 to 24 nucleotides. A "labeled probe" comprises an isolated nucleic acid probe attached to a detectable label or other reporter molecule. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al. (In Molecular Cloning: A Laboratory 5 Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). A "promoter" includes one or more nucleic acid sequences that direct transcription of a nucleic acid. A promoter includes nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter may also include distal 10 enhancer or repressor elements that can be located as much as several thousand base pairs from the start site of transcription. The term "purified" as it is used herein does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid (or -protein or other compound) preparation is one in which the specified molecule (or type of molecule) is more enriched than it is in 15 its generative environment, for instance within a cell or in a biochemical reaction chamber (as appropriate). A preparation of a "substantially pure" substance, for instance a substantially pure nucleic acid, may be purified such that the desired nucleic acid represents at least 50% of the total nucleic acid content of the preparation. In certain embodiments, a substantially pure preparation will represent at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% or more 20 desired molecule in the preparation. A "recombinant" nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic 25 engineering techniques. The term "residue" is used herein to refer to an amino acid (D or L), or an amino acid mimetic, that is incorporated into a peptide by an amide bond. As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid 30 mimetics). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those of ordinary skill in the art. The phrase "sequence identity" refers to the similarity between two nucleic acid sequences, or two amino acid sequences, and is expressed in terms of the similarity between the sequences. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); 35 the higher the percentage, the more similar the two sequences are. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman Adv. AppL. Math. 2: 482, 1981; Needleman & Wunsch J. Mol. Biol. 48: 443, 1970; Pearson & Lipman Proc. Natl. Acad. Sci. USA 85: 2444, 1988; Higgins & Sharp Gene, 73: 237-244, 1988; Higgins & Sharp CABIOS 5: 151- - 12 153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881-90, 1988; Huang et al. Computer Appis. in the Biosciences 8, 155-65, 1992; and Pearson et aL Meth. Mol. Bio. 24, 307-31, 1994. Altschul et al. (J. Mol Biol 215:403-410, 1990), presents a detailed consideration of sequence alignment methods and homology calculations. 5 The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blast, tblastn and tblastx. An alternative indication that two nucleic acid molecules are closely related is that the two 10 molecules hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5' C to 20' C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The T. is the temperature (under defined ionic strength and pH) at which 50% of the target sequence remains hybridized to a perfectly matched 15 probe or complementary strand. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Tijssen (Laboratory Techniques in Biochemistry and Molecular Biology Hybridization with Nucleic Acid Probes Part I, Chapter 2, Elsevier, New York, 1993). Nucleic acid molecules that hybridize under stringent conditions to a target sequence will typically hybridize to a 20 probe based on either an entire target protein encoding sequence, or selected portions of the encoding sequence, under wash conditions of 2 x SSC at 50' C. Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid 25 molecules that all encode substantially the same protein or the identical protein. Furthermore, one of ordinary skill in the art will recognize that individual substitutions, deletions or additions in the amino acid sequence of the protein, or in the nucleotide sequence encoding for the amino acids in the protein, which alter, add or delete a single amino acid or a small percentage of amino acids (in some instances less than 5%, or even less than 1%) in an encoded 30 sequence are conservatively modified variations, wherein the alterations result in the substitution of an amino acid with a chemically similar amino acid, and so long as the resultant variant still retains a substantial proportion of a property or activity, such as an immunostimulatory property (e.g., a protective immune response in a subject), of the base protein. Envisioned in specific embodiments are molecules in which there is no more than one amino acid substitution, no more than about three 35 substitutions, or about 5, 10, or even 20 substitutions, so long as the resultant variant retains a substantial proportion (e.g., at least 20%, at least 30%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or more) of an immunostimulatory or other property of the base protein. Some variant embodiments are expected to have greater immunostimulatory properties than the protein or peptide from which they are derived.
- 13 Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 5 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 10 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). The term "specific binding agent" as used herein refers to an agent that binds substantially only to a defined target. Thus a protein-specific binding agent binds substantially only the specified protein. The term "protein specific binding agent" includes anti-protein antibodies (and functional 15 fragments thereof) and other agents (such as soluble receptors) that bind substantially only to the specified protein. Anti-protein antibodies (such as anti-Acr antibodies) may be produced using standard - procedures described in a number of texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). The determination that a particular agent binds substantially only 20 to the specified protein, or component epitopes thereof, may readily be made by using or adapting routine procedures. One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988)). Western blotting may be used to determine that a given protein binding agent, such as an anti-Acr monoclonal antibody, binds substantially only to the specified protein. 25 Shorter fragments of antibodies can also serve as specific binding agents. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to Acr would be Acr-specific binding agents. These antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment 30 of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody, molecule; (3) (Fab') 2 , the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab') 2 , a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable 35 region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine.
- 14 A "specific binding partner" is a member of a pair of molecules (the "specific binding pair") capable of recognizing and binding to a structural aspect of another molecule by means of specific, noncovalent interactions that depend on the three-dimensional structures of the molecules involved. Typical pairs of specific binding partners include antigen/antibody, hapten/antibody, 5 hormone/receptor, nucleic acid strand/complementary nucleic acid strand, substrate/enzyme, inhibitor/enzyme, apoprotein/cofactor, carbohydrate/lectin, biotin/(strept)avidin, and virus/cellular receptor. A specific binding pair that includes at least one immunological molecule (such as an antibody or antigen) can be referred to as a specific immunological binding pair, and the 10 immunological molecule(s) as specific immunological binding partner(s). An example of a specific binding pair is a latency-specific binding pair, which includes a molecule that is a latency-specific molecule (such as a latency-specific antigen) and a molecule that is a specific binding partner for that latency-specific molecule. The phrase "specifically binds to an analyte" or "specifically immunoreactive with," when 15 referring to an antibody, refers to a binding reaction or interaction which is determinative of the presence of the analyte or epitope in a heterogeneous population of molecules such as proteins and other biological molecules. Thus, under designated immunoassay conditions, specified antibodies bind to a particular analyte or epitope and do not bind in a significant amount to other analytes or epitope present in the sample. A variety of immunoassay formats may be used to select antibodies 20 specifically immunoreactive with a particular analyte or epitope. For example, solid-phase ELISA immnunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane, Antibodies, A Laboratory Manual, CSHP, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. 25 The term "subject" as used herein refers to living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals. The term "subject" includes both human and veterinary subjects. When referring to cytokines or other biological materials, the term "steady state level" refers to the level of the cytokine or biological material produced in uninfected cells. 30 The term "synthetic polypeptide" refers to a polypeptide formed, in vitro, by joining amino acids in a particular order, using the tools of organic chemistry to form the peptide bonds. -A "transformed" cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with 35 viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. The term "vaccine" is used herein to mean a composition useful for stimulating a specific immune response in a vertebrate.
- 15 The term "vector" as used herein refers to a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. 5 Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for 10 description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 15 III. Description of several embodiments Provided herein in a first embodiment is an immunological assay method for detection of latent tuberculosis in a subject. Such methods include contacting a biological sample that may contain a first latency-specific binding partner (LSBP) from the subject with a corresponding LSBP, and detecting binding between the first LSBP and the corresponding LSBP, wherein such binding is 20 indicative of latent tuberculosis in the subject. In some specific examples of these methods, the first LSBP is an antibody, and the corresponding LSBP is a latency-specific M tuberculosis antigen (for instance, c.-crystallin (Acr) or an immunogenic fragment thereof). In other specific examples of the methods, the first LSBP is a latency-specific M tuberculosis antigen, and the corresponding LSBP is an antibody. In certain specific examples of the methods, the antigen is Acr or an immunogenic 25 fragment thereof. Another embodiment is a kit for the detection of latent tuberculosis in a subject. Such kits include at least one LSBP and instructions for carrying out an immunological assay method for detection of latent tuberculosis in a subject. This disclosure also provides a method of eliciting an immune response in a subject. The 30 methods include introducing into the subject an immune stimulatory amount of a M tuberculosis latency-specific antigen or immunogenic fragment thereof, or a nucleic acid molecule encoding such an antigen (e.g., Acr) or immunogenic fragment thereof. In certain specific examples, the method is a method of inhibiting or treating a latent tuberculosis infection in the subject In particular examples of the methods, the elicited immune response results in decreased susceptibility of the subject to 35 latent infection by M tuberculosis. This disclosure further provides a kit for eliciting an immune response in a subject. Such kits include an immune stimulatory amount of a M. tuberculosis latency-specific antigen or immunogenic fragment thereof, or a nucleic acid molecule encoding such an antigen or immunogenic fragment, and instructions for carrying out a method of eliciting an immune response in a subject.
-16 Particular examples of the kit include instructions for administering a component of the kit to a patient with a possible latent tuberculosis infection. Further embodiments provide in vitro granulomas that include peripheral blood mononuclear cells, autologous macrophages, and mycobacteria. In such granulomas, the peripheral blood 5 mononuclear cells are human peripheral blood mononuclear cells selected from the group consisting of monocytes, B lymphocytes, T lymphocytes, and combinations thereof. In certain examples of the granuloma, the mycobacteria are M. tuberculosis. Certain examples of the granuloma further include fibroblasts. Also provided is a method for producing an in vitro granuloma, which method involves 10 combining peripheral blood mononuclear cells, autologous macrophages, and mycobacteria in a low attachment container and incubating the combination for a sufficient amount of time to form the in vitro granuloma. In some, specific examples, fibroblasts are added to the combination. In some specific examples, exogenous cytokine(s) are added to the container in sufficient amount to enhance production of the in vitro granuloma. In some instances, the exogenous cytokine is IL-2, IFN-Y, 15 TNF-c, or a combination of two or more thereof. Another embodiment provided herein is a method of screening a tuberculosis drug candidate for anti-tuberculosis therapeutic activity. Such methods include combining the drug with an in vitro granuloma comprising peripheral blood mononuclear cells, autologous macrophages, and mycobacteria, and determining whether the drug inhibits mycobacterial viability. In some specific 20 examples, the peripheral blood mononuclear cells are human peripheral blood mononuclear cells selected from the group consisting of monocytes, B lymphocytes, T lymphocytes, and combinations thereof. Still another embodiment provided herein is a method of screening a tuberculosis drug candidate for anti-tuberculosis therapeutic activity that includes combining the drug with an in vitro 25 granuloma comprising peripheral blood mononuclear cells, autologous macrophages, and inactivated mycobacteria, and determining whether the drug inhibits reactivation of mycobacteria contained in the granuloma. In certain specific examples of the methods, the mycobacteria are M tuberculosis. Also provided is a method of screening a tuberculosis vaccine candidate that includes determining whether a mutant mycobacteria has a reduced ability, when compared against a wild type 30 mycobacteria, to induce latency, survive, reactivate or induce granuloma necrosis in an in vitro granuloma comprising peripheral blood mononuclear cells, autologous macrophages, and the mutant mycobacteria. In some specific examples of the methods, the in vitro granuloma further comprises fibroblasts. In certain examples of the methods, the mutant mycobacteria includes a mycobacteria strain having a mutation in a latency gene. In other examples, the mutant mycobacteria is a 35 Mycobacterium tuberculosis strain having a mutation in a gene selected from the group consisting of acr, a sigma factor gene, oxyR and aphC. In particular examples, the sigma factor gene is selected from the group consisting of sigF, sigC, and sigH. Additional embodiments are kits for producing an in vitro granuloma, including a culture medium and instructions for carrying out a method of screening a tuberculosis vaccine candidate that -17 includes determining whether a mutant mycobacteria has a reduced ability, when compared against a wild type mycobacteria, to induce latency, survive, reactivate or induce granuloma necrosis in an in vitro granuloma comprising peripheral blood mononuclear cells, autologous macrophages, and the mutant mycobacteria. In certain examples, the kit further includes a low attachment container and in 5 certain specific examples, the kit further includes an amount of a cytokine. IV. Production and Use of the In Vitro Granuloma Model This disclosure provides in this embodiment methods for producing and using an in vitro granuloma model, which model provides a consistent, replicable, and reliable laboratory system. The 10 in vitro granuloma can be used in many applications, for instance to study granuloma formation and maintenance, as well as to identify and characterize compounds that affect granuloma formation, maintenance, or reversion, and to study aspects of mycobacterial, including particularly mycobacterial latency. In general, the in vitro granuloma is formed by combining peripheral blood mononuclear 15 cells with macrophages and mycobacteria. The mixture of cells is incubated for a number of days to encourage formation of aggregates in the cell culture. Formation of the granulomas is further encouraged by use of low attachment containers in certain embodiments. After formation of cell aggregates, fibroblasts can optionally be added to the culture. In some embodiments, exogenous cytokines are added to the growth medium, for instance, IL-2, IFN-y, and/or TNF-cL, for instance 20 prior to infection with mycobacteria. By way of more specific example, autologous macrophages and mycobacteria are combined in one or more wells of a low attachment container, such as wells of a tissue culture dish treated to inhibit all attachment. Peripheral blood mononuclear cells (PBMCs), optionally 1 X 106 cells in a cell culture media (such as RPMI plus 10% human serum, HS (Lampire Biological Laboratories, 25 Pipersville, PA)), are combined with the macrophages and mycobacteria and incubated at a temperature at which the cells will grow for between 5 and 7 days. When aggregates reach a diameter of approximately 1 mm, fibroblasts (optionally human lung fibroblasts) may be added. Secretion of a variety of chemoattractant cytokines following phagocytosis of M iuberczdosis bacilli by the macrophage is important not only to the formation of the granuloma but 30 also to its maintenance. Because of this, progression of the in vitro granuloma can be monitored by measuring cytokine levels. For cytokine analysis, supernatants are harvested, filter sterilized, and assayed by a known technique such as ELISA. It is also beneficial in some embodiments to add exogenous cytokine to the medium, for instance during formation of the in vitro granuloma; in some instances, addition of cytokine enhances 35 aggregate formation. By way of example, IL-2 (at 10 units/ml, for instance), IFN-y (at 2 ng/ml, for instance), or TNF-a (at 50 ng/ml, for instance) (Endogen, Woburn, MA) or combinations of two or three cytokines are added to the cells prior to mycobacterial infection. When in vitro granulomas were maintained for longer periods of time, for instance for 9 days, cytokines could beneficially be - 18 added a second time. In some specific examples, the same amounts and kinds of cytokines were added. at day 5 after starting the cultures. Similarly, gene expression can be used to characterize the in vitro granuloma. For RT-PCR or RPA analysis, aggregates are collected, washed and the RNA extracted. For histopathology, cells 5 are fixed in a fixative, such as 10% formalin, and processed as tissue. The in vitro granuloma model of the present disclosure has a variety of uses. For example, the in vitro granuloma model can be used to analyze and characterize the process of granuloma formation and granuloma necrosis. It can also be also used to characterizeM tuberculosis genes that are differentially expressed when the mycobacterium is located inside a granuloma versus when the 10 mycobacterium in not located inside a granuloma. The model, for instance when employed without the addition of exogenous cytokines, is useful to characterize native cytokine production by the cells composing a granuloma. In one embodiment, the cytokine levels produced by the in vitro granuloma model are compared to the steady state levels of the cytokines. The granuloma model is also useful for analyzing mycobacterial 15 viability, as well as for pathophysiologic analyses. V. Drug and Immune-Stimulating Compound Candidate Screening The in vitro granuloma model described herein can be used for accurately screening candidate compounds, for instance, tuberculosis drug candidates. Methods of screening a 20 tuberculosis drug candidate include adding the drug candidate to the in vitro granuloma model and determining whether the drug kills mycobacteria contained in the granuloma or otherwise alters the physiology of the bacilli or other cells in the granuloma. The model is optionally used to screen drugs for use in treating latent mycobacterial infections. The in vitro granuloma model is also useful for screening immunostimulatory compounds, 25 for example tuberculosis vaccine candidates. By way of example, the in vitro granuloma model is used as a precursor to animal studies, for instance to test mutant strains of M tuberculosis that may be used as vaccines to prevent or reduce M tuberculosis infection or the reactivation of latent mycobacterial infections. Animal study precursors help to reduce the costs and negative connotations associated with animal experimentation. Examples of the in vitro granuloma model provided herein 30 are less expensive and much more rapid than conventional vaccine screening tests that rely on data generated from animal experiments. A mutant M tuberculosis strain can be constructed by methods known to those of skill in the art, for instance by inducing or screening for a mutation, such as a mutation in one of the latency genes selected from, but not limited to, acr, sigF, sigC, sigH, and other sigma factors, oxyR and 35 aphC. Examples of methods known to those of skill in the art include in vitro mutagenesis and in vivo mutagenesis. The mutation can be a deletion of all or part of one or more of these latency genes or an insertion or substitution in one or more of these latency genes. When referring to mycobacterial strains in general or an M. tuberculosis strain specifically, the terms "mutated" and "mutant" refer to strains having one or more mutations that inhibit or prevent the strain from shifting down into a - 19 dormant or latent state, or that inhibit or prevent the strain from reactivating after shifting down into a dormant or latent state. Dormancy is a state of slow or stopped bacterial replication, but with some ongoing metabolism, whereas reactivation involves bacterial replication and log phase biochemistry. In examples of methods for identifying mutated strains that can serve as vaccines, a mutated 5 mycobacterial strain as described herein is added to the in vitro granuloma model in place of a wild type mycobacteria strain. The efficacy of the mutated mycobacteria strain is determined based upon a reduced ability of the mutated strain to induce formation of a latent mycobacterial infection state, survive within the granuloma, reactivate from a latent state to an active state, and/or a reduced ability of the mutant strain to induce granuloma necrosis as compared to a wild-type mycobacterial strain. 10 Cytokine production in the in vitro granuloma containing the mutated mycobacteria strain also can be analyzed and compared to cytokine production in a control in vitro granuloma model containing a wild-type mycobacteria strain. VI. Identification of Latency-Specific Antigens 15 To gain insight into the molecular mechanisms of mycobacterial dormancy, and to provide molecules for detecting and/or tracking dormant infection, genes and proteins suspected of being involved in the adaptation to anoxia have been investigated. Results using reverse-transcription polymerase chain reaction (RT-PCR) technology (as described herein) have confirmed differential expression of various putative stress-response genes. Two of these genes that demonstrate increased 20 activity in the anaerobic (shift-down) granuloma model are involved in oxidative stress response, oxyR and aphC (Dhadayauthapani et al., J. Bacteriol. 178:3641-3649, 1996). The third encodes ot crystallin, an ATP-independent chaperon (Henriques et al., J. Bacter. 179:1887-1897, 1997; Horwitz, Proc. Nat'l. Acad Sci. USA 89:10449-10453, 1992), reported to be required for bacilli growth within macrophages (Yuan et al., Microbiol. 95, 16:9578-9583, 1998). 25 Latency-specific antigens in addition to those discussed in specific embodiments disclosed herein can be identified based on their preferential expression by latent mycobacteria, particularly M tuberculosis, in comparison to mycobacteria that are not in latent phase. By way of example, differential expression can be detected using two-dimensional gel electrophoresis of proteins extracted from latent and non-latent mycobacteria (e.g., bacteria cultured under anoxic conditions and 30 aerobic conditions). Alternatively, gene-chip (or cDNA microarray) analysis can be performed to detect preferential mRNA expression in latent (e.g., in vitro granuloma) versus non-latent (i.e., aerobic) cultures of mycobacteria. For instance, subtractive hybridization can be carried out by spotting all of the transcripts expressed in one culture or the other. The chip can then be probed using labeled (e.g., 35 with a cyanine dye) latent and labeled non-latent mRNA (or cDNA) pools, and differential expression detected using known techniques. The prototypical latency-specific antigen is oL-crystallin; its identification and characterization is described in more detail herein.
-20 VI. Detection of Latent Tuberculosis It has been surprisingly found that latent M tuberculosis can produce a high enough level of latency-specific antigens that such antigens, and/or antibodies reactive therewith, can be detected in 5 subjects with latent M. tuberculosis infection. With the demonstration herein that latent tuberculosis organisms produce specific antigens that elicit immunogenic responses in subjects, methods for the detection of latency-specific antigens, and/or antibodies to latency-specific antigens, and for the detection and/or diagnosis of latent infections, are now enabled. 10 Latency-specific antigens, or antibodies that recognize an epitope of a M tuberculosis latency-specific protein (such as Acr) can be detected in samples from a subject, for instance serum or other biological fluid, using known immunological techniques. The presence of such latency specific antigens or antibodies (e.g., circulating antibodies specific for an Acr epitope) indicates that the subject suffers from a latent tuberculosis infection. 15 Many techniques are commonly known in the art for the detection and quantification of antigen. Most commonly, purified antigen will be bound to a substrate, the antibody of the sample will bind via its Fab portion to this antigen, the substrate will then be washed and a second, labeled antibody will then be added which will bind to the Fc portion of the antibody that is the subject of the assay. The second, labeled antibody will be species specific, i.e., if the serum is from a human, the 20 second, labeled antibody will be anti-human-IgG antibody. The substrate will then be washed and the amount of the second, labeled antibody that has been bound will be detected and quantified by standard methods. Examples of methods for the detection of antibodies in biological samples, including methods employing dip strips or other immobilized assay devices, are disclosed for instance in the 25 following patents: U.S. Patents No. 5,965,356 (Herpes simplex virus type specific seroassay); 6,114,179 (Method and test kit for detection of antigens and/or antibodies); 6,077,681 (Diagnosis of motor neuropathy by detection of antibodies); 6,057,097 (Marker for pathologies comprising an auto immune reaction and/or for inflammatory diseases); and 5,552,285 (Immunoassay methods, compositions and kits for antibodies to oxidized DNA bases). 30 By way of example, a microsphere assay (also called flow beads assays) also can be used to detect Acr protein or another LSA in biological fluids (such as a culture supernatant from an in vitro latency model, or biological samples from a subject). This technology, as represented by systems developed by Luminex Corporation and other systems developed by Becton Dickinson, allows one to process a very small amount of sample, typically 20 pl, to detect one or several analytes. The 35 principle of this assay is based on the coupling of a "capture antibody" to microspheres containing specific amounts of a red dye and an infrared dye. After incubation of these microspheres with the sample, a secondary detection antibody coupled with biotin and streptavidin coupled with phycoerythrin (PE), the beads are analyzed with a flow cytometer. One laser detects the beads and a second one detects the intensity of the PE bound to those beads. This technology has been used to -21 detect cytokines in multiplex assays, serotyping of Streptococcus pneumonia, simultaneous measurement of human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP), simultaneous detection of serum IgG to Toxoplasma gondii, Rubella virus, Cytomegalovirus, and Herpes Simplex Virus Types 1 and 2 (see technical notes available from Luminex Corp., for instance at their Web-site 5 or through their catalog) In certain embodiments, a polyclonal rabbit antiserum is used to capture the Acr protein on the microspheres. In some embodiments, the secondary detection antibody is a monoclonal antibody to Acr. Secondary antibodies used in such methods can be coupled to, for instance, biotin. 10 VIII. Production of Latency-Specific Immunological Binding Partners Once a latency-specific M tuberculosis protein is identified, it is advantageous to produce that protein, and/or antibodies that specifically recognize one or more epitopes on the protein, in sufficient amounts to be used in immunological or other assays. Methods for production of proteins, and antibodies reactive with identified proteins, are well known to those of ordinary skill in the art. 15 The following methods are provided as representative examples, and should not be viewed as limiting. A. Production ofproteins Once a latency-specific protein is identified, it is a matter of well-known techniques to 20 determine the sequence that encodes the protein. For instance, the entire coding sequence of the M tuberculosis genome is known (Cole et al., Nature 393:537-544, 1998); this can be used to identify the gene that encodes an isolated latency-specific protein. The encoding sequence can then be used to produce quantities of protein in vitro. One skilled in the art will understand that there are myriad ways to express a recombinant 25 protein such that it can subsequently be purified. In general, an expression vector carrying the nucleic acid sequence that encodes the desired protein will be transformed into a microorganism for expression. Such microorganisms can be prokaryotic (bacteria) or eukaryotic (e.g., yeast). One appropriate species of bacteria is Escherichia coli (E. coh), which has been used extensively as a laboratory experimental expression system. Also, protein can be expressed using a viral (e.g., 30 vaccinia) based expression system. Protein can also be expressed in animal cell tissue culture, and such a system will be appropriate where animal-specific protein modifications are desirable or required in the recombinant protein, or in one portion of a fusion protein. Vectors suitable for stable transformation of culturable cells are also well known. Typically, such vectors include a multiple-cloning site suitable for inserting a cloned nucleic acid molecule, 35 such that it will be under the transcriptional control of 5' and 3' regulatory sequences. In addition, transformation vectors include one or more selectable markers; for bacterial transformation this is often an antibiotic resistance gene. Such transformation vectors typically also contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, and a - 22 transcription termination site, each functionally arranged in relation to the multiple-cloning site. For production of large amounts of recombinant proteins, an inducible promoter is preferred. This permits selective production of the recombinant protein, and allows both higher levels of production than constitutive promoters, and enables the production of recombinant proteins that may be toxic to 5 the expressing cell if expressed constitutively. In addition to these general guidelines, protein expression/purification kits are produced commercially. See, for instance, the QlAexpressTM expression system from QIAGEN (Chatsworth, CA) and various expression systems provided by InVitrogen (Carlsbad, CA). Depending on the details provided by the manufactures, such kits can be used for production and purification of 10 latency-specific proteins. One skilled in the art will understand that there are myriad ways to purify recombinant polypeptides, and such typical methods of protein purification may be used to purify latency-specific proteins. Such methods include, for instance, protein chromatographic methods including ion exchange, gel filtration, HPLC, monoclonal antibody affinity chromatography and isolation of 15 insoluble protein inclusion bodies after over production. In addition, purification affinity-tags, for instance a six-histidine sequence, may be recombinantly fused or linked to the protein and used to facilitate polypeptide purification. A specific proteolytic site, for instance a thrombin-specific digestion site, can be engineered into the protein between the tag and the remainder of the fusion to facilitate removal of the tag after purification, if sucli removal is desired. 20 Commercially produced protein expression/purification kits provide tailored protocols for the purification of proteins made using each system. See, for instance, the QL4expressTm expression system from QIAGEN (Chatsworth, CA) and various expression systems provided by InVitrogen (Carlsbad, CA). Where a commercial kit is employed to produce a functionalized TGF-p fusion protein, the manufacturer's purification protocol is a preferred protocol for purification of that 25 protein. For instance, proteins expressed with an amino-terminal hexa-histidine tag can be purified by binding to nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography matrix (The QL4expressionist, QIAGEN, 1997). If the recombinant latency-specific protein is produced in a secreted form, e.g., secreted into the milk of a transgenic animal, purification can be from the secreted fluid. Alternatively, 30 purification may be unnecessary if it is appropriate to apply the latency-specific protein directly to the subject in the secreted fluid (e.g., milk), for instance to induce an immunological response in a subject. B. Production of antibodies Monoclonal or polyclonal antibodies may be produced to M tuberculosis latency-specific 35 proteins, or to specific epitopes within such proteins. Optimally, antibodies raised against a latency specific protein would specifically detect that protein. That is, such antibodies would recognize and bind the Acr protein and would not substantially recognize or bind to other proteins found in a biological sample. The determination that an antibody specifically detects its target latency-specific protein is made by any one of a number of standard immunoassay methods; for instance, the Western -23 blotting technique (Sambrook et al., In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989). To determine that a given antibody preparation (such as one produced in a mouse) specifically detects the target protein by Western blotting, total cellular protein is extracted from cells 5 of a latent M tuberculosis preparation, such as granulomas, and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. The proteins are then transferred to a membrane (for example, nitrocellulose) by Western blotting, and the test antibody preparation is incubated with the membrane. After washing the membrane to remove non-specifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti-mouse antibody conjugated to an 10 enzyme such as alkaline phosphatase. Application of an alkaline phosphatase substrate 5-bromo-4 chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immunolocalized alkaline phosphatase. Antibodies that specifically detect the target latency specific protein will, by this technique, be shown to bind to the target latency-specific protein band (which will be localized at a given position on the gel determined by its molecular weight). Non 15 specific binding of the antibody to other proteins may occur and may be detectable as a weak signal on the Western blot. The non-specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific antibody-latency-specific protein binding. Substantially pure latency-specific protein suitable for use as an immunogen is isolated from 20 the transfected or transformed cells as described above. Concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms per milliliter. Monoclonal or polyclonal antibody to the protein can then be prepared as follows: 25 i. Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibody to epitopes of a latency-specific protein identified and isolated as described (e.g., Acr) can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256:495, 1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. 30 The mouse is then sacrificed, and the antibody-producing cells of the spleen are isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess un-fused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by 35 detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Enzymol. 70:419, 1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988).
- 24 ii. Polyclonal Antibody Production by Immunization Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein, which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many 5 factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with either inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appear to be most reliable. An effective immunization protocol for rabbits can be found in 10 Vaitukaitis et al. (J. Clin. Endocrinol. Metab. 33:988-991, 1971). Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony et al. (In Handbook ofExperimental Immunology, Wier, D. (ed.) chapter 19. Blackwell, 1973). Plateau 15 concentration of antibody is usually in the range of about 0.1 to 02 mg/ml of serum (about 12 pM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (Manual of Clinical Immunology, Ch. 42, 1980). iii. Antibodies Raised against Synthetic Peptides A third approach to raising antibodies against a latency-specific protein is to use synthetic 20 peptides synthesized on a commercially available peptide synthesizer based upon the predicted amino acid sequence of the latency-specific protein. By way of example only, polyclonal antibodies to specific peptides within Acr can be generated through well-known techniques by injecting rabbits with chemically synthesized peptide. iv. Antibodies Raised by Injection of Latency-Specific Protein Encoding Sequence 25 Antibodies may be raised against a latency-specific protein by subcutaneous injection of a DNA vector that expresses the latency-specific protein into laboratory animals, such as mice. Delivery of the recombinant vector into the animals may be achieved using a hand-held form of the Biolistic system (Sanford et al., Particulate Sci. Technol. 5:27-37, 1987) as described by Tang et al. (Nature 356:152-154, 1992). Expression vectors suitable for this purpose may include those that 30 express the Z47 encoding sequence under the transcriptional control of either the human p-actin promoter or the cytomegalovirus (CMV) promoter. Antibody preparations prepared against a latency-specific antigen or epitope of such are useful in quantitative immunoassays that determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of 35 antigen in a biological sample, as described herein. IX. Stimulation of immunological Responses to Latent Tuberculosis With the provision herein of antigens specific to latent tuberculosis infections, methods are now enabled for the stimulation of immune responses to such antigens in subjects. In certain -25 embodiments, such immune responses will be protective against formation of latent tuberculosis infection in the subject. Latency-specific proteins (e.g., Acr) can be used, for instance, as immunogenic agents in the treatment, amelioration, or prevention of latent tuberculosis. Subjects selected for this type of treatment are those who are known to have, or are suspected of having or are 5 at risk of suffering, a latent tuberculosis infection. An example of such a person is someone who has a positive tuberculin skin test, but has no or limited evidence of active disease (for example clinical symptoms such as fatigue, anorexia, weight loss, fever, nocturnal diaphoresis, cough, hemoptysis, or radiographic or other laboratory evidence recognized as indicative of active disease). The provided immunostimulatory proteins or peptides, derived from latency-specific 10 proteins (such as Acr) are combined with a pharmaceutically acceptable carrier or vehicle for administration as an immunostimulatory composition or a vaccine to human or animal subjects. In some embodiments, more than one protein or peptide fragment may be combined to form a single preparation. The immunogenic formulations may be conveniently presented in unit dosage form and 15 prepared using conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes 20 which render the formulation isotonic with the blood of the intended recipient; and aqueous and non aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous 25 injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by one of ordinary skill in the art. In certain embodiments, unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients particularly mentioned above, formulations encompassed heroin may include other 30 agents commonly used by one of ordinary skill in the art. The compositions provided herein, including those for use as immunostimulatory agents or vaccines, may be administered through different routes, such as oral, including buccal and sublingual, rectal, parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical. They may be administered in different forms, including but not limited to solutions, emulsions and suspensions, 35 microspheres, particles, microparticles, nanoparticles, and liposomes. The volume of administration will vary depending on the route of administration. By way of example, intramuscular injections may range from about 0.1 ml to 1.0 ml. Those of ordinary skill in the art will know appropriate volumes for different routes of administration.
- 26 The amount of protein in each vaccine dose is selected as an amount that induces an immunoprotective response without significant, adverse side effects. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Initial injections may range from about I pg to I mg, with some embodiments having a range of about 10 pg to 800 5 pg, and still other embodiments a range of from approximately 25 jig to 500 pg. Following an initial vaccination, subjects may receive one or several booster immunizations, adequately spaced. Booster injections may range from I pig to 1 mg, with other embodiments having a range of approximately 10 gg to 750 jg, and still others a range of about 50 sg to 500 pg. Periodic boosters at intervals of 1-5 years, for instance three years, may be desirable to maintain the desired levels of protective 10 immunity. As described in WO 95/01441, the course of the immunization may be followed by in vitro proliferation assays of PBL (peripheral blood lymphocytes) co-cultured with ESAT6 or ST-CF, and especially by measuring the levels of IFN-released from the primed lymphocytes. The assays are well known and are widely described in the literature, including in U.S. Patent Nos. 3,791,932; 15 4,174,384 and 3,949,064. A recent development in the field of immune stimulatory compounds (e.g., vaccines) is the direct injection of nucleic acid molecules encoding peptide antigens (broadly described in Janeway & Travers, Jinmunobiology: The immune System In Health and Disease, page 13.25, Garland Publishing, Inc., New York, 1997; and McDonnell & Askari, N. Engl. J Med. 334:42-45, 1996). 20 Plasmids that include nucleic acid molecules described herein, or that include a nucleic acid sequence encoding an immunogenic peptide or peptide fragment of a latency specific polypeptide (such as Acr) or derived from a latency specific polypeptide (for instance, as a fusion protein, may be utilized in such DNA vaccination methods. Thus, the terms "immunostimulatory preparation" and "vaccine" as used herein also include 25 nucleic acid vaccines in which a nucleic acid molecule encoding a latency-specific polypeptide (such as Acr), or a fragment thereof, is administered to a subject in a pharmaceutical composition. For genetic immunization, suitable delivery methods known to those skilled in the art include direct injection of plasmid DNA into muscles (Wolff et al., Hum. Mol. Genet. 1:363, 1992), delivery of DNA complexed with specific protein carriers (Wu et al., J Biol. Chem. 264:16985, 1989), co 30 precipitation of DNA with calcium phosphate (Benvenisty and Reshef, Proc. Nat. A cad. Sci. 83:9551, 1986), encapsulation of DNA in liposomes (Kaneda et al., Science 243:375, 1989), particle bombardment (Tang et al., Nature 356:152, 1992) and (Eisenbraun et al., DNA Cell Biol. 12:791, 1993), and in vivo infection using cloned retroviral vectors (Seeger et al., Proc. Nat!. Acad Sci. 81:5849, 1984). 35 Similarly, nucleic acid vaccine preparations can be administered via viral carrier. It is also contemplated that the provided immunostimulatory molecules and preparations can be administered to a subject indirectly, by first stimulating a cell in vitro, which stimulated cell is thereafter administered to the subject to elicit an immune response.
-27 X immunological and Pharmaceutical Compositions Immunological compositions, including immunological elicitor compositions and vaccines, and other pharmaceutical compositions containing latency-specific polypeptides or antigenic fragments thereof are useful for reducing, ameliorating, treating, or possibly preventing 5 mycobacterial infection, particularly latent M tuberculosis infection. One or more of the polypeptides are formulated and packaged, alone or in combination with adjuvants or other antigens, using methods and materials known to those skilled in the vaccine art. An immunological response of a subject to such an immunological composition may be used therapeutically or prophylactically, and in certain embodiments provides antibody immunity and/or cellular immunity such as that 10 produced by T lymphocytes such as cytotoxic T lymphocytes or CD4* T lymphocytes. To enhance immunogenicity, one or more immunogenic polypeptides or fragments (e.g., haptens) may be conjugated to a carrier molecule. Immunogenic carrier molecules include proteins, polypeptides or peptides such as albumin, hemocyanin, thyroglobulin and derivatives thereof, particularly bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), polysaccharides, 15 carbohydrates, polymers, and solid phases. Other protein-derived or non-protein-derived substances are known to those of ordinary skill in the art. An immunogenic carrier typically has a molecular weight of at least 1,000 Daltons, and in some embodiments greater than 10,000 Daltons. Carrier molecules often contain a reactive group to facilitate covalent conjugation to the hapten. The carboxylic acid group or amine group of amino acids or the sugar groups of glycoproteins are often 20 used in this manner. Carriers lacking such groups can often be reacted with an appropriate chemical to produce them. Alternatively, a multiple antigenic polypeptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide may be sufficiently antigenic to improve immunogenicity without the use of a carrier. The latency-specific polypeptides may be administered with an adjuvant in an amount 25 effective to enhance the immunogenic response against the conjugate. At this time, the only adjuvant widely used in humans has been alum (aluminum phosphate or aluminum hydroxide). Saponin and its purified component Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications have toxicities which limit their potential use in human vaccines. However, chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid 30 conjugates such as those described by Goodman-Snitkoff et al. (J. Immunol. 147:410-415, 1991), encapsulation of the conjugate within a proteoliposome as described by Miller et al. (J. Exp. Med 176:1739-1744, 1992), and encapsulation of the protein in lipid vesicles may also be useful. The compositions provided herein, including those formulated to serve as vaccines, may be stored at temperatures of from about -100' C to 4' C. They may also be stored in a lyophilized state 35 at different temperatures, including higher temperatures such as room temperature. The preparation may be sterilized through conventional means known to one of ordinary skill in the art. Such means include, but are not limited to filtration, radiation and heat. The preparations also may be combined with bacteriostatic agents, such as thimerosal, to inhibit bacterial growth.
-28 A variety of adjuvants known to one of ordinary skill in the art may be administered in conjunction with the protein(s) in the provided vaccine composition. Such adjuvants include but are not limited to the following: polymers, co-polymers such as polyoxyethylene-polyoxypropylene copolymers, including block co-polymers; polymer P1005; Freund's complete adjuvant (for animals); 5 Freund's incomplete adjuvant; sorbitan monooleate; squalene; CRL-8300 adjuvant; alum; QS 21, muramyl dipeptide; CpG oligonucleotide motifs and combinations of CpG oligonucleotide motifs; trehalose; bacterial extracts, including mycobacterial extracts; detoxified endotoxins; membrane lipids; or combinations thereof. In a particular embodiment, a vaccine is packaged in a single dosage for immunization by 10 parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. In certain embodiments, the vaccine is injected intramuscularly into the deltoid muscle. The vaccine may be combined with a pharmaceutically acceptable carrier to facilitate administration. The carrier is, for instance, water, or a buffered saline, with or without a preservative. The vaccine may be lyophilized for resuspension at the time of administration or in solution. 15 The carrier to which the polypeptide may be conjugated may also be a polymeric delayed release system. Synthetic polymers are particularly useful in the formulation of a vaccine to effect the controlled release of antigens. Microencapsulation of the polypeptide will also give a controlled release. A number of factors contribute to the selection of a particular polymer for microencapsulation. The reproducibility 20 of polymer synthesis and the microencapsulation process, the cost of the microencapsulation materials and process, the toxicological profile, the requirements for variable release kinetics and the physicochemical compatibility of the polymer and the antigens are all factors that must be considered. Examples of useful polymers are polycarbonates, polyesters, polyurethanes, polyorthoesters polyamides, poly-(dl-lactide-co-glycolide) (PLGA) and other biodegradable 25 polymers. Doses for human administration of a pharmaceutical composition or a vaccine may be from about 0.01 mg/kg to 10 mg/kg, for instance approximately 1 mg/kg. Based on this range, equivalent dosages for heavier (or lighter) body weights can be determined. The dose may be adjusted to suit the individual to whom the composition is administered, and may vary with age, weight, and 30 metabolism of the individual, as well as the health of the subject. Such determinations are left to the attending physician or another familiar with the subject and/or the specific situation. The vaccine may additionally contain stabilizers or physiologically acceptable preservatives, such as thimerosal (ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma Chemical Co., St. Louis, MO). 35 X. Kits Kits are provided which contain the necessary reagents for growing in vitro granulomas or for determining the presence (or absence) of a latency-specific antigen and/or antibody in a biological sample, using an immunological binding reaction. Instructions provided in the diagnostic kits can -29 include calibration curves, diagrams, illustrations, or charts or the like to compare with the determined (e.g., experimentally measured) values or other results. A. Kits for Growing In Vitro Granulomas 5 Kits for growing in vitro granulomas include, for instance, cell culture media (e.g., RPMI plus 10% human serum, HS) and optionally may include a low attachment container (e.g., a tissue culture dish treated to inhibit all attachment), a filter, and/or a fixative. Specific examples of such kits also include an amount of one or more cytokine, for instance IL-2, IFN-y, and/or TNF-a. Reagents supplied in the kits may be contained in separate containers. 10 The kits may also include means for granuloma analysis, for instance ELISA reagents, reagents for RT-PCR, and/or RPA reagents, which may also be provided in some kits in one or more separate containers. Cell culture, ELISA, RT-PCR, and RPA techniques are well known to those of ordinary skill in the art. Reaction vessels and auxiliary reagents such as buffers, enzymes, etc. may also be included 15 inthekits. Additional components in some kits include instructions for carrying out the cell culture and/or subsequent analysis. Where provided, instructions may allow the tester to grow in vitro granulomas and use them to identify latency-specific antigens and screen drugs and immunostimulatory compounds, such as vaccines. 20 B. Kits For Detection of Latency-Specific Antigen Kits for the detection of latency-specific M. tuberculosis protein expression include for instance at least one target protein specific binding agent (e.g., a polyclonal or monoclonal antibody or antibody fragment) and may include at least one control. The latency-specific protein specific binding agent and control may be contained in separate containers. 'Te kits may also include means 25 for detecting target protein:agent complexes, for instance the agent may be detectably labeled. If the detectable agent is not labeled, it may be detected by second antibodies or protein A, for example, which may also be provided in some kits in one or more separate containers. Such techniques are well known. Additional components in some kits include instructions for carrying out the assay. 30 Instructions will allow the tester to determine whether latency-specific protein expression levels are altered, for instance in comparison to a control sample. Reaction vessels and auxiliary reagents such as chro'mogens, buffers, enzymes, etc. may also be included in the kits. By way of example only, an effective and convenient immunoassay kit such as an enzyme linked immunosorbent assay can be constructed to test anti-Acr antibody in human serum. 35 Expression vectors can be constructed using the Acr cDNA to produce the recombinant Acr protein in either bacteria or baculovirus (as described above). By affinity purification, unlimited amounts of pure recombinant latency-specific protein (such as Acr) can be produced.
-30 C. Kits for Detection ofAntibody to Latency-Specifc Antigens Other examples of assay kits provide a recombinant latency-specific protein as an antigen and enzyme-conjugated goat anti-human IgG as a second antibody. Examples of such kits also can include one or more enzymatic substrates. Such kits can be used to test if a biological sample from a 5 subject contains antibodies against a latency-specific protein. The disclosure is further illustrated by the following non-limiting Examples. 10 EXAMPLES Example 1: Preparation and Characterization of an In Vitro Granuloma Model The principal defense of the human host against a mycobacterial infection is the formation of granulomas, which are compact, organized collections of activated macrophages, including 15 epithelioid and multinucleated giant cells, surrounded by T lymphocytes, and later by fibroblasts and collagen that aggregate around the macrophage core. The granuloma may prevent active (non-latent) disease by sequestering the invading organisms. If the granuloma is maintained, these bacteria may remain latent for many years. To study this process of granuloma formation and the granuloma's subsequent breakdown 20 when host defenses are compromised, an in vitro model was developed. This example provides a description of one method for producing the in vitro granuloma that can be used as a model system, as well as several methods used to characterize the model. In overview, human peripheral blood mononuclear cells, autologous macrophages and mycobacteria were combined in low attachment tissue culture dishes. The resulting aggregates were characterized using microscopy and 25 immunohistochemical staining. Cytokine production was assessed by ELISA and bacterial mRNA detected by RT-PCR. Peripheral blood mononuclear cells (1 x 106), autologous macrophages (I x 106) and mycobacteria (I x 10') were combined in low attachment tissue culture dishes (COSTARTM Utra Low Attachment Clusters, Costar Corp., Cambridge, MA) and incubated at 37 'C in 5% CO 2 . 30 Human peripheral blood mononuclear cells (PBMCs, 1 X 10' cells in RPMI plus 10% HS) were added after 24 hours, and the mixture incubated for 5-7 days. Aggregate formation was observed. Humaii-lung fibroblasts (from the cell line 33Lu) were added when the aggregates were approximately 1 mm in diameter. The aggregates were characterized using microscopy and immunohistochemical staining 35 with standard methodology. Small, rounded aggregate structures were formed in the cultures, which developed more defined edges with the addition of human lung fibroblasts. Microscopic examination of these aggregates using immunostaining found CD68* epithelioid macrophages and sparse, small round CD3 4 lymphocytes that, in complex, resembled small granulomas seen in clinicopathologic -31 specimens. Acid-fast staining bacteria were observed within and between the cells composing the granulomas (Figure 1). In addition to morphology, cytokine production was assessed by ELISA. In particular, cytokines that are known to be upregulated during early stage M tuberculosis infection were 5 analyzed. By 24 hours following infection, the aggregates were found to generate levels of TNF, IL-8, and IL-6 that were elevated well above levels found in uninfected control cells. After 48 hours levels of IFN were likewise increased above controls. This elevated cytokine production continued over the nine day duration of the experiment. Results also indicate increased levels of IL-2 and IL-12, 10 peaking at 48 hours, but remaining above control levels throughout the course of the experiment. All of these cytokines are detected at significantly higher levels in tuberculosis patients when compared to healthy controls. Example 2: Detection of Differential Transcription of Acr mRNA in a Granuloma Model 15 Using bacteria cultured in an anoxic chamber, M. tuberculosis genes were identified that were differentially expressed after much of the available oxygen had been utilized. The genes that were differentially expressed were acr, sigF, oxyR and aphC. Of these four genes, acr encodes a protein (ct-crystallin) that is secreted by the Mycobacterium. In order to confin that these genes are expressed in the in vitro granuloma model, RT-PCR 20 and RNA protection assays were performed. These assays showed that mRNA from mycobacterial genes acr, aphC, and sigF were transcribed. These transcripts were not found in uninfected in vitro granuloma controls. Typical results from representative experiments are shown in Figures 2 and 3, which are ribonuclease protection assay (RPA) blots. In Figure 2, acr mRNA was observed at all four time 25 points while RpoB mRNA was only observed in aerobically grown cultures. In Figure 3, acr and RpoB mRNA were observed both at 7- or 12-day incubations in the in vitro granuloma model, which is believed to indicate that aerobic bacilli were present in the granuloma. Example 3: Other In Vitro Latency Models . 30 The following example provides other in vitro latency models, which can be used, for example, to confirm results obtained with the in vitro granuloma model (e.g., to screen drugs and immunostimulatory compounds and to identify latency-specific antigens). Guinea pig aerosol infection model. 35 When infected by aerosol inoculation using a small number of M tuberculosis bacilli, it was observed that the bacteria cause formation of granulomas associated with the epithelial pneumocytes in the deep alveoli of the lung. Though these granulomas apparently are not able to completely contain the infection, and the bacteria eventually overwhelm this animal, there are a number of similarities with human granulomas. For example, these granulomas center on necrotic areas and -32 contain predominantly macrophages, including epithelioid and multinucleated giant cells and T lymphocytes. Differential transcription of the bacterial Acr gene has been detected in these granulomas. 5 In vitro anoxic chamber models. Wayne and Hayes developed an in vitro persistence model that subject M tuberculosis bacilli to gradual oxygen deprivation by incubation in sealed containers with controlled agitation (Wayne and Hayes, Infect Immun. 64:2062-2069, 1996). Growth under these conditions can be operationally divided into two non-replicating persistent (NRP) states: a microaerophilic state 10 associated with induction of glycine dehydrogenase activity (NRPI), and a subsequent lower oxygen state (NRP2), in which glycine dehydrogenase activity declines and alterations in drug susceptibility are manifest. Specifically, cells become resistant to ciprofloxicin and sensitive to metronidazole, possible due to changes in DNA superhelicity and cell permeability, respectively (Wayne and Hayes, Infect. Immun. 64:2062-2069, 1996). These observations correlate with the intractability of clinical 15 TB to single antibiotic therapy (Dickinson and Mitchison, Am. Rev. Respir. Dis. 123:367-371, 1981). A modified version of this sealed vessel has been developed (Figure 5), which allows monitoring of several environmental growth conditions (including optical density culture population, oxygen concentration, pH, and assaying of enzymes induced only under low oxygen tension), as well as providing containment of the pathogen and easy nucleic acid harvest by direct centrifugation of the 20 vessel. Using this system, it has been shown that M tuberculosis bacilli cease to replicate but remain metabolically active for several months under conditions of low oxygen (Figure 12). Example 4: Construction of Acr-FLAG fusion proteins. Oligonucleotide primers for polymerase chain reaction (PCR) were designed and generated. 25 These primers were designed to allow the amplification of the hspX gene from M tuberculosis (encoding Acr) with the addition of the FLAG (Sigma) epitope tag fused to amino- or carboxy terminus of Acr depending on which primer pair was used. Primer design also included the introduction of restriction endonuclease recognition sites to facilitate subsequent recombinant DNA methodologies involved in cloning and expression of these amplified sequences. Sequences of the 30 primers to generate the N-terminal FLAG-Acr fusion were SEQ ID NO: land SEQ ID NO: 2. Sequences of the primers to generate the C-terminal Acr-FLAG fusion were SEQ ID NO: 3 and SEQ IDNOI4. PCR reactions were performed using one microgram MTB H37Rv genomic DNA as template and 20 mM Tris-HCI, pH 8.4, 50 mM KCI, 1.5 mM MgCl 2 , and 2.5 units AmpliTaq (Gibco 35 BRL) thernostable DNA polymerase. PCR was performed with the following cycle parameters: 94 *C, 5 minutes (lX), 94 *C, 1 minute; 55 *C, 30 seconds; 72 *C, 2.5 minutes (2x), 94 *C, 1 minute; 60 *C, 30 seconds; 72 *C, 2.5 minutes (30x), 72 "C, 7 minutes (Ix). Following PCR amplification the amplified DNA fragments were purified and digested by restriction endonuclease, and ligated into - 33 likewise digested plasmid vector pMV261.1. Following ligation, the reactions were transformed into E. coli and positive colonies selected by antibiotic resistance. Selected colonies were screened for the presence of the recombinant insert by PCR, and sequence verification was achieved by DNA sequencing of each positive clone. Positive N-terminal and C-terminal Act-FLAG fusion plasmids 5 were subsequently transformed into both M. smegmatis and M tuberculosis and transformed colonies selected by antibiotic resistance. Expression of the fusion proteins was confirmed by Western blotting; representative blots are shown in Figure 6 (N-terminal fusion) and Figure 7 (C-terminal fusion). 10 Example 5: Immunoprecipitation ofAcr. Acr-FLAG fusion proteins were immunoprecipitated with a FLAG Immunoprecipitation Kit acquired from Sigma according to the manufacturer's instructions. Native Acr immunoprecipitations were performed on mycobacterial lysates using sepharose CL4B beads (Pharmacia) to which polyclonal rabbit antibodies generated against Acr had been conjugated according to the 15 manufacturer's instructions. Briefly, a pre-clearing step was performed to remove any proteins from the lysate that might non-specifically bind to the sepharose beads. This was accomplished by adding 50 tl of unconjugated sepharose to 975 si of TSA (0.01M Tris-HCl, pH 8; 0. 14M NaCl; 0.025% NaN 3 ) and 16 pl of MTB lysate (2.3 mg/mL) in a 1.5 mL microfuge tube. The solution was incubated for one hr at 4 *C with constant rocking. The solution was then centrifuged for 5 sec at 20 14,000 rpm to pellet the sepharose and the supernatant removed and transferred to a new microfuge tube. 25 pl of anti-Acr antibody conjugated sepharose beads were added to the supernatant and incubated for one hour at 4 "C with constant rocking. The solution was then centrifuged as previously described and the supernatant was removed and discarded. The sepharose pellet was then washed 4 times with 1 mL each of 1) 0.1% Triton X-100 in TSA, 2) 0.1% Triton X-100 in TSA, 3) 25 TSA, 4) 0.05M Tris-HCI, pH 6.8. Addition of each solution was followed by resuspension of the sepharose pellet, a 5-second centrifugation to repellet the sepharose, and removal of the wash supernatant. Finally, 40 1 of 2x gel loading buffer (Novex) was added to the sepharose pellet, and the suspension incubated for 1.5 hours at 56 *C. A final centrifugation was performed to re-pellet the sepharose and the supernatant (containing the immunoprecipitated Acr protein) was removed and 30 saved for analysis by Western blot. Example 6: Western Blot. Immunoprecipitated Acr samples or mycobacterial lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) with pre-cast 4-12% bis-Tris polyacrylamide 35 gradient gels (Novex) for one hour at 200 volts constant. Following electrophoresis, the resolved proteins were transferred to a pre-cut 0.2-micron polyvinylidene difluoride membrane (PVDF, Novex) according to the manufacturer's instructions by electrocapillary transfer at 150 milliamps constant for one hour. Dried membranes were prepared for Acr detection by immersion in methanol and unconjugated PVDF surfaces were blocked by incubation of the membrane in TBS Tween 20 - 34 containing 3% gelatin (BioRad) for one hour with constant rocking. The membrane was then washed 3 times for 10 minutes each with TBS + Tween 20 with constant rocking. Detection of Acr was accomplished by incubation of the membrane with a 1 to 5,000 dilution of polyclonal rabbit antibodies generated against Acr, or a 1:20,000 anti-FLAG M2 5 monoclonal antibody (Sigma), or a I to 250 dilution of rabbit anti-phosphotyrosine antibody (Zymed) in TBS + Tween 20 containing 1% gelatin for one hour with constant rocking. Subsequently, the membrane was washed as described above. A one-to-I 0,000 dilution of biotinylated goat-anti-rabbit IgG in TBS + Tween 20 with 1% gelatin was then applied to the blot, and incubated for one hour with constant rocking. Subsequently, the blot was washed as described above. A final incubation of 10 the blot with a I to 10,000 dilution of a streptavidin - alkaline phosphatase (AP) conjugate in TBS + Tween 20 was incubated with the blot for 30 minutes with constant rocking. Detection of the presence of AP was accomplished by the incubation of the blot in a solution of NBT+BCIP (Novex). The incubation was halted when the desired level of color development was achieved, and stopped by extensive washing of the blot in water. Blots were dried for archiving. 15 Figure 8 shows five strips cut from a Western blot prepared as described of whole cell mycobacteria cell lysates; the arrows indicate the location of recombinant Acr protein. The strips were developed with the indicated primary antibodies. In addition to the control antibody, this blot demonstrates the presence of tyrosine phosphorylation in the final strip. Figure 9 shows two Western blots prepared as described from culture supernatants of M 20 tuberculosis bacilli grown under the conditions indicated in the Brief Description of the Figures. Figure 9A was probed using rabbit anti-Acr antibody; Figure 9B is a control blot. Acr protein is detected in 7 day and 12 month anoxic cultures and in vitro granuloma. Lower molecular weight variants are observed in the 12 month and in vitro granuloma supernatants. For comparison, Figure I1 shows s a Coomassie stained SDS-PAGE gel of culture supematants from M tuberculosis bacilli 25 cultured under the indicated conditions. Example 7: 2- Dimensional Gel Electrophoresis. Lysates of M smegmatis and M tuberculosis carrying the Acr-FLAG fusion plasmids were prepared. Bacterial suspension cultures were pelleted by centrifugation (5,000 rpm, 10 minutes) and 30 the bacterial pellet washed with phosphate buffered saline (PBS). Pellets were then resuspended in 1 mL of 9M urea and the suspension transferred to 2 mL microfuge tubes containing approximately 200 il of 0.1 mm diameter glass beads (Biospec products). The cells were then lysed by rapid shaking (three times, one minute each) in a Minibeadbeater (Biospec products). Following lysis, the cellular debris and glass beads were peleted by centrifugation and the supernatant removed. Protein 35 quantification was performed using the colorimetric BioRad protein assay (BioRad). For resolution of the protein sample in the first dimension, 150 micrograms of protein was added to 92.5 pl of solubilization buffer (9M Urea, 140 mM DTT, 4% Triton X-100) and brought to a final volume of 185 pL with 9M urea. The suspension was incubated for one hour with constant rocking at room temperature. Following this incubation, 1 I Biolytes electrolyte solution (BioRad, - 35 0.2% final), and several crystals of Bromophenol Blue (Fisher) were added. Hydration of the IPG strips and application of the protein solution to the strips was carried out overnight in a IPG strip hydration apparatus (Pharmacia). Following hydration the strips were subjected to electrophoresis in a LKB MultiPhor I apparatus (Pharmacia) using a Pharmacia EPS 3500 XL electrophoresis 5 apparatus with the following parameters: 350V, 30 minutes, 350V-3500V (1.5 hour gradient), 3500V, 3.5 hours, to give a final running time of 15,200 Volt-hours. For running the second dimension, the resolved IPG strips were equilibrated in equilibration buffer (6M Urea, 0.375M Tris, pH 8.8, 2% SDS, and 20% Glycerol) containing 2% w/v DTT (10 minutes) and 2.5% iodoacetamide (10 minutes). The equilibrated strips were resolved on Criterion 10 (BioRad) 8-16% polyacrylamide gels according to the manufacturer's instructions for one hour at 200 volts constant. Figure 10 is a representative two-dimensional gel electrophoresis analysis of a sample taken from M tuberculosis grown under anoxic conditions. Acr protein is indicated by the circle. 15 Example 8: Detection of Acr Using polyclonal antibodies produced against the cloned M tuberculosis Acr protein, secreted bacterial Acr was detected in culture supematants from anoxic chamber and the in vitro granuloma model (Figure 9). Polyclonal antiserum to Acr protein was also used to detect mycobacteria in aerosol infected guinea pig lung granuloma tissue (Figure 1C). 20 Acr protein was detected in supernatants and cell lysates from anoxic but not aerobic grown M. tuberculosis (Figure 9). Acr was immunoprecipitated from M tuberculosis lysates. Changes in molecular weight of Acr were detected in anoxic or infected in vitro granulomas but not aerobic grown cultures (Figure 9). M. tuberculosis acr mRNA was detected in anoxic but not aerobic cultures and acr and eukaryotic actin mRNA in infected but not uninfected control guinea pig lungs 25 (Figure 4). This disclosure provides an in vitro granuloma model and methods of use, as well as immunological methods for the detection of latent tuberculosis in a subject. The in vitro granuloma model can be used, for example, to identify latency-specific antibodies and to screen drugs and 30 immunostimulatory compounds. The immunological methods can include, for example, using a latency-specific M tuberculosis antigen to detect a corresponding antibody from the subject, or using an antibody to detect the latency-specific antigen. The disclosure further provides methods for identifying latency-specific antigens (and their corresponding antibodies) for use in such methods, and specific latency-specific antigens such as ct-crystallin. It will be apparent that the precise details 35 of the methods described may be varied or modified without departing from the spirit of the described disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.
Claims (22)
1. An in vitro granuloma comprising peripheral blood mononuclear cells, macrophages, and mycobacteria. 5
2. An in vitro granuloma according to claim 1, wherein the peripheral blood mononuclear cells are human peripheral blood mononuclear cells selected from the group consisting of monocytes, B lymphocytes, T lymphocytes, and combinations thereof. 10
3. An in vitro granuloma according to claim I or claim 2, wherein the mycobacteria are M. tuberculosis.
4. An in vitro granuloma according to any one of claims 1 to 3, further 15 comprising fibroblasts.
5. A method for producing an in vitro granuloma, comprising combining peripheral blood mononuclear cells, macrophages, and mycobacteria in a low attachment container and incubating the combination for a sufficient amount of time to 20 form the in vitro granuloma.
6. A method according to claim 5, wherein fibroblasts are added to the combination. 25
7. A method according to claim 5, further comprising adding exogenous cytokine to the container in sufficient amount to enhance production of the in vitro granuloma.
8. A method according to claim 7, wherein the exogenous cytokine is IL-2, 30 IFN-y, TNF-a, or a combination of two or more thereof.
9. A method of screening a tuberculosis drug candidate for anti tuberculosis therapeutic activity, comprising combining the drug with an in vitro granuloma comprising peripheral blood mononuclear cells, macrophages, and 35 mycobacteria, and determining whether the drug inhibits mycobacterial viability. N:\Melboue\ Cse\Pat t\40000-.400Q\P40867 AU I\Specis\P49867.AU.I Specification 2007-1 1-16 doc 16/11/07 - 37
10. A method according to any one of claims 5 to 9, wherein the peripheral blood mononuclear cells are human peripheral blood mononuclear cells selected from the group consisting of monocytes, B lymphocytes, T lymphocytes, and combinations thereof. 5
11. A method of screening a tuberculosis drug candidate for anti tuberculosis therapeutic activity, comprising combining the drug with an in vitro granuloma comprising peripheral blood mononuclear cells, macrophages, and inactivated mycobacteria, and determining whether the drug inhibits reactivation of 10 mycobacteria contained in the granuloma.
12. A method according to any one of claims 5 to 11, wherein the mycobacteria are M tuberculosis. 15
13. A method of screening a tuberculosis vaccine candidate, comprising determining whether a mutant mycobacterium has a reduced ability, when compared against a wild type mycobacterium, to induce latency, survive, reactivate or induce granuloma necrosis in an in vitro granuloma comprising peripheral blood mononuclear cells, macrophages, and the mutant mycobacteria. 20
14. A method according to any one of claims 9 to 13, wherein the in vitro granuloma further comprises fibroblasts.
15. A method according to claim 13, wherein the mutant mycobacterium 25 comprises a mycobacterial strain having a mutation in a latency gene.
16. A method according to claim 13 or claim 15, wherein the mutant mycobacterium is a Mycobacterium tuberculosis strain having a mutation in a gene selected from the group consisting of acr, a sigma factor gene, oxyR and aphC. 30
17. A method according to claim 16, wherein the sigma factor gene is selected from the group consisting of sigF, sigC, and sigH.
18. A kit comprising a culture medium, when used in the method according 35 to any one of claims 5 to 8. 728351_1 (GHMattcrs) 5/03/10 - 38
19. A kit according to claim 18, further comprising a low attachment container.
20. A kit according to claim 18, further comprising an amount of a cytokine. 5
21. An in vitro granuloma according to any one of claims 1 to 4, or a method according to any one of claims 5 to 17, wherein the macrophages comprise autologous macrophages. 10
22. An in vitro granuloma according to claim 1, a method according to any one of claims 5, 9, 11 and 13, or a kit according to claim 18, substantially as herein described with reference to any one or more of the examples and/or figures. N.\Melbournc\Cases\Patcnt\49000.4999%P49867AU l\Specis\P49867 AU.I Specification 2007-11-16.doc 16/11/07
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2007234526A AU2007234526B2 (en) | 2001-01-08 | 2007-11-16 | Latent human tuberculosis model, diagnostic antigens, and methods of use |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US60/260,348 | 2001-01-08 | ||
US60/311,235 | 2001-08-09 | ||
AU2002237764A AU2002237764B2 (en) | 2001-01-08 | 2002-01-07 | Latent human tuberculosis model, diagnostic antigens, and methods of use |
AU2007234526A AU2007234526B2 (en) | 2001-01-08 | 2007-11-16 | Latent human tuberculosis model, diagnostic antigens, and methods of use |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2002237764A Division AU2002237764B2 (en) | 2001-01-08 | 2002-01-07 | Latent human tuberculosis model, diagnostic antigens, and methods of use |
Publications (2)
Publication Number | Publication Date |
---|---|
AU2007234526A1 AU2007234526A1 (en) | 2007-12-06 |
AU2007234526B2 true AU2007234526B2 (en) | 2010-04-01 |
Family
ID=38819951
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2007234526A Expired AU2007234526B2 (en) | 2001-01-08 | 2007-11-16 | Latent human tuberculosis model, diagnostic antigens, and methods of use |
Country Status (1)
Country | Link |
---|---|
AU (1) | AU2007234526B2 (en) |
-
2007
- 2007-11-16 AU AU2007234526A patent/AU2007234526B2/en not_active Expired
Also Published As
Publication number | Publication date |
---|---|
AU2007234526A1 (en) | 2007-12-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2002237764B2 (en) | Latent human tuberculosis model, diagnostic antigens, and methods of use | |
AU2002237764A1 (en) | Latent human tuberculosis model, diagnostic antigens, and methods of use | |
WO2021194940A1 (en) | Sars-cov-2-specific t cells and methods of treatment using them | |
JP2008515388A (en) | Diagnostic and therapeutic methods for Mycobacterium tuberculosis infection and reagents therefor | |
US8865422B2 (en) | Method for the diagnosis of tuberculosis | |
WO2013075608A1 (en) | Chimeric recombinant antigen and use thereof | |
Triccas et al. | Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein | |
HUE034632T2 (en) | Improved vaccine diagnostics | |
ZA200400843B (en) | Early detection of mycobacterial disease using peptides | |
WO1991014448A1 (en) | New proteins, peptides and corresponding dna or rna sequences and probes useful for diagnosing tuberculosis | |
US7807182B2 (en) | Early detection of mycobacterial disease using peptides | |
AU2007234526B2 (en) | Latent human tuberculosis model, diagnostic antigens, and methods of use | |
US20100209936A1 (en) | Novel recombinant 15-kDa polypeptide and use of same in detecting human infection with Bartonella henselae | |
US6572865B1 (en) | Mycobacterium tuberculosis DNA sequences encoding immunostimulatory peptides and methods for using same | |
US20090068218A1 (en) | Antigens for Vaccination Against and Detection of Mycoplasma Suis | |
US20240216507A1 (en) | Sars-cov-2-specific t cells and methods of treatment using them | |
JP2010209082A (en) | Detection marker, detection kit and detection method for idiopathic pulmonary fibrosis | |
WO2000073345A2 (en) | Antibodies specific for mycobacterial polypeptides and uses thereof | |
AU2005256177A1 (en) | Novel methods of diagnosis of treatment of P. aeruginosa infection and reagents therefor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FGA | Letters patent sealed or granted (standard patent) | ||
MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |