Cover Page
The handle
http://hdl.handle.net/1887/77741
holds various files of this Leiden University
dissertation.
Author: Beyranvand Nejad, E.
&KDSWHU
'HPDUFDWHGWKUHVKROGVRIWXPRUVSHFL¿F
&'7FHOOVHOLFLWHGE\0&09EDVHGYDFFLQH
YHFWRUVSURYLGHUREXVWFRUUHODWHVRISURWHFWLRQ
Chapter 5
$EVWUDFW
%DFNJURXQG 7KHFDSDFLW\RIF\WRPHJDORYLUXV &09 WRHOLFLWORQJODVWLQJVWURQJ7FHOOUHVSRQVHVDQGWKH DELOLW\WRHQJLQHHUWKHJHQRPHRIWKLV'1$YLUXVSRVLWLRQV&09EDVHGYDFFLQHYHFWRUVKLJKO\ VXLWDEOHDVDFDQFHUYDFFLQHSODWIRUP'H¿QHGLPPXQHWKUHVKROGVIRUWXPRUSURWHFWLRQDQGWKH IDFWRUVD൵HFWLQJVXFKWKUHVKROGVKDYHQRWZHOOEHHQLQYHVWLJDWHGLQFDQFHULPPXQRWKHUDS\ :HKHUHGHWHUPLQHGXVLQJ&09DVDYDFFLQHSODWIRUPZKHWKHUFULWLFDOWKUHVKROGVRIYDFFLQH VSHFL¿F7FHOOUHVSRQVHVFDQEHHVWDEOLVKHGWKDWUHODWHWRWXPRUSURWHFWLRQDQGZKLFKIDFWRUV FRQWUROVXFKWKUHVKROGV 0HWKRGV:H JHQHUDWHG &09EDVHG YDFFLQH YHFWRUV H[SUHVVLQJ WKH ( HSLWRSH DQG WHVWHG WKHVH LQ SUHFOLQLFDOPRGHOVRI+39LQGXFHGFDQFHU9DFFLQDWLRQZDVDSSOLHGYLDGL൵HUHQWGRVHVDQG URXWHV LQWUDSHULWRQHDO ,3 VXEFXWDQHRXV 6& DQGLQWUDQDVDO ,1 7KHPDJQLWXGHNLQHWLFV DQGSKHQRW\SHRIWKHFLUFXODWLQJWXPRUVSHFL¿F&'7FHOOUHVSRQVHZHUHGHWHUPLQHG0LFH ZHUHVXEVHTXHQWO\FKDOOHQJHGZLWKWXPRUFHOOVDQGWKHWXPRUSURWHFWLRQZDVPRQLWRUHG 5HVXOWV
,PPXQL]DWLRQ ZLWK &09EDVHG YDFFLQHV YLD WKH ,3 RU 6& URXWH HOLFLWLQJ YDFFLQHLQGXFHG &'7FHOOUHVSRQVHVRI!RIWKHWRWDOFLUFXODWLQJ&'7FHOOSRSXODWLRQIXOO\SURWHFWV PLFHDJDLQVWOHWKDOWXPRUFKDOOHQJH+RZHYHUORZGRVHLQRFXODWLRQVYLDWKH,3RU6&URXWHRU ,1YDFFLQDWLRQHOLFLWHGYDFFLQHLQGXFHG&'7FHOOUHVSRQVHVWKDWGLGQRWUHDFKSURWHFWLYH WKUHVKROGVIRUWXPRUSURWHFWLRQ,QDGGLWLRQZKHUHDVZHDNSUHH[LVWLQJLPPXQLW\GLGQRW DOWHUWKHSURWHFWLYHWKUHVKROGVRIWKHYDFFLQHVSHFL¿F7FHOOUHVSRQVHIROORZLQJVXEVHTXHQW LPPXQL]DWLRQZLWK&09EDVHGYDFFLQHYHFWRUVVWURQJSUHH[LVWLQJLPPXQLW\LQKLELWHGWKH GHYHORSPHQWRIYDFFLQHLQGXFHG7FHOOVDQGWKHLUFRQWURORQWXPRUSURJUHVVLRQ &RQFOXVLRQV
7KLV VWXG\ KLJKOLJKW WKH H൵HFWLYHQHVV RI &09EDVHG YDFFLQH YHFWRUV DQG VKRZV WKDW GHPDUFDWHGWKUHVKROGVRIYDFFLQHVSHFL¿F7FHOOVFRXOGEHGH¿QHGWKDWFRUUHODWHWRWXPRU SURWHFWLRQ7RJHWKHUWKHVHUHVXOWVPD\KROGLPSRUWDQFHIRUFDQFHUYDFFLQHGHYHORSPHQWWR DFKLHYHKLJKH൶FDF\LQYDFFLQHUHFLSLHQWV
.H\ZRUGV
5
Chapter 5
%DFNJURXQG
Chapter 5
7FHOOUHVSRQVHVLQWKHSRSXODWLRQUDQJLQJIURPEDUHO\GHWHFWDEOHWRRIWKHPHPRU\ FRPSDUWPHQW ZH DOVR DGGUHVVHG WKH LPSDFW RI SUHH[LVWLQJ LPPXQLW\ RQ WKH SURWHFWLYH WKUHVKROGV >@ 'H¿QLQJ SURWHFWLRQ WKUHVKROGV FRXOG EH KLJKO\ UHOHYDQW LQ GHWHUPLQLQJ FRUUHODWHVRISURWHFWLRQFRQVLGHUHGDVDFUXFLDOHOHPHQWLQYDFFLQHGHYHORSPHQWDVLWSURYLGHV WKHOHYHORIYDFFLQHH൶FDF\LQYDFFLQHUHFLSLHQWV
:HIRXQGWKDW&09EDVHGYDFFLQHVHOLFLWLQJFOHDUO\GLVFHUQLEOH&'7FHOOUHVSRQVHVLQWKH EORRG !RIWKHWRWDO&'7FHOOSRSXODWLRQ IXOO\SURWHFWHGPLFHDJDLQVWOHWKDOWXPRU FKDOOHQJH ZKLOH UHODWLYHO\ ORZ UHVSRQVHV GR QRW SURYLGH SURWHFWLRQ 3UHYLRXV H[SRVXUH WR &09UHVXOWLQJLQDZHDNLPPXQHUHVSRQVHGLGQRWLPSDFWYDFFLQHH൶FDF\KRZHYHUVWURQJ SUHH[LVWLQJ LPPXQLW\ KDPSHUHG WKH LQGXFWLRQ RI YDFFLQHLQGXFHG 7 FHOOV FRQVHTXHQWO\ UHVXOWLQJ LQ ORVV RI WXPRU SURWHFWLRQ 7KH UHVXOWV KLJKOLJKW WKH SUHPLVH RI &09EDVHG YDFFLQHVDQGSXWIRUZDUGWKHSRWHQWLDORIVXFKYDFFLQHVLQKDOWLQJWXPRUGHYHORSPHQWEDVHG RQFRUUHODWHVRISURWHFWLRQWKDWDUHHVWDEOLVKHGRQFDUHIXOGHWHUPLQDWLRQRIYDFFLQHLQGXFHG LPPXQHUHVSRQVHV
0HWKRGV
0LFH
5
Chapter 5 UHVLVWDQFHJURZQDQGVXEMHFWHGWRDVHFRQGWUDQVIRUPDWLRQWRUHSODFHWKHJDO.NDQFDVVHWWH ZLWKWKH¿QDOLQVHUW7KHVHLQFOXGHGDQQHDOHGROLJRVXVHGIRU,(SHSWLGHIXVLRQRUWKH3&5 SURGXFWIRU0 ).%3> @DQG+39((LQVHUWLRQVFRQWDLQLQJWKHGHVLUHGPRGL¿FDWLRQ ZLWK WKH VDPH 0&09 ÀDQNLQJ KRPRORJ\ WR LQVHUW WKH JDO.NDQ FDVVHWWH 5HFRPELQDQW EDFWHULDZHUHFRXQWHUVHOHFWHGRQFKORUDPSKHQLFROGHR[\JDODFWRVH '2* PLQLPDOPHGLD SODWHVZLWKJO\FHURODVWKHFDUERQVRXUFH0&09%$&FRQVWUXFWVZHUHFKDUDFWHUL]HGE\ UHVWULFWLRQGLJHVW3&5VFUHHQLQJDQG6DQJHUDQG1*6VHTXHQFLQJ9LUXVZDVUHFRQVWLWXWHG E\HLWKHU/LSRIHFWDPLQH 7KHUPR)LVKHU6FLHQWL¿F WUDQVIHFWLRQRUHOHFWURSRUDWLRQ 9DQG X) RI1,+7V7LVVXH FXOWXUHGHULYHG VWRFNV RI WKH 0&09 YHFWRUV ZHUH DPSOL¿HG DQG WLWHUHG LQ 1,+ 7FHOOVJURZQLQFRPSOHWHJURZWKPHGLD '0(0)%636* ).%3WDJJHGYLUXVHVZHUH JURZQLQFRPSOHWHJURZWKPHGLDVXSSOHPHQWHGZLWK6KLHOGDWD¿QDOFRQFHQWUDWLRQRI X0DQGDGGHGHYHU\K> @&HOOIUHHYLUXVZDVREWDLQHGIURPVXSHUQDWDQWRILQIHFWHG FHOOVFODUL¿HGDWUSPIRUPLQDQGYLUXVZDVSHOOHWHGDWUSPIRUKWKURXJK DVRUELWROFXVKLRQ 'VRUELWRO07ULVS+P00J&O 9LUXVSHOOHWZDV UHVXVSHQGHGLQ3%6)RUYLUXVTXDQWL¿FDWLRQSODTXHDVVD\VZHUHSHUIRUPHGLQZHOOSODWHV E\LQIHFWLRQZLWKDSSURSULDWHVHULDOYLUXVGLOXWLRQLQP/RIPHGLDDQGWKHQLQFXEDWHGDW &IRUKURFNLQJ)ROORZLQJLQFXEDWLRQWKHLQIHFWHGFHOOVZHUHRYHUODLGZLWKP/FRPSOHWH PHGLDVXSSOHPHQWHGZLWKFDUER[\PHWK\OFHOOXORVH$IWHUWRGD\VWKHFHOOVZHUH¿[HGLQ IRUPDOGHK\GHLQ3%6DQGVWDLQHGZLWKDTXHRXVPHWK\OHQHEOXH7KHSODTXHV ZHUH FRXQWHG E\ OLJKW PLFURVFRS\ 0XOWLVWHS YLUXV UHSOLFDWLRQ FXUYHV ZHUH SHUIRUPHG LQ 1,+7FHOOVDW02,LQZHOOSODWHVUHSOLFDWHVSHUYLUXVSHUWLPHSRLQW9LUXVZDV LQFXEDWHGDW&IRUKZDVKHGWLPHVZLWK3%6DQGWKHQP/RIPHGLDZDVDGGHG 6XSHUQDWDQWZDVKDUYHVWHGDWDQGGD\VSRVWLQIHFWLRQVWRUHGDWí&DQGWLWHUHG E\SODTXHDVVD\).%3WDJJHGYLUXVHVZHUHJURZQLQFRPSOHWHJURZWKPHGLDVXSSOHPHQWHG ZLWK6KLHOGDWD¿QDOFRQFHQWUDWLRQRIX0DQGDGGHGHYHU\K
Chapter 5
FXOWXUHWXPRUFHOOOLQHV&HOOVZHUHFXOWXUHGLQDKXPLGL¿HGLQFXEDWRUDW&DQG&2 0\FRSODVPDWHVWVWKDWZHUHIUHTXHQWO\SHUIRUPHGIRUDOOFHOOOLQHVE\3&5ZHUHQHJDWLYH 7UHDWPHQW VFKHGXOH RI H[SHULPHQWV DUH LQGLFDWHG LQ WKH UHVSHFWLYH ¿JXUHV DQG OHJHQGV 0LFHZHUHYDFFLQDWHGZLWK0&09YHFWRUVYLDWKHLQWUDSHULWRQHDO ,3 LQWUDQDVDO ,1 RU VXEFXWDQHRXV 6& URXWHZLWKWKHLQGLFDWHGLQRFXOXPVL]H,QWXPRUH[SHULPHQWVPLFHZHUH LQRFXODWHGVXEFXWDQHRXVO\LQWKHÀDQNZLWK±î7&WXPRUFHOOVî&WXPRU FHOOVRUZLWKî0&29$LQȝO3%6FRQWDLQLQJ%6$RQGD\7XPRU VL]HZDVPHDVXUHGWZRWLPHVDZHHNXVLQJDFDOLSHU0LFHZHUHHXWKDQL]HGZKHQWXPRUVL]H UHDFKHG!PPLQYROXPHRUZKHQPLFHORVWRYHU!RIWKHLUWRWDOERG\ZHLJKW UHODWLYHWRLQLWLDOERG\PDVV ,QYLYRDQWLERG\XVDJH &'7FHOOGHSOHWLQJPRQRFORQDODQWLERGLHV FORQH ZHUHSXUFKDVHGIURP%LR;&HOO :HVW/HEDQRQ1+8QLWHG6WDWHV DQGDGPLQLVWHUHG,3WZLFHZHHNO\ ȝJPRXVH IRU ± ZHHNV &' 7 FHOO GHSOHWLRQ ZDV VWDUWHG GD\V EHIRUH WXPRU FKDOOHQJH 'HSOHWLRQ ZDVFKHFNHGE\VWDLQLQJIRU&'DQG&'PDUNHUH[SUHVVLRQIROORZHGE\ÀRZF\WRPHWULF DQDO\VLV
)ORZF\WRPHWU\
%ORRGFROOHFWLRQDQGSURFHVVLQJZDVSHUIRUPHGDVGHVFULEHG>@&HOOVZHUHUHVXVSHQGHG LQ VWDLQLQJ EX൵HU 3%6 )&6 VRGLXP D]LGH DQG LQFXEDWHG ZLWK YDULRXV ÀXRUHVFHQWO\ ODEHOOHG DQWLERGLHV GHWHFWLQJ &' FORQH ± &'/ FORQH 0(/ &' FORQH ,0 ./5* FORQH ) &' FORQH $ &' FORQH $5 $QWLERGLHVZHUHREWDLQHGIURPH%LRVFLHQFH 6DQ'LHJR&$8QLWHG6WDWHV %'%LRVFLHQFHV 6DQ -RVH &$ 8QLWHG 6WDWHV DQG %LROHJHQG 6DQ 'LHJR &$ 8QLWHG 6WDWHV )RU GHDG FHOO H[FOXVLRQ $PLQRDFWLQRP\FLQ ' ,QYLWURJHQ &DUOVEDG &$ 8QLWHG 6WDWHV ZDV XVHG 7R PHDVXUH WKH 0&09VSHFL¿F DQG WXPRU DQWLJHQVSHFL¿F 7 FHOO UHVSRQVHV 3( DQG$3&ODEHOOHG FODVV ,UHVWULFWHG PXOWLPHUV WHWUDPHUV RU GH[WUDPHUV ZLWK WKH SHSWLGH HSLWRSHV29$± 6,,1)(./ +39( ± 5$+<1,97) 0&090 ± +*,51$6), 0&090± 66330)59 ZHUHXVHG7HWUDPHUVZHUHSURGXFHGDV GHVFULEHG>@DQGGH[WUDPHUVZHUHREWDLQHGIURP,PPXGH[6DPSOHVZHUHDQDO\]HGZLWKD %'/65,,RU/65)RUWHVVDÀRZF\WRPHWHUDQGUHVXOWVZHUHDQDO\]HGXVLQJ)ORZ-RVRIWZDUH 7UHH6WDU$VKODQG258QLWHG6WDWHV ,QYLYRF\WRWR[LFLW\DVVD\
6SOHQRF\WHV RI QDwYH &' /\ PLFH ZHUH LVRODWHG DQG ORDGHG ZLWK 0+& FODVV ,
UHVWULFWHG( ±RU29$±SHSWLGHVIRUKDW&DWWKH¿QDOFRQFHQWUDWLRQRIȝJPO
5
Chapter 5 ZHUHODEHOOHGZLWKKLJKDQGORZFRQFHQWUDWLRQVRI&)6( ,QYLWURJHQ UHVSHFWLYHO\1H[W îSHSWLGHSXOVHGFHOOVZHUHSRROHGDQGLQMHFWHGLQWUDYHQRXV ,9 YLDWKHUHWURRUELWDO URXWHLQWRWKHPLFHWKDWKDYHEHHQYDFFLQDWHGGD\VHDUOLHUZLWK0&09EDVHGYDFFLQHV $IWHUKVSOHHQVRIWKHUHFLSLHQWPLFHZHUHLVRODWHGVWDLQHGIRU&'DQGVXEMHFWHG WR ÀRZ F\WRPHWU\7KH F\WRWR[LF FDSDFLW\ ZDV FDOFXODWHG UHODWLYH WR QDwYH PLFH E\ XVLQJ WKHIROORZLQJIRUPXOD >± SHUFHQWDJHRISXOVHGSHSWLGHLQLQIHFWHGPLFHSHUFHQWDJHRI XQSXOVHGSHSWLGHLQLQIHFWHGPLFH SHUFHQWDJHRISXOVHGSHSWLGHLQQDLYHPLFHSHUFHQWDJHRI XQSXOVHGSHSWLGHLQQDLYHPLFH @î(Q]\PHOLQNHG,PPXQR6SRW (/,6SRW DVVD\
6SOHQRF\WHV RI LQGLYLGXDO YDFFLQDWHG PLFH ZHUH LVRODWHG DQG FXOWXUHG RQ (/,6SRW SODWHV 5 ' V\VWHPV 0LQQHDSROLV 01 SUHFRDWHG ZLWK DQWL,)1Ȗ DQWLERGLHV 7KH LQGLFDWHG
SHSWLGHV ( ±5$+<1,97) RUDSRRORISHSWLGHVVSDQQLQJWKH(VHTXHQFHPHUV
RYHUODSSLQJ E\ ZHUH DGGHG DW ȝJPO K ODWHU FHOOV ZHUH UHPRYHG DQG VHFRQGDU\ DQWLERGLHVDGGHG3ODWHVZHUHGHYHORSHGDFFRUGLQJWRWKHPDQXIDFWXUHVLQVWUXFWLRQV6SRWV ZHUHFRXQWHGXVLQJDQ$,'(/,6SRWUHDGHU
6WDWLVWLFDODQDO\VLV
6WDWLVWLFDO DQDO\VHV ZHUH SHUIRUPHG XVLQJ *UDSK3DG 3ULVP /D -ROOD &$ 8QLWHV 6WDWHV 6XUYLYDOGDWDZHUHDQDO\]HGE\.DSODQ0HLHUDQGWKHORJUDQN 0DQWHO&R[ WHVW6WDWLVWLFDO VLJQL¿FDQFH ZDV GHWHUPLQHG E\ 0DQQ :KLWQH\ 3YDOXHV RI ZHUH FRQVLGHUHG VWDWLVWLFDOO\VLJQL¿FDQW
5HVXOWV
/RFDWLRQDQGVL]HRIIRUHLJQVHTXHQFHVLQ&09EDVHGYDFFLQHYHFWRUVLPSDFW YDFFLQHHOLFLWHG7FHOOUHVSRQVHVDQGDVVRFLDWHGWXPRUSURWHFWLRQ
7R GHWHUPLQH H൵HFWLYH UHJLRQV WR LQVHUW IRUHLJQ VHTXHQFHV ZH JHQHUDWHG UHFRPELQDQW 0&09EDVHGYDFFLQHYHFWRUVFRQWDLQLQJWKHHQWLUH(DQG(DQWLJHQVRIKXPDQSDSLOORPD YLUXVW\SH +39 ,QWKHVHYHFWRUVWKH((JHQHVUHSODFHGH[RQRI,( 0&09 ,(((IXOOOHQJWKUHSODFH RU((ZDVSODFHGLQWKHHQGSDUWRIWKH,(UHJLRQ 0&09 ,(((IXOOOHQJWKIXVLRQ )LJD 7RDQDO\]HWKH7FHOOUHVSRQVLYHQHVVWRWKHLQVHUWHG IRUHLJQVHTXHQFHVLQGXFHGE\WKHVHYDFFLQHYHFWRUVZH,3YDFFLQDWHGPLFHDQGPHDVXUHG WKH (VSHFL¿F 7 FHOO UHVSRQVHV E\ ,)1Ȗ (/,6327 IROORZLQJ UHVWLPXODWLRQ ZLWK WKH
LPPXQRGRPLQDQWSHSWLGHHSLWRSH( ±RUZLWKDSRRORIRYHUODSSLQJSHSWLGHVFRYHULQJWKH
Chapter 5
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
5$+<1,97) ( ± IXVHGWRWKHFDUER[\OWHUPLQXVRIWKH0&09,(JHQH 0&09
,(( &OHDUO\PLFHLPPXQL]HGZLWK0&09,((UHVXOWHGLQWKHKLJKHVW(VSHFL¿F 7FHOOUHVSRQVH a± DQGYDFFLQDWLRQZLWK0&09,((IXOO\SURWHFWHGPLFHDJDLQVW RWKHUZLVHOHWKDO7&WXPRUFKDOOHQJH )LJFDQGG ,QFRQFOXVLRQERWKWKHORFDWLRQDQG VL]H RI LQVHUWHG IRUHLJQ VHTXHQFHV LPSDFW WKH YDFFLQHHOLFLWHG7 FHOO UHVSRQVH RI 0&09 YDFFLQHYHFWRUVDQGWKLVFRUUHVSRQGVWRSURWHFWLRQDJDLQVWWXPRURXWJURZWK
7KHPDJQLWXGHRILQÀDWLRQDU\7FHOOUHVSRQVHVHOLFLWHGE\UHFRPELQDQW0&09 EDVHG YDFFLQH YHFWRUV LV LQÀXHQFHG E\ WKH LQRFXODWLRQ URXWH DQG GRVH DQG GHWHUPLQHVYDFFLQHHႈFDF\
7R LQYHVWLJDWH WKH LPSDFW RI WKH URXWH RI LQRFXODWLRQ RQ WKH DSSOLFDWLRQ RI 0&09EDVHG YDFFLQHYHFWRUVIRUYDFFLQHHOLFLWHGLPPXQLW\DJDLQVWFDQFHUZHFRPSDUHGWKHPDJQLWXGH DQGNLQHWLFVRIWKHDQWLJHQVSHFL¿F&'7FHOOUHVSRQVHLQWKHEORRGXSRQ,3,1DQG6& LQRFXODWLRQ7KH(VSHFL¿F&'7FHOOUHVSRQVHVHOLFLWHGZLWK0&09,((LQFUHDVHG JUDGXDOO\ WR a DIWHU ,3 LPPXQL]DWLRQ ZKLOH WKH UHVSRQVHV ZHUH VWHDGLO\ a DIWHU 6&LQRFXODWLRQ8SRQ,1LQRFXODWLRQKRZHYHUWKH(VSHFL¿F&'7FHOOUHVSRQVHVZHUH
PLQXWHWKURXJKRXWLQIHFWLRQ )LJDDQGE 7KH0±HSLWRSHVSHFL¿F&'
7FHOO UHVSRQVH DIWHU ,3 DQG 6& LQRFXODWLRQ VKRZHG VLPLODU PDJQLWXGH DQG NLQHWLFV DV WKH UHVSRQVHWRWKH(HSLWRSHLQWKHVDPHYHFWRU )LJDDQGE 8SRQ,1LPPXQL]DWLRQWKH 0VSHFL¿F&'7FHOOUHVSRQVHZDVEDUHO\GHWHFWDEOHDQGGLGQRWVKRZVLJQVRILQÀDWLRQ 8SRQLPPXQL]DWLRQVYLDWKH,3DQG6&URXWHVWKHQRQLQÀDWLRQDU\UHVSRQVHWRWKH0 ± HSLWRSHVKRZHGWKHW\SLFDOUHVSRQVHSDWWHUQRIUDSLGH[SDQVLRQIROORZHGE\VZLIWFRQWUDFWLRQ DQGVWDEOHPHPRU\IRUPDWLRQ )LJDDQGE ,1YDFFLQDWLRQLQGXFHGRQO\a0 VSHFL¿F&'7FHOOVDQGGXULQJWKHPHPRU\SKDVHWKLVUHVSRQVHZDVFORVHWRWKHGHWHFWLRQ OLPLW )LJDDQGE
5
Chapter 5
)LJXUH3RVLWLRQDQGHSLWRSHFRQWH[WRIKHWHURORJRXVDQWLJHQVLQ0&09YHFWRUHGYDFFLQHVLPSDFWYDFFLQHHOLFLWHG 7 FHOO UHVSRQVHV DQG DVVRFLDWHG WXPRU SURWHFWLRQ D 6FKHPDWLF RI 0&09,((( IXOO OHQJWK UHSODFHIXVLRQ DQG0&9,((7KH0&09JHQRPHUHJLRQDWNE±FRUUHVSRQGVWRWKH0&09JHQHLH HQODUJHG &RQVWUXFWV HQFRGLQJ WKH (( IXVLRQ SURWHLQ RU WKH ( HSLWRSHZHUH JHQHUDWHG E\ %$& UHFRPELQHHULQJ '1$ VHTXHQFHVHQFRGLQJWKHIXOOOHQJWK((IXVLRQSURWHLQZHUHLQVHUWHGHLWKHUWRUHSODFHWKHLHJHQH IXOOOHQJWK UHSODFH RULQIUDPHDWWKHYHU\HQGRIWKHLHJHQH IXOOOHQJWKIXVLRQ 7KH( ±HSLWRSH5$+<1,97)ZDVIXVHG WRWKHHQGRILHEF&%/PLFHZHUHYDFFLQDWHG,3ZLWKî3)80&09,((IXOOOHQJWKIXVLRQ 0&09,(( IXOO OHQJWK UHSODFH RU 0&09,(( 6SOHQRF\WHV ZHUH KDUYHVWHG E RU F ZHHNV DIWHU YDFFLQDWLRQDQGVWLPXODWHGZLWKWKHLPPXQRGRPLQDQW( ±SHSWLGHRUZLWKDSRRORIPHUSHSWLGHVFRYHULQJWKH HQWLUH(SURWHLQIRUK,)1ȖSURGXFWLRQZDVPHDVXUHGE\(/,6SRWDVVD\6KRZQLVWKHQXPEHURIVSRWVSHUî FHOOV6(0F)UHTXHQF\RI( ±VSHFL¿F7FHOOVLGHQWL¿HGE\0+&FODVV,PXOWLPHUVZLWKLQWKHWRWDO&' FHOOSRSXODWLRQDWZHHNVSRVWYDFFLQDWLRQ'DWDUHSUHVHQWVPHDQYDOXHV6(0 Q ±PLFHSHUJURXS 3 3 G7XPRURXWJURZWKRI7&WXPRUFHOOVLQPLFHFKDOOHQJHGZLWK7&WXPRUFHOOVZHHNVDIWHU YDFFLQDWLRQZLWKî3)80&09,((IXOOOHQJWKUHSODFH0&09,((IXOOOHQJWKIXVLRQRU0&09 ,(( ±PLFHSHUJURXS 7KHQXPEHURIWXPRUIUHHWRWDOPLFHLVLQGLFDWHGLQHDFKWXPRURXWJURZWKJUDSK 7/10 MCMV-IE2-E6/7 full length fusion
Days after TC-1 tumor challenge
0 300 200 100 0 10 40 T u mor size (mm 2) 20 30 1/10 MCMV-IE2-E6/7
full length replace MCMV-IE2-E7 5/5 MCMV-IE2-E6/E7 full length replace
(235 kb) 50 100 150 200
E6/E7 full length
ie2
MCMV-IE2-E6/E7 full length fusion
(235 kb) 50 100 150 200
E6/E7 full length
ie2 MCMV-IE2-E7 (235 kb) 50 100 150 200 ie2 RAHYNIVTF % E7-specific CD8 + T cells ** 0 4 8 12 2 6 10 MCMV-IE2-E6/7 full length fusion MCMV-IE2-E6/7 full length replace
MCMV-IE2-E7 * * spots per 5x10 5 cells 0 400 800 200 600 E749-57 E7 pool ** ** * * * * spots per 5x10 5 cells E749-57 E7 pool MCMV-IE2-E6/7 full length fusion MCMV-IE2-E6/7 full length replace
100 200 50 150 0 * * 10 weeks 21 weeks A C B D
Chapter 5 A E7 M38 M45 control Day 8 Day 64 0.49 5.78 1.27 6.70 3.12 0.50 0.04 0.03 CD8 MHC multimer 0 103 104 105 0 103 104 105 B C
Days after vaccination
% of EM cells within
MHC multimer
+ T cells
Days after vaccination E7 E7 0 10 20 30 40 50 60 70 80 0 20 100 40 60 80 0 10 20 30 40 50 60 70 80 M38 0 10 20 30 40 50 60 70 80 0 100 20 60 40 80 M38 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 80 M45 0 10 20 30 40 50 60 70 0 20 100 60 40 80 Day 0
MCMV-IE2-E7 SC Tracking E7-specific CD8
+ T cell response in blood
MCMV-IE2-E7 IN SC IN IP % MHC multimer + cells within CD8 + T cells 0.1 10 0.01 1 0.1 10 0.01 1 0.1 10 0.01 1 M45 SC IN IP MCMV-IE2-E7 IP no vaccination
)LJXUH 9DFFLQDWLRQ ZLWK UHFRPELQDQW 0&09 YHFWRUV YLD WKH ,3 DQG 6& URXWH LQGXFHV SRWHQW VSHFL¿F7 FHOO UHVSRQVHV0LFHZHUHYDFFLQDWHGZLWK0&09,((YLD,36&RU,1URXWHRUNHSWXQYDFFLQDWHG FRQWURO DQG WKH&'7FHOOUHVSRQVHVZHUHH[DPLQHGLQEORRGD5HSUHVHQWDWLYHÀRZF\WRPHWU\SORWVIRU0+&FODVV,PXOWLPHU VWDLQLQJWR(0DQG0HSLWRSHVLQEORRGRI0&09,((LQIHFWHGPLFHYLD,3GXULQJDFXWHSKDVH GD\ SRVWYDFFLQDWLRQ DQGPHPRU\SKDVH GD\SRVWYDFFLQDWLRQ 1XPEHUVUHSUHVHQWWKHSHUFHQWDJHRI$JVSHFL¿F &' 7 FHOOV ZLWKLQ WKH WRWDO &' 7 FHOO SRSXODWLRQ &RQWURO ÀRZ F\WRPHWU\ SORWV VKRZ WKH VWDLQLQJ IRU ( PXOWLPHULQXQYDFFLQDWHGPLFH)ORZF\WRPHWU\SORWVVKRZVLPLODUQXPEHUVRIWRWDOFHOOVLQHDFKSORWH[FHSWIRU FRQWUROE)UHTXHQF\RI$JVSHFL¿F&'7FHOOVWR(0DQG0HSLWRSHVRYHUWLPH
5
Chapter 5 F 7DNHQWRJHWKHUWKHVHGDWDVKRZWKDWLPPXQL]DWLRQZLWKUHFRPELQDQW0&09YHFWRUVYLD WKH,3DQG6&URXWHLQGXFHVSURPLQHQW(0OLNH&'7FHOOUHVSRQVHVDOEHLWZLWKGL൵HUHQW NLQHWLFVZKLOH,1LQIHFWLRQUHVXOWHGLQZHDNUHVSRQVHVDWEHVW 1H[WZHLQYHVWLJDWHGWKHLQÀXHQFHRIWKHURXWHRILPPXQL]DWLRQDQGWKHUHVXOWLQJYDFFLQH VSHFL¿F 7 FHOO UHVSRQVH HOLFLWHG E\ WKH 0&09 YHFWRUV RQ H൶FDF\ LQ WXPRU FKDOOHQJH H[SHULPHQWV :H YDFFLQDWHG PLFH ZLWK 0&09,(( YLD WKH ,3 ,1 RU 6& URXWHV DQG FKDOOHQJHGWKHVHPLFHZLWK7&WXPRUFHOOV:KLOH7&WXPRUVJUHZRXWSURJUHVVLYHO\ LQ DOO QDwYH PLFH LPPXQL]DWLRQ ZLWK 0&09,(( YLD WKH ,3 DQG 6& URXWH LQGXFHG FRPSOHWHSURWHFWLRQDJDLQVW7&WXPRUFKDOOHQJH )LJD &RQYHUVHO\,1LPPXQL]DWLRQ ZLWK0&09,((SURWHFWHGRQO\RIWKHPLFH )LJD 1RWDEO\WKH,1LPPXQL]HG PLFHWKDWZHUHSURWHFWHGKDGVWURQJHU(VSHFL¿F&'7FHOOUHVSRQVHVDVFRPSDUHGWR WKHXQSURWHFWHGJURXS )LJE 7KXV0&09YHFWRUVLQGXFLQJFLUFXODWLQJWXPRUVSHFL¿F &'7FHOOUHVSRQVHV!SURYLGHVIXOOSURWHFWLRQZKLOHUHVSRQVHVEHORZWKLVWKUHVKROG UHVXOWVLQORVVRISURWHFWLRQ,PPXQL]DWLRQVZLWK0&09EDVHGYDFFLQHYHFWRUVYLDURXWHVRI YDFFLQDWLRQWKDWLQGXFHODUJHWXPRUVSHFL¿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± 6,,1)(./ HSLWRSH IURPRYDOEXPLQ 29$ ,PPXQL]DWLRQZLWK0&09,(29$LQGXFHG29$VSHFL¿F&' 7FHOOUHVSRQVHVDJDLQVW29$±WKDWZHUHVOLJKWO\KLJKHUWKDQWKHUHVSRQVHDJDLQVW(
Chapter 5
QRWSURWHFWHGZKHUHDV0&09,(29$LPPXQL]DWLRQSURWHFWVDJDLQVW0&29$ )LJD DQGE 9LFHYHUVD0&09,((LPPXQL]DWLRQGRHVQRWSURYLGHSURWHFWLRQDJDLQVW0& 29$ )LJ E ZKLOH WKLV YDFFLQH YHFWRU SURYLGHV SURWHFWLRQ DJDLQVW (H[SUHVVLQJ7& WXPRUFHOOV )LJVDD 7RIXUWKHUGHPRQVWUDWHVSHFL¿FWDUJHWPHGLDWHGNLOOLQJLQGXFHG E\WKH0&09EDVHGYDFFLQHYHFWRUVZHSHUIRUPHGLQYLYRF\WRWR[LFLW\DVVD\V7DUJHWFHOOV SXOVHGZLWKYDFFLQHVSHFL¿FDQWLJHQRUFRQWURODQWLJHQZHUHLQMHFWHGLQWRPLFHSUHYLRXVO\ YDFFLQDWHGZLWK0&09,(( )LJF RU0&09,(29$ )LJG )LJXUH,PPXQL]DWLRQZLWK0&09EDVHGYDFFLQHYHFWRUVWKDWLQGXFHVXEVWDQWLDOWXPRUVSHFL¿F7FHOOUHVSRQVHV YLDGL൵HUHQWURXWHVRILQIHFWLRQFRQWUROWXPRURXWJURZWKD7&WXPRURXWJURZWKRIXQYDFFLQDWHGPLFHDQGRI PLFHSUHYLRXVO\YDFFLQDWHGZLWK0&09,((YLDWKH,36&RU,1URXWH7KHQXPEHURIWXPRUIUHHWRWDOPLFHLV LQGLFDWHGLQHDFKJUDSKE7KHSHUFHQWDJHRI(VSHFL¿F&'7FHOOVZLWKLQWKHWRWDO&'7FHOOSRSXODWLRQLQ WKHEORRG \D[LV SORWWHGDJDLQVWWKH(VSHFL¿F&'7FHOOUHVSRQVHUDQNHGIURPORZWRKLJK [D[LV RIWKHPLFH WKDWZHUHHLWKHUWXPRUIUHH WXPRU RUWXPRUSRVLWLYH WXPRU F7KHPLFHWKDWFOHDUHGWKH7&WXPRULQ$ZHUH UHFKDOOHQJHGZLWKî&WXPRUFHOOV7KHQXPEHURIWXPRUIUHHWRWDOPLFHLVLQGLFDWHGLQHDFKJUDSK'DWDDUH UHSUHVHQWDWLYHRIWZRLQGHSHQGHQWH[SHULPHQWVZLWKVLPLODUUHVXOWV A B C MCMV-IE2-E7 IP 11/11 MCMV-IE2-E7 SC 6/6 MCMV-IE2-E7 IN 3/3 Day -35 0
MCMV-IE2-E7 SC TC-1 tumor challenge
MCMV-IE2-E7 IP
no vaccination
C3 tumor re-challenge in mice that cleared TC-1 tumor
(shown in C) Tracking tumor
outgrowth (shown in A) and T cell response
MCMV-IE2-E7 IN
70
unvaccinated 0/10
Days after re-challenge with C3 tumor cells
T u mor size (mm 2) 0 20 40 60 0 40 120 60 80 100 20 unvaccinated 0/10
Days after TC-1 tumor challenge
T u mor size (mm 2) 0 20 40 60 0 40 120 60 80 100 20 11/11 MCMV-IE2-E7 IP MCMV-IE2-E7 SC 6/6 3/6 MCMV-IE2-E7 IN 0.01 0.1 1 10 % E7-specific CD8 + T cells
ranked E7-specific CD8+ T cell response
Tumor -Tumor +
5
Chapter 5
)LJXUH 6SHFL¿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î3)80&09,(( F RU0&09,(29$ G YLD6&$IWHU GD\V VSOHQRF\WHVIURPQDwYHPRXVHZHUHSXOVHGZLWKVSHFL¿FSHSWLGH ( ± F RU29$± G RUXQVSHFL¿FSHSWLGH DQGODEHOOHGZLWKKLJKDQGORZFRQFHQWUDWLRQVRI&)6(UHVSHFWLYHO\7KHQFHOOVZHUHDGRSWLYHO\WUDQVIHUUHGWR WKH0&09YDFFLQDWHGPLFHRUQDwYHPLFHDVFRQWURO2QHGD\ODWHUVSOHHQVRIWKHPLFHZHUHDQDO\]HGIRUWKH SHUFHQWDJHRINLOOLQJDVGHVFULEHGLQPDWHULDOVDQGPHWKRGV 105 CFSE % of max 104 103 102 0 40 60 80 100 20 MCMV-IE2-E7 Naive 42.3% killing 105 CFSE % of max 104 103 102 0 40 60 80 100 20 Naive MCMV-IE2-OVA 93.4% killing A C D OVA257-264 E7 49-57 OVA257-264 OVA257-264 OVA257-264 E749-57 E749-57 E749-57 Day -50 0 MCMV-IE2-OVA
MC38-OVA tumor challenge MCMV-IE2-E7
no vaccination
Tracking tumor outgrowth B
Days after TC-1 tumor challenge
T u mor size (mm 2) 0 20 40 60 0 40 120 60 80 100 20 Day -35 0 TC-1 tumor challenge MCMV-IE2-E7 no vaccination
Tracking tumor outgrowth
MCMV-IE2-E7 IP + anti-CD8 mAb MCMV-IE2-OVA MCMV-IE2-OVA 0/10 MCMV-IE2-E7 + anti-CD8 mAb 0/10 MCMV-IE2-E7 8/8 unvaccinated 0/11
Days after MC38-OVA tumor challenge
Chapter 5
%RWK0&09EDVHGYDFFLQHYHFWRUVLQGXFHGH[FOXVLYHNLOOLQJRIWDUJHWFHOOVSUHVHQWLQJWKH YDFFLQHVSHFL¿F DQWLJHQ 7KXV YDFFLQHLQGXFHG VSHFL¿F &' 7 FHOO UHVSRQVHV DJDLQVW DQWLJHQVH[SUHVVHGE\WKHWXPRUDUHUHTXLUHGWRLQGXFHSURWHFWLYHLPPXQLW\DQGUHVSRQVHV DJDLQVWWKHYHFWRUDORQHGRQRWSURYLGHSURWHFWLRQ
7R VXEVWDQWLDWH ZKHWKHU WKH PDJQLWXGH RI WKH FLUFXODWLQJ YDFFLQHVSHFL¿F &' 7 FHOO UHVSRQVH UHODWHV WR WXPRU SURWHFWLRQ ZH ,3 DQG 6& LPPXQL]HG PLFH ZLWK ORZ LQRFXOXP GRVDJHVWRDFKLHYHUHVSRQVHVEHORZWKHWKUHVKROG )LJD ,QIHFWLRQZLWKDKLJKGRVHRI 0&09,((YLDWKH,3DQG6&URXWHLQGXFHGDVH[SHFWHGVWURQJHU&'7FHOOUHVSRQVHV ! DJDLQVWWKH(HSLWRSHFRPSDUHGWRLQRFXOXPGRVDJHVZLWKIROGOHVVYLUXV ZKLFKUHVXOWHGLQUHVSRQVHV LQWKHEORRG )LJE 7KHKLJKHUUHVSRQVHLQPDJQLWXGH FRUUHODWHGWRDKLJKHUSHUFHQWDJHRI(0OLNHSKHQRW\SHZLWKLQWKHWRWDO$JVSHFL¿F&'7 FHOOSRSXODWLRQ )LJE ,PSRUWDQWO\LPPXQL]DWLRQZLWKKLJKGRVH0&09,((YLDWKH ,3DQG6&URXWHSURWHFWHGPLFHIURPWXPRURXWJURZWK )LJFDQGG*URXSDQG ZKLOH LPPXQL]DWLRQZLWKWKHORZGRVHUHVXOWHGLQOHVVSURWHFWLRQ )LJFDQGG*URXSDQG*URXS ,QWKH6&EXWQRWLQWKH,3ORZGRVHLPPXQL]HGJURXSVRPHPLFHZHUHSURWHFWHGGHVSLWH FLUFXODWLQJ(VSHFL¿F&'7FHOOVEHORZWKHWKUHVKROG)XUWKHUPRUHVLPLODUWRWKHGDWD VKRZQLQ)LJ,1LPPXQL]DWLRQZLWKî3)80&09,((HOLFLWHGDUHVSRQVHRIa DQGUHVXOWHGLQSDUWLDOSURWHFWLRQ VXUYLYDO )LJDG*URXS $JDLQWKH,1 LPPXQL]HGPLFHWKDWZHUHSURWHFWHGKDG(VSHFL¿F&'7FHOOUHVSRQVHV!ZKLOHWKH XQSURWHFWHGJURXSKDGUHVSRQVHV GDWDQRWVKRZQ :KHQDIROGORZHUYDFFLQH GRVH 3)8 IRUWKH,1URXWHZDVXVHGZKLFKHOLFLWHGDORZHUUHVSRQVH a DOOWKH PLFHVXFFXPEHGWRWKHWXPRUFKDOOHQJH )LJDG*URXS 7DNHQWRJHWKHUWKHVHGDWD LQGLFDWH WKDW WKH PDJQLWXGH DQG (0 SKHQRW\SH RI WKH YDFFLQHLQGXFHG &' 7 FHOOV DUH FRQWUROOHGE\WKHURXWHRIYDFFLQDWLRQDQGLQRFXOXPGRVDJHDQGDUHSULQFLSDOGHWHUPLQDQWV IRUWKHH൶FDF\RI0&09EDVHGYDFFLQHYHFWRUVDJDLQVWWXPRUV
,PPXQLW\WRUHLQIHFWLRQFDQKDPSHUWKHIRUPDWLRQRIYDFFLQHLQGXFHG&' 7FHOOUHVSRQVHV
Chapter 5 )LJXUH7KHLQRFXOXPGRVDJHRIUHFRPELQDQW0&09EDVHGYDFFLQHYHFWRUVGHWHUPLQHVWKHPDJQLWXGHRIDQWL WXPRU7FHOOUHVSRQVHDQGSURWHFWLRQH൶FDF\D6FKHPDWLFRIWKHH[SHULPHQW0LFHZHUHLQIHFWHGZLWKLQGLFDWHG GRVHVDQGURXWHVRUNHSWXQYDFFLQDWHG$IWHUGD\VDOOWKHPLFHZHUHFKDOOHQJHGZLWK7&WXPRUFHOOV7KHWXPRU RXWJURZWKDQGVXUYLYDORIWKHPLFHZDVIROORZHGIRUGD\VE3HUFHQWDJHRI(VSHFL¿FDQGWKHSHUFHQWDJHRI(0 OLNHFHOOVZLWKLQWKH(VSHFL¿F&'7FHOOVRIWKHYDFFLQDWHGPLFHFDQGG7&WXPRURXWJURZWK F DQGVXUYLYDO G JUDSKVRIPLFHLPPXQL]HGZLWK0&09,(( VFKHPDWLFVKRZQLQD 7KHQXPEHURIWXPRUIUHHWRWDOPLFH LVLQGLFDWHGDERYHHDFKWXPRURXWJURZWKJUDSKLQ F 7XPRURXWJURZWKZDVIROORZHGIRUGD\V7KHQXPEHURI WXPRUIUHHWRWDOPLFHLVLQGLFDWHG 3 3 3
Figure 5, Beyranvand Nejad et al.
A
D C
Day -35 0
Group 1: no vaccination
Tracking tumor outgrowth and T cell response TC-1 tumor challenge
MCMV-IE2-E7 SC 5x105 PFU/mouse Group 3:
MCMV-IE2-E7 IN 1x105 PFU/mouse Group 4:
Group 2: MCMV-IE2-E7 IP 1x105 PFU/mouse
MCMV-IE2-E7 SC 500 PFU/mouse Group 6:
Group 5: MCMV-IE2-E7 IP 100 PFU/mouse
MCMV-IE2-E7 IN 500 PFU/mouse Group 7: 3/12 Group 6 0/12 Group 7 Days after TC-1 tumor challenge
T u mor size (mm 2) 0 20 40 60 0 40 120 60 80 100 20 Group 1 0/12 Group 2 10/10 Group 3 11/11 5/11 Group 4 0/12 Group 5 0 40 60 80 100 20 0 20 40 60 80
Days after TC-1 tumor challenge
Percent survival Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 *** * B % E7-specific CD8 + T cells 0 10 20 30 0 60 100 20 40 80 % EM cells within MHC multimer + CD8 + T cells Group 3 Group 4 Group 2 Group 6 Group 7 Group 5 ***
Days after vaccination Days after vaccination
0 1 0 2 0 3 0 4 0
5
Chapter 5 )LJXUH3UHH[LVWLQJLPPXQLW\DJDLQVWWKH0&09YHFWRUD൵HFWVWKHVWUHQJWKRI7FHOOUHVSRQVHVDQGDQWLWXPRU H൶FDF\D3HUFHQWDJHRI29$0DQG(VSHFL¿F&'7FHOOVLGHQWL¿HGXVLQJ0+&FODVV,PXOWLPHUV0LFH ZHUHLQIHFWHGZLWKî3)8YLD,3RU,10&09,(29$DWGD\$IWHUGD\VWKHPLFHZHUHYDFFLQDWHG ZLWKî3)80&09,((YLD,3URXWHRUZLWKî0&09,((YLDWKH6&URXWHDVVKRZQLQWKH VFKHPDWLF7FHOOUHVSRQVHVZHUHIROORZHGIRUGD\VLQEORRG'DWDUHSUHVHQWPHDQYDOXHV6(0EDQGF7XPRU RXWJURZWK E DQGVXUYLYDO F RIPLFHLQIHFWHGZLWKî3)80&09,(29$YLD,3RU,1DQGYDFFLQDWHGZLWK î3)80&09,((YLD,3RU,1URXWHRUZLWKî0&09,((YLD6&DVVKRZQLQWKHVFKHPDWLF $IWHUGD\VDOOWKHYDFFLQDWHGDQGFRQWUROPLFHZHUHFKDOOHQJHGZLWKî7&WXPRUFHOOV7KHQXPEHURI WXPRUIUHHWRWDOPLFHLVLQGLFDWHGDERYHHDFKWXPRURXWJURZWKJUDSKG7KHSHUFHQWDJHRI(VSHFL¿F&'7FHOOV ZLWKLQWKHWRWDO&'7FHOOSRSXODWLRQLQWKHEORRG \D[LV SORWWHGDJDLQVWWKH(VSHFL¿F&'7FHOOUHVSRQVH UDQNHGIURPORZWRKLJK [D[LV RIWKHPLFHWKDWZHUHHLWKHUWXPRUIUHH WXPRU RUWXPRUSRVLWLYH WXPRU 'DWD DUHUHSUHVHQWDWLYHRIWZRLQGHSHQGHQWH[SHULPHQWVZLWKVLPLODUUHVXOWV A C 0/9 0/6Days after TC-1 tumor challenge
T u mor size (mm 2) 0 20 40 60 0 40 120 60 80 100 20 1/6 Group 4 Day 0 TC-1 tumor challenge -70 -35
Group 6: MCMV-IE2-OVA IP MCMV-IE2-E7 IP Group 4: MCMV-IE2-OVA IP MCMV-IE2-E7 IN Group 5: MCMV-IE2-OVA IP MCMV-IE2-E7 SC Group 1: no infection no vaccination
Tracking tumor outgrowth and T cell response B
Day 0 35
Days after infection
% MHC multimer
+ cells
within CD8
+ T cells
Group 3: MCMV-IE2-OVA IN MCMV-IE2-E7 SC Group 2: MCMV-IE2-OVA IN MCMV-IE2-E7 IP
Group 7: MCMV-IE2-OVA IP MCMV-IE2-E7 IP
100 PFU/mouse Group 3 Group 5 6/6 6/6 6/10 Group 2 Group 7 Group 1 Group 6 0/10 0 40 60 80 100 20 0 20 40 60
Days after TC-1 tumor challenge
Percent survival Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 M38 0 10 20 3040 50 60 70 80 0.1 10 0.01 1 Group 1 Group 2 Group 3 OVA 0 10 2030 4050 60 7080 0.1 10 0.01 1 E7
Tracking T cell response
0 .01 0 .1 1 1 0 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7
ranked E7-specific CD8+ T cell response
% E7-specific CD8
+ T cells
Tumor -Tumor +
D Group 1: MCMV-IE2-OVA IN MCMV-IE2-E7 IP Group 2: MCMV-IE2-OVA IN MCMV-IE2-E7 SC
Group 3: MCMV-IE2-OVA IP MCMV-IE2-E7 SC
Chapter 5 6LQJOHF\FOHUHSOLFDWLRQLVVXႈFLHQWIRUWKHDQWLWXPRUHႈFDF\RI0&09EDVHG YDFFLQHYHFWRUV 1RQDWWHQXDWHG&09PD\FDXVHGLVHDVHLQLPPXQRVXSSUHVVHGLQGLYLGXDOV7RIDFLOLWDWHWKH WUDQVODWLRQRI&09EDVHGYDFFLQHYHFWRUVLQWRWKHFOLQLFZHJHQHUDWHGDQDWWHQXDWHG( HSLWRSHH[SUHVVLQJ0&09YHFWRUE\IXVLQJWKH).%3GHJUDGDWLRQGRPDLQWRWKHHVVHQWLDO 0 SURWHLQ 0&090 ).%3( ZKLFKUHVWULFWVYLUDOUHSOLFDWLRQWRDVLQJOHF\FOHLQ DEVHQFHRIWKHVWDELOL]LQJOLJDQG6KLHOGVLPLODUWRFRUUHVSRQGLQJ+&09).%3FRQVWUXFWV WDUJHWLQJ WKH KRPRORJRXV 8/ JHQH $GGLWLRQDO ¿OH )LJXUH6 9DFFLQDWLRQ ZLWK WKH VSUHDGGH¿FLHQW 0&090 ).%3( HOLFLWHG (VSHFL¿F &' 7 FHOO UHVSRQVHV WKDW ZHUH ! RI WKH WRWDO &' 7 FHOO SRSXODWLRQ DW ZHHNV DQG HYHQ XS WR PRQWKV DIWHUYDFFLQDWLRQ )LJDDQGE ,QDGGLWLRQWKH(VSHFL¿F&'7FHOOVGLVSOD\HGDQ(0 SKHQRW\SHDWODWHWLPHSRLQWVSRVWLPPXQL]DWLRQ )LJF ,3LPPXQL]DWLRQZLWK0&09 0 ).%3(EXWQRWZLWKDFRQWUROVLQJOHF\FOHYLUXVH[SUHVVLQJWKHLPPXQRGRPLQDQW +69J%HSLWRSH 0&090 ).%3J% SUHYHQWHG7&WXPRUSURJUHVVLRQDWZHHNV ZHHNVDQGPRQWKVSRVWYDFFLQDWLRQLQGLFDWLQJORQJODVWLQJVSHFL¿FDQWLWXPRULPPXQLW\ )LJG ,QDVLPLODUIDVKLRQ0&090 ).%3(YDFFLQDWLRQSUHYHQWHGRXWJURZWKRI &DQG7&WXPRUVLQPRVWPLFH )LJH$GGLWLRQDO¿OH )LJXUH6$DQGGDWDQRWVKRZQ /RZ OHYHO SUHH[LVWLQJ LPPXQLW\ HOLFLWHG YLD ,1 LQIHFWLRQ ZLWK 0&09,(29$ GLG QRW LPSDFWWKHDQWLWXPRUH൶FDF\RI0&090 ).%3( )LJH ,QPLFHWKDWVXFFXPEHG WRWKHWXPRUFKDOOHQJHWKH(VSHFL¿F&'7FHOOUHVSRQVHVZHUHDOO ZKLOHDOO H[FHSWRQH RIWKHSURWHFWHGPLFHKDGUHVSRQVHV! )LJI
5
Chapter 5
&09EDVHG YHFWRUV DUH WKXV DEOH WR JHQHUDWH GH¿QHG WKUHVKROGV RI FLUFXODWLQJ YDFFLQH VSHFL¿F&'7FHOOVWKDWSURYLGHORQJODVWLQJWXPRUSURWHFWLRQ7KHIDFWRUVWKDWGHWHUPLQH WKLVWKUHVKROGVXFKDVYLUXVGRVHURXWHRIYDFFLQDWLRQDQGSUHH[LVWLQJLPPXQLW\KDYHWR EH H[WHQVLYHO\ VWXGLHG WR FRQVLGHU WKH GHYHORSPHQW RI H൵HFWLYH &09EDVHG YDFFLQHV LQFDQFHUSDWLHQWV&09VDUHODUJHYLUXVHV !.E HQFRGLQJPRUHWKDQSURWHLQV $OWKRXJK ODUJH YLUDO YDFFLQH YHFWRUV HOLFLWLQJ VWURQJ7 FHOO UHVSRQVHV FRXOG SRVVLEO\ OHDG WR FRPSHWLWLRQ IRU DQWLJHQSUHVHQWDWLRQ DQG ³VSDFH´ZKLFK PD\ OLPLW DGGLWLRQDO 7 FHOO UHVSRQVHVWKLVVHHPVQRWWRKDPSHUYDFFLQHVSHFL¿F7FHOOUHVSRQVHVHOLFLWHGE\&09%RWK LQPLFHDQGPRQNH\V&09VKDYHVKRZQWREHYHU\SRWHQWLQGXFHUVRI7FHOOUHVSRQVHVWR DQWLJHQVLQ6,90\FREDFWHULXPWXEHUFXORVLV(EROD>±@DQGVHYHUDOFDQFHUVLQFOXGLQJ PHODQRPDSURVWDWHFDQFHUDQG+39FDQFHU XQSXEOLVKHGGDWD.ODXV)UK :LWKUHVSHFW WRVSDFHOLPLWDWLRQIRUH൵HFWRUPHPRU\7FHOOV7KHUHLVHYLGHQFHLQPLFHDQGKXPDQWKDW VXSHULQIHFWLRQV DUH DEOH WR DGG PRUH 7 FHOOV WR WKH PHPRU\ SRRO > @$OWKRXJK WKH SHUFHQWDJHRIDJLYHQ7FHOOSRSXODWLRQFDQGHFUHDVHZKHQPRUHPHPRU\7FHOOVDUHDGGHG WRWKHPHPRU\SRROWKHDEVROXWHQXPEHUVRIDJLYHQ7FHOOSRSXODWLRQJHQHUDOO\GRHVQRW UHGXFH 'H¿QHGWKUHVKROGVIRUSURWHFWLRQEDVHGRQGLVHDVHVSHFL¿F7FHOOVKDYHEHHQVFDUFHO\UHSRUWHG 6FKPLGWHWDOUHSRUWHGWKDWSURWHFWLRQDJDLQVWPDODULDUHTXLUHGDODUJHWKUHVKROGLQPHPRU\ &'7FHOOIUHTXHQFLHVLH!VSHFL¿F7FHOOVRIWKHWRWDO&'7FHOOSRSXODWLRQLQEORRG >@+HUHZHIRXQGWKDWDWKUHVKROGRI!VSHFL¿FEORRG&'7FHOOVOLQNVWRIXOOWXPRU SURWHFWLRQ7KHVKDUSWKUHVKROGZHREVHUYHGVXJJHVWWKDWWKHHOLFLWHGWXPRUVSHFL¿F7FHOOV KDYHDFRQWUROOHGFDSDFLW\WRNLOOWDUJHWFHOOVZKLFKLVLQOLQHZLWKVWXGLHVVKRZLQJWKDWWKHLQ YLYRNLOOLQJFDSDFLW\RI&'7FHOOVLVPDUNHG\HWUHVWULFWHG> @7KLVGHPDUFDWHGWKUHVKROG LVFOHDUO\UHDFKHGXSRQYDFFLQDWLRQZLWKKLJKGRVHLQRFXOXPVRI0&09YHFWRUVGHOLYHUHG YLDWKH,3DQG6&URXWHDQGSURWHFWVDJDLQVWWKHGHYHORSPHQWRIVXEFXWDQHRXVWXPRUV2Q WKH RWKHU KDQG LPPXQL]DWLRQ YLD WKH ,1 URXWH FRQVLGHUHG DV DQDWXUDO URXWH RI LQIHFWLRQ >@ UHVXOWHG LQ PLQRU YDFFLQHVSHFL¿F UHVSRQVHV DQG KHQFH ORZHU SURWHFWLRQ +RZHYHU ,1LPPXQL]DWLRQZDVVXSHULRULQSURWHFWLRQDJDLQVWUHVSLUDWRU\SDWKRJHQVFRPSDUHGWR,3 LPPXQL]DWLRQGXHWRHQKDQFHGLQGXFWLRQRIWLVVXHUHVLGHQWPHPRU\ 750 7FHOOV>@,W LVWKXVFRQFHLYDEOHWKDWOXQJWXPRUVDUHEHWWHUFRQWUROOHGIROORZLQJYDFFLQDWLRQYLDWKH,1 URXWH,QOLQHZLWKWKLVDUHWKHREVHUYDWLRQVWKDWXSRQ6&LPPXQL]DWLRQVRPHPLFH ± DUHSURWHFWHGGHVSLWHWKDWWKHWXPRUVSHFL¿F&'7FHOOUHVSRQVHLQWKHEORRGLVEHORZWKH WKUHVKROG,QWKHVHPLFHWKH6&LPPXQL]DWLRQPD\KDYHOHGWRLQGXFWLRQRIVNLQ750FHOOV NQRZQWREHDEOHWRSURYLGHWXPRUSURWHFWLRQ>@
Chapter 5 A G B 4 weeks MCMV-M79-FKBP-gB 13 months 0 200 300 100 0 10 20 30 40 T u mor size (mm 2)
Days after TC-1 tumor challenge
0/10 0/14 12/15 21 weeks 5/5 D MCMV-M79-FKBP-E7 CD127 KLRG1 0 104 105 105 104 10-3 0 103 E749-57 spots per 5x10 5 cells 0 200 400 500 100 300 E7 pool
4 weeks 21 weeks 13 months
% of MHC multimer + cells within CD8 + cells 0 4 2 6
4 weeks 21 weeks 13 months
MCMV-M79-FKBP-E7 MCMV-M79-FKBP-gB MCMV-M79-FKBP-E7 0/5
unvaccinated MCMV-M79-FKBP-E7
5/5
E Day -70 -35 0
Group 3: MCMV-IE2-OVA IN MCMV-M79-FKBP-E7 IP
C3 tumor challenge
Group 2: no infection MCMV-M79-FKBP-E7 IP
Group 1: no infection no vaccination Tracking tumor
outgrowth and T cell response
Day 0 8
Group 1:
Tracking tumor outgrowth Group 2:
Group 3: TC-1 tumor challenge
Group 4: Group 5: no vaccination MCMV-M79-FKBP-gB IP MCMV-M79-FKBP-E7 IP MCMV-IE2-E7 IP MCMV-IE2-E7 SC
Days after TC-1 tumor challenge 0 10 20 40 0 100 250 150 200 50 30 0/5 0/4 0/5 T u mor size (mm 2) 2/9 0/8
Group 1 Group 2 Group 3 Group 4 Group 5
0/5 Group 1 7/10 Group 2 8/10 Group 3
Days after C3 tumor challenge
T u mor size (mm 2) 0 40 120 60 80 100 20 0 20 40 60 80 C MCMV-M79-FKBP-E7 MCMV-M79-FKBP-E7 73.3 6.67 1.11 18.9 Day 60 Day 7 71.1 3.77 15.1 9.43 F % E7-specific CD8 + T cells Tumor -Tumor +
ranked E7-specific CD8+ T cell response
5
Chapter 5 TXDOLWDWLYHO\GL൵HUHQWIURP&07FHOOVZLWKUHVSHFWWRWKHLULPPHGLDWHH൵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¿F7FHOOUHVSRQVHVGXHWRDQWLYHFWRULPPXQLW\ WKHUHE\UHGXFLQJWKHH൶FDF\RI0&09EDVHGYDFFLQHYHFWRUV,QFRQWUDVWLQGXFWLRQRISUH H[LVWLQJLPPXQLW\E\WKHQDWXUDO,1URXWHHQDEOHVVX൶FLHQWIRUPDWLRQRIYDFFLQHVSHFL¿F7 FHOOVXSRQUHLQRFXODWLRQOHDGLQJWRWXPRUSURWHFWLRQ,QVXSSRUWRIWKHFRQFOXVLRQWKDW&09V FDQ RYHUFRPH QDWXUDO LPPXQLW\ LV DOVR WKH REVHUYDWLRQ WKDW VWUDLQV RI +&09 KDYH EHHQ UHSRUWHGWRIUHTXHQWO\UHLQIHFWVHURSRVLWLYHLQGLYLGXDOV>@0RUHRYHUQDWXUDOO\LQIHFWHG VHURSRVLWLYHUKHVXVPDFDTXHVFDQEHUHDGLO\UHLQIHFWHGZLWKUKHVXV&09 5K&09 HYHQ ZKHQJLYHQ6&DWYHU\ORZGRVH> @$OVR5K&096,9YHFWRULPPXQL]HGPRQNH\VFDQEH UHSHDWHGO\UHLQIHFWHGYLDWKH6&URXWH>@,QWKLVDQLPDOPRGHOWKHDELOLW\WRUHLQIHFWKDV EHHQVKRZQWREHLQSDUWGXHWRWKHH൵HFWLYHLQKLELWLRQRI0+&,DQWLJHQSUHVHQWDWLRQ> @ ,WPD\EHSRVVLEOHWRHQJLQHHUUHFRPELQDQW+&09YHFWRUVWKDWDUHDEOHWRLQGXFHVWURQJ DQGSURWHFWLYHLPPXQHUHVSRQVHVGHVSLWHSUHH[LVWLQJLPPXQLW\)RUUKHVXVPDFDTXHVLWZDV
)LJXUH 6LQJOH F\FOH UHSOLFDWLRQ RI 0&09 EDVHG YHFWRU YDFFLQH LV VX൶FLHQW WR LQGXFH DQWLWXPRU H൵HFWV LQ SURSK\ODFWLFDQGWKHUDSHXWLFVHWWLQJV D0LFHZHUHYDFFLQDWHG,3ZLWKî3)80&090 ).%3(RUFRQWUROYLUXV 0&090 ).%3J% 6SOHQRF\WHVZHUHKDUYHVWHGZHHNVDQGPRQWKVDIWHUYDFFLQDWLRQDQGVWLPXODWHGZLWKGRPLQDQWSHSWLGHRI (RUSRROHG(SHSWLGHVIRUK D ,)1ȖSURGXFWLRQZDVPHDVXUHGE\(/,6327DVVD\6KRZQLVWKHQXPEHURI VSRWVSHUîFHOOV6(0E)UHTXHQF\RI( ±VSHFL¿F&'7FHOOVZLWKLQWRWDO&'7FHOOSRSXODWLRQDW ZHHNVDQGPRQWKVSRVWYDFFLQDWLRQ'DWDUHSUHVHQWVPHDQYDOXHV6(0 Q PLFHSHUJURXS F./5* DQG&'PDUNHUH[SUHVVLRQRQ(VSHFL¿F&'7FHOOVDWGD\DQGSRVWYDFFLQDWLRQZLWK0&090 ).%3(G7&WXPRURXWJURZWKRIWKHPLFHWKDWZHUHYDFFLQDWHGZLWKî3)80&090 ).%3(RU FRQWUROYLUXV0&090 ).%3J%DWZHHNVDQGPRQWKVEHIRUHWXPRUFKDOOHQJH7XPRURXWJURZWKZDV IROORZHGIRURUGD\VHDQGI&WXPRURXWJURZWKRIWKHPLFHZHUHLQIHFWHGZLWKî3)80&09,( 29$YLD,1URXWHRUNHSWXQLQIHFWHG$IWHUGD\VWKHPLFHZHUHYDFFLQDWHGZLWKî3)80&090 ).%3 ,3RUNHSWXQYDFFLQDWHG0LFHZHUHFKDOOHQJHGZLWK&WXPRUFHOOVDIWHUGD\V7XPRURXWJURZWKZDVIROORZHG IRUGD\V H 7KHQXPEHURIWXPRUIUHHPLFHIURPWKHWRWDOPLFHLVLQGLFDWHGDERYHHDFKWXPRURXWJURZWKJUDSK I7KHSHUFHQWDJHRI(VSHFL¿F&'7FHOOVZLWKLQWKHWRWDO&'7FHOOSRSXODWLRQLQWKHEORRG \D[LV SORWWHG DJDLQVWWKH(VSHFL¿F&'7FHOOUHVSRQVHUDQNHGIURPORZWRKLJK [D[LV RIWKHPLFHWKDWZHUHHLWKHUWXPRU IUHH WXPRU RUWXPRUSRVLWLYH WXPRU I J0LFHZHUHFKDOOHQJHGZLWK7&WXPRUFHOOVDQGDIWHUGD\VZKHQ WXPRUVZHUHSDOSDEOHPLFHZHUHWUHDWHGZLWKî3)80&09P ).%3(RUFRQWUROYLUXV0&09P
Chapter 5
5
Chapter 5$FNQRZOHGJHPHQWV
7KHDXWKRUVDFNQRZOHGJH6X]DQQH:HOWHQDQG$QNH5HGHNHUIRUDGYLFHDQGSUDFWLFDOKHOS 7KHDXWKRUVZRXOGOLNHWRWKDQN-DQ:RXWHU'ULMIKRXWDQG.HHV)UDQNHQIRUFRQVWUXFWLQJ WHWUDPHUV )XQGLQJChapter 5 3XEOLVKHU¶V1RWH 6SULQJHU1DWXUHUHPDLQVQHXWUDOZLWKUHJDUGWRMXULVGLFWLRQDOFODLPVLQSXEOLVKHGPDSVDQG LQVWLWXWLRQDOD൶OLDWLRQV $XWKRUGHWDLOV
'HSDUWPHQW RI ,PPXQRKHPDWRORJ\ DQG %ORRG 7UDQVIXVLRQ /HLGHQ 8QLYHUVLW\ 0HGLFDO &HQWHU $OELQXVGUHHI =$ /HLGHQ 7KH 1HWKHUODQGV 'HSDUWPHQW RI 0HGLFDO 2QFRORJ\/HLGHQ8QLYHUVLW\0HGLFDO&HQWHU/HLGHQ7KH1HWKHUODQGV9LU%LRWHFKQRORJ\ 3RUWODQG 25 86$ 'HSDUWPHQW RI 9DFFLQRORJ\ DQG $SSOLHG 0LFURELRORJ\ +HOPKROW] &HQWUHIRU,QIHFWLRQ5HVHDUFK%UDXQVFKZHLJ*HUPDQ\,QVWLWXWHIRU9LURORJ\+DQQRYHU 0HGLFDO 6FKRRO +DQQRYHU *HUPDQ\ *HUPDQ &HQWUH IRU ,QIHFWLRQ 5HVHDUFK '=,) 3DUWQHUVLWH+DQQRYHU%UDXQVFKZHLJ*HUPDQ\
5
Chapter 5
5HIHUHQFHV
5HGHNHU $ $UHQV 5 ,PSURYLQJ $GRSWLYH 7 &HOO 7KHUDS\ 7KH 3DUWLFXODU 5ROH RI 7 &HOO &RVWLPXODWLRQ&\WRNLQHVDQG3RVW7UDQVIHU9DFFLQDWLRQ)URQW,PPXQRO 6KDUPD3$OOLVRQ-37KHIXWXUHRILPPXQHFKHFNSRLQWWKHUDS\6FLHQFH ± YDQGHU%XUJ6+$UHQV52VVHQGRUS)YDQ+DOO70HOLHI&-9DFFLQHVIRUHVWDEOLVKHGFDQFHU RYHUFRPLQJWKHFKDOOHQJHVSRVHGE\LPPXQHHYDVLRQ1DW5HY&DQFHU 3ORWNLQ6$9DFFLQHV FRUUHODWHVRIYDFFLQHLQGXFHGLPPXQLW\&OLQ,QIHFW'LV ± .DUUHU86LHUUR6:DJQHU02[HQLXV$+HQJHO+.RV]LQRZVNL8+HWDO0HPRU\LQÀDWLRQ FRQWLQXRXVDFFXPXODWLRQRIDQWLYLUDO&'7FHOOVRYHUWLPH-,PPXQRO ± 2¶+DUD*$:HOWHQ63.OHQHUPDQ3$UHQV50HPRU\7FHOOLQÀDWLRQ XQGHUVWDQGLQJFDXVHDQG H൵HFW7UHQGV,PPXQRO ±
6\OZHVWHU$: 0LWFKHOO %/ (GJDU -%7DRUPLQD & 3HOWH & 5XFKWL ) HW DO %URDGO\ WDUJHWHG KXPDQF\WRPHJDORYLUXVVSHFL¿F&'DQG&'7FHOOVGRPLQDWHWKHPHPRU\FRPSDUWPHQWVRI H[SRVHGVXEMHFWV-([S0HG ± :HOWHQ635HGHNHU$7RHV5($UHQV59LUDOSHUVLVWHQFHLQGXFHVDQWLERG\LQÀDWLRQZLWKRXW DOWHULQJDQWLERG\DYLGLW\-9LURO ± .OHQHUPDQ32[HQLXV$7FHOOUHVSRQVHVWRF\WRPHJDORYLUXV1DW5HY,PPXQRO ± 5RVV6$$URUD11RYDN=)RZOHU.%%ULWW:-%RSSDQD6%&\WRPHJDORYLUXVUHLQIHFWLRQVLQ KHDOWK\VHURLPPXQHZRPHQ-,QIHFW'LV ± 7UJRYFLFK-.LQFDLG07KRPDV$*ULHVVO0=LPPHUPDQ3'ZLYHGL9HWDO&\WRPHJDORYLUXV 5HLQIHFWLRQV6WLPXODWH&'70HPRU\,QÀDWLRQ3/R62QH H
.HQQHVRQ $ &DQQRQ 0- 5HYLHZ DQG PHWDDQDO\VLV RI WKH HSLGHPLRORJ\ RI FRQJHQLWDO F\WRPHJDORYLUXV &09 LQIHFWLRQ5HY0HG9LURO ±
%RUVW(0%HQNDUWHN&0HVVHUOH08VHRIEDFWHULDODUWL¿FLDOFKURPRVRPHVLQJHQHUDWLQJWDUJHWHG PXWDWLRQVLQKXPDQDQGPRXVHF\WRPHJDORYLUXVHV&XUU3URWRF,PPXQRO &KDSWHU 8QLW +DQVHQ6* )RUG-&/HZLV069HQWXUD$% +XJKHV &0 &R\QH-RKQVRQ/HWDO3URIRXQG
HDUO\ FRQWURO RI KLJKO\ SDWKRJHQLF 6,9 E\ DQ H൵HFWRU PHPRU\ 7FHOO YDFFLQH 1DWXUH ±
7VXGD < &DSRVLR 3 3DUNLQV &- %RWWR 6 0HVVDRXGL , &LFLQ6DLQ / HW DO $ UHSOLFDWLQJ F\WRPHJDORYLUXVEDVHGYDFFLQHHQFRGLQJDVLQJOH(ERODYLUXVQXFOHRSURWHLQ&7/HSLWRSHFRQIHUV SURWHFWLRQDJDLQVW(ERODYLUXV3/R61HJO7URS'LV H
%HYHUOH\ 3& 5X]VLFV = +H\ $ +XWFKLQJV & %RRV 6 %ROLQJHU % HW DO $ QRYHO PXULQH F\WRPHJDORYLUXV YDFFLQH YHFWRU SURWHFWV DJDLQVW 0\FREDFWHULXP WXEHUFXORVLV - ,PPXQRO ± 0DU]L$0XUSK\$$)HOGPDQQ)3DUNLQV&-+DGGRFN(+DQOH\3:HWDO&\WRPHJDORYLUXV EDVHGYDFFLQHH[SUHVVLQJ(ERODYLUXVJO\FRSURWHLQSURWHFWVQRQKXPDQSULPDWHVIURP(ERODYLUXV LQIHFWLRQ6FL5HS +DQVHQ6*=DN'(;X*)RUG-&0DUVKDOO((0DORXOL'HWDO3UHYHQWLRQRIWXEHUFXORVLVLQ UKHVXVPDFDTXHVE\DF\WRPHJDORYLUXVEDVHGYDFFLQH1DW0HG ± (UNHV'$;X*'DVNDODNLV&=XUEDFK.$:LOVNL1$0RJKEHOL7HWDO,QWUDWXPRUDOLQIHFWLRQ ZLWK PXULQH F\WRPHJDORYLUXV V\QHUJL]HV ZLWK 3'/ EORFNDGH WR FOHDU PHODQRPD OHVLRQV DQG LQGXFHORQJWHUPLPPXQLW\0RO7KHU ±
;X*6PLWK7*UH\)+LOO$%&\WRPHJDORYLUXVEDVHGFDQFHUYDFFLQHVH[SUHVVLQJ753LQGXFH UHMHFWLRQRIPHODQRPDLQPLFH%LRFKHP%LRSK\V5HV&RPPXQ ±
Chapter 5
4LX=+XDQJ+*UHQLHU-03HUH]2$6PLORZLW]+0$GOHU%HWDO&\WRPHJDORYLUXVEDVHG YDFFLQHH[SUHVVLQJDPRGL¿HGWXPRUDQWLJHQLQGXFHVSRWHQWWXPRUVSHFL¿F&' 7FHOOUHVSRQVH DQGSURWHFWVPLFHIURPPHODQRPD&DQFHU,PPXQRO5HV ±
.O\XVKQHQNRYD (1 .RXLDYVNDLD '9 3DUNLQV &- &DSRVLR 3 %RWWR 6$OH[DQGHU 5% HW DO$ F\WRPHJDORYLUXVEDVHG YDFFLQH H[SUHVVLQJ D VLQJOH WXPRUVSHFL¿F &' 7FHOO HSLWRSH GHOD\V WXPRUJURZWKLQDPXULQHPRGHORISURVWDWHFDQFHU-,PPXQRWKHU ± 7UVDQ79XNRYLF.)LOLSRYLF3%UL]LF$//HPPHUPDQQ1$:6FKREHU.HWDO&\WRPHJDORYLUXV YHFWRUH[SUHVVLQJ5$(JDPPDLQGXFHVHQKDQFHGDQWLWXPRUFDSDFLW\RIPXULQH&' 7FHOOV (XU-,PPXQRO ± :HOWHQ635HGHNHU$)UDQNHQ./%HQHGLFW&$<DJLWD+:HQVYHHQ)0HWDO&'&' FRVWLPXODWLRQFRQWUROV7FHOOLPPXQLW\GXULQJDFXWHDQGSHUVLVWHQWF\WRPHJDORYLUXVLQIHFWLRQ- 9LURO ± 'HNKWLDUHQNR,5DWWV5%%ODWQLN5/HH/1)LVFKHU6%RUNQHU/HWDO3HSWLGHSURFHVVLQJLV FULWLFDOIRU7FHOOPHPRU\LQÀDWLRQDQGPD\EHRSWLPL]HGWRLPSURYHLPPXQHSURWHFWLRQE\&09 EDVHGYDFFLQHYHFWRUV3/R63DWKRJ H :DUPLQJ6&RVWDQWLQR1&RXUW'/-HQNLQV1$&RSHODQG1*6LPSOHDQGKLJKO\H൶FLHQW%$& UHFRPELQHHULQJXVLQJJDO.VHOHFWLRQ1XFOHLF$FLGV5HV H 'HNKWLDUHQNR,-DUYLV0$5X]VLFV=&LFLQ6DLQ/7KHFRQWH[WRIJHQHH[SUHVVLRQGH¿QHVWKH LPPXQRGRPLQDQFHKLHUDUFK\RIF\WRPHJDORYLUXVDQWLJHQV-,PPXQRO ± 3HUQJ<&4LDQ=)HKU$5;XDQ%<X'7KHKXPDQF\WRPHJDORYLUXVJHQH8/ LVUHTXLUHG IRUWKHDFFXPXODWLRQRIODWHYLUDOWUDQVFULSWV-9LURO ±
/LQ .< *XDUQLHUL )* 6WDYHOH\2¶&DUUROO .) /HYLWVN\ +, $XJXVW -7 3DUGROO '0 HW DO 7UHDWPHQWRIHVWDEOLVKHGWXPRUVZLWKDQRYHOYDFFLQHWKDWHQKDQFHVPDMRUKLVWRFRPSDWLELOLW\FODVV ,,SUHVHQWDWLRQRIWXPRUDQWLJHQ&DQFHU5HV ±
YDQ'XLNHUHQ6)UDQVHQ0)5HGHNHU$:LHOHV%3ODWHQEXUJ*.UHEEHU:-HWDO9DFFLQH LQGXFHG H൵HFWRUPHPRU\ &'7 FHOO UHVSRQVHV SUHGLFW WKHUDSHXWLF H൶FDF\ DJDLQVW WXPRUV - ,PPXQRO ±
)HOWNDPS 0& 6PLWV +/ 9LHUERRP 03 0LQQDDU 53 'H -RQJK %0 'ULMIKRXW -: HW DO 9DFFLQDWLRQ ZLWK F\WRWR[LF 7 O\PSKRF\WH HSLWRSHFRQWDLQLQJ SHSWLGH SURWHFWV DJDLQVW D WXPRU LQGXFHGE\KXPDQSDSLOORPDYLUXVW\SH WUDQVIRUPHGFHOOV(XU-,PPXQRO ± 5HGHNHU$:HOWHQ63%DHUW059ORHPDQV6$7LHPHVVHQ006WDDO)-HWDO7KHTXDQWLW\RI DXWRFULQH,/JRYHUQVWKHH[SDQVLRQSRWHQWLDORI&'7FHOOV-,PPXQRO ± $UHQV5/RHZHQGRUI$5HGHNHU$6LHUUR6%RRQ/.OHQHUPDQ3HWDO'L൵HUHQWLDO%&' FRVWLPXODWRU\UHTXLUHPHQWVIRUVWDEOHDQGLQÀDWLRQDU\PRXVHF\WRPHJDORYLUXVVSHFL¿FPHPRU\ &'7FHOOSRSXODWLRQV-,PPXQRO ±
$OWPDQ -' 0RVV 3$+ *RXOGHU 3-5 %DURXFK '+ 0F+H\]HU:LOOLDPV 0* %HOO -, HW DO 3KHQRW\SLFDQDO\VLVRIDQWLJHQVSHFL¿F7O\PSKRF\WHV6FLHQFH ±
5
Chapter 5 2GXUR-'5HGHNHU$/HPPHUPDQQ1$(EHUPDQQ/0DUDQGX7)'HNKWLDUHQNR,HWDO0XULQH F\WRPHJDORYLUXVLQIHFWLRQYLDWKHLQWUDQDVDOURXWHR൵HUVDUREXVWPRGHORILPPXQLW\XSRQPXFRVDO &09LQIHFWLRQ-*HQ9LURO0RUDELWR .0 5XFNZDUGW 75 5HGZRRG $- 0RLQ 60 3ULFH '$ *UDKDP %6 ,QWUDQDVDO DGPLQLVWUDWLRQ RI 569 DQWLJHQH[SUHVVLQJ 0&09 HOLFLWV UREXVW WLVVXHUHVLGHQW H൵HFWRU DQG H൵HFWRUPHPRU\&'7FHOOVLQWKHOXQJ0XFRVDO,PPXQRO ±
*DOYH]&DQFLQR)/RSH](0HQDUHV('LD];)ORUHV&&DFHUHV3HWDO9DFFLQDWLRQLQGXFHG VNLQUHVLGHQW PHPRU\ &' 7 FHOOV PHGLDWH VWURQJ SURWHFWLRQ DJDLQVW FXWDQHRXV PHODQRPD 2QFRLPPXQRORJ\ H
5HGHNHU$5HPPHUVZDDO(%0YDQGHU*UDFKW(7,:HOWHQ630+ROOW7.RQLQJ)HWDO7KH FRQWULEXWLRQ RI F\WRPHJDORYLUXV LQIHFWLRQ WR LPPXQH VHQHVFHQFH LV VHW E\ WKH LQIHFWLRXV GRVH )URQW,PPXQRO
3DUGLHFN ,1 %H\UHQG * 5HGHNHU $ $UHQV 5 &\WRPHJDORYLUXV LQIHFWLRQ DQG SURJUHVVLYH GL൵HUHQWLDWLRQRIH൵HFWRUPHPRU\7FHOOV)5HV
$SSD\9'XQEDU35&DOODQ0.OHQHUPDQ3*LOOHVSLH*03DSDJQR/HWDO0HPRU\&' 7 FHOOV YDU\ LQ GL൵HUHQWLDWLRQ SKHQRW\SH LQ GL൵HUHQW SHUVLVWHQW YLUXV LQIHFWLRQV 1DW 0HG ±
5HGHNHU$ :HOWHQ 63$UHQV 5 9LUDO LQRFXOXP GRVH LPSDFWV PHPRU\ 7FHOO LQÀDWLRQ (XU - ,PPXQRO ±
%DOH-)-U3HWKHUDP6-6RX]D,(0XUSK-5&\WRPHJDORYLUXVUHLQIHFWLRQLQ\RXQJFKLOGUHQ- 3HGLDWU ±
*RU]HU , .HUVFKQHU + 5HGOEHUJHU)ULW] 0 3XFKKDPPHU6WRFNO ( +XPDQ F\WRPHJDORYLUXV +&09 JHQRW\SHSRSXODWLRQVLQLPPXQRFRPSHWHQWLQGLYLGXDOVGXULQJSULPDU\+&09LQIHFWLRQ -&OLQ9LURO ± +DQVHQ6*3RZHUV&-5LFKDUGV59HQWXUD$%)RUG-&6LHVV'HWDO(YDVLRQRI&'7FHOOV LVFULWLFDOIRUVXSHULQIHFWLRQE\F\WRPHJDORYLUXV6FLHQFH ± +DQVHQ6*9LHYLOOH&:KL]LQ1&R\QH-RKQVRQ/6LHVV'&'UXPPRQG''HWDO(൵HFWRU PHPRU\7FHOOUHVSRQVHVDUHDVVRFLDWHGZLWKSURWHFWLRQRIUKHVXVPRQNH\VIURPPXFRVDOVLPLDQ LPPXQRGH¿FLHQF\YLUXVFKDOOHQJH1DW0HG ±
+DQVHQ 6* :X +/ %XUZLW] %- +XJKHV &0 +DPPRQG .% 9HQWXUD $% HW DO %URDGO\ WDUJHWHG &' 7 FHOO UHVSRQVHV UHVWULFWHG E\ PDMRU KLVWRFRPSDWLELOLW\ FRPSOH[ ( 6FLHQFH ±
0XUUD\ 6( 1HVWHUHQNR 3$ 9DQDUVGDOO $/ 0XQNV 0: 6PDUW 60 9H]LURJOX (0 HW DO )LEUREODVWDGDSWHGKXPDQ&09YDFFLQHVHOLFLWSUHGRPLQDQWO\FRQYHQWLRQDO&'7FHOOUHVSRQVHV LQKXPDQV-([S0HG :DQJ;0HVVHUOH06DSLQRUR56DQWRV.+RFNQHOO3.-LQ;HWDO0XULQHF\WRPHJDORYLUXV DERUWLYHO\LQIHFWVKXPDQGHQGULWLFFHOOVOHDGLQJWRH[SUHVVLRQDQGSUHVHQWDWLRQRIYLUDOO\YHFWRUHG JHQHV-9LURO ± 7DQJ40DXO**0RXVHF\WRPHJDORYLUXVFURVVHVWKHVSHFLHVEDUULHUZLWKKHOSIURPDIHZKXPDQ F\WRPHJDORYLUXVSURWHLQV-9LURO ± YDQGHU%XUJ6+.ZDSSHQEHUJ.02¶1HLOO7%UDQGW500HOLHI&-+LFNOLQJ-.HWDO3UH FOLQLFDOVDIHW\DQGH൶FDF\RI7$&,1DUHFRPELQDQW+39/((IXVLRQSURWHLQYDFFLQHLQ KRPRORJRXVDQGKHWHURORJRXVSULPHERRVWUHJLPHQV9DFFLQH ± %DOGZLQ3-YDQGHU%XUJ6+%RVZHOO&02൵ULQJD5+LFNOLQJ-.'REVRQ-HWDO9DFFLQLD H[SUHVVHGKXPDQSDSLOORPDYLUXVDQGHDQGHDVDWKHUDSHXWLFYDFFLQDWLRQIRUYXOYDODQG YDJLQDOLQWUDHSLWKHOLDOQHRSODVLD&OLQ&DQFHU5HV ±
Chapter 5
*DOOLYHUWL*7LFKHW0'RPLQJRV3HUHLUD6+DXHUW61DUGHOOL+DHÀLJHU'6ZDUW]0$HWDO 1DQRSDUWLFOH FRQMXJDWLRQ RI KXPDQ SDSLOORPDYLUXV (ORQJ SHSWLGHV HQKDQFHV WKHUDSHXWLF YDFFLQHH൶FDF\DJDLQVWVROLGWXPRUVLQPLFH&DQFHU,PPXQRO5HV ± 3HQJ64LX-<DQJ$<DQJ%-HDQJ-:DQJ-:HWDO2SWLPL]DWLRQRIKHWHURORJRXV'1$
SULPHSURWHLQERRVWUHJLPHQVDQGVLWHRIYDFFLQDWLRQWRHQKDQFHWKHUDSHXWLFLPPXQLW\DJDLQVW KXPDQSDSLOORPDYLUXVDVVRFLDWHGGLVHDVH&HOO%LRVFLHQFH
'RPLQJRV3HUHLUD 6 'HUUH /:DUSHOLQ'HFUDXVD] / +DHÀLJHU -$ 5RPHUR 3 -LFKOLQVNL 3 HW DO,QWUDYDJLQDODQGVXEFXWDQHRXVLPPXQL]DWLRQLQGXFHGYDFFLQHVSHFL¿F&'7FHOOVDQGWXPRU UHJUHVVLRQLQWKHEODGGHU-8URO ± $QGUHZV'0(VWFRXUW0-$QGRQLRX&(:LNVWURP0(.KRQJ$9RLJW9HWDO,QQDWHLPPXQLW\ GH¿QHVWKHFDSDFLW\RIDQWLYLUDO7FHOOVWROLPLWSHUVLVWHQWLQIHFWLRQ-([S0HG ± .HQWHU**:HOWHUV0-9DOHQWLMQ$5/RZLN0-%HUHQGVYDQGHU0HHU'09ORRQ$3HWDO YDFFLQDWLRQ DJDLQVW +39 RQFRSURWHLQV IRU YXOYDU LQWUDHSLWKHOLDO QHRSODVLD 1 (QJO - 0HG ±
5
Chapter 5
6XSSOHPHQWDU\¿JXUHV
)LJXUH6,PPXQL]DWLRQZLWK0&09,(29$LQGXFHVYDFFLQHVSHFL¿F&'7FHOOUHVSRQVHV $ 0LFHZHUH LQIHFWHGZLWKî3)80&09,(29$YLD,3RUîYLD6&)UHTXHQF\RIDQWLJHQVSHFL¿F&'7FHOOVIRU WKH0DQG0HSLWRSHVRI0&09DQGRIWKHLQVHUWHG29$DQWLJHQZHUHLGHQWL¿HGXVLQJ0+&FODVV,PXOWLPHUV 'DWDUHSUHVHQWVPHDQYDOXHV6(0 Q PLFHSHUJURXS 'DWDDUHUHSUHVHQWDWLYHRIWZRLQGHSHQGHQWH[SHULPHQWV % 0&29$WXPRURXWJURZWKRIXQYDFFLQDWHGPLFHDQGRIPLFHSUHYLRXVO\YDFFLQDWHGZLWK0&09,( 29$YLDWKH,3RU6&URXWH0LFHZHUHYDFFLQDWHGZLWKî3)80&09,(29$YLD,3RUîYLD6&$IWHU GD\V 0&09,(29$,3 RUGD\V 0&09,(29$6& PLFHZHUHFKDOOHQJHGZLWKî
0&29$WXPRUFHOOV7KHWXPRURXWJURZWKZDVIROORZHGIRUGD\V7KHQXPEHURIWXPRUIUHHWRWDOPLFHLV LQGLFDWHGLQHDFKJUDSK'DWDDUHUHSUHVHQWDWLYHRIWZRLQGHSHQGHQWZLWKVLPLODUUHVXOWV (36NE
A
Days after vaccination
% of MHC multimer + cells within CD8 + T cells OVA 0 10 20 30 40 M38 0 10 20 30 40 IP M45 0 10 20 30 40 SC 0.1 10 0.01 1 0.1 10 0.01 1 0.1 10 0.01 1 MCMV-IE2-OVA IP 8/8
Days after MC38-OVA tumor challenge
Chapter 5
)LJXUH66LQJOHF\FOHUHSOLFDWLRQRI0&09EDVHGYHFWRUVLQGXFHVDQWLWXPRUH൵HFWV $ 6XUYLYDORIWKHPLFH ZKLFK ZHUH LQIHFWHG ZLWK î 3)8 0&09,(29$ ,1 RU NHSW XQLQIHFWHG$IWHU GD\V WKH PLFH ZHUH
YDFFLQDWHGZLWKî3)80&090 ).%3,3RUNHSWXQYDFFLQDWHG0LFHZHUHFKDOOHQJHGZLWK&WXPRU
FHOOVDIWHUGD\V6XUYLYDOZDVIROORZHGIRUGD\V % 6XUYLYDORIWKHPLFHZKLFKZHUHFKDOOHQJHGZLWK7&
WXPRUFHOOVDQGDIWHUGD\VZKHQWXPRUVZHUHSDOSDEOHPLFHZHUHWUHDWHGZLWKî3)80&090 ).%3(
RUFRQWUROYLUXV0&090 ).%3J%YLD,3î3)80&09,((YLD,3RUî3)80&09,(( YLD6&RUNHSWXQYDFFLQDWHG6XUYLYDOJURZWKZDVIROORZHGIRUGD\V 3 3 3 (36NE
A
Days after TC-1 tumor challenge
Percent survival 0 40 60 80 100 20 0 20 60 80 Group 1 Group 2 Group 3 40 Group 4 Group 5 * *** *** Group 1 Group 2 ** Group 3
Days after C3 tumor challenge
Percent survival 0 40 60 80 100 20 0 20 40 60 80 ns B Day 0 8 Group 1:
Tracking tumor outgrowth Group 2:
Group 3: TC-1 tumor challenge Group 4: Group 5: no vaccination MCMV-M79-FKBP-gB IP MCMV-M79-FKBP-E7 IP MCMV-IE2-E7 IP MCMV-IE2-E7 SC Day -70 -35 0
Group 3: MCMV-IE2-OVA IN MCMV-M79-FKBP-E7 IP
C3 tumor challenge Group 2: no infection MCMV-M79-FKBP-E7 IP
Group 1: no infection no vaccination Tracking tumor
