JVI Accepts, published online ahead of print on 3 December 2014 J. Virol. doi:10.1128/JVI.02190-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

ͳ

6WUXFWXUDO)OH[LELOLW\RID&RQVHUYHG%URDGO\1HXWUDOL]LQJ(SLWRSHLQ

ʹ

+HSDWLWLV&9LUXV*O\FRSURWHLQ(

͵



Ͷ

$QQDOLVD0HRODDE $OH[DQGHU:7DUUFG 3DWULFN(QJODQGHI/XNH:0HUHGLWKJ&3DWULFN

ͷ

0F&OXUHFG6WHYHQ.+)RXQJK-DQH$0F.HDWLQJJ-RQDWKDQ.%DOOFG)HOL[$5H\DEDQG

͸

7KRPDV.UH\DE

͹



ͺ ͻ

D

,QVWLWXW 3DVWHXU 8QLWp GH 9LURORJLH 6WUXFWXUDOH 'HSDUWPHQW 9LURORJLH 3DULV

)UDQFH

ͳͲ

E

ͳͳ

F

ͳʹ

%LRPHGLFDO5HVHDUFK8QLW7KH8QLYHUVLW\RI1RWWLQJKDP4XHHQ¶V0HGLFDO&HQWUH

&1568053DULV)UDQFH

6FKRRORI/LIH6FLHQFHVDQGG7KH1RWWLQJKDP'LJHVWLYH'LVHDVHV&HQWUH

ͳ͵

1RWWLQJKDP8QLWHG.LQJGRP

ͳͶ

H

ͳͷ

,QWHUDFWLRQV3DULV)UDQFH

ͳ͸

I

ͳ͹

J

,QVWLWXW 3DVWHXU 3ODWH)RUPH GH %LRSK\VLTXH GHV 0DFURPROpFXOHV HW GH OHXUV

&1568053DULV)UDQFH +HSDWLWLV&5HVHDUFK*URXS&HQWUHIRU+XPDQ9LURORJ\8QLYHUVLW\RI

ͳͺ

%LUPLQJKDP%LUPLQJKDP8QLWHG.LQJGRP

ͳͻ

K

ʹͲ

&DOLIRUQLD8QLWHG6WDWHVRI$PHULFD

ʹͳ ʹʹ

 :RUGFRXQWDEVWUDFW

ʹ͵

:RUGFRXQWWH[W

ʹͶ ʹͷ

 

ʹ͸

5XQQLQJ+HDG6WUXFWXUDOO\IOH[LEOH+&9(QHXWUDOL]LQJHSLWRSH

ʹ͹

7KHVHDXWKRUVFRQWULEXWHGHTXDOO\WRWKLVZRUN

ʹͺ



ʹͻ

$GGUHVVFRUUHVSRQGHQFHWRWNUH\#SDVWHXUIU



'HSDUWPHQWRI3DWKRORJ\6WDQIRUG8QLYHUVLW\6FKRRORI0HGLFLQH6WDQIRUG

ͳ

͵Ͳ

Abstract

͵ͳ

1HXWUDOL]LQJ DQWLERGLHV Q$EV  WDUJHWLQJ JO\FRSURWHLQ ( DUH LPSRUWDQW WR FRQWURO

͵ʹ

KHSDWLWLV&YLUXV+&9 LQIHFWLRQ2QHFRQVHUYHGDQWLJHQLFVLWHDPLQRDFLGV

͵͵

  LV GLVRUGHUHG LQ WKH UHSRUWHG ( VWUXFWXUH EXW D V\QWKHWLF SHSWLGH PLPLFNLQJ

͵Ͷ

WKLVVLWHIRUPVDȕKDLUSLQLQFRPSOH[ZLWKWKUHHLQGHSHQGHQWQ$EV2XUVWUXFWXUHRI

͵ͷ

WKH VDPH SHSWLGH LQ FRPSOH[ ZLWK Q$E  GHPRQVWUDWHV D VWULNLQJO\ GLIIHUHQW

͵͸

H[WHQGHGFRQIRUPDWLRQ:HDOVRVKRZWKDWUHVLGXHVDUHHVVHQWLDOIRUYLUXV

͵͹

HQWU\EXWQRWIRU(IROGLQJ7RJHWKHUZLWKWKHQHXWUDOL]LQJFDSDFLW\RIWKH)DE

͵ͺ

WKLVLQGLFDWHVDQXQH[SHFWHGVWUXFWXUDOIOH[LELOLW\ZLWKLQWKLVHSLWRSH1$EVDQG

͵ͻ

$3 UHFRJQL]LQJ WKH H[WHQGHG DQG ȕKDLUSLQ FRQIRUPDWLRQ UHVSHFWLYHO\  GLVSOD\

ͶͲ

VLPLODU QHXWUDOL]LQJ DFWLYLW\ GHVSLWH FRQYHUVH ELQGLQJ NLQHWLFV 2XU UHVXOWV VXJJHVW

Ͷͳ

WKDW +&9 XWLOL]HV FRQIRUPDWLRQDO IOH[LELOLW\ DV LPPXQH HYDVLRQ VWUDWHJ\

Ͷʹ

FRQWULEXWLQJWRWKHOLPLWHGLPPXQRJHQLFLW\RIWKLVHSLWRSHLQSDWLHQWVVLPLODUWRWKH

Ͷ͵

FRQIRUPDWLRQDOIOH[LELOLW\GHVFULEHGIRURWKHUHQYHORSHGDQGQRQHQYHORSHGYLUXVHV

ͶͶ



Ͷͷ





ʹ

Ͷ͸

Importance

Ͷ͹

$SSUR[LPDWHO\  PLOOLRQ SHRSOH ZRUOGZLGH DUH LQIHFWHG ZLWK +HSDWLWLV & YLUXV

Ͷͺ

+&9 DQGQHXWUDOL]LQJDQWLERGLHVSOD\DQLPSRUWDQWUROHLQFRQWUROOLQJUHSOLFDWLRQ

Ͷͻ

RI WKLV PDMRU KXPDQ SDWKRJHQ :H VKRZ KHUH WKDW RQH RI WKH PRVW FRQVHUYHG

ͷͲ

DQWLJHQLF VLWHV ZLWKLQ WKH PDMRU JO\FRSURWHLQ ( DPLQR DFLGV    WKDW LV

ͷͳ

GLVRUGHUHG LQ WKH UHFHQWO\ UHSRUWHG FU\VWDO VWUXFWXUH RI DQ ( FRUH IUDJPHQW  FDQ

ͷʹ

DGRSW GLIIHUHQW FRQIRUPDWLRQV LQ WKH FRQWH[W RI WKH LQIHFWLRXV YLUXV SDUWLFOH

ͷ͵

5HFRPELQDQW )DE IUDJPHQWV UHFRJQL]LQJ GLIIHUHQW FRQIRUPDWLRQV RI WKLV DQWLJHQLF

ͷͶ

VLWH KDYH VLPLODU QHXWUDOL]DWLRQ DFWLYLW\ LQ VSLWH RI FRQYHUVH NLQHWLF ELQGLQJ

ͷͷ

SDUDPHWHUV 2I QRWH DQ DQWLERG\ UHVSRQVH WDUJHWLQJ WKLV DQWLJHQLF UHJLRQ LV OHVV

ͷ͸

IUHTXHQWWKDQWRRWKHUPRUHLPPXQRJHQLFUHJLRQVLQ(2XUUHVXOWVVXJJHVWWKDWWKH

ͷ͹

REVHUYHGFRQIRUPDWLRQDOIOH[LELOLW\LQWKLVFRQVHUYHGDQWLJHQLFUHJLRQFRQWULEXWHVWR

ͷͺ

WKHHYDVLRQ RIWKHKXPRUDOKRVWLPPXQHUHVSRQVHIDFLOLWDWLQJ FKURQLFLW\DQGYLUDO

ͷͻ

VSUHDGRI+&9ZLWKLQDQLQIHFWHGLQGLYLGXDO

͸Ͳ



͸ͳ





͵

͸ʹ

Introduction

͸͵

$QHVWLPDWHGPLOOLRQSHRSOHZRUOGZLGHDUHLQIHFWHGE\+&9DQGWKHPDMRULW\

͸Ͷ

RI LQIHFWHG SDWLHQWV   GHYHORS FKURQLF LQIHFWLRQ WKDW OHDGV WR SURJUHVVLYH

͸ͷ

OLYHUGLVHDVH 0DMRUDGYDQFHVLQ+&9WKHUDS\GXULQJWKHODVWGHFDGHUHVXOWHGLQ

͸͸

FRPELQDWLRQWKHUDSLHVRIGLUHFWDFWLQJDQWLYLUDOV'$$V ZLWKVXVWDLQHGYLURORJLFDO

͸͹

UHVSRQVHUDWHV!UHYLHZHGLQ 1HYHUWKHOHVVWKHODFNRIDYDLODELOLW\RIWKLV

͸ͺ

+&9WKHUDS\LQGHYHORSLQJFRXQWULHVLOOXVWUDWHWKHXUJHQWQHHGWRGHVLJQDVDIHDQG

͸ͻ

HIILFLHQW+&9YDFFLQHDSURFHVVWKDWLVKDPSHUHGE\RXUOLPLWHGXQGHUVWDQGLQJRI

͹Ͳ

WKHNH\HSLWRSHVLQGXFLQJDSURWHFWLYHQHXWUDOL]LQJLPPXQHUHVSRQVH

͹ͳ



͹ʹ

7KH PDMRULW\ RI QHXWUDOL]LQJ DQWLERGLHV Q$EV  LGHQWLILHG WR GDWH WDUJHW WKH PDMRU

͹͵

HQYHORSH JO\FRSURWHLQ ( UHYLHZHG E\   ZKLFK ELQGV WKH FHOOXODU UHFHSWRUV

͹Ͷ

&' DQG VFDYHQJHU UHFHSWRU %, 65%,     7KH JO\FRSURWHLQ FRQWDLQV

͹ͷ

K\SHUYDULDEOH UHJLRQV +95  WHUPHG +95 +95 DQG LJ95 LQWHUJHQRW\SLF

͹͸

YDULDEOHUHJLRQ WKHGHOHWLRQRIZKLFKGRHVQRWDIIHFWWKHRYHUDOOJO\FRSURWHLQ

͹͹

FRQIRUPDWLRQ7KHVWUXFWXUDOO\IOH[LEOH+95ORFDWHGDWWKH1WHUPLQXVRI( LV

͹ͺ

GLVSHQVDEOHIRUYLUXVLQIHFWLYLW\LQFKLPSDQ]HHV 5HFHQWVWUXFWXUDOVWXGLHVKDYH

͹ͻ

VKRZQWKDW+&9(KDVDFRUHIUDJPHQWZLWKDQ,JVXSHUIDPLO\IROGIODQNHGE\D

ͺͲ

IURQW DQG D EDFN OD\HU FRQWDLQLQJ ȕVKHHWV UDQGRP FRLOV DQG VKRUW ĮKHOLFHV 

ͺͳ

 2IQRWHERWKVWUXFWXUHVZHUHREWDLQHGXVLQJDQ(IUDJPHQWODFNLQJWKH+95

ͺʹ

WKXVDQLQWHUDFWLRQRIWKH+95ZLWKWKH(FRUHFDQQRWEHH[FOXGHG

ͺ͵



ͺͶ

7KHQHXWUDOL]LQJDQWLERG\$5&ELQGVWRDODUJHSDUWRIWKHIURQWOD\HUDD 

ͺͷ

DQG UHVLGXHV ZLWKLQ WKH &' ELQGLQJ ORRS DD  )XUWKHU LQVLJKWV LQWR

ͺ͸

UHFRJQLWLRQ RI ( QHXWUDOL]LQJ HSLWRSHV FDPH IURP VWUXFWXUDO VWXGLHV WKDW FR



Ͷ

ͺ͹

FU\VWDOOL]HG )DE IUDJPHQWV GHULYHG IURP Į( Q$EV UHFRJQL]LQJ WZR UHJLRQV

ͺͺ

FRPSULVLQJ UHVLGXHV  DQG  UHVSHFWLYHO\ LQ FRPSOH[ ZLWK WKHLU

ͺͻ

UHVSHFWLYH HSLWRSH SHSWLGHV   ,Q WKHVH FRPSOH[HV WKH SHSWLGH DD

ͻͲ

DGRSWV D VKRUW ĮKHOLFDO FRQIRUPDWLRQ ZLWK H[WHQGHG VHJPHQWV LQ HLWKHU GLUHFWLRQ

ͻͳ

 DQGZDVSURSRVHGWRDGRSWWZRGLVFUHWHFRQIRUPDWLRQVLQWKHFRQWH[WRIWKH

ͻʹ

YLUDO SDUWLFOH EDVHG RQ WKHVH SHSWLGH VWUXFWXUHV    7KH VHJPHQW FRPSULVLQJ

ͻ͵

DD DGRSWV D ȕKDLUSLQ LQ FRPSOH[ ZLWK WKUHH LQGHSHQGHQW EURDGO\ Q$EV

ͻͶ

+&9$3+X%Y VXJJHVWLQJDIOH[LEOHIODSOLNHVWUXFWXUH 

ͻͷ

7KHDQWLJHQLFVLWHDDFRQWDLQVKLJKO\FRQVHUYHGHSLWRSHVWDUJHWHGE\P$EV

ͻ͸

QHXWUDOL]LQJ +&9 VWUDLQV RI DOO PDMRU JHQRW\SHV   DQG LV SRVLWLRQHG

ͻ͹

GRZQVWUHDP RI WKH +95 $OO GHVFULEHG HSLWRSHV LQFOXGH D WU\SWRSKDQ UHVLGXH DW

ͻͺ

SRVLWLRQ  WKDW SOD\V D FULWLFDO UROH LQ &' UHFRJQLWLRQ   QRQHWKHOHVV D

ͻͻ

VXUSULVLQJO\ ZHDN LPPXQH UHVSRQVH DJDLQVW WKLV DQWLJHQLF VLWH ZDV UHSRUWHG IRU

ͳͲͲ

LQIHFWHGSDWLHQWV 

ͳͲͳ



ͳͲʹ

+HUHZHUHSRUWWKH FU\VWDOVWUXFWXUH RIWKH DDHSLWRSH LQ FRPSOH[ZLWKWKH

ͳͲ͵

QHXWUDOL]LQJĮ+&9(DQWLERG\XQH[SHFWHGO\UHYHDOLQJDQH[WHQGHGSHSWLGH

ͳͲͶ

FRQIRUPDWLRQWKDWLVVWULNLQJO\GLIIHUHQWIURPWKHSUHYLRXVO\UHSRUWHGȕKDLUSLQ:H

ͳͲͷ

GHPRQVWUDWHWKDWWKHVHJPHQWDDLVQRWUHTXLUHGIRUWKHQDWLYHRYHUDOOIROGRI

ͳͲ͸

D VROXEOH ( HFWRGRPDLQ EXW HVVHQWLDO IRU LQIHFWLYLW\ RI +&9SS DQG +&9FF $

ͳͲ͹

FRPSDUDWLYH IXQFWLRQDO DQDO\VLV RI )DE  DQG )DE $3  H[HPSODU\ IRU Q$EV

ͳͲͺ

UHFRJQL]LQJWKHȕKDLUSLQUHYHDOVVLPLODUQHXWUDOL]DWLRQDFWLYLWLHVIRUERWKQ$EVLQ

ͳͲͻ

VSLWH RI VWULNLQJO\ GLIIHUHQW ELQGLQJ NLQHWLFV ,Q FRPELQDWLRQ ZLWK WKH QHXWUDOL]LQJ

ͳͳͲ

DFWLYLW\ RI P$E  RXU UHVXOWV LOOXVWUDWH WKH VWUXFWXUDO IOH[LELOLW\ RI WKLV UHJLRQ



ͷ

ͳͳͳ

ZLWKLQ +&9 ( DQG SURYLGH QRYHO LQVLJKWV LQWR WKH UHFRJQLWLRQ RI FRQVHUYHG

ͳͳʹ

QHXWUDOL]LQJHSLWRSHVZLWKLPSOLFDWLRQVIRUYDFFLQHGHVLJQ

ͳͳ͵



ͳͳͶ

Materials and methods

ͳͳͷ



ͳͳ͸

Production and purification of recombinant Fabs and glycoproteins

ͳͳ͹

6\QWKHWLF JHQHV RI WKH )DE FRGLQJ UHJLRQV RI P$EV  DQG $3 ZHUH FORQHG

ͳͳͺ

LQWR D 'URVRSKLOD 6 )DE H[SUHVVLRQ YHFWRU GHVFULEHG SUHYLRXVO\   +&9 (

ͳͳͻ

IXOOOHQJWKHFWRGRPDLQV( HFWRGRPDLQODFNLQJWKHK\SHUYDULDEOHUHJLRQV(

ͳʹͲ

ǻ+95  DPLQR DFLGV  RI WKH +&9 SRO\SURWHLQ  DQG HFWRGRPDLQ ODFNLQJ

ͳʹͳ

WKH 1WHUPLQDO  UHVLGXHV V(  IURP VWUDLQ 8.1% ZHUH H[SUHVVHG LQ

ͳʹʹ

'URVRSKLOD6FHOOVDV SUHYLRXVO\GHVFULEHG %ULHIO\'URVRSKLOD6FHOOV

ͳʹ͵

ZHUH WUDQVIHFWHG DV UHSRUWHG SUHYLRXVO\ DPSOLILHG DQG IRU ODUJHVFDOH SURGXFWLRQ

ͳʹͶ

LQGXFHGZLWKȝ0&G&ODWDGHQVLW\RIDSSUR[LPDWHO\[FHOOVPOSHUPOIRU

ͳʹͷ

GD\V3URWHLQVZHUHSXULILHGE\DIILQLW\FKURPDWRJUDSK\IURPWKHVXSHUQDWDQWXVLQJ

ͳʹ͸

D6WUHS7DFWLQ6XSHUIORZFROXPQIROORZHGE\VL]HH[FOXVLRQFKURPDWRJUDSK\XVLQJ

ͳʹ͹

D 6XSHUGH[ FROXPQ 3XUH PRQRPHULF SURWHLQV ZHUH FRQFHQWUDWHG WR

ͳʹͺ

DSSUR[LPDWHO\PJPO

ͳʹͻ



ͳ͵Ͳ

Neutralizing capacity of recombinant Fab fragments and infectivity of

ͳ͵ͳ

HCVcc and HCVpp ȟaa384-425 deletion mutants

ͳ͵ʹ

+&9FFRI+-)+DQG--)+ZHUHSURGXFHGDVSUHYLRXVO\GHVFULEHG $

ͳ͵͵

PXWDQW --)+ +&9 FORQH ODFNLQJ +95 DD  ZDV JHQHUDWHG XVLQJ

ͳ͵Ͷ

SODVPLG S)/--)+ DV WHPSODWH   DQG WKH 4 6'0 NLW 1(% SULPHUV



͸

ͳ͵ͷ

DYDLODEOH RQ UHTXHVW  +XK FHOOV ZHUH FXOWXUHG LQ '0(0 )&6 DQG

ͳ͵͸

KDUYHVWHG DW   DQG  KRXUV IROORZLQJ WUDQVIHFWLRQV 16$ H[SUHVVLRQ DV DQ

ͳ͵͹

LQGLUHFW LQGLFDWRU IRU UHSOLFDWLRQWUDQVODWLRQ ZDV DVVHVVHG E\ LPPXQRVWDLQLQJ RI

ͳ͵ͺ

HOHFWURSRUDWHGFHOOVXVLQJP$E(6XSHUQDWDQWVZHUHWKHQXVHGWRLQIHFWQDwYH

ͳ͵ͻ

FHOOVLQIHFWLYLW\EHLQJDVVHVVHGE\16$LPPXQRVWDLQLQJDIWHUKRXUV

ͳͶͲ

1HXWUDOL]DWLRQDVVD\VZHUHSHUIRUPHGE\PL[LQJ))8RI+&9FFLQWKHDEVHQFH

ͳͶͳ

RU SUHVHQFH RI WKH LQGLFDWHG FRQFHQWUDWLRQV RI DQWLERGLHV IRU K EHIRUH DGGLQJ WR

ͳͶʹ

+XKFHOOVDNLQGJLIWIURP&5LFH5RFNHIHOOHU8QLYHUVLW\ DQGLQFXEDWLRQIRU

ͳͶ͵

KDWƒ&&HOOVZHUHWKHQIL[HGVWDLQHGIRU16$DQGWKHQXPEHURILQIHFWHG

ͳͶͶ

IRFL GHWHUPLQHG E\ FRXQWLQJ RI ,) SRVLWLYH FHOOV 3HUFHQWDJH LQIHFWLRQ ZDV

ͳͶͷ

GHWHUPLQHGE\FRPSDULVRQWRWKHQXPEHURIFHOOVLQIHFWHGHLWKHULQWKHSUHVHQFHRI

ͳͶ͸

FRQWURO,J*RUDFRQWUROLQWKHDEVHQFHRI,J*

ͳͶ͹

51$WUDQVFULSWVZHUHSURGXFHGDQGHOHFWURSRUDWHGLQWR+XKFHOOVDVSUHYLRXVO\

ͳͶͺ

GHVFULEHG$SODVPLGHQFRGLQJWKH((SULPDU\LVRODWH8.1$ZDVXVHGDV

ͳͶͻ

WHPSODWH IRU SURGXFWLRQ RI +&9 SVHXGRSDUWLFOHV FRQWDLQLQJ ZW RU ǻ+95 ( DV

ͳͷͲ

SUHYLRXVO\GHVFULEHG ,QIHFWLYLW\LQ+XKFHOOVZDVGHWHUPLQHGDIWHUKRXUV

ͳͷͳ

E\UHDGLQJOXFLIHUDVHDFWLYLW\LQO\VHGFHOOV3URPHJD 

ͳͷʹ



ͳͷ͵

Peptides and complex formation

ͳͷͶ

$ V\QWKHWLF SHSWLGH FRPSULVLQJ UHVLGXHV  4/,171*6:+91  RI WKH

ͳͷͷ

JHQRW\SH D VWUDLQ *ODVJRZ DFFHVVLRQ QXPEHU $@ RU)DEIUDJPHQWH RU&%+

ʹͶ͵

' GLUHFWHGDJDLQVW+&9(ZDVDGGHG$IWHUH[WHQVLYHZDVKLQJWKHFRPSOH[

ʹͶͶ

ZDVHOXWHGDQGDQDO\VHGE\6'63$*(IROORZHGE\&RRPDVVLH%OXHVWDLQLQJ

ʹͶͷ



ʹͶ͸

Data deposition.

ʹͶ͹

7KH DWRPLF FRRUGLQDWHV DQG VWUXFWXUH IDFWRUV IRU WZR FU\VWDO VWUXFWXUHV ZHUH

ʹͶͺ

GHSRVLWHG LQ WKH 3URWHLQ 'DWD %DQN ZZZSGERUJ XQGHU WKH DFFHVVLRQ QXPEHUV

ʹͶͻ

:+7DQG:+ N7H UHG  WR 

ͺ͵ͳ

N7H EOXH @ DFURVV WKH PROHFXODU VXUIDFH RI WKH SDUDWRSH FDOFXODWHG XVLQJ WKH

ͺ͵ʹ

DGDSWLYH 3RLVVRQ%ROW]PDQQ VROYHU '  RU DFFRUGLQJ WR D QRUPDOL]HG

ͺ͵͵

K\GURSKRELFLW\VFDOHIURPZKLWHK\GURSKRELF WREULJKW\HOORZK\GURSKLOLF ( 

ͺ͵Ͷ



ͺ͵ͷ





͵ͳ

ͺ͵͸

Figure 4. Relevant conformations of the antigenic site aa412-423.

ͺ͵͹

$ &RPSDWLELOLW\RIWKH)DESHSWLGHFRPSOH[VWUXFWXUHZLWKWKHSRVLWLRQRI1

ͺ͵ͺ

OLQNHG JO\FDQV DWWDFKHG WR 1 DQG 1 RI WKH QDWLYH JO\FRSURWHLQ &DUWRRQ

ͺ͵ͻ

UHSUHVHQWDWLRQRIWKHSHSWLGHUDPSFRORUHGIURP EOXHWRUHGWKURXJK\HOORZIURP

ͺͶͲ

1WR&WHUPLQXVZLWKWKHWZRDVSDUDJLQHVLGHFKDLQVVKRZQDVVWLFNVDQGWKH1'

ͺͶͳ

DWRPV WR ZKLFK WKH VXJDU FKDLQV DUH OLQNHG FRORUHG LQ EOXH +\SRWKHWLFDO JO\FDQ

ͺͶʹ

FKDLQVFRQWDLQLQJWZR1DFHW\OJOXFRVDPLQDQGRQH PDQQRVHPRLHW\OLJKWRUDQJH 

ͺͶ͵

DUH PRGHOHG WR YLVXDOL]H WKH H[WHQGHG FRQIRUPDWLRQ RI WKH QDWLYH JO\FRSURWHLQ

ͺͶͶ

UHTXLUHG IRU  ELQGLQJ %&  &RPSDULVRQ RI ERWK SHSWLGH FRQIRUPDWLRQV

ͺͶͷ

REVHUYHGIRUDD7KHEDFNERQHDWRPVRIWKHSHSWLGHVERXQGWR)DE% 

ͺͶ͸

DQG)DE+&9&  DVDQH[DPSOHIRUWKHȕKDLUSLQFRQIRUPDWLRQRIDD

ͺͶ͹

DUH VKRZQ DV VWLFNV DQG FRORUHG E\ DWRPW\SH JUHHQ UHG DQG EOXH IRU FDUERQ

ͺͶͺ

R[\JHQ DQG QLWURJHQ UHVSHFWLYHO\  WR LOOXVWUDWH WKH GLIIHUHQFHV LQ WKH EDFNERQH

ͺͶͻ

FRQIRUPDWLRQREVHUYHGIRUWKHWZRVWUXFWXUHV7KHILYHK\GURJHQERQGVVWDELOL]LQJ

ͺͷͲ

WKHEDFNERQHFRQIRUPDWLRQLQERWKVWUXFWXUHVDUHLQGLFDWHGDVGRWWHGOLQHV

ͺͷͳ



ͺͷʹ



ͺͷ͵

Figure 5. Surface plasmon resonance (SPR) analysis of Fab binding to

ͺͷͶ

aa412-423.

ͺͷͷ

7R IDFLOLWDWH GLUHFW FRPSDULVRQ RI ELQGLQJ NLQHWLFV WR GLIIHUHQW )DE IUDJPHQWV WKH

ͺͷ͸

ELQGLQJ UHVSRQVH ZDV QRUPDOL]HG LQ HDFK FDVH DV GHVFULEHG LQ 0DWHULDOV DQG

ͺͷ͹

0HWKRGVDQGH[SUHVVHGDVRFFXSDQF\RIELQGLQJVLWHV$ 5HDOWLPH635DQDO\VLV

ͺͷͺ

RIWKHLQWHUDFWLRQRIWKHHSLWRSHSHSWLGHVGHULYHGIURPJHQRW\SHDVWUDLQ*ODVJRZ

ͺͷͻ

EODFNDQGJUHHQIRUDQG$3UHVSHFWLYHO\ DQG+OLJKWJUH\DQGUHGIRU

ͺ͸Ͳ

 DQG $3 UHVSHFWLYHO\  ZLWK LPPRELOL]HG )DEV 7KH UHVSHFWLYH HSLWRSH



͵ʹ

ͺ͸ͳ

SHSWLGHZDVLQMHFWHGRYHUDVXUIDFHRIFRYDOHQWO\LPPRELOL]HG)DEDWDIORZUDWHRI

ͺ͸ʹ

 ȝ/PO DQG WKH ELQGLQJ UHVSRQVH LQ UHVRQDQFH XQLWV 58  ZDV UHFRUGHG DV D

ͺ͸͵

IXQFWLRQ RI WLPH 7R FRPSHQVDWH IRU WKH VPDOO VLJQDO GXH WR WKH ORZ PROHFXODU

ͺ͸Ͷ

ZHLJKW RI WKH SHSWLGH OLJDQG RI aN'  WKUHH LQGHSHQGHQW H[SHULPHQWV ZHUH

ͺ͸ͷ

SHUIRUPHG DQG WKH PHDQ YDOXH ZLWK VWDQGDUG HUURUV LV SUHVHQWHG LQ 7DEOH  7KH

ͺ͸͸

VKRZQUHSUHVHQWDWLYHDVVRFLDWLRQGLVVRFLDWLRQWLPHFRXUVHSURILOHFRUUHVSRQGVWRWKH

ͺ͸͹

LQMHFWLRQRIQ0RISHSWLGHRYHUHDFKRIWKH)DEVLOOXVWUDWLQJWKHGLIIHUHQWNLQHWLF

ͺ͸ͺ

SURSHUWLHVRIWKHWZR)DESHSWLGHFRPSOH[HV

ͺ͸ͻ

%&  5HDOWLPH 635 DQDO\VLV RI WKH ELQGLQJ RI )DE $3 %  DQG  &  WR

ͺ͹Ͳ

LPPRELOL]HG +&9 ( HFWRGRPDLQ )DEV ZHUH LQMHFWHG RYHU D +&9 V(

ͺ͹ͳ

LPPRELOL]HG XVLQJ DQ DQWLVWUHSWDJ DQWLERG\ DW D IORZ UDWH RI  ȝ/PO DQG WKH

ͺ͹ʹ

ELQGLQJ UHVSRQVH LQ UHVRQDQFH XQLWV 58  ZDV UHFRUGHG DV D IXQFWLRQ RI WLPH

ͺ͹͵

5HSUHVHQWDWLYH DVVRFLDWLRQGLVVRFLDWLRQ WLPHFRXUVH SURILOHV FRUUHVSRQGLQJ WR WKH

ͺ͹Ͷ

LQMHFWLRQ RI )DEV Q0  RYHU IXOOOHQJWK LPPRELOL]HG +&9 V( RU QDWLYH DQG

ͺ͹ͷ

GHQDWXUHG+&9V(ǻ+95

ͺ͹͸



ͺ͹͹

Figure 6. Effect of aa412-425 deletion on protein folding and infectivity.

ͺ͹ͺ

$  3XOOGRZQ DVVD\ RI WKH FRQIRUPDWLRQGHSHQGHQW QRQQHXWUDOL]LQJ KXPDQ

ͺ͹ͻ

DQWLERG\ &%+'   E\ V( ǻ+95 DQG )DE V( ǻ+95 FRPSOH[

ͺͺͲ

LQGLFDWLQJ WKDW )DE  ELQGLQJ GRHV QRW DIIHFW WKH RYHUDOO JO\FRSURWHLQ IROG % 

ͺͺͳ

(OXWLRQ SURILOH RI V( 8.1%  IURP D 6XSHUGH[ VL]H H[FOXVLRQ

ͺͺʹ

FROXPQ& 3XOOGRZQDVVD\RI&'/(/&%+')DEDQGH)DEE\V(

ͺͺ͵

UHYHDOVDQLQWDFWRYHUDOOJO\FRSURWHLQIROGEXWDVHYHUHO\LPSDLUHGELQGLQJWRWKH

ͺͺͶ

&' ELQGLQJ VLWH '*  ,QIHFWLYLW\ RI GHOHWLRQ PXWDQWV LQ +&9FF DQG +&9SS

ͺͺͷ

PRGHOV RI LQIHFWLRQ '  7KH (( JHQHV RI WKH KLJKO\ LQIHFWLRXV SULPDU\ LVRODWH



͵͵

ͺͺ͸

8.1$ ZHUH XVHG DV WHPSODWH IRU GHOHWLRQ RI DPLQR DFLGV  +&9

ͺͺ͹

SVHXGRSDUWLFOHV +&9SS  SRVVHVVLQJ ZW DQG PXWDQW JO\FRSURWHLQV ZHUH XVHG WR

ͺͺͺ

LQIHFW +XK FHOOV ,QIHFWLYLW\ PHDVXUHPHQWV ZHUH SHUIRUPHG ZLWK D OXFLIHUDVH

ͺͺͻ

UHSRUWHU JHQH 2QO\ WDUJHW FHOOV LQRFXODWHG ZLWK ZLOGW\SH +&9SS H[KLELWHG

ͺͻͲ

OXFLIHUDVH DFWLYLW\ (  :HVWHUQ EORW DQDO\VLV RI +&9SS SDUWLFOHV XVLQJ WKH OLQHDU

ͺͻͳ

DQWL( P$E $/3 UHYHDOHG VLPLODU ( LQFRUSRUDWLRQ OHYHOV IRU ZW DQG PXWDQW

ͺͻʹ

+&9SS XSSHU SDQHO ODQH  DQG  UHVSHFWLYHO\  7KH DPRXQW RI SDUWLFOHV ZDV

ͺͻ͵

YHULILHGE\:HVWHUQEORWDQDO\VLVXVLQJDQDQWL*DJDQWLERG\ORZHUSDQHO )* 

ͺͻͶ

$SODVPLGHQFRGLQJDFKLPHULF--)+YLUXVZDVXVHGDVWHPSODWHIRUGHOHWLRQRI

ͺͻͷ

DPLQRDFLGV51$WUDQVFULSWVRIZWDQGPXWDQWYLUXVHVZHUHHOHFWURSRUDWHG

ͺͻ͸

LQWR+XKFHOOV5HVXOWVDUHSUHVHQWHGDVPHDQDQGVWDQGDUGGHYLDWLRQRIGXSOLFDWH

ͺͻ͹

H[SHULPHQWV )  51$ UHSOLFDWLRQ ZDV DVVHVVHG E\ LQWUDFHOOXODU VWDLQLQJ IRU WKH

ͺͻͺ

SUHVHQFHRI+&916DLQHOHFWURSRUDWHGFHOOV%RWKFRQVWUXFWVUHSOLFDWHGVLPLODUO\

ͺͻͻ

ZLWKWKHGHOHWLRQPXWDQWKDYLQJVOLJKWO\HQKDQFHGUHSOLFDWLRQ* &HOOVXSHUQDWDQWV

ͻͲͲ

KDUYHVWHGDQGKRXUVDIWHUWUDQVIHFWLRQZHUHDVVHVVHGIRUWKHSUHVHQFHRI

ͻͲͳ

LQIHFWLRXV YLUXV E\ LQIHFWLQJ QDwYH +XK FHOOV ,QIHFWLRXV ZLOGW\SH YLUXV ZDV

ͻͲʹ

SURGXFHGZKLOHQRLQIHFWLYLW\ZDVREVHUYHGIRUWKHPXWDQWYLUXV

ͻͲ͵



ͻͲͶ







͵Ͷ

ͻͲͷ

Table 1. IC50-Values for 3/11 and AP33 Fabs and mAbs.

ͻͲ͸

 

ͻͲ͹

͹͹

͸

͵Ȁͳͳ ƒ„

ͲǤͶ͵Ɋ‰ȀŽ ͺǤͶͺ

ʹǤͷ͹Ɋ‰ȀŽ ͷͲǤ͸ͷ

͵Ȁͳͳ„

ͲǤ͵ͻɊ‰ȀŽ ʹǤ͸

ͳǤͺͻɊ‰ȀŽ ͳʹǤ͸

͵͵ ƒ„

ͲǤͷ͹Ɋ‰ȀŽ ͳͲǤͻͺ

ͲǤͺ͵Ɋ‰ȀŽ ͳͷǤͻͺ

͵͵„

ͲǤʹ͵Ɋ‰ȀŽ ͳǤͷ͵

ͳǤͲͷɊ‰ȀŽ ͹

‘–”‘Ž ‰ 

͸ͳǤ͹͸Ɋ‰ȀŽ ͶͳͳǤ͹͵

ͳͲ͹Ǥ͵Ɋ‰ȀŽ ͹ͳͷǤ͵͵





͵ͷ

ͻͲͺ

Table 2. Data collection and refinement statistics.

ͻͲͻ



)DE SHSWLGH 3'%:+