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Discovery of dormancy associated antigens of Mycobacterium tuberculosis : novel targets for the development of post-exposure or therapeutic tuberculosis vaccines

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(1)Discovery of dormancy associated antigens of Mycobacterium tuberculosis : novel targets for the development of post-exposure or therapeutic tuberculosis vaccines Lin, M.Y.. Citation Lin, M. Y. (2009, December 15). Discovery of dormancy associated antigens of Mycobacterium tuberculosis : novel targets for the development of postexposure or therapeutic tuberculosis vaccines. Retrieved from https://hdl.handle.net/1887/14507 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/14507. Note: To cite this publication please use the final published version (if applicable)..

(2)  Chapter5 TcellrecognitionoftheHspXproteinof Mycobacteriumtuberculosiscorrelateswithlatent M.tuberculosisinfectionbutnotwith  M.bovisBCGvaccination         .  ‡‹‡‡ ‡Ž—ǡƒ›‘—‰‹ǡ”‹•–ƒǤ˜ƒ‡‹Œ‰ƒƒ”†‡ǡŽ‹ƒ‡ǤǤ‡›–‡ǡ‡‡•ǤǤǤ ”ƒ‡ǡ‘ ǤǤ––‡Š‘ˆˆǡƒ†‹ Š°ŽǤŽ‡‹  ‡’ƒ”–‡–‘ˆ

(3) —‘Š‡ƒ–‘Ž‘‰›ƬŽ‘‘†”ƒ•ˆ—•‹‘ƒ†‡’ƒ”–‡–‘ˆ

(4) ˆ‡ –‹‘—•‹•‡ƒ•‡•ǡ ‡‹†‡‹˜‡”•‹–›‡†‹ ƒŽ‡–”‡ǡ–Š‡‡–Š‡”Žƒ†• InfectImmun.2007Jun;75(6):29142921.

(5) Chapter5. Abstract —”‹‰ •–ƒ–‹‘ƒ”› ‰”‘™–Š ‘” ‹ ˜‹–”‘ ‘†‹–‹‘• ‹‹ ‹‰ ”‡Ž‡˜ƒ– ƒ•’‡ –• ‘ˆ Žƒ–‡ › •— Š ƒ• Š›’‘š‹ƒǡ –Š‡ •’ ’”‘–‡‹ ȋ˜ʹͲ͵ͳ Ȍ ‹• •’‡ ‹ˆ‹ ƒŽŽ› —’”‡‰—Žƒ–‡† „› M. tuberculosisǤ

(6)  –Š‹• •–—†› ™‡ ‘’ƒ”‡†  ‡ŽŽ ”‡•’‘•‡• ƒ‰ƒ‹•– •’ ƒ† –Š‡ •‡ ”‡–‡† M. tuberculosis ’”‘–‡‹ ‰ͺͷ ȋ˜ͳͺͺ͸ Ȍǡ ‹ –—„‡” —Ž‘•‹• ȋȌ ’ƒ–‹‡–•ǡ –—„‡” —Ž‹ •‹ –‡•– ’‘•‹–‹˜‡ ‹†‹˜‹†—ƒŽ• ȋΪȌǡ  Ǧ˜ƒ ‹ƒ–‡† ‹†‹˜‹†—ƒŽ• ƒ† Š‡ƒŽ–Š› ‡‰ƒ–‹˜‡ ‘–”‘Ž•Ǥ

(7) ǦJ ”‡•’‘•‡• ƒ‰ƒ‹•– •’ ™‡”‡ •‹‰‹ˆ‹ ƒ–Ž› Š‹‰Š‡” ‹ M.tuberculosis‡š’‘•‡†‹†‹˜‹†—ƒŽ•–Šƒ‹M.tuberculosis—‡š’‘•‡† ˜ƒ ‹‡‡•Ǥ

(8)  ‘–”ƒ•–ǡ ‘ •— Š †‹ˆˆ‡”‡ ‡• ™‡”‡ ˆ‘—† ™‹–Š ”‡•’‡ – –‘  ‡ŽŽ ”‡•’‘•‡• ƒ‰ƒ‹•– ‰ͺͷǤ Š‡”‡ˆ‘”‡ǡ  Ǧ„ƒ•‡† ˜ƒ ‹‡• ‘–ƒ‹‹‰ ”‡Ž‡˜ƒ– ˆ”ƒ‰‡–• ‘ˆ •’ǡ ƒ› ‹†— ‡‹’”‘˜‡†”‡•’‘•‡•ƒ‰ƒ‹•––Š‹•Žƒ–‡ ›ƒ–‹‰‡Ǥ‘‹†‡–‹ˆ›”‡Ž‡˜ƒ–  Žƒ••

(9) Ǧƒ† Žƒ••

(10)

(11) Ǧ”‡•–”‹ –‡† •’Ǧ•’‡ ‹ˆ‹  ‡ŽŽ‡’‹–‘’‡•ǡ™‡‹—‹œ‡† ǦʹȀ„ ƒ† Ǧ͵Ǥ„Ͳ –”ƒ•‰‡‹  ȋ–‰Ȍ ‹ ‡ ™‹–Š •’Ǥ ™‘ ‡™  ‡ŽŽ ‡’‹–‘’‡• ™‡”‡ ‹†‡–‹ˆ‹‡†ǡ ’ͻͳǦͳͲͷ ƒ† ’͵ͳǦͷͲǡ ”‡•–”‹ –‡† ˜‹ƒ ǦȗͲʹͲͳ ƒ† ǦͳȗͲ͵Ͳͳǡ ”‡•’‡ –‹˜‡Ž›Ǥ Š‡•‡ ‡’‹–‘’‡• ™‡”‡ ”‡ ‘‰‹œ‡† „› Š—ƒ  ‡ŽŽ• ƒ• ™‡ŽŽǡ —†‡”Ž‹‹‰ –Š‡”‡Ž‡˜ƒ ‡‘ˆ •’ ‡ŽŽ”‡ ‘‰‹–‹‘„‘–Š‹˜‹˜‘ƒ†‹˜‹–”‘Ǥ

(12) Ž‹‡™‹–Š–Š‡†ƒ–ƒ ‹ Š—ƒ•ǡ   ‹—‹œƒ–‹‘ ‘ˆ „‘–Š –‰ •–”ƒ‹• †‹† ‘– Ž‡ƒ† –‘  ‡ŽŽ ”‡•’‘•‡• ƒ‰ƒ‹•– •’Ǧ†‡”‹˜‡† ‡’‹–‘’‡•ǡ ™Š‡”‡ƒ• ‘Žƒ–‡ › ƒ–‹‰‡• ™‡”‡ ‡ˆˆ‹ ‹‡–Ž› ”‡ ‘‰‹œ‡†ǤŠ‡•‡†ƒ–ƒ•—’’‘”––Š‡‘–‹‘–Šƒ– ˜ƒ ‹ƒ–‹‘’‡”•‡†‘‡•‘–‹†— ‡  ‡ŽŽ ”‡•’‘•‡• ƒ‰ƒ‹•– –Š‡ Žƒ–‡ › ƒ–‹‰‡ǡ •’Ǥ Š—•ǡ ™‡ •—‰‰‡•– –Šƒ– •—„—‹– ˜ƒ ‹‡• ‹ ‘”’‘”ƒ–‹‰ •’ ƒ†Ȁ‘” ‘–Š‡” Žƒ–‡ › ƒ–‹‰‡•ǡ ƒ• ™‡ŽŽ ƒ• ”‡ ‘„‹ƒ–   •–”ƒ‹• ‡š’”‡••‹‰ Žƒ–‡ › ƒ–‹‰‡• ‡‡† –‘ „‡ ‘•‹†‡”‡† ƒ• ‡™ ˜ƒ ‹‡• ƒ‰ƒ‹•–Ǥ. ͳͲͶ.

(13) TcellresponsestoM.tuberculosisDosRantigenHspX. Introduction —„‡” —Ž‘•‹•ȋȌ‹•‘‡‘ˆ–Š‡Ž‡ƒ†‹‰ ƒ—•‡•‘ˆ‘”–ƒŽ‹–›ȋ–™‘‹ŽŽ‹‘ƒ—ƒŽŽ›Ȍˆ”‘ ƒ‹ˆ‡ –‹‘—•†‹•‡ƒ•‡ǡ’ƒ”–‹ —Žƒ”Ž›‹–Š‡†‡˜‡Ž‘’‹‰™‘”Ž†ȋ͵͸ȌǤ

(14) –‹•‡•–‹ƒ–‡†–Šƒ–ʹ „‹ŽŽ‹‘’‡‘’Ž‡ƒ”‡Žƒ–‡–Ž›‹ˆ‡ –‡†™‹–ŠMycobacteriumtuberculosisȋM.tuberculosisȌǡ –Š‡‡–‹‘Ž‘‰‹ ƒ‰‡–‘ˆ™Š‹ Š ƒ—•‡•ƒ –‹˜‡†‹•‡ƒ•‡‹ͷǦͳͲΨ‘ˆ‹ˆ‡ –‡†‹†‹˜‹†—ƒŽ•Ǥ ‘•– ‹†‹˜‹†—ƒŽ• ‹‹–‹ƒŽŽ› ‘–”‘Ž –Š‡ ‹ˆ‡ –‹‘ „› ‘—–‹‰ ‡ŽŽǦ‡†‹ƒ–‡† ‹—‹–›Ǥ ‘™‡˜‡”ǡ ”‡•‹†—ƒŽ › ‘„ƒ –‡”‹ƒ ”‡ƒ‹ ˜‹ƒ„Ž‡ ˆ‘” ƒ› ›‡ƒ”• ‹ Š‡ƒŽ–Š› ‹—‘ ‘’‡–‡–Š‘•–•‹–Š‡ƒ„•‡ ‡‘ˆ†‹•‡ƒ•‡ȋͶȌƒ†–Š‡ƒŒ‘”‹–›‘ˆ ‘–ƒ‰‹‘—• †‹•‡ƒ•‡ ƒ•‡•™‹ŽŽƒ”‹•‡ˆ”‘–Š‹•‡‘”‘—••‘—” ‡‘ˆŽƒ–‡–‹ˆ‡ –‹‘Ǥ Š‡ —””‡–Ž› ƒ˜ƒ‹Žƒ„Ž‡ ˜ƒ ‹‡ ƒ‰ƒ‹•– ǡ M. bovis  ǡ ‹• Žƒ”‰‡Ž› ‹‡ˆˆ‡ –‹˜‡ ƒ– ’”‘–‡ –‹‰ ƒ‰ƒ‹•– ’—Ž‘ƒ”› †‹•‡ƒ•‡ ‹ ƒ†—Ž–• ȋͳ͸ǡ ʹ͵ȌǤ Š‡ ’”‡ ‹•‡ ƒ–—”‡ ‘ˆ –Š‡  ‡ŽŽ ”‡•’‘•‡ ‡‡†‡† ˆ‘” ’”‘–‡ –‹‘ ƒ‰ƒ‹•– ƒ†—Ž– ’—Ž‘ƒ”›  ‹• ‹ ‘’Ž‡–‡Ž› †‡ˆ‹‡†ǡƒ•‹•–Š‡ ƒ—•‡ˆ‘”–Š‡Žƒ ‘ˆ’”‘–‡ –‹‘„› ˜ƒ ‹ƒ–‹‘ȋ͵ȌǤ Š‡ ͳ͸ƒ Š‡ƒ– •Š‘  ’”‘–‡‹ •’ ȋ˜ʹͲ͵ͳ Ȍ ‹• ”‡“—‹”‡† ˆ‘” › ‘„ƒ –‡”‹ƒŽ ’‡”•‹•–‡ ‡™‹–Š‹–Š‡ƒ ”‘’Šƒ‰‡ƒ†‹•ƒ†‘‹ƒ–’”‘–‡‹’”‘†— ‡††—”‹‰•–ƒ–‹  ‰”‘™–Š‘”—†‡”‘š›‰‡†‡’”‹˜ƒ–‹‘ȋ͵͹ȌǤ†‡”–Š‡•‡ ‘†‹–‹‘•ǡ‹– ƒƒ ‘—–ˆ‘” —’–‘ʹͷΨ‘ˆ–Š‡–‘–ƒŽ„ƒ ‹ŽŽƒ”›’”‘–‡‹‡š’”‡••‹‘Ǥ

(15) –‹•’”‘’‘•‡†–Šƒ– •’’Žƒ›•ƒ ƒ –‹˜‡”‘Ž‡‹•Ž‘™‹‰–Š‡‰”‘™–Š‘ˆM.tuberculosis‹˜‹˜‘‹‡†‹ƒ–‡Ž›ƒˆ–‡”‹ˆ‡ –‹‘ǡ ƒ•M.tuberculosis—–ƒ–•Žƒ ‹‰–Š‡Š•’‰‡‡‡šŠ‹„‹–‡†‹ ”‡ƒ•‡†‰”‘™–Š„‘–Š‹ ‹ ‡ ƒ† ‹ ƒ ”‘’Šƒ‰‡• ȋʹͶȌǤ

(16)  ƒ††‹–‹‘ –‘ –Š‡ ’”‡•‡ ‡ ‘ˆ •’‡ ‹ˆ‹  Š—‘”ƒŽ ”‡•’‘•‡•ƒ‰ƒ‹•– •’‹•‡”ƒ‘ˆ ƒ˜‹–ƒ”›’ƒ–‹‡–•ȋʹͻȌǡ ‡ŽŽ”‡•’‘•‡•–‘ •’ ™‡”‡ƒŽ•‘ˆ‘—†–‘„‡ƒ••‘ ‹ƒ–‡†™‹–ŠŽƒ–‡–M.tuberculosis‹ˆ‡ –‹‘ȋͳ͵ǡͳͶȌ’‘‹–‹‰ –‘–Š‡‹’‘”–ƒ ‡‘ˆ •’ƒ•ƒƒ–‹‰‡‹ –ƒ”‰‡–‘ˆ‹—‡”‡•’‘•‡•†—”‹‰Žƒ–‡– ‹ˆ‡ –‹‘Ǥ ‹ ‡ ‡™ ˜ƒ ‹‡• ‘–ƒ‹‹‰ ”‡Ž‡˜ƒ– ˆ”ƒ‰‡–• ‘ˆ •’ǡ ƒ› ‹†— ‡ ‹’”‘˜‡† ”‡•’‘•‡•ƒ‰ƒ‹•––Š‹•Žƒ–‡ ›ƒ–‹‰‡ǡ™‡Šƒ˜‡‰‡‡”ƒ–‡†ƒ† Šƒ”ƒ –‡”‹œ‡† •’Ǧ •’‡ ‹ˆ‹ ǡŠ—ƒͺΪƒ†ͶΪ ‡ŽŽ•ǡ”‡•–”‹ –‡†„› ‘‘  Žƒ••

(17) ƒ† Žƒ••

(18)

(19)  ƒŽŽ‡Ž‡•Ǥ

(20) ƒ††‹–‹‘ǡˆ”‘M.tuberculosis‹ˆ‡ –‡†‹†‹˜‹†—ƒŽ•ȋƒ –‹˜‡ƒ•™‡ŽŽƒ• Žƒ–‡– ‹ˆ‡ –‹‘Ȍ ƒ†   ˜ƒ ‹ƒ–‡† ‹†‹˜‹†—ƒŽ• ™‹–Š ƒ† ™‹–Š‘—– ‡š’‘•—”‡ –‘ M. tuberculosis ™‡”‡ ‡šƒ‹‡† ˆ‘” –Š‡‹” ‹ ˜‹–”‘ ”‡•’‘•‡ –‘ •’Ǥ ‹ƒŽŽ›ǡ –Š‡ ‡ˆˆ‡ – ‘ˆ  Ǧ‘” •’ ‹—‹œƒ–‹‘ ‘‹†— ‡†‹—‹–›ƒ‰ƒ‹•– •’™ƒ• ƒƒŽ›œ‡† ‹ Ǧ ʹȀ„ƒ† Ǧ͵Ǥ„Ͳ–”ƒ•‰‡‹ ȋ–‰Ȍ‹ ‡Ǥ. ͳͲͷ.

(21) Chapter5. MaterialsandMethods Antigens   ȋM. bovis „ƒ ‹ŽŽ—• ƒŽ‡––‡ —±”‹ǡ ƒ‹•Š ͳ͵͵ͳȌ ™ƒ• ’—” Šƒ•‡† ˆ”‘ –Š‡ –ƒ–‡• ‡”—

(22) •–‹–—–‡ ȋ‘’‡Šƒ‰‡ǡ ‡ƒ”Ȍǡ ‹ŽŽ‡† M. tuberculosis ͵͹˜ •‘‹ ƒ–‡ ™ƒ• ‘„–ƒ‹‡† ˆ”‘ ”Ǥ Ǥ ˜ƒ ‘‘Ž‹‰‡ ȋ

(23) ǡ Š‡ ‡–Š‡”Žƒ†•ȌǤ Š‡ ƒ–‹‰‡ ͺͷ ‰‡‡ ȋ˜ͳͺͺ͸ Ȍƒ†–Š‡Š•’‰‡‡ȋ˜ʹͲ͵ͳ Ȍ‘ˆM.tuberculosis™‡”‡ƒ’Ž‹ˆ‹‡† „›ƒ† Ž‘‡† „› ƒ–‡™ƒ› ‡ Š‘Ž‘‰› ȋ

(24) ˜‹–”‘‰‡ǡ ƒ ‹‡‰‘ǡ Ȍ ‹ ƒ „ƒ –‡”‹ƒŽ ‡š’”‡••‹‘ ˜‡ –‘” ‘–ƒ‹‹‰ ƒ Ǧ–‡”‹ƒŽ Š‹•–‹†‹‡ –ƒ‰Ǥ Š‡ ’”‘–‡‹• ™‡”‡ ‘˜‡”‡š’”‡••‡† ‹ Escherichiacoliʹͳȋ͵Ȍƒ†’—”‹ˆ‹‡†ǡƒ•†‡• ”‹„‡†ȋͳ͹ȌǤ  Studysubjects Š‡•–—†›‹ Ž—†‡†͸͵‹†‹˜‹†—ƒŽ•ǡƒ‘‰•–™Š‘ͳ͹’ƒ–‹‡–•ǡͳͺ–—„‡” —Ž‹•‹ –‡•– ȋȌ ’‘•‹–‹˜‡ ‹†‹˜‹†—ƒŽ•ǡ ͳ͹   ˜ƒ ‹ƒ–‡† ‹†‹˜‹†—ƒŽ• ƒ† ͳͳ ‘Ǧ  ˜ƒ ‹ƒ–‡†ǡ  ‡‰ƒ–‹˜‡ǡ Š‡ƒŽ–Š› —– Š ‘–”‘Ž•Ǥ ŽŽ ‹†‹˜‹†—ƒŽ• ‰ƒ˜‡ ‹ˆ‘”‡† ‘•‡–„‡ˆ‘”‡„Ž‘‘†•ƒ’Ž‹‰ǤŠ‡•–—†›’”‘–‘ ‘Ž™ƒ•ƒ’’”‘˜‡†„›–Š‡‹•–‹–—–‹‘ƒŽ ”‡˜‹‡™„‘ƒ”†‘ˆ–Š‡‡‹†‡‹˜‡”•‹–›‡†‹ ƒŽ‡–”‡Ǥ  ELISPOTassayforsinglecellIFNJJrelease ‡‘—• „Ž‘‘† ™ƒ• ‘„–ƒ‹‡† ˆ”‘ •–—†› ’ƒ”–‹ ‹’ƒ–• ‹ Š‡’ƒ”‹‹œ‡† –—„‡• ƒ†  ‹•‘Žƒ–‡†„› ‹ ‘ŽŽ†‡•‹–› ‡–”‹ˆ—‰ƒ–‹‘ǤȋͳšͳͲ͸Ȍ™‡”‡’—Ž•‡†ȋͳ͸ŠȌ‹ͶͺǦ™‡ŽŽ ’Žƒ–‡•™‹–Šƒ–‹‰‡ȋͳͲρ‰ȀŽȌ‹

(25) ȋ‹ˆ‡‡ Š‘Ž‘‰‹‡•Ȍ™‹–ŠͳͲΨ’‘‘Ž‡†Š—ƒ •‡”—Ǥ  Ǧ„ƒ ‡† ͻ͸Ǧ™‡ŽŽ ’Žƒ–‡• ȋ

(26) Ͷͷǡ ‹ŽŽ‹’‘”‡ǡ ‡†ˆ‘”†ǡ Ȍ ™‡”‡ ’”‡Ǧ ‘ƒ–‡†™‹–Šͷρ‰ȀŽƒ–‹Ǧ

(27) Ǧɀ„ͳǦͳȋƒ„–‡ Šǡ–‘ Š‘Žǡ™‡†‡Ȍǡ™ƒ•Š‡†͸š ™‹–Š

(28) ƒ†„Ž‘ ‡†ȋʹŠȌ™‹–Š

(29)  ‘–ƒ‹‹‰ͳͲΨ Ǥ–‹‰‡Ǧ’—Ž•‡† ȋʹǤͷšͳͲͷ ‡ŽŽ•Ȁ™‡ŽŽǢͳͷͲPŽȌ™‡”‡‹ —„ƒ–‡†‹–Š‡ Ǧ„ƒ ‡†ͻ͸Ǧ™‡ŽŽ’Žƒ–‡•ƒ–͵͹ιǡ ͷΨʹǤˆ–‡”ͳ͸Š’Žƒ–‡•™‡”‡™ƒ•Š‡†ȋͲǤͲͷΨ™‡‡ǦʹͲȌǡ‹ —„ƒ–‡†™‹–ŠͳͲͲρŽ „‹‘–‹›Žƒ–‡† ƒ–‹Ǧ

(30) Ǧɀ „ ȋͲǤ͵ ρ‰ȀŽȌ ˆ‘” ͵ Š ƒ– ǡ ™ƒ•Š‡†ǡ ‹ —„ƒ–‡† ™‹–Š •–”‡’–ƒ˜‹†‹ǦƒŽƒŽ‹‡ ’Š‘•’Šƒ–ƒ•‡ ‘Œ—‰ƒ–‡ ȋͳǣͳͲͲͲǢ ƒ„–‡ ŠȌ ˆ‘” ʹ Šǡ ™ƒ•Š‡† ƒ† ‹ —„ƒ–‡† ™‹–Š ͳͲͲ ρŽ Ȁ

(31)  •—„•–”ƒ–‡ ȋ‹‰ƒǡ –Ǥ ‘—‹•ǡ ȌǤ Š‡ ”‡ƒ –‹‘ ™ƒ• •–‘’’‡†„›ƒ††‹–‹‘‘ˆ™ƒ–‡”ǤŽƒ–‡•™‡”‡†”‹‡†ƒ†ƒƒŽ›œ‡†—•‹‰ƒ‡‹••š‹‘’Žƒʹ ‹ ”‘• ‘’‡ ƒ†  

(32)  •‘ˆ–™ƒ”‡ ȋƒ”Ž‡‹•• ‹•‹‘ǡ ƒŽŽ„‡”‰‘‘•ǡ ‡”ƒ›ȌǤ  ’‘•‹–‹˜‡”‡•’‘•‡–‘ƒ–‹‰‡™ƒ•–ƒ‡ƒ•–™‹ ‡–Š‡„ƒ ‰”‘—†Ǥ  Syntheticpeptides ͳͷǦ‡” ƒ† ʹͲǦ‡” ’‡’–‹†‡• ™‡”‡ •›–Š‡•‹œ‡† „› •‹—Ž–ƒ‡‘—• —Ž–‹’Ž‡ ’‡’–‹†‡ •›–Š‡•‹•ƒ•†‡• ”‹„‡†ȋ͵ͲȌǤ ‘‘‰‡‡‹–›™ƒ• ‘ˆ‹”‡†„›ƒƒŽ›–‹ ƒŽ ƒ†„›ƒ•• •’‡ –”‘‡–”›Ǥ Ž—‘”‡• ‡ ‡ Žƒ„‡Ž‡† ’‡’–‹†‡• ™‡”‡ •›–Š‡•‹œ‡† ƒ• †‡• ”‹„‡† ’”‡˜‹‘—•Ž› ȋͳͻȌǤ  HLADR/peptidebindingassay • ƒ •‘—” ‡ ‘ˆ Ǧ ‘Ž‡ —Ž‡• Ǧ–”ƒ•ˆ‘”‡†  Ž›’Š‘„Žƒ•–‘‹† ‡ŽŽ Ž‹‡• ȋǦ ȌŠ‘‘œ›‰‘—•ˆ‘” Ǧ™‡”‡—•‡†Ǥ Ǧ‘Ž‡ —Ž‡•™‡”‡’—”‹ˆ‹‡†„›ƒˆˆ‹‹–› Š”‘ƒ–‘‰”ƒ’Š›ƒ†’‡’–‹†‡„‹†‹‰–‘’—”‹ˆ‹‡† Ǧ͵‘Ž‡ —Ž‡•ȋ͸ͲǦ͸ͲͲȌ™ƒ• †‡–‡”‹‡†ƒ•†‡• ”‹„‡†ȋʹͳȌǤ••–ƒ†ƒ”†ˆŽ—‘”‡• ‡–’‡’–‹†‡•‹ˆŽ—‡œƒŠ‡ƒ‰‰Ž—–‹‹ ȋ Ȍ’͵ͲͺǦ͵ͳͻȋȌ™ƒ•—•‡†ˆ‘” Ǧͳƒ† Ǧʹǡƒ†Š•’͸ͷ’͵Ǧ ͳͲ͸.

(33) TcellresponsestoM.tuberculosisDosRantigenHspX. ͳ͵ ȋ

(34) Ȍ ˆ‘” Ǧ͵Ǥ ‡’–‹†‡ „‹†‹‰ ƒˆˆ‹‹–› ™ƒ• †‡ˆ‹‡† ƒ• Š‹‰Š ȋ

(35) ͷͲ δ ͳɊȌǡ‹–‡”‡†‹ƒ–‡ȋͳɊǦͳͲɊȌǡ™‡ƒȋͳͲɊǦͳͲͲɊȌ‘”ƒ„•‡–ȋεͳͲͲɊȌȋʹͳȌǤ  Generationandepitopemappingofhuman,HspXreactiveCD8+Tcelllines  ȋͳšͳͲͷ ‡ŽŽ•Ȁ ™‡ŽŽȌ †‡”‹˜‡† ˆ”‘ ƒ ǦȗͲʹͲͳΪ †‘‘” ™‡”‡ •–‹—Žƒ–‡† ™‹–Š ’‡’–‹†‡ ’‘‘Ž• ‘–ƒ‹‹‰ ˆ‘—” ͳͷǦ‡” •’ ’‡’–‹†‡• ‘˜‡”Žƒ’’‹‰ ͳͲ  ȋͳͲ ρ‰Ȁ Ž ‘ˆ ‡ƒ Š’‡’–‹†‡Ȍˆ‘”͹†ƒ›•‹

(36) ȋͳͲΨ Ȍ‹–Š‡’”‡•‡ ‡‘ˆ”Ǧ

(37) ͹ȋͷ‰ȀŽǡ‹‘ ƒ”–ƒȌ ƒ†”Ǧ

(38) ͳͷȋͷ‰ȀŽǡ‹‘ ƒ”–ƒȌ‹ͻ͸™‡ŽŽ•”‘—†„‘––‘’Žƒ–‡•Ǥˆ–‡”ʹ†ƒ›•”Ǧ

(39) ʹȋʹͷȀ Žǡ‡–—•Ȍ™ƒ•ƒ††‡†–‘–Š‡ —Ž–—”‡•Ǥ  IntracellularIFNJJstaining ‡˜‡ †ƒ›• Žƒ–‡”ǡ ‡ŽŽ• ™‡”‡ ‘ŽŽ‡ –‡† ƒ† ‘Ǧ —Ž–—”‡† ˆ‘” ͸ Š ™‹–Š –Š‡ ǦȗͲʹͲͳǦ ’‘•‹–‹˜‡Ǧ ǡ–Šƒ–Šƒ†„‡‡’—Ž•‡†‘˜‡”‹‰Š–™‹–Š•‹‰Ž‡ •’’‡’–‹†‡•‹

(40)  ȋͳͲΨ Ȍƒ†™ƒ•Š‡†–™‹ ‡ ™‹–Š

(41) ȋͳͲΨ ȌǤ —”‹‰–Š‡ Žƒ•– ʹ Š‘ˆ ‘Ǧ —Ž–—”‡ǡ ”‡ˆ‡Ž†‹  ™ƒ• ƒ††‡† ȋͳͲ ρ‰Ȁ Žǡ ‹‰ƒȌǤ ‡ŽŽǦ•—”ˆƒ ‡ •–ƒ‹‹‰ ™ƒ• ’‡”ˆ‘”‡† —•‹‰ ͳͻǦ

(42) ǡ ͶǦ‡” ƒ† ͺǦ ȋƒŽŽ ‡ –‘ ‹ ‹•‘ǡ Ȍ ƒˆ–‡” ™Š‹ Š –Š‡ ‡ŽŽ• ™‡”‡’‡”‡ƒ„‹Ž‹œ‡†™‹–Š‡”„—ˆˆ‡”ȋŠƒ”‹‰‡ǡȌƒ†•–ƒ‹‡†™‹–Šƒ–‹Ǧ

(43) ǦJǦ ȋ‡ –‘‹ ‹•‘ǡȌǤ–‹—Žƒ–‹‘™‹–Š’Š‘”„‘Ž›”‹•–ƒ–‡ƒ ‡–ƒ–‡Ǧ‹‘‘› ‹‡™ƒ•—•‡† ƒ•ƒ’‘•‹–‹˜‡ ‘–”‘Žƒ†—•–ƒ‹‡† ‡ŽŽ•ƒ•ƒ‡‰ƒ–‹˜‡ ‘–”‘ŽǤ  Generationandepitopemappingofhuman,HspXreactiveCD4+Tcelllines  ȋͳǤͷ š ͳͲ͸ ‡ŽŽ•Ȁ ™‡ŽŽȌ‘ˆŠ‡ƒŽ–Š› Š—ƒ†‘‘”• ™‡”‡•–‹—Žƒ–‡†™‹–Š •’ ȋͳͲ P‰ȀŽȌ‹

(44) ȋͳͲΨŠ—ƒ•‡”—Ȍ‹ʹͶǦ™‡ŽŽ’Žƒ–‡•Ǥˆ–‡” ͸†ƒ›•”Ǧ

(45) ǦʹȋʹͷȀ Ž ȋŠ‹”‘ǡȌ™ƒ•ƒ††‡†ƒ† ‡ŽŽ•™‡”‡ƒ††‹–‹‘ƒŽŽ› —Ž–—”‡†ˆ‘”ͳͲ–‘ͳͷ†ƒ›•ǡˆ”‘œ‡ƒ† •–‘”‡†‹Ž‹“—‹†‹–”‘‰‡—–‹Žˆ—”–Š‡”—•‡Ǥ —ƒͶΪ ‡ŽŽŽ‹‡•ȋͳǤͷšͳͲͷ ‡ŽŽ•Ȁ™‡ŽŽȌ ƒ† Ǧƒ– Š‡†‹””ƒ†‹ƒ–‡†ȋͷšͳͲͷ ‡Ž•Ȁ™‡ŽŽȌ™‡”‡‹ —„ƒ–‡†™‹–Š‡‹–Š‡” •’ȋͳͲǡͳǤͲǡͲǤͳ‘”ͲǤͲͳP‰ȀŽȌ‘” •’Ǧ†‡”‹˜‡†’‡’–‹†‡•ȋͳͲǡͳǤͲǡͲǤͳ‘”ͲǤͲͳP‰Ȁ ŽȌ‹ˆŽƒ–Ǧ„‘––‘ͻ͸Ǧ™‡ŽŽ’Žƒ–‡•Ǥˆ–‡”͹ʹŠǡͲǤͷɊ‹‘ˆȏ͵ Ȑ–Š›‹†‹‡™ƒ•ƒ††‡†–‘‡ƒ Š ™‡ŽŽǤ ˆ–‡” ͳͺ Šǡ ‡ŽŽ• ™‡”‡ ‘ŽŽ‡ –‡† ‘ ‰Žƒ•• ˆ‹„‡” ˆ‹Ž–‡” •–”‹’• ƒ† –Š‡ ”ƒ†‹‘ƒ –‹˜‹–› ‹ ‘”’‘”ƒ–‡† ‹–‘ –Š‡  ™ƒ• †‡–‡”‹‡† „› Ž‹“—‹† • ‹–‹ŽŽƒ–‹‘ ‘—–‹‰Ǥ ‡•—Ž–• ƒ”‡ –Š‡‡ƒ‘ˆ–”‹’Ž‹ ƒ–‡ —Ž–—”‡•Ǥ™‡”‡δʹͲΨǤ  HLAtransgenic(tg)mice ǦͳȗͲ͵ͲͳȀ–‰ǡ—”‹‡ Žƒ••

(46)

(47) †‡ˆ‹ ‹‡–ȋ Ǧ͵Ǥ„ͲȌ‹ ‡™‡”‡‰‡‡”ƒ–‡† ƒ•†‡• ”‹„‡†ȋͳͺȌǤ—”‹‰„”‡‡†‹‰ǡ•™‡”‡–›’‡†ˆ‘”‡š’”‡••‹‘ƒ†•‡‰”‡‰ƒ–‹‘‘ˆ –Š‡–”ƒ•‰‡‡„›ˆŽ‘™ ›–‘‡–”›ƒ†ȋʹ͹ȌǤ ǦʹȀ„‹ ‡‡š’”‡••‡†ǡ‹ƒ††‹–‹‘ –‘ –Š‡ —”‹‡ Žƒ••

(48)  ƒŽŽ‡Ž‡• ʹǦ„ ƒ† ʹǦ„ǡ ƒ Š‹‡”‹  ǦȗͲʹͲͳȀ„ ‰‡‡ ‡ ‘†‹‰ ʹǦ„D͵†‘ƒ‹ƒ†–Š‡ ǦȗͲʹͲͳDͳƒ†Dʹ†‘ƒ‹•ȋʹͲȌǤ ǦʹȀ„ ‹ ‡ ™‡”‡ „”‡† —†‡” •’‡ ‹ˆ‹  ’ƒ–Š‘‰‡Ǧˆ”‡‡ ‘†‹–‹‘• ƒ– Ǧ  ȋ‡‹†‡ǡ Š‡ ‡–Š‡”Žƒ†•ȌǤ—”ˆƒ ‡‡š’”‡••‹‘‘ˆ ǦȗͲʹͲͳȀ„‘Ž‡ —Ž‡™ƒ• ‘ˆ‹”‡†„›ˆŽ‘™ ›–‘‡–”›Ǥ  Immunizations —Ž•‹‘• ‘’”‹•‹‰‡“—ƒŽ˜‘Ž—‡•‘ˆ •’”‡ ‘„‹ƒ–’”‘–‡‹‹ƒ†‹ ‘’Ž‡Ǧ –‡ ”‡—†̵• ƒ†Œ—˜ƒ– ȋ

(49) Ǣ 

(50) ǡ ‡–”‘‹–ǡ 

(51) Ȍ ™‡”‡ ’”‡’ƒ”‡† ƒ† ƒ†‹‹•–‡”‡† ƒ• •—„ —–ƒ‡‘—• ȋ•Ǥ ǤȌ ‹Œ‡ –‹‘• ‹–‘ –Š‡ ˆŽƒ• ȋ‹ –‘–ƒŽ ͳͲ Ɋ‰ ’”‘–‡‹ ’‡” ‘—•‡ǡ  α ͶȌǤ. ͳͲ͹.

(52) Chapter5. ‹˜‡M.bovis ȋ‘–”‡ƒŽ•–”ƒ‹Ȍ™ƒ•†‹Ž—–‡†™‹–ŠǤƒ Š‘—•‡™ƒ••Ǥ Ǥ‹Œ‡ –‡† ™‹–ŠͷͲPŽƒ”‘—†‡ƒ ŠŠ‹†Ž‡‰ȋ‹–‘–ƒŽͳšͳͲ͸ Ȁ‘—•‡ȌǤ‡˜‡†ƒ›•’‘•–Ǧ‹Œ‡ –‹‘ǡ •’Ž‡‡• ™‡”‡ ”‡‘˜‡† ƒ† ‡ŽŽ •—•’‡•‹‘• ’”‡’ƒ”‡† ˆ‘” ‹ ˜‹–”‘ —Ž–—”‡Ǥ ‘”  ‹—‹œƒ–‹‘•‹ ‡™‡”‡‹Œ‡ –‡†‹–”ƒ—• —Žƒ”Ž›͵–‹‡•ȋƒ–͵Ǧ™‹–‡”˜ƒŽ•Ȍ‹„‘–Š “—ƒ†”‹ ‡’•ȋʹšͷͲPŽȌ™‹–Š •’’Žƒ•‹†ȋͳ‰ȀŽȌ‘” ‘–”‘Žȋ‡’–›˜‡ –‘”Ȍ‹ Ǥ’Ž‡‘ ›–‡•™‡”‡Šƒ”˜‡•–‡†–Š”‡‡™‡‡•ƒˆ–‡”–Š‡Žƒ•–‹Œ‡ –‹‘Ǥ  Invitroculture ’Ž‡‘ ›–‡• ˆ”‘ ‡ƒ Š ‘—•‡ ȋͳǤͷ š ͳͲͷ ‡ŽŽ•Ȁ ™‡ŽŽȌ ™‡”‡ •–‹—Žƒ–‡† ‹ –”‹’Ž‹ ƒ–‡ —Ž–—”‡• ™‹–Š ƒ–‹‰‡ ‹ ”‘—† „‘––‘ ͻ͸Ǧ™‡ŽŽ ’Žƒ–‡• ‹ ʹͲͲ PŽ 

(53)  ͳ͸ͶͲ ȋ‹ˆ‡ ‡ Š‘Ž‘‰‹‡•Ȍ •—’’Ž‡‡–‡† ™‹–Š ʹ Ǧ‰Ž—–ƒ‹‡ ȋ‹ˆ‡ ‡ Š‘Ž‘‰‹‡•Ȍǡ ͳͲͲ ȀͳͲͲ Ɋ‰ȀŽ ’‡‹ ‹ŽŽ‹Ȁ •–”‡’–‘› ‹ •‘Ž—–‹‘ ȋ‹ˆ‡ ‡ Š‘Ž‘‰‹‡•Ȍ ƒ† ͳͲ Ψ Š‡ƒ–Ǧ‹ƒ –‹˜ƒ–‡†  ȋ‹ˆ‡ ‡ Š‘Ž‘‰‹‡•ȌǤ •’ ƒ† M. tuberculosis •‘‹ ƒ–‡ ™‡”‡ –‡•–‡† ƒ– ͷ P‰Ȁ Žǡ ’‡’–‹†‡• ƒ– ʹͷ P‰Ȁ ŽǤ ˆ–‡” ͸ †ƒ›•ǡ ͲǤͷɊ‹‘ˆ ȏ͵ Ȑ–Š›‹†‹‡ ™ƒ•ƒ††‡†–‘ ‡ƒ Š ™‡ŽŽǤ ˆ–‡”ͳͺŠ ‡ŽŽ•™‡”‡ ‘ŽŽ‡ –‡†‘‰Žƒ••ˆ‹„‡”ˆ‹Ž–‡”•–”‹’•ƒ†–Š‡”ƒ†‹‘ƒ –‹˜‹–›‹ ‘”’‘”ƒǦ –‡†‹–‘–Š‡™ƒ•†‡–‡”‹‡†„›Ž‹“—‹†• ‹–‹ŽŽƒ–‹‘ ‘—–‹‰Ǥ‡•—Ž–•ƒ”‡–Š‡‡ƒ‘ˆ –”‹’Ž‹ ƒ–‡ —Ž–—”‡•Ǥ™‡”‡δʹͲΨǤ  ELISAmurineIFNJJ ’Ž‡‘ ›–‡• ™‡”‡ ”‡•—•’‡†‡† ‹ 

(54) Ǧͳ͸ͶͲ ‡†‹— ȋ‹ˆ‡ ‡ Š‘Ž‘‰‹‡•ǡ ‘ ˜‹ŽŽ‡ǡ ǡ Ȍ •—’’Ž‡‡–‡† ™‹–Š ͳͲΨ Š‡ƒ–Ǧ‹ƒ –‹˜ƒ–‡†  ȋ

(55)   ‹‘• ‹‡ ‡•  ǡ ™‹–œ‡”Žƒ†Ȍ™‡”‡ƒ††‡†–‘ͻ͸Ǧ™‡ŽŽǦ„‘––‘’Žƒ–‡•ȋ‘”‹‰ǤǤ‹ˆ‡ ‹‡ ‡•ǡŠ‡ ‡–Š‡”Žƒ†•Ȍ ƒ† •–‹—Žƒ–‡† ‹ –”‹’Ž‹ ƒ–‡• ™‹–Š ƒ–‹‰‡•Ǥ ˆ–‡” ͹ʹ Š‘—”•ǡ —Ž–—”‡ •—’‡”ƒ–ƒ–• ™‡”‡ –ƒ‡ ƒ† ‡˜ƒŽ—ƒ–‡† ˆ‘” –Š‡‹”

(56) ǦJ ‘–‡– —•‹‰ —”‹‡

(57) ǦJ ›–‘‡–̻ 

(58)  ‹– ȋ‹‘•‘—” ‡ǡ ƒƒ–‹ŽŽ‘ǡ ǡ ȌǤ Š‡ ƒ••ƒ› ™ƒ• ’‡”ˆ‘”‡† ƒ ‘”†‹‰–‘–Š‡ƒ—ˆƒ –—”‡”‰—‹†‡Ž‹‡•Ǥ  Cytotoxicityassays Š‡Š—ƒǦ ȋ ǦȗͲʹͲͳǡǦ͹ǡǦ™͹Ȍ™ƒ•‹ —„ƒ–‡†ƒ–͵͹ιˆ‘”ͳŠ‘—” ™‹–ŠͲǤͳ‹ƒʹͷͳ”Ͷȋ‡”•ŠƒǡȌǡ™ƒ•Š‡†ƒ†’Žƒ–‡†™‹–Š’‘‘Ž‡†•’Ž‡‘ ›–‡• ˆ”‘‹—‹œ‡†‹ ‡ȋαͶȌǡ‹–”‹’Ž‹ ƒ–‡•‹ͻ͸Ǧ™‡ŽŽ”‘—†Ǧ„‘––‘‡†’Žƒ–‡•ȋʹͷͲͲ ‡ŽŽ•Ȁ™‡ŽŽȌ–‘‰‡–Š‡”™‹–Š‡†‹—ǡ’‡’–‹†‡ȋʹP‰Ȍ‘”ͷΨ”‹–‘ǦͳͲͲȋʹͲȌǤˆ–‡”͸Š •—’‡”ƒ–ƒ–• ™‡”‡ Šƒ”˜‡•–‡† ƒ† Ψ •’‡ ‹ˆ‹  Ž›•‹• ™ƒ• ƒŽ —Žƒ–‡† ƒ•ǣ ȏȋ”‡Ž‡ƒ•‡ Ȃ •’‘–ƒ‡‘—•”‡Ž‡ƒ•‡ȌȀȋƒš‹—”‡Ž‡ƒ•‡Ȃ•’‘–ƒ‡‘—•”‡Ž‡ƒ•‡ȌȐšͳͲͲΨǤ  Statisticalanalysis ‹ˆˆ‡”‡ ‡•‹

(59) ǦJ”‡•’‘•‡•–‘ •’„‡–™‡‡†‹ˆˆ‡”‡––‡•–‰”‘—’•™‡”‡ƒƒŽ›œ‡† ™‹–Š–Š‡–™‘Ǧ–ƒ‹Ž‡†ƒǦŠ‹–‡›–‡•–ˆ‘”‘Ǧ’ƒ”ƒ‡–”‹ †‹•–”‹„—–‹‘—•‹‰ ”ƒ’Š ƒ† ”‹• ȋ˜‡”•‹‘ ͶȌǤ PǦ˜ƒŽ—‡• ™‡”‡ ‘””‡ –‡† ˆ‘” —Ž–‹’Ž‡ ‘’ƒ”‹•‘•Ǥ Š‡ •–ƒ–‹•–‹ ƒŽ•‹‰‹ˆ‹ ƒ ‡Ž‡˜‡Ž—•‡†™ƒ•PδͲǤͲͷǤ . ͳͲͺ.

(60) TcellresponsestoM.tuberculosisDosRantigenHspX. Results TcellresponsesagainstHspXdonotcorrespondtoBCGvaccinationperse

(61) †‹˜‹†—ƒŽ•™‹–Š†‹ˆˆ‡”‡–•–ƒ‰‡•‘ˆM.tuberculosis‹ˆ‡ –‹‘™‡”‡’”‡˜‹‘—•Ž›”‡’‘”–‡† –‘ ”‡•’‘† †‹ˆˆ‡”‡–‹ƒŽŽ› –‘ •’ ȋͳͶȌǤ ‹ ‡ –Š‡ ‡ˆˆ‡ – ‘ Ǧ ‡ŽŽ ”‡•’‘•‡• ‹   ˜ƒ ‹ƒ–‹‘ ‹ ƒ”‡ƒ• ™Š‡”‡  ‹• ƒ† ‘– ‡†‡‹  ƒ› ˜ƒ”› ȋͷȌǡ ™‡ ƒƒŽ›œ‡† •’ ”‡•’‘•‡•‹

(62) Ǧɀ

(63) ƒ••ƒ›•—•‹‰ˆ”‘ Ǧ˜ƒ ‹ƒ–‡†‹†‹˜‹†—ƒŽ•ƒ† ˆ”‘ ‘Ǧ  ˜ƒ ‹ƒ–‡†ǡ Š‡ƒŽ–Š› ‘–”‘Ž• ‹ ƒ††‹–‹‘ –‘  ’ƒ–‹‡–• ƒ† Ϊ ‹†‹˜‹†—ƒŽ•ȋ ‹‰—”‡ͳȌǤ  p=0.005 p=0.0081 p=0.0016. IFN-JJ (SFC / 10 6 PBMC). 10 3. HspX. 10 2 10 1 10 0 10 -1 10 -2. TB. TST+. BCG/ ESAT-6/CFP-10 M. tub positive exposed. n=17. n=18. n= 9. BCG/. HC. ESAT-6/CFP-10 M. tub unexposed negative. n=8. n=11. IFN-JJ (SFC / 10 6 PBMC). 10 3. Ag85B. 10 2 10 1 10 0 10 -1 10 -2. TB. TST+. BCG/ ESAT-6/CFP-10 M. tub positive exposed. n=17. n=18. n= 9. BCG/. HC. ESAT-6/CFP-10 M. tub negative unexposed. n=8. n=11. Figure 1. IFNJ production against HspXandAg85BofM.tuberculosisby PBMC of 17 TB patients (TB), 18 tuberculin skin test positive (TST+) individuals, 17 BCG vaccinated individuals (nine in vitro positive for ESAT6 and/or CFP10 and eight in vitronegativeforESAT6and/orCFP 10 ), and 11 nonBCG vaccinated, healthy controls (HC). Spot forming cells (SFC) per 106 PBMC corrected for medium values are given on the y axis. Median values are indicated by horizontal lines. P values are calculated by the nonparametric MannWhitneyUtest.Tcellresponses were considered positive if the mediumcorrectednumberofSFCwas >10/106cells..  ƒ•‡†‘–Š‡‹”‹˜‹–”‘

(64) ǦJ”‡•’‘•‡–‘–Š‡M.tuberculosisǦ•’‡ ‹ˆ‹ ’”‘–‡‹•Ǧ͸ ƒ†Ȁ ‘”  ǦͳͲ ȋ͵ʹȌǡ ™‡ †‹˜‹†‡†   ˜ƒ ‹‡• ‹–‘ M. tuberculosis—‡š’‘•‡† ‹†‹˜‹†—ƒŽ•ȋδͳͲ•’‘–Ǧˆ‘”‹‰ ‡ŽŽ•ȏ ȐȀͳͲ͸ǢαͺȌƒ†‹†‹˜‹†—ƒŽ•™‹–ŠŽ‹‡Ž› ‡š’‘•—”‡ –‘ M. tuberculosis ȋε ͳͲ  Ȁ ͳͲ͸ Ǣ  α ͻǢ ”ƒ‰‹‰ ˆ”‘ ͳ͸ –‘ ͳͳ͸  ȀͳͲ͸ȌǤ     ͳͲͻ.

(65) Chapter5. •’ ‹†— ‡† Š‹‰Š —„‡”• ‘ˆ

(66) ǦJ ‡†‹ƒ •’Ȁ ‡•–‰”‘—’ ’”‘†— ‹‰  ‡ŽŽ• ‹ ‰ͺͷ˜ƒŽ—‡ –Š”‡‡ ‘ˆ –Š‡ ˆ‹˜‡ ‰”‘—’• ’ƒ–‹‡–• ͲǤͷͷ ȋǡ Ϊ ƒ†   Ϊ  ͲǤ͵͵ ˜ƒ ‹‡‡• ”‡•’‘†‹‰ –‘ a  ǡǦ͸Ȁ ǦͳͲ’‘•‹–‹˜‡ ͲǤ͵ͷ Ǧ͸ ƒ†Ȁ‘”  ǦͳͲȌǤ b  ǡǦ͸Ȁ ǦͳͲ‡‰ƒ–‹˜‡ ͲǤͲ͸Ͳ Š‡•‡  ‡ŽŽ ”‡•’‘•‡• ‘–”‘Ž•ǡM.tuberculosis —‡š’‘•‡† ͲǤͲͲ͸ʹ †‹ˆˆ‡”‡†•‹‰‹ˆ‹ ƒ–Ž›ȋPζ a InvitroresponsivetoESAT6and/orCFP10 ͲǤͲͲͺͳȌ ˆ”‘  Ǧ b InvitrounresponsivetoESAT6and/orCFP10 ˜ƒ ‹ƒ–‡† ‹†‹˜‹†—ƒŽ• ™‹–Š‘—–ƒ”‡•’‘•‡–‘Ǧ͸ƒ†Ȁ‘” ǦͳͲƒ†ˆ”‘Š‡ƒŽ–Š› ‘–”‘Ž•Ǥ•ƒ ‘–”‘Žǡ –Š‡—„‡”‘ˆ

(67) Ǧɀ’”‘†— ‹‰ ‡ŽŽ•‹†— ‡†„›–Š‡•‡ ”‡–‡†‰ͺͷ™ƒ•ƒƒŽ›•‡†‹ –Š‡ •ƒ‡ ‹†‹˜‹†—ƒŽ•Ǥ

(68) –‡”‡•–‹‰Ž›ǡ ‘ •‹‰‹ˆ‹ ƒ–†‹ˆˆ‡”‡ ‡•„‡–™‡‡–Š‡ˆ‹˜‡ ‡†‹— ‰”‘—’•™‡”‡†‡–‡ –‡†‹–Š‡—„‡”‘ˆ ͲǤ͸Ψ   ‹†— ‡† „› ‰ͺͷǤ Š‹• ‹†‹ ƒ–‡• –Šƒ– –Š‡ Žƒ  ‘ˆ

(69) ǦJ ’”‘†— –‹‘ ‹ ”‡•’‘•‡–‘ •’‹ ˜ƒ ‹‡‡•ƒ† ‘–”‘Ž•‹•‘– ƒ—•‡†„›–Š‡ƒ„•‡ ‡‘ˆ ƒ–‹Ǧ› ‘„ƒ –‡”‹ƒŽ ”‡•’‘•‡•Ǥ Š‡ ‡†‹ƒ ˜ƒŽ—‡• ‘ˆ –Š‡ •’Ȁ‰ͺͷ •’ ’ͺ͸ǦͳͲͲ ”‡•’‘•‡ ”ƒ–‹‘ ™‡”‡ ͲǤͲ͸Ͳ ƒ† ͲǤͲͲ͸ʹ ͲǤ͵Ψ ‹  Ǧ˜ƒ ‹ƒ–‡†ǡ MǤ tuberculosis —‡š’‘•‡† ‹†‹˜‹†—ƒŽ• ƒ† –Š‡ Š‡ƒŽ–Š› ‘–”‘Ž‰”‘—’ǡ”‡•’‡ –‹˜‡Ž›ǡ™Š‡”‡ƒ•M. tuberculosis‹ˆ‡ –‡†‘”‡š’‘•‡†‰”‘—’• Šƒ† ͳͲǦˆ‘Ž† Š‹‰Š‡” •’Ȁ‰ͺͷ ”‡•’‘•‡ ”ƒ–‹‘• ”ƒ‰‹‰ „‡–™‡‡ ͲǤͷͷ ƒ†ͲǤ͵͵ȋƒ„Ž‡ͳȌǤ •’ ’ͻͳǦͳͲͷ ͵Ǥ͸Ψ  HLAA*0201restrictedTcell epitopes ‹ ‡ ‘—–‹‰ ‡˜‹†‡ ‡ ˆ”‘ Š—ƒ •–—†‹‡•ƒ†—”‹‡‘†‡Ž•‘ˆ’‘‹–• –‘™ƒ”† ƒ ”‘Ž‡ ‘ˆ ͺΪ  ‡ŽŽ• ‹ ‘–”‘ŽŽ‹‰ ȋŽƒ–‡–Ȍ M. tuberculosis

(70) ǦJ ‹ˆ‡ –‹‘ ȋʹʹǡ ʹͺǡ ͵ͳȌǡ ™‡ •‡– ‘—– –‘ ‹†‡–‹ˆ› Ǧ Žƒ••

(71) Ǧ”‡•–”‹ –‡†  ‡ŽŽ Figure 2. Intracellular IFNJ staining of HLAA*0201+, ”‡•’‘•‡• –‘ –Š‡ •’ M. tuberculosis CD8+ human T cell lines generated by stimulation of with seven peptide pools, each containing four Žƒ–‡ › ƒ–‹‰‡Ǥ  †‡”‹˜‡† ˆ”‘ ƒ PBMC HspX 15mers. T cell lines were cocultured for 6 h with Ϊ •’ ”‡•’‘†‹‰ ǦȗͲʹͲͳ  †‘‘” the HLAA*0201positive EBVBLCL JY, that had been ™‡”‡ •–‹—Žƒ–‡† ™‹–Š •‡˜‡ ’‡’–‹†‡ pulsedovernightwithonesingleHspXpeptide(indicated ’‘‘Ž•ǡ‡ƒ Š ‘–ƒ‹‹‰ˆ‘—” •’Ǧ†‡”‹˜‡† on the left). Cells were incubated with PElabeled anti IFN and APClabeled antiCD8 MAb. For the analysis, ͳͷǦ‡” ’‡’–‹†‡• ™‹–Š ƒ ‘˜‡”Žƒ’ ‘ˆ ͳͲ only lymphocytes were used with theexclusion of CD19 ƒ‹‘ ƒ ‹†•Ǥ ˆ–‡” ͹ †ƒ›•ǡ ‡ŽŽ• ™‡”‡ positivecellstoavoidcontaminationoftheJYcells.. ͺ. Table1.HspX/Ag85Bratios. ͳͳͲ.

(72) TcellresponsestoM.tuberculosisDosRantigenHspX. ‘ŽŽ‡ –‡† ƒ† ‘Ǧ —Ž–—”‡† ˆ‘” ͸ Š ™‹–Š ǦȗͲʹͲͳǦ’‘•‹–‹˜‡ Ǧǡ –Šƒ– Šƒ† „‡‡ ’—Ž•‡† ‘˜‡”‹‰Š– ™‹–Š •‹‰Ž‡ •’ ’‡’–‹†‡•ǡ ƒ† ‹†— –‹‘ ‘ˆ ‹–”ƒ ‡ŽŽ—Žƒ”

(73) ǦJ ™ƒ• ƒ••‡••‡† „›  ƒƒŽ›•‹• ȋ ‹‰—”‡ ʹȌǤ

(74) –”ƒ ‡ŽŽ—Žƒ”

(75) ǦJ ’”‘†— ‹‰ ͺΪ  ‡ŽŽ• ™‡”‡ †‡–‡ –‡†‹–Š‡ ‡ŽŽŽ‹‡‰‡‡”ƒ–‡†™‹–Š’‘‘ŽͷǤ

(76) –Š‹•’‘‘Ž‘Ž›‘‡’‡’–‹†‡ǡ •’’ͻͳǦ ͳͲͷǡ‹†— ‡†•‹‰‹ˆ‹ ƒ–Ž‡˜‡Ž•‘ˆ‹–”ƒ ‡ŽŽ—Žƒ”

(77) Ǧɀ’”‘†— –‹‘ȋ ‹‰—”‡ʹȌǤ  MurineHLAA*0201restrictedepitopes ǦʹȀ„ ‹ ‡ ”‡’”‡•‡– ƒ •—‹–ƒ„Ž‡ ‘†‡Ž ˆ‘” –Š‡ ‹†— –‹‘ ƒ† †‡–‡ –‹‘ ‘ˆ Š‹‰Š ƒˆˆ‹‹–› ǦȗͲʹͲͳǦ”‡•–”‹ –‡†ͺΪ”‡•’‘•‡•‹˜‹˜‘ȋͳͳǡʹͲȌǤŠ—•ǡ–Š‡•‡‹ ‡ ™‡”‡‹—‹œ‡†–Š”‡‡–‹‡•™‹–Š’Žƒ•‹†‡ ‘†‹‰hspX‘”‡’–›˜‡ –‘”Ǥ Š”‡‡ ™‡‡• ƒˆ–‡” –Š‡ Žƒ•–  ‹Œ‡ –‹‘ǡ •’Ž‡‘ ›–‡• ™‡”‡ ‹ —„ƒ–‡† ™‹–Š ’‡’–‹†‡ ’—Ž•‡†ǡͷͳ”ǦŽƒ„‡Ž‡† ǦȗͲʹͲͳǦ’‘•‹–‹˜‡  ‡ŽŽ•ȋ™Š‹ Š‡š’”‡•• ǦȗͲʹͲͳ„—–‘– ʹǦ„ ‘” ʹǦ„Ȍ‹‘”†‡”–‘ƒƒŽ›œ‡–Š‡‹” ›–‘Ž›–‹ ’‘–‡–‹ƒŽǤ  ‡ŽŽ•’—Ž•‡†™‹–Š •’ ’ͻͳǦͳͲͷ™‡”‡Ž›•‡†„›•’Ž‡‘ ›–‡•‘ˆ‹—‹œ‡†‹ ‡ȋ ‹‰—”‡͵ȌǤ’Ž‡‘ ›–‡•†‡”‹˜‡† ˆ”‘‹ ‡‹—‹œ‡†•‹‹Žƒ”Ž›™‹–Š‡’–›˜‡ –‘”†‹†‘–•Š‘™ƒ›Ž›•‹•ȋ†ƒ–ƒ ‘– •Š‘™ȌǤ Š‡•‡ †ƒ–ƒ •Š‘™ –Šƒ– –Š‡ •ƒ‡ ǦȗͲʹͲͳǦ”‡•–”‹ –‡† ›–‘–‘š‹   Ž›’Š‘ ›–‡ȋȌ‡’‹–‘’‡‹•‹†‡–‹ˆ‹‡†‹ ǦȗͲʹͲͳŠ—ƒ•ƒ†‹ ǦʹȀ„Ǥ Š‡”‡Žƒ–‹˜‡Ž›Ž‘™Ž‡˜‡Ž‘ˆŽ›•‹•„›–Š‡”ƒ‹•‡†‹ ǦʹȀ„‹ ‡ȋƒš‹ƒŽʹͷΨȌ ‘—Ž†„‡†—‡–‘–Š‡’”‡•‡ ‡‘ˆ–Š‡ Š‹‡”‹  ǦȗͲʹͲͳȀ„‰‡‡‹–Š‡–‰‹ ‡ǤŠ‹• ‡š’”‡••‡•–Š‡ ʹǦ„D͵Ǧƒ†–Š‡ ǦȗͲʹͲͳDͳƒ†Dʹ‘Ž‡ —Ž‡•ƒŽŽ‘™‹‰‡ˆˆ‹ ‹‡– ͺ ‹–‡”ƒ –‹‘ ‘ˆ —”‹‡ ͺΪ  ‡ŽŽ• ƒ† –Š—• ‹–‡”ƒ –• Ž‡•• ‡ˆˆ‹ ‹‡–Ž› ™‹–Š –Š‡ Š—ƒ ǦȗͲʹͲͳ‘Ž‡ —Ž‡‡š’”‡••‡†‘–Š‡ –ƒ”‰‡– ‡ŽŽ•Ǥ E:T. % specific lysis. 30. 100:1 50:1 25:1 12.5:1. 20. 10. 131-144. 126-140. 121-135. 116-130. 111-125. 106-120. 96-110. 101-115. 91-105. 81-95. 86-100. 76-90. 71-85. 65-80. 61-75. 55-70. 51-65. 46-60. 41-55. 36-50. 31-45. 26-40. 21-35. 16-30. 6-20. 11-25. 1-15. 0. overlapping hspX peptides Figure3.CytotoxicactivityofsplenocytesderivedfromHLAA2/Kbmice,immunizedwithHspXplasmidDNA.M. tuberculosisderived peptides used to pulse HLAA*0201+ target cells (JY) are indicated on the x axis. The percentagespecificlysisisindicatedontheyaxis.E:T,effectortotargetratio.. . ͳͳͳ.

(78) Chapter5. HLADRbindingofHspXderivedpeptidesandHLADRrestrictedTcellepitopes ”‡˜‹‘—•Ž›ǡ •‡˜‡”ƒŽ •–—†‹‡• Šƒ˜‡ •Š‘™ –Šƒ– †‹ˆˆ‡”‡– ”‡‰‹‘• ‘ˆ –Š‡ M. tuberculosis •’ Žƒ–‡ › ƒ–‹‰‡ ƒ ’”‘˜‘‡ Š—ƒ ͶΪ  ‡ŽŽ ”‡•’‘•‡•Ǥ •’‡ ‹ƒŽŽ› –Š‡ ‹—‘†‘‹ƒ–”‡‰‹‘’ͻͳǦͳͲͷ‹•’‡”‹••‹˜‡Ž›”‡ ‘‰‹œ‡†ǡ•‹ ‡’‡’–‹†‡‡’‹–‘’‡• ™‡”‡ †‡ˆ‹‡† ‹ –Š‡ ‘–‡š– ‘ˆ ǦͳȗͲͳͲͳǡ ǦͳȗͳͳͲͳǡ Ǧͳȗͳ͵Ͳͳǡ Ǧ ͳȗͳͷͲͳǡƒ†ͳȗͲͶͲͳȋͳǡʹȌǤ–Š‡”’‡’–‹†‡•‡“—‡ ‡•”‡ ‘‰‹œ‡†„›Š—ƒ ‡ŽŽ• ƒ”‡ǣ ’ʹͳǦͶͲ ȋ ǦͳȗͲͶͲͳȌ ȋͳȌǡ ’ʹͳǦʹͻ ƒ† ’ͳʹͲǦͳʹͺ ȋ„‘–Š ǦȗͲʹͲͳȌ ȋͳͲȌǤ ‘ •–—†› ͶΪ  ‡ŽŽ ”‡•’‘•‡• –‘ •’ǡ ™‡ ˆ‹”•– ƒ••‡••‡† Ǧ„‹†‹‰ ƒˆˆ‹‹–› ‘ˆ •’Ǧ†‡”‹˜‡† ’‡’–‹†‡•Ǥ ‡ ƒ‹‘ ƒ ‹†• ‘˜‡”Žƒ’’‹‰ ʹͲǦ‡” ’‡’–‹†‡•ǡ ‘˜‡”‹‰ –Š‡ ‡–‹”‡ •‡“—‡ ‡ ‘ˆ –Š‡ •’ ’”‘–‡‹ ‘ˆ M. tuberculosis ™‡”‡ —•‡† –‘ ƒƒŽ›œ‡ ’‡’–‹†‡ „‹†‹‰ –‘ ’—”‹ˆ‹‡†  ‘Ž‡ —Ž‡• ȋƒ„Ž‡ ʹȌǤ Ǧͳ ƒ† Ǧʹ •Š‘™‡† •‹‹Žƒ” „‹†‹‰ ƒˆˆ‹‹–‹‡• ˆ‘” –Š‡ •’ ’‡’–‹†‡•ǡ ’ƒ”–‹ —Žƒ”Ž› ’ͳͳǦ͵Ͳ ƒ† ’ͻͳǦͳͲͷ „‘—† ™‹–Š Š‹‰Šƒˆˆ‹‹–›–‘–Š‡•‡ƒŽŽ‡Ž‡•Ǥ Ǧ͵ǡ‘–Š‡‘–Š‡”Šƒ†ǡ„‘—†‡•’‡ ‹ƒŽŽ›™‡ŽŽ–‘’͵ͳǦ ͷͲǡ ™Š‹ Š ™ƒ• ‘ˆ‹”‡† „› –Š‡ ’”‡•‡ ‡ ‘ˆ –Š‡ Ǧ͵ ’‡’–‹†‡ „‹†‹‰ ‘–‹ˆ ȋʹͳȌ ™‹–Šȋ‡— ‹‡Ȍƒ–’‘•‹–‹‘ǡȋ Ž—–ƒƒ–‡Ȍƒ–’‘•‹–‹‘Ϊʹƒ†ȋ›•‹‡Ȍƒ–’‘•‹–‹‘ ΪͶȋƒ„Ž‡ʹȌǤ •’ʹͲǦ‡”•™‡”‡—•‡†ˆ‘”‡’‹–‘’‡ƒ’’‹‰‹–Š”‡‡Š—ƒǡ •’Ǧ•’‡ ‹ˆ‹  ‡ŽŽŽ‹‡• ˆ”‘ ‹†‹˜‹†—ƒŽ• Š‘‘œ›‰‘—• ˆ‘” ǦͳȗͲͳͲͳǡ ǦȗͲͳͷͲͳ ‘” ǦȗͲ͵Ͳͳ ȋ ‹‰—”‡ ͶȌǤ Š‡ ǦͳȗͲͳͲͳΪ ‡ŽŽŽ‹‡”‡ ‘‰‹œ‡†’ͻͳǦͳͲͷȋ ‹‰—”‡ͶȌǡ™Š‹ ŠƒŽ•‘„‹†•™‹–ŠŠ‹‰Š ƒˆˆ‹‹–›–‘„‘–Š Ǧͳƒ† ǦʹǤ‘–Š‡”’‡’–‹†‡–Šƒ–„‘—†™‹–ŠŠ‹‰Šƒˆˆ‹‹–›–‘ –Š‡•‡ ƒŽŽ‡Ž‡•ǡ ’ʹͳǦͶͲ ȋƒ„Ž‡ ʹȌǡ ‹†— ‡† Š‹‰Š ’”‘Ž‹ˆ‡”ƒ–‹˜‡ ”‡•’‘•‡•ǡ ™Š‡”‡ƒ• ’͹ͳǦͻͲǡ ™Š‹ Š„‘—†™‹–ŠŽ‘™‡”ƒˆˆ‹‹–›ǡ‹†— ‡†‘†‡”ƒ–‡”‡•’‘•‡•‹–Š‡ ǦͳȗͲͳͷͲͳΪ  ‡ŽŽ Ž‹‡ ȋ ‹‰—”‡ ͶȌǤ Š‡ Ǧ͵Ǧ”‡•–”‹ –‡† ‡’‹–‘’‡ ‹†‡–‹ˆ‹‡† Š‡”‡ǡ ’͵ͳǦͷͲ ȋ ‹‰—”‡ ͶȌǡ ‘””‡•’‘†‡†™‡ŽŽ™‹–Š–Š‡Š‹‰Š„‹†‹‰ƒˆˆ‹‹–›ˆ‘” Ǧ͵ƒ•†‡• ”‹„‡†ˆ‘”‘–Š‡” Ǧ͵Ǧ”‡•–”‹ –‡†ǡ› ‘„ƒ –‡”‹ƒŽ’‡’–‹†‡‡’‹–‘’‡•ȋͳͺȌǤ ‡ŽŽ”‡•’‘•‡•™‡”‡ ǦǦ ”‡•–”‹ –‡†ƒ•„•†‹”‡ –‡†ƒ‰ƒ‹•– ǦȋͺǤͳͳǤʹȌ†‡ ”‡ƒ•‡†’”‘Ž‹ˆ‡”ƒ–‹‘‘ˆƒŽŽ–Š”‡‡  ‡ŽŽŽ‹‡•ȋ†ƒ–ƒ‘–•Š‘™ȌǤ  Table2.HLADRbindingofoverlappingHspXpeptidesa ‹‘ƒ ‹†•. ‡“—‡ ‡. ͳǦʹͲ ͳͳǦ͵Ͳ 2140 3150 ͶͳǦ͸Ͳ ͷͳǦ͹Ͳ ͸ͳǦͺͲ ͹ͳǦͻͲ ͺͳǦͳͲͲ 91110 ͳͲͳǦͳʹͲ ͳͳͳǦͳ͵Ͳ ͳʹͳǦͳͶͲ ͳʹͷǦͳͶͶ.          .        LEK      

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(89) 

(90) . a. Tcellepitopesandassociatedvaluesaredepictedinboldface.. ͳͳʹ.

(91) ͷͲȋɊȌ ǦǣȗͲͳͲͳ. ȗͳͷͲͳ. ȗͲ͵Ͳͳ. ͳͷ δͲǤ͹ ͷ ʹͶ ͺ ͳʹ ͷ ͳͶ ͸ <0.7 εͳͲͲ εͳͲͲ εͳͲͲ εͳͲͲ. ʹʹ δͲǤ͹ 0.9 ͵ͷ ͳ͵ ͳ͹ ͵ ͻ ͳ <0.7 εͳͲͲ ͹Ͳ εͳͲͲ εͳͲͲ. εͳͲͲ Ͷʹ ͸ͺ 0.5 Ͷͳ ͵ͷ Ͷ εͳͲͲ εͳͲͲ Ͷͷ εͳͲͲ εͳͲͲ εͳͲͲ εͳͲͲ.

(92) TcellresponsestoM.tuberculosisDosRantigenHspX. MurineHLADR3restrictedepitopes ‘ ƒ††”‡•• –Š‡ ‹ ˜‹˜‘ ‹—‘‰‡‹ ‹–› ƒ† ‡’‹–‘’‡ •’‡ ‹ˆ‹ ‹–› ‘ˆ –Š‡ •’ ’”‘–‡‹ǡ ™‡ Šƒ˜‡ —•‡† Ǧ͵Ǥ„Ͳ –‰ ‹ ‡Ǥ ˆ–‡” ‹—‹œƒ–‹‘ ‘ˆ –Š‡ ‹ ‡ ™‹–Š •’ ’”‘–‡‹ ‹

(93) ǡ•’Ž‡‘ ›–‡•™‡”‡”‡•–‹—Žƒ–‡†‹˜‹–”‘™‹–Š–Š‡‘˜‡”Žƒ’’‹‰ •’ʹͲǦ‡”’‡’–‹†‡•Ǥ ‹ ‡–Š‡ Ǧ͵Ǥ„Ͳ‹ ‡ƒ”‡†‡˜‘‹†‘ˆƒ›—”‹‡ Žƒ••

(94)

(95) ‘Ž‡ —Ž‡•‡š’”‡••‡†ƒ––Š‡ ‡ŽŽ •—”ˆƒ ‡ǡ ƒŽŽ ͶΪ  ‡ŽŽ• ƒ”‡ ”‡•–”‹ –‡† „› Ǧ͵Ǥ ‹‰—”‡ Ͷ •Š‘™• –Šƒ– —”‹‡ Ǧ͵Ǧ”‡•–”‹ –‡† ‡ŽŽ•”‡ ‘‰‹œ‡–Š‡•ƒ‡‹—‘†‘‹ƒ– •’’‡’–‹†‡ȋ’͵ͳǦͷͲȌ ƒ•Š—ƒ Ǧ͵Ǧ”‡•–”‹ –‡† ‡ŽŽ•ǡ‹†‹ ƒ–‹‰–Šƒ–’͵ͳǦͷͲ‹•ƒ‹˜‹˜‘’”‘ ‡••‡†ƒ† ’”‡•‡–‡† Ǧ͵Ǧ”‡•–”‹ –‡† ‡ŽŽ‡’‹–‘’‡Ǥ A 35000. C. HLA-DR1. 10000. A. C. HLA-DR3. 25000 7500. 125-144. 121-140. 111-130. 91-110. 101-120. 71-90. 81-100. 61-80. 51-70. 41-60. Overlapping HspX peptides. overlapping HspX peptides. B 10000. 31-50. 21-40. 1-20. 11-30. 125-144. 121-140. 111-130. 91-110. 101-120. 71-90. 81-100. 61-80. 51-70. 41-60. 31-50. 21-40. 0. 1-20. 0. 11-30. 2500. HspX. 5000. HspX. 5000. medium. cpm. 10000. medium. cpm. 15000. D. HLA-DR2. 150000. B. HLA-DR3.Ab 0 tg. D. 100000. 7500. 125-144. 121-140. 111-130. 91-110. 101-120. 81-100. 71-90. 61-80. 51-70. 41-60. 31-50. 21-40. 11-30. 1-20. HspX. 125-144. 121-140. 111-130. 91-110. overlapping HspX peptides. 101-120. 81-100. 71-90. 61-80. 51-70. 41-60. 31-50. 21-40. 11-30. 0. 1-20. 0. HspX. 5000. medium. cpm. 10000. 2500. medium. cpm. 50000. 5000. Overlapping HspX peptides. Figure 4. T cell proliferation of HLADR1+ (A), DR2+ (B) or DR3+ (C) human T cell lines responsive to the HspX protein, or of splenocytes from HLADR3.Ab0 tg mice immunized with recombinant protein of HspX (D). Tcellproliferation(incountsperminute[cpm])correctedformediumvaluesisshownontheyaxis.TheSEM were<12%.TheaminoacidnumberingoftheoverlappingHspXpeptidesfromTable2isgivenonthexaxis..  BCGimmunizationofHLAA2/KbandHLADR3.Ab0doesnotinduceTcell responsesagainstHspX ‹ ‡   ˜ƒ ‹ƒ–‡†ǡ M. tuberculosis —‡š’‘•‡† ‹†‹˜‹†—ƒŽ• ‹ ƒ ”‘••Ǧ•‡ –‹‘ƒŽ ‘’ƒ”‹•‘†‹†‘–•Š‘™ ‡ŽŽ”‡•’‘•‡•†‹”‡ –‡†ƒ‰ƒ‹•– •’ȋ ‹‰—”‡ͳƒ†ȋ͵͵Ȍǡ ™‡†‡ ‹†‡†–‘‡˜ƒŽ—ƒ–‡–Š‹•‹ Ǧ–‰‹ ‡Ǥ’Ž‡‘ ›–‡•™‡”‡Šƒ”˜‡•–‡†–‡†ƒ›•’‘•–  Ǧ˜ƒ ‹ƒ–‹‘ǡƒ†ƒƒŽ›œ‡†ˆ‘”–Š‡‹”ƒ„‹Ž‹–›–‘Ž›•‡–Š‡Š—ƒ–ƒ”‰‡– ‡ŽŽ ǡ™Š‹ Š ‡š’”‡••‡• ǦȗͲʹͲͳǤ ƒ”‰‡– ‡ŽŽ• ™‡”‡ ’—Ž•‡† ™‹–Š ‘‡ ‘ˆ •‡˜‡ ‘™ Ǧ ȗͲʹͲͳǦ‡’‹–‘’‡•ȋͳͲP‰ȀŽ’‡”’‡’–‹†‡ȌǣŠ•’͸ͷ’͵͸ͻǦ͵͹͹ȋͳͳȌǡ‰ͺͷ’ͳͶ͵Ǧͳͷʹƒ†. ͳͳ͵.

(96) Chapter5. . ͳͳͶ. 85B p51-70. 85B. hsp65. hsp65 p3-13. HspX p31-50. BCG. HspX. PHA. M.tub. medium. IFN-JJ (pg/ml). % specific lysis. ‰ͺͷ ’ͳͻͻǦʹͲ͹ȋʹͲȌǡ •’ ’ͻͳǦͳͲͷ ȋ–Š‡ ’”‡•‡– •–—†›Ȍǡ •’ ’ʹͳǦʹͻ ƒ† •’ ’ͳʹͲǦͳʹͺȋͳͲȌ‘”–Š‡ ǦʹǦ„‹†‹‰‹ˆŽ—‡œƒƒ–”‹š’‡’–‹†‡ǣ Ž—’ͷͺǦ͸͸ȋ͵ͶȌǤ Š‡Š•’͸ͷƒ†‰ͺͷ‡’‹–‘’‡•™‡”‡•–”‘‰Ž›”‡ ‘‰‹œ‡†„›‹ƒ†‘•‡Ǧ†‡’‡†‡– ˆƒ•Š‹‘ȋ ‹‰—”‡ͷȌǤ ‘™‡˜‡”ǡ‘‡‘ˆ–Š‡–ƒ”‰‡– ‡ŽŽ•’—Ž•‡†™‹–Š •’’‡’–‹†‡•™‡”‡ Ž›•‡†Ǥ‘Ž›•‹•™ƒ•‘„•‡”˜‡†ˆ‘” ‘–”‘Ž ‡ŽŽ•ǣ•’Ž‡‘ ›–‡•’—Ž•‡†™‹–Š ǦȗͲʹͲͳǦ „‹†‹‰‹ˆŽ—‡œƒ˜‹”—•’ͷͺǦ͸͸ȋ ‹‰—”‡ͷȌ‘”•’Ž‡‘ ›–‡•†‡”‹˜‡†ˆ”‘—‹—‹œ‡† ʹȀ„‹ ‡ȋ†ƒ–ƒ‘–•Š‘™ȌǤ‹‹Žƒ”Ž›ǡ™‡‹˜‡•–‹‰ƒ–‡†–Š‡ ‡ŽŽ”‡•’‘•‡•‹ Ǧ ͵Ǥ„Ͳ –‰ ‹ ‡ ƒˆ–‡” ‹—‹œƒ–‹‘ ™‹–Š  Ǥ ’Ž‡‘ ›–‡• ‘ˆ  Ǧ‹—‹œ‡† ‹ ‡ ™‡”‡”‡•–‹—Žƒ–‡†‹˜‹–”‘™‹–Š–Š‡ •’’”‘–‡‹ǡ‹–• Ǧ͵Ǧ”‡•–”‹ –‡†‡’‹–‘’‡’͵ͳǦ ͷͲǡ M. tuberculosis Š•’͸ͷǡ ‹–• Ǧ͵Ǧ”‡•–”‹ –‡† ‡’‹–‘’‡ ’͵Ǧͳ͵ǡ ‰ͺͷ ‘” ‹–• Ǧ ͵Ǧ”‡•–”‹ –‡†‡’‹–‘’‡’ͷͳǦ͹Ͳȋ ‹‰—”‡ͷȌǤ‰ƒ‹ǡ‘

(97) ǦJ”‡•’‘•‡•™‡”‡†‡–‡ –‡† ‹ ”‡•’‘•‡ –‘ •’ ‘” ‹–• Ǧ͵Ǧ”‡•–”‹ –‡† ‡’‹–‘’‡Ǥ

(98)  ‘–”ƒ•–ǡ „‘–Š ‘–Š‡” ’”‘–‡‹•ƒ†–Š‡‹” Ǧ͵Ǧ”‡•–”‹ –‡†‡’‹–‘’‡•‹†— ‡†•‹‰‹ˆ‹ ƒ–Ž‡˜‡Ž•‘ˆ

(99) ǦJǤ‘  ‡ŽŽ ”‡•’‘•‡• ‘—Ž† „‡ †‡–‡ –‡† ƒ‰ƒ‹•– ƒ› ‘ˆ –Š‡ ƒ–‹‰‡ ‹ ‘ Ǧ‹—‹œ‡† Ǧ͵Ǥ„Ͳ‹ ‡Ǥ   A B E:T ratio  100 2000 100:1 1500 BCG  50:1 mock 1000 25:1  75 800 5:1  600  50  400  25 200   0 0  hsp65 85B 85B HspX HspX HspX Flu 369-377 143-152 199-207 91-105 21-29 120-128 58-66    bmice,immunizedwithBCG.Tendaysafter Figure5.(A)CytotoxicactivityofsplenocytesderivedfromHLAA2/K  immunization, splenocytes were harvested and analyzed for their ability to lyse human HLAA*0201positive  target cells (JY). HLAA*0201restricted peptides used to pulse target cells are indicated on the x axis. The HLA  A*0201bindingpeptideofinfluenzaAmatrix(F5866)wasusedasacontrol.E:T,effectortotargetratio.The sequences of peptides used in the CTL assay were as follows: hsp65p369377: KLAGGVAVI, Ag85B p143152:  FIYAGSLSAL,Ag85Bp199207:KLVANNTRL,HspXp91105:SEFAYGSFVRTVSLP,HspXp2129:LFAAFPSFA,HspX  p120128:GILTVSVAVandinfluenzaAmatrixp5866:GILGFVFTL.(B).IFNproductionofsplenocytesofHLA  0miceimmunizedwithBCGinPBS(BCG)orwithPBSonly(mock).Theantigensusedforinvitrochallenge DR3.Ab  aregivenonthexaxis..

(100) TcellresponsestoM.tuberculosisDosRantigenHspX. Discussion ‹ ‡   ˜ƒ ‹ƒ–‹‘ †‘‡• ‘– ’”‘–‡ – ƒ‰ƒ‹•– ”‡ƒ –‹˜ƒ–‹‘ ‘ˆ ǡ › ‘„ƒ –‡”‹ƒŽ ƒ–‹‰‡•–Šƒ–‹†— ‡†‹ˆˆ‡”‡–‹ƒŽ ‡ŽŽ”‡•’‘•‡•‹‹†‹˜‹†—ƒŽ•Žƒ–‡–Ž›‹ˆ‡ –‡†™‹–ŠM. tuberculosis ‘’ƒ”‡† –‘ —‡š’‘•‡†  Ǧ˜ƒ ‹ƒ–‡† ‹†‹˜‹†—ƒŽ• ƒ› Š‡Ž’ –‘ ‹†‡–‹ˆ› ’”‘–‡ –‹˜‡ƒ–‹‰‡•ƒ†‹—‡”‡•’‘•‡•ƒ‰ƒ‹•–Ǥ— Šƒ–‹‰‡•ƒ›„‡ƒ’’Ž‹‡† ‹†‡˜‡Ž‘’‡–‘ˆ’‘•–Ǧ‡š’‘•—”‡˜ƒ ‹‡•ƒ•™‡ŽŽƒ•ˆ‘”‡™•’‡ ‹ˆ‹ †‹ƒ‰‘•–‹ –‘‘Ž•Ǥ › ‡ƒ• ‘ˆ ™Š‘Ž‡ ‰‡‘‡  ‹ ”‘ƒ””ƒ› ‡š’”‡••‹‘ ’”‘ˆ‹Ž‹‰ ƒ† ’”‘–‡‘‹  ƒƒŽ›•‹•Ͷͺ•‘Ǧ ƒŽŽ‡†Žƒ–‡ ›ƒ–‹‰‡•Šƒ˜‡„‡‡‹†‡–‹ˆ‹‡†ȋ͵ͷȌǤ

(101) ƒ’”‡˜‹‘—••–—†›ǡ ‘—”‰”‘—’ƒƒŽ›œ‡†–Š‡‹—‘‰‡‹ ‹–›‘ˆʹͷ‘ˆ–Š‡•‡Žƒ–‡ ›ƒ–‹‰‡•ƒ†‘„•‡”˜‡† •–”‘‰

(102) ǦJ”‡•’‘•‡•’ƒ”–‹ —Žƒ”Ž›‹Žƒ–‡–Ž›‹ˆ‡ –‡†‹†‹˜‹†—ƒŽ•ȋʹͷȌǤ

(103) –Š‡’”‡•‡–•–—†›™‡Šƒ˜‡ˆ‘ —••‡†‘–Š‡Žƒ–‡ ›ƒ–‹‰‡ •’ǡƒ•‹–‹•”‡“—‹”‡† ˆ‘” „ƒ –‡”‹ƒŽ ‰”‘™–Š ™‹–Š‹ –Š‡ ƒ ”‘’Šƒ‰‡ ƒ† ‹• ’”‡†‘‹ƒ–Ž› ’”‡•‡– †—”‹‰ •–ƒ–‹‘ƒ”›‰”‘™–Š‘ˆM.tuberculosisȋ͵͹ȌǤ‘”‡‘˜‡”ǡ•–”‘‰ ‡ŽŽ”‡•’‘•‡•–‘ •’‹ ˆ”‹ ƒ’‘’—Žƒ–‹‘•™‡”‡‘„•‡”˜‡†™Š‹ Š™‡”‡‘•–Ž›”‡•–”‹ –‡†–‘Žƒ–‡–Ž›‹ˆ‡ –‡† ‹†‹˜‹†—ƒŽ• ȋͳͶȌǤ

(104)  ƒ††‹–‹‘ǡ  ‡ŽŽ• ˆ”‘  ’ƒ–‹‡–• ”‡ ‘‰‹œ‹‰ –Š‡ •’ ’”‘–‡‹ •Š‘™‡†ƒ•™‹– Šˆ”‘ŠͲ–‘™ƒ”†•Šͳƒˆ–‡” Š‡‘–Š‡”ƒ’›ǡ‹†‹ ƒ–‹‰–Š‡‹”’‘–‡–‹ƒŽ –‘‹†— ‡’”‘–‡ –‹˜‡ ‡ŽŽ”‡•’‘•‡•ȋͻǡͳͷȌǤŠ‡•‡ Šƒ”ƒ –‡”‹•–‹ •‘ˆ •’ƒ‡‹–ƒ ‹–‡”‡•–‹‰ –ƒ”‰‡– ˆ‘” ’‘•–Ǧ‡š’‘•—”‡  ˜ƒ ‹ƒ–‹‘ ƒ• ™‡ŽŽ ƒ• ˆ‘” –Š‡ ’‘••‹„Ž‡ †‹ƒ‰‘•‹•‘ˆ’”‡ Ž‹‹ ƒŽ‹ˆ‡ –‹‘Ǥ ‡ Š‡”‡ †‡• ”‹„‡ •’Ǧ•’‡ ‹ˆ‹   ‡ŽŽ ”‡•’‘•‡• ‹ M. tuberculosis ȋŽ‹‡Ž›ȌǦ‡š’‘•‡† ‹†‹˜‹†—ƒŽ•ȋ’ƒ–‹‡–•ǡΪƒ•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽ•ǡ ˜ƒ ‹‡‡•™‹–Š’‘•‹–‹˜‡ Ǧ͸ƒ†Ȁ‘” ǦͳͲ ‡ŽŽ”‡•’‘•‡•Ȍ‹ ‘’ƒ”‹•‘–‘M.tuberculosis—‡š’‘•‡† ‹†‹˜‹†—ƒŽ• ȋ  ˜ƒ ‹‡‡• ƒ† Š‡ƒŽ–Š› ‘–”‘Ž• Žƒ ‹‰ ƒ  ‡ŽŽ ”‡•’‘•‡ –‘ Ǧ͸ ƒ†Ȁ ‘”  ǦͳͲȌǤ —” ”‡•—Ž–• •Š‘™ –Šƒ– ‘•– ȋʹͶ ‘ˆ ͵Ͷǡ ȏ͹ͳΨȐȌǡ M. tuberculosis ‹ˆ‡ –‡† ‘” Ȃ‡š’‘•‡† ‹†‹˜‹†—ƒŽ• ”‡•’‘†‡† ™‡ŽŽ ‹ –Š‡ 

(105)  ƒ••ƒ› –‘ •’ ™Š‡”‡ƒ••’‘”ƒ†‹ ƒ†•‹‰‹ˆ‹ ƒ–Ž›Ž‘™‡””‡•’‘•‡•™‡”‡‘„•‡”˜‡†‹M.tuberculosis —‡š’‘•‡† ‹†‹˜‹†—ƒŽ• ‹ Ž—†‹‰   ˜ƒ ‹ƒ–‡† ‹†‹˜‹†—ƒŽ• ™‹–Š‘—– ƒ› ‘™ ‡š’‘•—”‡–‘M.tuberculosis.Š‹••—‰‰‡•–•–Šƒ– ˜ƒ ‹ƒ–‹‘ƒŽ‘‡†‘‡•‘–‹†— ‡  ‡ŽŽ”‡•’‘•‡•ƒ‰ƒ‹•––Š‡ •’ƒ–‹‰‡Ǥ‹‹Žƒ”ˆ‹†‹‰• ‘‡ˆ”‘ƒ•–—†›‹Š‡. ƒ„‹ƒ™Š‡”‡‡‘ƒ–ƒŽ ‹—‹œƒ–‹‘†‹†‘–Ž‡ƒ†–‘

(106) ǦJ”‡•’‘•‡•–‘ •’‘”  ǦͳͲǡ ™Š‡”‡ƒ• –Š‡•‡ ’”‘–‡‹• ™‡”‡ ™‡ŽŽǦ”‡ ‘‰‹œ‡† ‹ M. tuberculosisǦ‡š’‘•‡† Š‘—•‡Š‘Ž† ‘–ƒ –•ƒ†Š‡ƒŽ–Š ƒ”‡™‘”‡”•ȋ͵͵ȌǤ ‡˜‡”ƒŽ •–—†‹‡• Šƒ˜‡ ‹†‹ ƒ–‡† –Šƒ–   ƒ ‡š’”‡•• –Š‡ •’ Š‘‘Ž‘‰—‡ †—”‹‰ ‘š›‰‡†‡’Ž‡–‹‘ȋ͸ǡͳʹǡʹ͸ȌǢŠ‘™‡˜‡”ǡ‘—”†ƒ–ƒ•—‰‰‡•––Šƒ–˜ƒ ‹ƒ–‹‘™‹–Š ‹ Š—ƒ• †‘‡• ‘– ‹†— ‡ ‹—‡ ”‡•’‘•‡• ƒ‰ƒ‹•– •’Ǥ ‡ Š›’‘–Š‡•‹œ‡ –Šƒ– –Š‡ ‡š’”‡••‹‘ ‘ˆ •’ „›   ‹ ˜‹˜‘ ƒˆ–‡” ˜ƒ ‹ƒ–‹‘ ‹• Ž‹‹–‡† ƒ† ‹•—ˆˆ‹ ‹‡– –‘ ‹†— ‡ƒ‹—‡”‡•’‘•‡Ǥ ‘ ‘ˆ‹”–Š‹• ƒ••—’–‹‘ ‹ ˜‹˜‘ǡ ™‡ ƒ’’Ž‹‡†   ‹—‹œƒ–‹‘–‘ƒ Ǧ Žƒ••

(107)  ƒ† Ǧ Žƒ••

(108)

(109)  –‰ ‘—•‡ ‘†‡Ž• Ǧ ǦʹȀ„ ƒ† Ǧ͵Ǥ„Ͳǡ ”‡•’‡ –‹˜‡Ž›Ǧ ™Š‹ Š ’”‘˜‹†‡ ’‘™‡”ˆ—Ž ‘†‡Ž• –‘ Š‡Ž’ Šƒ”ƒ –‡”‹œ‹‰ ‹ ˜‹˜‘  ‡ŽŽ ”‡•’‘•‡• ƒ‰ƒ‹•– › ‘„ƒ –‡”‹ƒŽƒ–‹‰‡•‹–Š‡ ‘–‡š–‘ˆ ’‘Ž›‘”’Š‹•ȋͳͺǡʹͲȌǤ —”–Š‡”‘”‡ǡ–Š‡ ǦȗͲʹͲͳ ƒ† ǦͳȗͲ͵Ͳͳ ƒŽŽ‡Ž‡• ƒ”‡ ƒŒ‘”  ƒŽŽ‡Ž‡• ƒ• ˆ”‡“—‡ ‹‡• ƒ”‡ ͶʹΨ ƒ† ʹͶΨǡ ”‡•’‡ –‹˜‡Ž› ‹ ƒ— ƒ•‹ƒǡ ˆ”‹ ƒȀ ˆ”‘Ǧƒ”‹„„‡ƒǡ ƒ† ”‹‡–ƒŽ ’‘’—Žƒ–‹‘•ȋͺȌǤ. ͳͳͷ.

(110) Chapter5. ’Ž‡‘ ›–‡• †‡”‹˜‡† ˆ”‘  Ǧ‹—‹œ‡† ǦȗͲʹͲͳǤ„ ‹ ‡ ˆƒ‹Ž‡† –‘ Ž›•‡ –ƒ”‰‡– ‡ŽŽ• ’—Ž•‡† ™‹–Š ǦȗͲʹͲͳǦ”‡•–”‹ –‡† •’ ’‡’–‹†‡•ǡ ™Š‡”‡ƒ• –ƒ”‰‡– ‡ŽŽ• ’—Ž•‡† ™‹–Š ǦȗͲʹͲͳǦ”‡•–”‹ –‡†‡’‹–‘’‡•†‡”‹˜‡†ˆ”‘Š•’͸ͷ‘”‰ͺͷ‘ˆM.tuberculosis ™‡”‡ Ž›•‡† —’ –‘ ͷͶΨ ȋ ‹‰—”‡ ͷȌǤ ‹‹Žƒ”Ž› •’Ž‡‘ ›–‡• ˆ”‘  Ǧ‹—‹œ‡† ͵Ǥ„Ͳ ‹ ‡ ™‡”‡ ŠƒŽŽ‡‰‡† ‹ ˜‹–”‘ ™‹–Š •‡˜‡”ƒŽ › ‘„ƒ –‡”‹ƒŽ ƒ–‹‰‡ ƒ† –Š‡‹” Ǧ͵Ǧ”‡•–”‹ –‡† ’‡’–‹†‡•Ǥ Š‡”‡ƒ• ‹—‹œƒ–‹‘ ‘ˆ –Š‡ Ǧ͵Ǥ„Ͳ ‹ ‡ ™‹–Š –Š‡ •’ ’”‘–‡‹ ‹†— ‡† ”‡•’‘•‡• ƒ‰ƒ‹•– •’ ’͵ͳǦͷͲ ƒ† –Š‡ •’ ’”‘–‡‹ ȋ ‹‰—”‡ ͶȌǡ •— Š ”‡•’‘•‡• ™‡”‡ ‘– †‡–‡ –‡† ƒˆ–‡”   ‹—‹œƒ–‹‘ ‘ˆ Ǧ ͵Ǥ„Ͳ ‹ ‡ ȋ ‹‰—”‡ ͷȌǤ

(111)  ‘–”ƒ•–ǡ „‘–Š –Š‡ ›–‘•‘Ž‹  Š•’͸ͷ ƒ† –Š‡ •‡ ”‡–‡† ‰ͺͷ‘ˆM.tuberculosisƒ†–Š‡‹””‡•’‡ –‹˜‡ Ǧ͵Ǧ”‡•–”‹ –‡†‡’‹–‘’‡•ǡ’͵Ǧͳ͵ƒ† ’ͷͳǦ͹Ͳǡ‹†— ‡†•‹‰‹ˆ‹ ƒ–Ž‡˜‡Ž•‘ˆ

(112) ǦJ‹–Š‡•‡‹ ‡ǡƒˆ‹†‹‰ ‘•‹•–‡–™‹–Š‘—” ’”‡˜‹‘—•ˆ‹†‹‰•ȋͳͺȌǤŠ‡•‡†ƒ–ƒ‹†‹ ƒ–‡–Šƒ– ‹—‹œƒ–‹‘†‘‡•‘–’”‘˜‘‡ ‡ŽŽ ”‡•’‘•‡• ƒ‰ƒ‹•– –Š‡ •’ Žƒ–‡ › ƒ–‹‰‡ǡ ™Š‹Ž‡ ‹– ‹• ’‘••‹„Ž‡ –‘ ‹†— ‡ ͶΪǦ ƒ†ͺΪ ‡ŽŽ”‡•’‘•‡•–‘ •’—•‹‰’”‘–‡‹Ǧ‘”˜ƒ ‹ƒ–‹‘ǤŠ—•ǡ–Š‡•‡†ƒ–ƒ •—‰‰‡•– –Šƒ– ‡š’”‡••‹‘ ‘ˆ •’ „›   ƒˆ–‡” ‹ ˜‹˜‘ ˜ƒ ‹ƒ–‹‘ ‹• ’”‘„ƒ„Ž› Ž‘™ ‘’ƒ”‡† –‘ –Š‡ ‡š’”‡••‹‘ ‘ˆ ‹–• Š‘‘Ž‘‰—‡ ‹ M. tuberculosis —†‡” Žƒ–‡– ‘†‹–‹‘• ƒ† –Š—• ™‹ŽŽ ‘– Ž‡ƒ† –‘ ƒ •‹‰‹ˆ‹ ƒ– ‹—‡ ”‡•’‘•‡ †‹”‡ –‡† ƒ‰ƒ‹•– •’Ǥ Š‹••–—†›•Š‘™•–Šƒ– ˜ƒ ‹ƒ–‹‘ƒŽ‘‡†‘‡•‘–‹†— ‡ ‡ŽŽ”‡•’‘•‡•ƒ‰ƒ‹•– –Š‡ •’ƒ–‹‰‡„—––Šƒ– •’‹•ƒ‹—‘‰‡‹ ƒ–‹‰‡–Šƒ–Šƒ”„‘”••‡˜‡”ƒŽ ‡ŽŽ ‡’‹–‘’‡•Ǥ Š—•ǡ ™‡ ƒ–‹ ‹’ƒ–‡ –Šƒ– ‹’”‘˜‡† ȋ Ȍ ˜ƒ ‹‡•ǡ ‡š’”‡••‹‰ ”‡Ž‡˜ƒ– ˆ”ƒ‰‡–•‘ˆM.tuberculosisŽƒ–‡ ›ƒ–‹‰‡•ȋ͹ǡʹͷȌǡ ƒ›Šƒ˜‡’‘–‡–‹ƒŽƒ•˜ƒ ‹‡• ƒ‰ƒ‹•–Žƒ–‡–Ǥ . Acknowledgements Š‹• •–—†› ™ƒ• •—’’‘”–‡† „› –Š‡ ‡–Š‡”Žƒ†• ‡’”‘•› ‡Ž‹‡ˆ ‘—†ƒ–‹‘ ȋȌǡ –Š‡  ‹‡ ‡ƒ†‡ Š‘Ž‘‰›ˆ‘”‡˜‡Ž‘’‡–’”‘‰”ƒ‘ˆ–Š‡—”‘’‡ƒ‘—‹–›ȋȌǡ –‹ Š–‹‰ ‹ ”‘„‹‘Ž‘‰› ‡‹†‡ ƒ† –Š‡ —”‘’‡ƒ ‘‹••‹‘ ȋ͸–Š ”ƒ‡™‘” ”‘‰”ƒ‡ ‘–”ƒ –‘Ǥ ǦǦʹͲͲ͵ǦͷͲ͵͵͸͹ȌǤ‡™‘—Ž†Ž‹‡–‘–ŠƒǤ”‹•ˆ‘” ‘ŽŽ‡ –‹‰’ƒ–‹‡–ƒ–‡”‹ƒŽƒ†”Ǥ Ǥ••‡†‘”’ˆ‘” ”‹–‹ ƒŽŽ›”‡ƒ†‹‰–Š‹•ƒ—• ”‹’–Ǥ. ͳͳ͸.

(113) TcellresponsestoM.tuberculosisDosRantigenHspX. References ͳǤ . ʹǤ. . ͵Ǥ. . ͶǤ. . ͷǤ. . ͸Ǥ. . ͹Ǥ. . ͺǤ. . ͻǤ. . ͳͲǤ. . ͳͳǤ. . ͳʹǤ. . ͳ͵Ǥ. . ͳͶǤ. . ͳͷǤ. . ͳ͸Ǥ. . ͳ͹Ǥ. . ͳͺǤ. ‰”‡™ƒŽƒǡ ǤǤƒ†Ǥ Ǥ‹Ž‹•‘ǤͳͻͻͺǤ‹ˆˆ‡”‡–‹ƒŽ”‡‰—Žƒ–‹‘‘ˆŠͳƒ†Šʹ ‡ŽŽ•„›’ͻͳǦͳͳͲƒ† ’ʹͳǦͶͲ ’‡’–‹†‡• ‘ˆ –Š‡ ͳ͸Ǧ ƒŽ’ŠƒǦ ”›•–ƒŽŽ‹ ƒ–‹‰‡ ‘ˆ Mycobacterium tuberculosisǤ Clin. Exp. ImmunolǤͳͳͶǣ͵ͻʹǦ͵ͻ͹Ǥ ‰”‡™ƒŽƒǡ Ǥ Ǥ ƒ† Ǥ Ǥ ‹Ž‹•‘Ǥ ͳͻͻͻǤ

(114) ˆŽ—‡ ‡ ‘ˆ Ǧ ‘ –Š‡ ’Š‡‘–›’‡ ‘ˆ ͶΪ  Ž›’Š‘ ›–‡• •’‡ ‹ˆ‹  ˆ‘” ƒ ‡’‹–‘’‡ ‘ˆ –Š‡ ͳ͸Ǧƒ ƒŽ’ŠƒǦ ”›•–ƒŽŽ‹ ƒ–‹‰‡ ‘ˆ Mycobacterium tuberculosisǤEur.J.ImmunolǤʹͻǣͳ͹ͷ͵Ǧͳ͹͸ͳǤ †‡”•‡ǡ Ǥ ƒ† Ǥ Ǥ ‘Š‡”–›Ǥ ʹͲͲͷǤ Š‡ •— ‡•• ƒ† ˆƒ‹Ž—”‡ ‘ˆ   Ǧ ‹’Ž‹ ƒ–‹‘• ˆ‘” ƒ ‘˜‡Ž –—„‡” —Ž‘•‹•˜ƒ ‹‡ǤNat.Rev.MicrobiolǤ͵ǣ͸ͷ͸Ǧ͸͸ʹǤ ”‡†ǡǤǤƒ† ǤǤ˜ƒ‹••‡ŽǤʹͲͲʹǤ˜‹†‡ ‡‘ˆ‡†‘‰‡‘—•”‡ƒ –‹˜ƒ–‹‘‘ˆ–—„‡” —Ž‘•‹•ƒˆ–‡”ƒŽ‘‰ ’‡”‹‘†‘ˆŽƒ–‡ ›ǤJ.Infect.DisǤͳͺ͸ǣͺ͹͸Ǧͺ͹͹Ǥ Žƒ ǡ Ǥ Ǥǡ ‡‹”ǡ Ǥ Ǥǡ Ž‘›†ǡ Ǥǡ Ž‹••ǡ Ǥǡ ƒ”†‘”ˆˆǡ Ǥ Ǥǡ ”ƒ’‹ǡ Ǥ Ǥǡ ‰™‹”ƒǡ Ǥǡ ‹ ŠƒŽ‹ǡ Ǥǡ ƒœƒ”‡–ŠǡǤǡŽƒ ™‡ŽŽǡ ǤǤǡ”ƒ•‘ǡǤǡŠƒ‰—Ž—ƒǡǤǤǡ‘‘˜ƒǡǤǡ ƒ”ƒǡǤǡ‹‰ǡǤǡ ‹‡ǡǤǤǡ ƒ† ‘ ”‡ŽŽǡ Ǥ Ǥ ʹͲͲʹǤ  Ǧ‹†— ‡† ‹ ”‡ƒ•‡ ‹ ‹–‡”ˆ‡”‘Ǧ‰ƒƒ ”‡•’‘•‡ –‘ › ‘„ƒ –‡”‹ƒŽ ƒ–‹‰‡• ƒ† ‡ˆˆ‹ ƒ › ‘ˆ   ˜ƒ ‹ƒ–‹‘ ‹ ƒŽƒ™‹ ƒ† –Š‡ ǣ –™‘ ”ƒ†‘‹•‡† ‘–”‘ŽŽ‡† •–—†‹‡•Ǥ Lancet͵ͷͻ:ͳ͵ͻ͵ǦͳͶͲͳǤ ‘‘ǡ Ǥǡ Ǥ ‹ǡ Ǥ ‹ǡ ƒ† Ǥ ‹ Ǥ ʹͲͲͳǤ ”‘–‡‹• ‘ˆ Mycobacterium bovis   ‹†— ‡† ‹ –Š‡ ƒ›‡ †‘”ƒ ›‘†‡ŽǤJ.BacteriolǤͳͺ͵ǣʹ͸͹ʹǦʹ͸͹͸Ǥ ‘•Š‘ˆˆǡ Ǥ

(115) Ǥ ƒ† Ǥ Ǥ ƒ””›ǡ

(116)

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(118) Ǥ ʹͲͲͷǤ —„‡” —Ž‘•‹• Ǧ ‡–ƒ„‘Ž‹• ƒ† ”‡•’‹”ƒ–‹‘ ‹ –Š‡ ƒ„•‡ ‡ ‘ˆ ‰”‘™–ŠǤNat.Rev.MicrobiolǤ͵ǣ͹ͲǦͺͲǤ ”‘™ǡ Ǥǡ Ǥ ‘Ž‡•ǡ Ǥ Ǥ ”‘™ǡ Ǥ ‘–”‡”ƒ•ǡ ƒ† Ǥ Ǥ ƒ˜ƒ””‡–‡Ǥ ʹͲͲͲǤ Ǧǡ Ǧ ƒ† Ǧ ƒ–‹‰‡ ˆ”‡“—‡ ‹‡•‘ˆ–Š‡‘†‘‘”†Ž‘‘†ƒ—‹–•†‹ˆˆ‡”ˆ”‘–Š‘•‡ˆ‘—†‹‡•–ƒ„Ž‹•Š‡†„‘‡ƒ””‘™ †‘‘””‡‰‹•–”‹‡•ǤBoneMarrowTransplantǤʹͷǣͶ͹ͷǦͶͺͳǤ ƒ ƒ‘ǡ Ǥǡ Ǥ ‡”ƒ˜‹‰Ž‹ƒǡ Ǥ ‹‡Ž‹ǡ Ǥ ‘ƒ‘ǡ Ǥ ‹–‘‡ǡ ƒ† Ǥ ƒŽ‡”‘Ǥ ʹͲͲͷǤ ŠͲ –‘ Šͳ •™‹– Š ‘ˆ Ͷ  ‡ŽŽ Ž‘‡• •’‡ ‹ˆ‹  ˆ”‘ –Š‡ ͳ͸Ǧƒ ƒ–‹‰‡ ‘ˆ Mycobacterium tuberculosis ƒˆ–‡” •— ‡••ˆ—Ž –Š‡”ƒ’›ǣŽƒ ‘ˆ‹˜‘Ž˜‡‡–‘ˆ‡’‹–‘’‡”‡’‡”–‘‹”‡ƒ† ǦǤImmunol.LettǤͻͺǣʹͷ͵ǦʹͷͺǤ ƒ ƒ‘ǡ Ǥǡ Ǥ ‹Žƒ‘ǡ Ǥ Ǥ ‹ǡ Ǥ ‹‰ƒǡ Ǥ

(119) ˜ƒ›‹ǡ Ǥ Ǥ ”‡•›ǡ Ǥ ‹‡Ž‹ǡ ƒ† Ǥ ƒŽ‡”‘Ǥ ʹͲͲʹǤ

(120) †‡–‹ˆ‹ ƒ–‹‘ ‘ˆ ‡’‹–‘’‡• ‘ˆ Mycobacterium tuberculosis ͳ͸Ǧƒ ’”‘–‡‹ ”‡ ‘‰‹œ‡† „› Š—ƒ Ž‡—‘ ›–‡ƒ–‹‰‡ǦȗͲʹͲͳͺȋΪȌŽ›’Š‘ ›–‡•ǤJ.Infect.DisǤͳͺ͸ǣͻͻͳǦͻͻͺǤ Šƒ”‘ǡ ǤǡǤ ‡Ž—ǡǤ—†„ƒ ǡǤ‹”œƒ‹ǡǤǤ‹‡ŠŽǡǤ ǤƒŽ„‡”‰ǡǤ Š‘—”ǡǤ —”‹‰— Š‹ǡǤǤ˜ƒ ‡‹Œ‰ƒƒ”†‡ǡ Ǥ Ǥ ”‹ŒˆŠ‘—–ǡ Ǥ ‡‡ƒǡ Ǥ Ǥ ˜ƒǡ Ǥ ••‡†‘”’ǡ Ǥ Ǥ ––‡Š‘ˆˆǡ ƒ† Ǥ ‹‡••Ž‹‰Ǥ ʹͲͲͳǤŠ‡‹†‡–‹ˆ‹ ƒ–‹‘‘ˆƒ ‘‘’ƒ–Š‘‰‡Ǧ•’‡ ‹ˆ‹   Žƒ••

(121) ȗͲʹͲͳǦ”‡•–”‹ –‡† ›–‘–‘š‹  ‡ŽŽ ‡’‹–‘’‡‡ ‘†‡†™‹–Š‹–Š‡Š‡ƒ–•Š‘ ’”‘–‡‹͸ͷǤEur.J.Immunol.31ǣ͵͸ͲʹǦ͵͸ͳͳǤ —‹‰ŠƒǡǤ Ǥƒ†ǤǤ’”‡ƒ†„—”›ǤͳͻͻͺǤ› ‘„ƒ –‡”‹ƒŽ•–ƒ–‹‘ƒ”›’Šƒ•‡‹†— ‡†„›Ž‘™‘š›‰‡ –‡•‹‘ǣ ‡ŽŽ™ƒŽŽ–Š‹ ‡‹‰ƒ†Ž‘ ƒŽ‹œƒ–‹‘‘ˆ–Š‡ͳ͸Ǧ‹Ž‘†ƒŽ–‘ƒŽ’ŠƒǦ ”›•–ƒŽŽ‹Š‘‘Ž‘‰ǤJ.BacteriolǤ ͳͺͲǣͺͲͳǦͺͲͺǤ ƒ˜‹†‘™ǡǤǡƒƒ—Œ‹ƒǡ ǤǤǡŠ‹ǡǤǡƒ˜‹ƒ”ǡ Ǥǡ —‘ǡǤǡ—‰ǡǤǡƒ’Žƒǡ Ǥǡ‡œ‹‡•ǡǤǡƒ† ‡ƒ”‘ǡǤ Ǥ ʹͲͲͷǤ –‹„‘†› ’”‘ˆ‹Ž‡• Šƒ”ƒ –‡”‹•–‹  ‘ˆ Mycobacterium tuberculosis ‹ˆ‡ –‹‘ •–ƒ–‡Ǥ Infect.Immun. ͹͵:͸ͺͶ͸Ǧ͸ͺͷͳǤ ‡‹••‹‡ǡǤǡǤǤ‡›–‡ǡǤ„‡„‡ǡǤƒ••‹‡ǡǤ•‡ˆˆƒǡ Ǥ„ƒ–‡ǡ Ǥ Ž‡– Š‡”ǡǤ™‹ƒˆ‡ǡǤǤ ‹ŽŽǡǤ ”‘‘‡•ǡ Ǥ‘‘ǡǤ—ŽƒǡǤǤ”‡†ǡǤŽ‡‹ǡǤ Ǥ––‡Š‘ˆˆǡǤ†‡”•‡ǡƒ†ǤǤ‘Š‡”–›ǤʹͲͲ͸Ǥ ‡ ‘‰‹–‹‘‘ˆ•–ƒ‰‡Ǧ•’‡ ‹ˆ‹ › ‘„ƒ –‡”‹ƒŽƒ–‹‰‡•†‹ˆˆ‡”‡–‹ƒ–‡•„‡–™‡‡ƒ —–‡ƒ†Žƒ–‡–‹ˆ‡ –‹‘• ™‹–ŠMycobacteriumtuberculosisǤClin.VaccineImmunolǤͳ͵ǣͳ͹ͻǦͳͺ͸Ǥ ‹‡Ž‹ǡ ǤǡǤ‹‰ŠǡǤ’ƒŽŽ‡ǡǤ‘ƒ‘ǡǤ‹–‘‡ǡ Ǥ‹”‡ ‹ǡ Ǥ ”‹• ‹ƒǡǤǤ‹ǡǤƒ–‹‹ǡǤƒŽ‡”‘ǡƒ† Ǥ

(122) ˜ƒ›‹ǤʹͲͲͲǤŠƒ‰‡‘ˆŠͲ–‘Šͳ ‡ŽŽǦ ›–‘‹‡’”‘ˆ‹Ž‡ˆ‘ŽŽ‘™‹‰–—„‡” —Ž‘•‹• Š‡‘–Š‡”ƒ’›ǤScand. J.ImmunolǤͷʹǣͻ͸ǦͳͲʹǤ ‘Š‡”–›ǡǤǤƒ†Ǥ†‡”•‡ǤʹͲͲͷǤƒ ‹‡•ˆ‘”–—„‡” —Ž‘•‹•ǣ‘˜‡Ž ‘ ‡’–•ƒ†”‡ ‡–’”‘‰”‡••Ǥ Clin.Microbiol.RevǤͳͺǣ͸ͺ͹Ǧ͹ͲʹǤ ”ƒ‡ǡ Ǥ Ǥǡ Ǥ Ǥ ‹‡•–”ƒǡ Ǥ Ǥ ˜ƒ ‡‹Œ‰ƒƒ”†‡ǡ Ǥ —„”‘–‘ǡ Ǥ Ǥ †‡ǡ Ǥ Ǥ ––‡Š‘ˆˆǡ ƒ† Ǥ Ǥ ”‹ŒˆŠ‘—–Ǥ ʹͲͲͲǤ —”‹ˆ‹ ƒ–‹‘ ‘ˆ Š‹•Ǧ–ƒ‰‰‡† ’”‘–‡‹• „› ‹‘„‹Ž‹œ‡† Š‡Žƒ–‡ ƒˆˆ‹‹–› Š”‘ƒ–‘‰”ƒ’Š›ǣ –Š‡„‡‡ˆ‹–•ˆ”‘–Š‡—•‡‘ˆ‘”‰ƒ‹ •‘Ž˜‡–ǤProteinExpr.PurifǤͳͺǣͻͷǦͻͻǤ. ‡Ž—ǡǤǡǤƒ‡ŒƒǡǤǤ˜ƒ‡‹Œ‰ƒƒ”†‡ǡǤƒ‡ŽŽ‹ǡǤ„‘—Ǧ‡‹†ǡ ǤǤŠ‘Ž‡ǡǤǤ†‡”‹‡•ǡǤǤƒ˜‹†ǡ ƒ† Ǥ Ǥ ––‡Š‘ˆˆǤ ͳͻͻͺǤ

(123) †‡–‹ˆ‹ ƒ–‹‘ ‘ˆ  Žƒ••

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(125) Ǧ”‡•–”‹ –‡† †‡–‡”‹ƒ–• ‘ˆ Mycobacterium tuberculosisǦ†‡”‹˜‡†’”‘–‡‹•„›—•‹‰ Ǧ–”ƒ•‰‡‹ ǡ Žƒ••

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(127) Ǧ†‡ˆ‹ ‹‡–‹ ‡ǤProc.Natl.Acad.Sci.U.S. AͻͷǣͳͲ͹ͻ͹ǦͳͲͺͲʹǤ. ͳͳ͹.

(128) Chapter5  .     .   .          . ͳͻǤ ‡Ž—ǡ Ǥǡ Ǥ Ǥ ˜ƒ ‡‹Œ‰ƒƒ”†‡ǡ Ǥ Ǥ ”‹ŒˆŠ‘—–ǡ ƒ† Ǥ Ǥ ––‡Š‘ˆˆǤ ͳͻͻͷǤ Ž‹’ „‹†• –‘  Žƒ••

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(130)  —•‹‰ ‡–Š‹‘‹‡Ǧ„ƒ•‡†ǡ ƒŽŽ‡Ž‡Ǧ†‡’‡†‡– ‘–‹ˆ• ƒ• ™‡ŽŽ ƒ• ƒŽŽ‡Ž‡Ǧ‹†‡’‡†‡– •—’‡”‘–‹ˆ•Ǥ Mol. ImmunolǤ͵ʹǣͻ͹ͷǦͻͺͳǤ ʹͲǤ ‡Ž—ǡǤǡǤǤ˜ƒ‡‹Œ‰ƒƒ”†‡ǡǤǤ ”ƒ‡ǡ ǤǤ”‹ŒˆŠ‘—–ǡǤ̵‘—œƒǡǤ‡ ‡”ǡǤ —›‰‡ǡƒ†Ǥ Ǥ ––‡Š‘ˆˆǤ ʹͲͲͲǤ

(131) †‡–‹ˆ‹ ƒ–‹‘ ‘ˆ ƒŒ‘” ‡’‹–‘’‡• ‘ˆ Mycobacterium tuberculosis  ͺͷ –Šƒ– ƒ”‡ ”‡ ‘‰‹œ‡† „› ǦȗͲʹͲͳǦ”‡•–”‹ –‡† ͺΪ  ‡ŽŽ• ‹ Ǧ–”ƒ•‰‡‹  ‹ ‡ ƒ† Š—ƒ•. J. ImmunolǤ ͳ͸ͷǣ͸Ͷ͸͵Ǧ͸Ͷ͹ͳǤ ʹͳǤ ‡Ž—ǡǤǡǤǤ˜ƒ‡‹Œ‰ƒƒ”†‡ǡǤ‘—–Š™‘‘†ǡǤ•‡”‘ˆˆǡ ǤǤ”‹ŒˆŠ‘—–ǡǤǤ†‡”‹‡•ǡǤ Ǥ––‡Š‘ˆˆǡ ƒ†Ǥ‡––‡ǤͳͻͻͶǤ Ǧ͵‘Ž‡ —Ž‡• ƒ„‹†’‡’–‹†‡• ƒ””›‹‰–™‘ƒŽ–‡”ƒ–‹˜‡•’‡ ‹ˆ‹ •—„‘–‹ˆ•ǤJ. ImmunolǤͳͷʹǣͷ͹ͶʹǦͷ͹ͶͺǤ ʹʹǤ ”‘–œ‡ǡ Ǥ Ǥ ƒ† Ǥ Ǥ ‡™‹•‘ŠǤ ʹͲͲͷǤ ‘Ž‡ ‘ˆ ͺΪ  Ž›’Š‘ ›–‡• ‹ ‘–”‘Ž ‘ˆ Mycobacterium tuberculosis‹ˆ‡ –‹‘Ǥ‹ ”‘„‡•Ǥ

(132) ˆ‡ –Ǥ͹ǣ͹͹͸Ǧ͹ͺͺǤ ʹ͵Ǥ —’–‡ǡǤǤͳͻͻͺǤƒ ‹‡–”‹ƒŽ•ƒ‰ƒ‹•–Ž‡’”‘•›ǤInt.J.Lepr.OtherMycobact.DisǤ͸͸ǣͷͺ͹ǦͷͺͻǤ ʹͶǤ —ǡ Ǥǡ Ǥ ‘˜ƒŠ‡†œƒ†‡Šǡ Ǥ Ǥ –‘‡”ǡ ƒ† Ǥ Ǥ ‘ƒ–‡•Ǥ ʹͲͲ͸Ǥ ‡Ž‡–‹‘ ‘ˆ –Š‡ Mycobacterium tuberculosis ƒŽ’ŠƒǦ ”›•–ƒŽŽ‹ǦŽ‹‡ Š•’ ‰‡‡ ƒ—•‡• ‹ ”‡ƒ•‡† „ƒ –‡”‹ƒŽ ‰”‘™–Š ‹ ˜‹˜‘Ǥ Infect. ImmunǤ ͹Ͷǣͺ͸ͳǦͺ͸ͺǤ ʹͷǤ ‡›–‡ǡ Ǥ Ǥǡ Ǥ Ǥ ‹ǡ Ǥ Ǥ ”ƒ‡ǡ Ǥ Ǥ ”‹‰‰‡ǡ Ǥ ”‹•ǡ Ǥ Ǥ ˜ƒ ‡‹Œ‰ƒƒ”†‡ǡ Ǥ

(133) Ǥ ‘•—‹Žǡ Ǥ ‡Ž†‹‰ŠǡǤ†‡”•‡ǡ ǤǤ Š‘‘Ž‹ǡǤǤ”‡†ǡǤ Ǥ––‡Š‘ˆˆǡƒ†ǤǤŽ‡‹ǤʹͲͲ͸Ǥ —ƒǦ ‡ŽŽ ”‡•’‘•‡• –‘ ʹͷ ‘˜‡Ž ƒ–‹‰‡• ‡ ‘†‡† „› ‰‡‡• ‘ˆ –Š‡ †‘”ƒ › ”‡‰—Ž‘ ‘ˆ Mycobacterium tuberculosisǤMicrobes.InfectǤͺǣʹͲͷʹǦʹͲ͸ͲǤ ʹ͸Ǥ ‹ǡ Ǥǡ Ǥ Ž‡—–‡”‹‘ǡ Ǥ —––‡”ǡ Ǥ —”—‰ƒ•—Ǧ‡‹ǡ ƒ† Ǥ ‹ Ǥ ͳͻͻͻǤ š›‰‡ †‡’Ž‡–‹‘Ǧ‹†— ‡† †‘”ƒ ›‹Mycobacteriumbovis ǤJ.BacteriolǤͳͺͳǣʹʹͷʹǦʹʹͷ͸Ǥ ʹ͹Ǥ ƒ„‘œ›ǡ Ǥ Ǥǡ ǤǤƒ‹• ŠǡǤŠ‡‰ǡǤ‘•‰”‘˜‡ǡǤǤ ”‹ˆˆ‹–Š•ǡ ǤǤ—–Š”ƒǡƒ†ǤǤƒ˜‹†Ǥͳͻͻ͸Ǥ Ǧͺ –”ƒ•‰‡‹  ‹ ‡ ƒ”‡ Š‹‰ŠŽ› •—• ‡’–‹„Ž‡ –‘ ‘ŽŽƒ‰‡Ǧ‹†— ‡† ƒ”–Š”‹–‹•ǣ ƒ ‘˜‡Ž ‘†‡Ž ˆ‘” Š—ƒ’‘Ž›ƒ”–Š”‹–‹•ǤJ.Exp.MedǤͳͺ͵ǣʹ͹Ǧ͵͹Ǥ ʹͺǤ ƒ–ŠƒǡǤǤǡǤǤ‹Ž‹•‘ǡǤ Ǥ‹Ž‹•‘ǡǤƒ–‹ˆǡ Ǥ Šƒ‡ǡ Ǥƒ•˜‘ŽǡǤǤ ‹ŽŽǡƒ†ǤƒŽ˜ƒ‹Ǥ ʹͲͲͲǤ ‹‰Š ˆ”‡“—‡ ‹‡• ‘ˆ ‹” —Žƒ–‹‰

(134) Ǧ‰ƒƒǦ•‡ ”‡–‹‰ ͺ ›–‘–‘š‹   ‡ŽŽ• •’‡ ‹ˆ‹  ˆ‘” ƒ ‘˜‡Ž   Žƒ••

(135) Ǧ”‡•–”‹ –‡†Mycobacteriumtuberculosis‡’‹–‘’‡‹M.tuberculosisǦ‹ˆ‡ –‡†•—„Œ‡ –•™‹–Š‘—– †‹•‡ƒ•‡.Eur.J.Immunol.͵Ͳǣʹ͹ͳ͵Ǧʹ͹ʹͳǤ ʹͻǤ ƒ”–ƒ‹ǡǤ ǤǡǤǤŽƒ›†‡ǡǤǤ‹‰ŠǡǤƒƒŽǡƒ† ǤǤ‡Ž‹•Ž‡ǤʹͲͲ͸Ǥ‹•‡ƒ•‡–ƒ–‡‹ˆˆ‡”‡–‹ƒ–‹‘ƒ†

(136) †‡–‹ˆ‹ ƒ–‹‘ ‘ˆ —„‡” —Ž‘•‹• ‹‘ƒ”‡”• ˜‹ƒ ƒ–‹˜‡ –‹‰‡ ””ƒ› ”‘ˆ‹Ž‹‰Ǥ Mol. Cell ProteomicsǤ ͷǣʹͳͲʹǦʹͳͳ͵Ǥ ͵ͲǤ ‡‹š‡‹”ƒǡǤǡǤǤ‡ Š—‹Œ•‡ǡǤ Ǥ†‡‘‹‰ǡǤǤ ƒŽ‡–‹Œǡƒ† ǤǤ”‹ŒˆŠ‘—–ǤʹͲͲʹǤŠ‡—•‡‘ˆ ƒ•ƒ‘ǦƒŽ‘†‘”‘—•• ƒ˜‡‰‡”‹ ‘ Ǧ„ƒ•‡†’‡’–‹†‡•›–Š‡•‹•ǤProteinPept.LettǤͻǣ͵͹ͻǦ͵ͺͷǤ ͵ͳǤ ˜ƒ ‹š–‡”‡ǡ Ǥ Ǥǡ Ǥ Ǥ ƒ••‹†›ǡ Ǥ Ǥ ‡†‡‰ƒƒ”†ǡ Ǥ Ǥ ‰‰‡”ǡ ƒ† Ǥ †‡”•‡Ǥ ʹͲͲͲǤ ‘–”‘Ž ‘ˆ Žƒ–‡– Mycobacterium tuberculosis ‹ˆ‡ –‹‘ ‹• †‡’‡†‡– ‘ ͺ  ‡ŽŽ•Ǥ Eur. J. ImmunolǤ ͵Ͳǣ͵͸ͺͻǦ ͵͸ͻͺǤ ͵ʹǤ ˜ƒ‹š–‡”‡ǡǤǤǡƒ˜ǡǤǡ‰‰‡”ǡǤǤǡ‘ŽŽ‘ ǡ Ǥǡƒ††‡”•‡ǡǤʹͲͲͲǤ‹ƒ‰‘•‹•‘ˆ–—„‡” —Ž‘•‹• „ƒ•‡†‘–Š‡–™‘•’‡ ‹ˆ‹ ƒ–‹‰‡•Ǧ͸ƒ† ͳͲǤClin.Diagn.LabImmunol.͹:ͳͷͷǦͳ͸ͲǤ ͵͵Ǥ ‡‡ƒ•ǡ ǤǡǤǤ–ƒǡ Ǥ‹ŽŽƒŠǡǤ ‹‡Ž†‹‰ǡǤǤŽ†‡”•‘ǡǤǤ‡‹›ǡǤƒŽ‡ƒ•ǡǤǤ †ƒǡǤ ‹‡Šƒ”†–ǡ ƒ† Ǥ ƒ” Šƒ–Ǥ ʹͲͲͶǤ

(137) —‡ ”‡•’‘•‡• –‘ › ‘„ƒ –‡”‹ƒŽ ƒ–‹‰‡• ‹ –Š‡ ƒ„‹ƒ ’‘’—Žƒ–‹‘ǣ‹’Ž‹ ƒ–‹‘•ˆ‘”˜ƒ ‹‡•ƒ†‹—‘†‹ƒ‰‘•–‹ –‡•–†‡•‹‰ǤInfect.ImmunǤ͹ʹǣ͵ͺͳǦ͵ͺͺǤ ͵ͶǤ ‹–‹‡ŽŽ‘ǡ Ǥǡ Ǥ ƒ” Š‡•‹‹ǡ Ǥ —”œ‡ǡ Ǥ Ǥ Š‡”ƒǡ ƒ† Ǥ Ǥ Š‡•—–Ǥ ͳͻͻͳǤ ƒŽ›•‹• ‘ˆ –Š‡ Ǧ ”‡•–”‹ –‡† ‹ˆŽ—‡œƒǦ•’‡ ‹ˆ‹  ›–‘–‘š‹   Ž›’Š‘ ›–‡ ”‡•’‘•‡ ‹ –”ƒ•‰‡‹  ‹ ‡ ƒ””›‹‰ ƒ Š‹‡”‹  Š—ƒǦ‘—•‡ Žƒ••

(138) ƒŒ‘”Š‹•–‘ ‘’ƒ–‹„‹Ž‹–› ‘’Ž‡šǤJ.Exp.MedǤͳ͹͵ǣͳͲͲ͹ǦͳͲͳͷǤ ͵ͷǤ ‘•—‹Žǡ Ǥ

(139) Ǥǡ Ǥ  Šƒ’’‹‰‡”ǡ Ǥ Ǥ ‹• ‘–‹ǡ Ǥ

(140) Ǥ ƒ””‡ŽŽǡ Ǥ Ǥ ‘Ž‰ƒ‘˜ǡ Ǥ Ǥ Š‡”ƒǡ ƒ† Ǥ Ǥ  Š‘‘Ž‹Ǥ ʹͲͲ͵Ǥ

(141) Š‹„‹–‹‘ ‘ˆ ”‡•’‹”ƒ–‹‘ „› ‹–”‹  ‘š‹†‡ ‹†— ‡• ƒ Mycobacterium tuberculosis †‘”ƒ ›’”‘‰”ƒǤJ.Exp.MedǤͳͻͺǣ͹ͲͷǦ͹ͳ͵Ǥ ͵͸Ǥ  Ǥ Ž‘„ƒŽ –—„‡” —Ž‘•‹• ‘–”‘Žǡ —”˜‡‹ŽŽƒ ‡ǡ ’Žƒ‹‰ǡ ˆ‹ƒ ‹‰ ’Ǥ ͳǦʹͶ͹ǦʹͶ͹Ǥ  ǡ ‡‡˜ƒǡ ™‹–œ‡”Žƒ†Ǥǡ ͵͹Ǥ —ƒǡǤǡǤǤ”ƒ‡ǡǤǤ‹’•‘ǡǤǤŠ—ǡǤ Ǥ ‹ ‡›ǡǤǤŠ‡”ƒǡƒ†ǤǤƒ””›ǡ

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(144) ǤͳͻͻͺǤŠ‡ ͳ͸Ǧƒ ƒŽ’ŠƒǦ ”›•–ƒŽŽ‹ ȋ ”Ȍ ’”‘–‡‹ ‘ˆ Mycobacterium tuberculosis ‹• ”‡“—‹”‡† ˆ‘” ‰”‘™–Š ‹ ƒ ”‘’Šƒ‰‡•Ǥ”‘ ǤNatl.Acad.Sci.U.S.Aͻͷǣͻͷ͹ͺǦͻͷͺ͵Ǥ. ͳͳͺ.

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