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    US - 2010 - 0120092 - A1.pdfpatente HVB PDF

    Uploaded by

    Emanuel Pomacco

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    © All Rights Reserved
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    c») United States cz) Patent Application Publication (1) Pub. No.: US 2010/0120092 AI Grgacie et al US 201001200921 (43) Pub. Date: May 13, 2010 (sa) sy RECOMBINANT PROTEINS AND VIRUS LIKE PARTICLES COMPRISING LANDS POLYPEPTIDES OF AVIAN HEPADNAVIRIDAE AND METHODS, NUCLEIC ACID CONSTRUCTS, VECTORS. AND HOST CELLS FOR PRODUCING SAME Jnventors: Elizabeth Vera Ludmilla Gracie, ‘Victoria (AU); David Andrew “Anderson, Vicioria (AU): Paton LLoke, Victoria (AU): Robin Fredrle Anders, Vicloria (AU) Corespondence Address: SCULLY SCOTT MURPHY & PRESS 4400 GARDEN CITY PLAZA, SUITE 300 GARDEN CITY, NY 11830 (US) PC (73) Assignee: HEPGENICS PTY LTD., Malvern, Victoria (AU) (21) Appl.No. 121439,268 (2) PCT Fite Aug, 29, 2007 (86) PCTNo. PCTAU2007/001241 5371 (0, ().(4)Date: Jan. 13,2010 G0) Foreign Application Priority Data Avg, 30,2006 (AU) 2006904731 Aug. 31,2006 (AU) 2006904757 Ick C13P 21/04 (2006.01) CRN 700 (2006.01) CRN 5907 (01001) cay 510 (2006.01) CRN 119 (2006.01) CRN 1968 (2006.01) corn 2104 (2006.01) us.cl 4435/69.7, 435/235, 1; 435/348 "435/340; 438/366; 435/255.1; 435/320.1, 530/234 on ABSTRACT The specification discloses chimeric or recombinant virus like particles comprising () S polypeptide of an avian hep ‘adnavirus and Gi) a chimetie fision protein comprising polypeptide oF interest covalently attached to a patile-aso ating portion of L polypeptide of an avian hepadnavirus, ‘wherein the polypeptide of interes comprises transmer brane domain or @ protein binding. domain or motif and ‘wherein the chimerie fusion protein Tarther comprises a se ‘ond o further polypeptide of interes comprising a transien brane domain andar « protein binding domain or motif, wherein the second of further polypeptide is associated with the virus-like particle via non-peptide bonds. I is proposed that such VLPs morelosely resemble the naturally occurring configuration ofartigenie complexes of pathogens. The ei tice vinis-lke particles a illustrated sng viral envelope proteins from mensles, hepatitis C virus, inlvenza.A and FIV ‘and by polyproteias trem Plasmodium surtice proveins [Nacleie acid constrcts, vectors, host cells comprising same and methods of producing virus-like particles and nucleic ‘cid constructs are also described, 3° ABV) | RSs Patent Application Publication May 13,2010 Sheet 1 of 151 US 2010/0120092 AI fasion PCR prod. — ; i = Cy Si HCV E2 ectodomain: DHBV Lipres) | (@.0384-465/nue 1492-1736) act oat [a m7 / SH1 pw [DHBY sequence from nt 727-2817] IRE sites shown are unique sites PMDLwt m8 sCDLERASS Figure 1 Patent Application Publication May 13,2010 Sheet 2 of 151 US 2010/0120092 AI [+ pres domain ——+ ——— $ domain ———1 a: fs a {far}——=7]—~ I, 82 a.a, of E2 ectodomain(a.a384-465) cobe2.4es Y Tl}: as} 0s sig B. C. L-£2.465 chimera is translocated D, L-E2.465 chimera is assembled ‘across the ER membrane into particles trypsin NP-4O + oF a: LER A65— ee LE2465 — | stietnannttel - norosomesprofease protein ase rtcles pried by sedimentation rote * Pen rough 20% seas export from ca) particle Figure 2 US 2010/0120092 A1 May 13,2010 Sheet 3 of 151 Patent Application Publication beabebebbe S5yevoeer] qqoebbbeee e6Goe6e036 y6a30080ee qebbober3e 2632022062 o4qqeobebe 263ee995e3 2e99230896 poeageee3e 2930655062 3299363962 qBBboeqoee 9926999963 a3eqej0Re2 ebe6beqObH 2666965663 S36qeooe6e 2090326326 966322038 obeoee 6653 qaq0072ebb 2635722085 2990859039 byebe393ee qo6b6e2b3e eebbezobo Boeesso080 peooeeee33 aeeeeebeob pbebe166e6 9222622939 beqebbqoro 2630965339 363698078 6226600693 eeeobeooqe qeeqeaqoob pobeebqe30 qooqeqseee eeeoobeo03 3999299083 peoqeqeaoe qooojo3ebe eqoooo bebe Seeeebeebe 5eBS6e0q0H pqeqeeeieb Beebebhi5q qeebbeboee Hebbhbeozee Beqoeoreoo oppooeerz] opeobbbbje 2076622833 qobbeooreq oeee3 3666 oebyqqeqee e6B0339e32 Hheowbz ee qeoowoobeo peee65699 ebeqo0PPho 636besojee 3003028036 6302993639 aqeeeeroby Beoa300989 aaqbeeobbe Baoeqeeebe BaeqaaeoR 3208025633, 3aeqeee365 6236620935 e6bqqeeebo Beooeq beer 965839020 Bef0736e36 oabeeeoq36 30P 6635939 oorbeoR02 3366930236 peoeaqe0bb eqqobeerh3 (6£8100 “ON “¥ OTC SA) eouanbas ouronas TIM} ATHC peeqebebbe 9356320863 Sbebbeebey qeoeeoqeeo qeoeeeqoqe eqbezeeoqe 3999449e]9 9933999635, B6qeEGEDo 3e066e3059 9299939990 oebeebeb65 oereebeqoe peoferbq3e 9633969903 9659399693 3380706380 196 106 Tre. Tel Te 199 T09 Ts Ter 12 TS TOE The 18 Tet 19 Figure 3 US 2010/0120092 A1 May 13,2010 Sheet 4 of 151 Patent Application Publication oeeqoeerqe ze30630622 eeb339e0b5 eebeeoqeey e6azebeooe eeeqeebeey 036039608 3306036030 3936609605 qo6beobeob 9333e65e09 eqeberoo33 Bez0b6eb5e 2899965995 eeqe636e09 ee66530930 epoeooe0e ooebeebbeb 3239632908 qSyeesoeve 6636133e6e Byoebbeeg3 3366039033 eebbeq3be6 pe00920963 qozeee6003 6632299495 2962396399, 9622656635 qobobyeeob Sy0eqeeRbe eq027 22656 696596e00e e003e60ebb eebbooeoe 9993026636 BoeqeIOOIe 29363225} pe32e 3532 vebeeebbae ebeeqeeeby peoeagaqaq 9999399339 2660300063 eeebbe1997 3ee9909903 9996330385 peereebibe qoq3eeRee 593300035 eBeozbeooe BBoo0rROR7 Beeebeeber e00996R039 9339399305 qeqeezbbee pebb0eb qe Seoseeebee Pe63650939 9906303520 eb7e2663e7e 9299802999 2992302565 3072978993 Se360"39e9 ees03039be 593633033 By0363eeee qeq06003R0 o6er6646e9 979359069 2q226ebevo eqaqoeee36 3089399999 7653eeb eS Saqeserb3e qqooboeoe Boepobeq1e qaoeoqaqqoe 9329939030 peeoebbord 99096629993 2093030382 9903092985 33905ee365 Seeberbqq0 oebeGo309 4993999937, 97995ee69 99396993 peooeeqoe} 6333263333 eqSeeerqee oqazeeeeba eerorb3490 e06oee3999 6633360889 3333000620 3092363063 2900266993 e0D06e6 639 6966366978 qoR33e 6609 9999930888 yoqo3beoqe 996999999 Boesbeeeee peroqe}3e6 Tr0oz T86T Te6T 198T Tost THT Te9T Te9T T9ST LOST LbPT TeeT Tze 192T Lozi TPIT Tg0 TZ0T Figure 3 continued US 2010/0120092 A1 May 13,2010 Sheet 5 of 151 Patent Application Publication peBoosee00 Bebeebj06e eqeqabyobe eeh33e0303, 6633233906 qebjebeooe 9639039e63 9036023399 Byeqohbe32 ebyo0390ee qqeroeqebo eqqbobeeae eeoo6e3346 3636e0606 o6eq6yb0e qaqbeeq bey eBBeboeobe Geeqqeoqee 2653639308 26e3330608 3092632953 2999629800 qeqbbeeoob peooqaeqea eoeob6e003 92eR990893 9969236333 3999692633 e6bbo6bo08 9639999895 ebbeeebeed 9998330303 Boobeqeeeb pqovesb33e Beee305326 Bqee008e33 2969295633 3996306266 eqerobeees eon0bboeby peopeesobb yoee0g 3263 eqorebbeoq oqezeoo078 qo300;6eee eeooeoeeeg 3666008003 3e067 20665 6393936 e08 2603953903, po6e6e2092. yoe3520e6 9399939999 9303030900 9bqo0zee3e 3363099629 9923393096 qBqeoeqeqe qeeobobbye oobeeeqeeb qeebezob50 392999ebeb 3630030279, poroRqoese eebeeezeZo ehzebeqVee 9632203272 3636269353 oovoeaqeeb e9e9290993 yeeeobeboo 3920663209 jaboeooree 5ebeo 66ee oeoeego008 eeqqzerezby eqaebooqe3 s6aqqooeqe 2492999399 9663033025 aqe6eoqeee e09333033 663729293 3630633208 ee33022663 3obboeb6a9 993939235 e33boebeoq qqebqeeqeb 2999999969 qobyebe0ey 5363665300 TOOE Tp6z Tesz Tzez T9LZ TOLe TH9Z Tasz Te2se T9be Tope Twez T87z lezz T9TZ Tot Figure 3 continued US 2010/0120092 A1 May 13,2010 Sheet 6 of 151 Patent Application Publication yoo 339 300 Beo aaa 530 390 ¥ 705 939 bee eRe s 903 eeb B99 L 208 2 265 eb s 303, 269 qee oee s 209 266 a 609 Pb 203 B2e 66a 2 265 @ 99 vee 233 beb poe a ees L ae ee beb 633, eae bob oo poe 998 908 TIT/TETT aoa os eb eb Seb 16/TLOT i o 296 ebe bbe TL/TtOT HL OO Bye Boe eo 1/156 boo 4” oe 686 vee Te/t68 woman 660 v6e 036 TI/TEs, a eeb ebb 636 e909 orb 609 oe 606 yee o7e a Be 9 8 6 a ebe a eed = eeb eo o 220 oeb 293 ea3, bbe eee 266 v 28 a 220 206 e0 (PSZT STE g 1283s S108 BE 1 341045) (1G SA) aouonbos | ATHA 3e0 koe 32 360 960 TOT/TOTT Ig 0 aze Geb ees 18/TPOT 2 ON 2665 Be qee 19/T86 a of th 230 BOR wD Tb/126 Om peo ove P27 12/198 oo fh eeo 665 BIO 1/108 Figure 4 TSz/Tsst Tee/TZgT TY T RK Bow T € 29 4 9 D9 MH @€ D9 FS dD A KR GE 399 H6e 939 9e] 99k 663 999 393 BBB v00 263 eB Bbq 699 063 303 ebb e325 0e7 3VO US 2010/0120092 A1 TEZ/TéyT T2Z/T9bT 4 6 rt 60 ¥ 9 £ G@ 0 4 ¥ D9 0 W 4 D9 Hw as Ss OD 303 O3e eed 096 BBB yok Aeh eed 939 JOH Of] eeD H3e vee BBG Bee woo 493 Abe T1Z/TEbT TOz/TObT. Ts rT A M M GQ T 8 8 T TT T TH tt Tt a 999 303 Jae BBa 662 663 3e6 eyo H6e She 630 eye ee eID 39e eRe PIP 533 623 033 T6T/TLET Tal/TyEt 4 s A T 2 D9 T T 9 ¥ T I 5 9 @ Fb 9 8S K ¥ 433 De 226 639 292 PBB o2e eID 966 JOH e39 wIe e6H 666 932 ooR 366 493 BIS eve May 13,2010 Sheet 7 of 151 TLT/TTET T9T/TBZT MoM T @ @ 2 ¥ FT A ad AY 4@ FT S OR Tat See Bee 633 999 999 Joe eee 2QP 635 vOD 636 60h woo eeb voy Sed 9} LAD BOD 4eO TST/TSZt IbT/tezt bo 8 T T S$ 6 N 9 T T &@€ GQoaoaoatx # 6 Joe 6e6 939 029 303 Beo yee ebb 649 939 vod Yeh 926 666 200 PRO BAD bee HH2 beo TEt/T6TL TeT/T9TT Patent Application Publication Figure 4 continued US 2010/0120092 A1 2 Fe a g ¢ L Boe pee ¥ 226 P02 s 903 2eb Oe 1 239 eed Bor 233 908 T 9 @ 4b 390 266 333 bor eqa 62 1 2 993933 439 998 B94 LoTR 1 1 Boe 639 323 690 629. TEE/TELT A T 2 6 936 030 Boe evo TTE/TELT WYoa ds 306 936 939 409 T6Z/TLOT a Ss ¥ & 238 B03 ob 654 TL2/TI9T a wo YA 265 206 e06 276 * § 5eq 36e Lov poe 906 uo eee Boo oT BPO 939 14 eq0 639 u bee 303 326 bee cae 903 693 266 B33 a aaa 902 290 243 930 1 a9 oe 632 aqaqe 233, Yos 1 326 303 839 TZE/T9LT pe Bye 669 ee TOE/TOLT Ts 8 B39 BO} 43 18Z/TH9T Los 9 30e 303 666 19Z/T8ST Iota oqe o3e 333 Figure 3 continued Patent Application Publication May 13,2010 Sheet 9 of 151 US 2010/0120092 AI 1 pres domain ———-_———— s domain —————+ is [5}—r —ememmeem §7 2.2. of E2 ectodomain(a.a384-465) pores V SEE EEE [TR—s, a 82 2.2, of E2 ectodomain(a.a384-465) commana r eg nel ng mn Figure 5 Patent Application Publication May 13,2010 Sheet 10 of 151 US 2010/0120092 AI 163 a.a. HBV preS domain coves _¥ r ig ——————— 163 a.a, HBV preS domain cousrmeamesY ey i ct faq. —_}—5, 230 a.a. MSP2 Counnan io ae t+ In B, Figure 5 continued Patent Application Publication May 13,2010 Sheet 11 of 151 US 2010/0120092 AI A. SigLATM1-£2.661 membrane fraction pee 120 * Bm gyn om 66 - 40 B. Lata t-Msp2 membrane fraction in 0 eam 100 © ea ° 50 mp 0 Figure 6 Patent Application Publication May 13,2010 Sheet 12 of 151 US 2010/0120092 AI A. DLS VEPs:suctose step gradient profile i i he s+ ree 23) 46 617, si 9) 10) 41) 10% 0% B. _ DLATMI-22.465 VLPs: sucrose step gradient profile Lama ao E24es 234667 8 90H 70% 7 20% (C, DLATML-HpreS VLPs: sucrose step gradient profile LATML, poy HpreS ” s> ee 2 34c 8 6 21008 70%: 7 20% Figure 7 Patent Application Publication May 13,2010 Sheet 13 of 151 US 2010/0120092 AI rat sera:sequential bleeds pre 1 2 ad 3 preS reactivity Figure 8 Patent Application Publication May 13, 2010 Sheet 14 of 151 US 2010/0120092 A1 zB | Bx oo, 26S 6 3833) aos3 3 ooo m oa = i w N o an >5 33 2 2f f I a an co °o Ss Ns We a 2 eo co = qo £ = oa eee 2 S o wee te) 9 oP 8) oe a) oc oo a a ee os (0Z9-0S7a0) eouequosqy Figure 9 Patent Application Publication May 13, 2010 Sheet 15 of 151 US 2010/0120092 A1 2. o wo 3 HS Q E ~~ 3 =3 ai 25 i 3 » og | 2n ee Da oo a2 0 - 3 No 3 2 O . = + = az a3 a= 13 o (0-60)) 2438 za-nuy Figure 10 US 2010/0120092 A1 May 13,2010 Sheet 16 of 151 Patent Application Publication [sna jue Pr) swe yw ewe! zw Two! a 6 sz SOP d1A-23 ADH Big Gry 6izo (uoneinus d7A-24) esuodsey asog dA LOdSI74 Nal Puwesd 73-nhuy S]]99 WON W/DIAS Figure 11 Patent Application Publication May 13, 2010 Sheet 17 of 151 US 2010/0120092 A1 Figure 12 Patent Application Publication May 13, 2010 Sheet 18 of 151 US 2010/0120092 A1 Figure 13 US 2010/0120092 A1 seuporsey SaVT-13 Figure 14 May 13,2010 Sheet 19 of 151 Patent Application Publication mee ZaJO woResO|suEN ¢_FBEABPD TqJO UoRPoO|sURMPBEATA|S —¢_ a ane a : Spndad rouis ‘pndod yousis wry SL 199 ese ove 261 peg LI eo Se— he eZ upeuopore> 13 uewops Aggy ”~ 4 Vv yonysuoo $q/ZATA Wapuey Jo oeUIaYOS US 2010/0120092 A1 May 13,2010 Sheet 20 of 151 Patent Application Publication sd-eala tod a Ss ca Ta uopesy wwe fT wo uorsng, b 7H — a opw —— oH Ta ST Tessess Figure 15 Patent Application Publication May 13, 2010 Sheet 21 of 151 US 2010/0120092 A1 A B Cc poiB 1E2-DS + poi pcDNA-HCON MAD anti-S MAb anti-B Goat anti £2 Figure 16 Patent Application Publication May 13,2010 Sheet 22 of 151 US 2010/0120092 AI A ' - {| Sool wT oo” £16208 0.44 a2 o.of 012345678 9401112 Fractions Probedwith: — goateE2 Mab o€1 Mab oS Figure 17 Patent Application Publication May 13, 2010 Sheet 23 of 151 US 2010/0120092 A1 35: A 30. 25 20 op 1s: 10 os: 00. ee WP wre 0.251 0.20 oas! os! 0.00 ELE iP wr Figure 18 US 2010/0120092 A1 May 13,2010 Sheet 24 of 151 Patent Application Publication £42eM peeideld 9 “ON esnow ‘gol pasiuNWul! ATA € J8A ZdSWN Ul AANOBe ZASIN-BUY wugz9-0S7 GO eouequosqy Figure 19 Patent Application Publication May 13,2010 Sheet 25 of 151 US 2010/0120092 AI Figure 20 Patent Application Publication May 13, 2010 Sheet 26 of 151 US 2010/0120092 A1 Anti-MSP2 titration gn = Anti-MSP2 titre (log1Q) nw a Rm Rw Be won fl 1 — os pas —s PrebleedWeek3 Week6 Week7 fj Mean of six mice Figure 21 Patent Application Publication May 13, 2010 Sheet 27 of 151 US 2010/0120092 A1 Anti-MSP2 titres in MSP2 VLP immunised rabbits 5 4s a4 S35 7 g [DPrebleed, 23 EB | oetceas | g EB ||510c3 2 | gas | FA |(netccas Z: Hl F : HA h 1 HI | H ‘ABDE2-R2 — ABOESD-RD — ABOGAOL-RY —ABOEAOD-R2 ABOSAOD-RE ABORADE-RA Figure 22 Patent Application Publication May 13,2010 Sheet 28 of 151 US 2010/0120092 AI = canis A TSE —— ‘CD61 Capture ELISA with reactivity against HSS 25 (lysate) Bioast (1094) vue nee (sou) > crimowo HR? ‘CDOS Capture ELISA with reactivity ogatnet 7C12 1/2 (soul) Figure 23 Patent Application Publication May 13,2010 Sheet 29 of 151 US 2010/0120092 AI Anti-E2 in mice (1 pg VLPs, cell cult derived) eee J g 825) Ra a 2p el (mPrebleed a |BBleed 1 § i [abies eo { 5 E0575 t otal, i M1 M2 M3 M4 M5 Mé M7 M8 M9 M10 M11 M17 Figure 24 Patent Application Publication May 13,2010 Sheet 30 of 151 US 2010/0120092 AI VEZ cell culure VLP IFK-g ELISPOT results E1E2-VLPs - Interferon gamma ELISPOT response "eee culpa LPFG ELISPOT ona | vol “| ' i i a E2-VLPs - Interferon gamma ELISPOT response Figure 25 Patent Application Publication May 13, 2010 Sheet 31 of 151 US 2010/0120092 A1 Immunofluorescence images of transfected cells E1E2S CO E1E2S HCON (tv) Non transfected (ve) Figure 26 Patent Application Publication —- May 13,2010 Sheet 32 of 151 US 2010/0120092 A1 A EIE2 +S EIE2CO+S EIE2 — E1EZCO bank MW 1234567 8 12:13:14 15 age 8 9 10 4412 13. Figure 27 Patent Application Publication May 13,2010 Sheet 33 of 151 US 2010/0120092 A1 E1E2-VLPs Figure 28 Patent Application Publication May 13,2010 Sheet 34 of 151 US 2010/0120092 AI Influenza HA VLPs Influenza HA ectodomain HSecto-S Hl oxoV stanemenbranedomain i Influenza HA sy. with own transmembrane domain HE nyg-S Cortransfectionof 2837 cells orco-transformation of yeast cels with one of the above HAS fusion constructs | Ki) ardan Seoressin plasmeutive ViP expression | Figure 29 Patent Application Publication May 13,2010 Sheet 35 of 151 US 2010/0120092 AI atggagaaaatagtgcttctttttgcaatagtcagtcttgttaaa agtgatcagatttgcattggttaccatgcaaacaactcgacagag caggttgacacaataatggaaaagaacgttactgttacacatgeco caagacatactggaaaagacacacaatgggaagctctgcegatcta gatggagtgaagcctctaattttgagagattgtagtgtagctgga tggctcctcggaaacccaatgtgtgacgaattcatcaatgtgecg gaatggtcttacatagtggagaaggecaatccagtcaatgacctc tgttacccaggggatttcaatgactatgaagaattgaaacaccta ttgagcagaataaaccattttgagaaaattcagatcatccccaaa agttcttggtccagtcatgaagecetcattgggggtgagctcagca tgtccataccagggaaagtectectttttcagaaatgtggtatgg cttatcaaaaagaacagtacatacccaacaataaagaggagctac aataataccaaccaagaagatcttttggtactgtgggggattcac catcctaatgatgoggcagagcagacaaagctctatcaaaaccca accacctatatttccgttgggacat caacactaaaccagagattg gtaccaagaatagctactagatccaaagtaaacgggcaaagtgga aggatggagttcttctggacaattttaaaaccgaatgatgcaatc aacttcgagagtaatggaaatttcattgetccagaatatgcatac aaaattgtcaagaaaggggactcaacaattatgaaaagtgaattg gaatatggtaactgcaacaccaagtgtcaaactccaatgggggcg ataaactctagcatgocattccacaatatacacectctcaccate ggggaat gocccaaatatgtgaaatcaaacagattagtcottgeg actgggctcagaaataccoctcaaagagagagaagaggattattt ggagctatagcaggttttatagagggaggatggcagggaatggta gatagttggtatgggtaccaccatagcaacgagcaggggagtggg tacgct gcagacaaagaatccactcaaaaggcaatagatggagtc accaataaggtcaactcgattattgacaaaatgaacactcagttt gaggecgttggaagggaatttaacaacttagaaaggagaatagag aatttaaacaagaagatggaagacgggttcctagatgtctggact tataatgctgaacttctagttctcatggaaaatgagagaactcta gactttcatgactcaaatgtcaagaacctttacgacaaggtccga ctacagcttagggataatgcaaaggagctgggtaacggttgtttc gagttctatcataaatgtyataatgaatgtatggaaagtgtaaga Figure 30 Patent Application Publication —- May 13,2010 Sheet 36 of 151 US 2010/0120092 A1 aacggaacgtatgactacccgcagtattcagaagaagcaagacta aaaagagaggaaataagtggagtaaaattggaatcaataggaatt TCACCT TAACGCAATTAGCCACGCTGTCTGCT CTTTTTTACAAG [AGTTA Figure 30 continued Patent Application Publication May 13,2010 Sheet 37 of 151 US 2010/0120092 AI atggagaaaatagtgcttetttttgcaatagtcagtettgttaaa agtgatcagatttgcattggttaccatgcaaacaactcgacagag caggttgacacaataatggaaaagaacgttactgttacacatgec caagacatactggaaaagacacacaatgggaagetctgegatcta gatggagtgaagectctaatittgagagattgtagtgtagctaga tggetecteggaaacccaatgtgtgacgaattcatcaatgtgccg gaatggtcttacatagtggagaaggecaatccagtcaatgacctc tgttacccaggggatttcaatgactatgaagaattgaaacaccta ttgagcagaataaaccattttgagaaaattcagatcatccccaaa agttcttggtccagtcatgaagectcattgggggtgagetcagca tgtecataccagggaaagtcctoctttttcagaaatgtggtatgg cttatcaaaaagaacagtacatacccaacaataaagaggagctac aataataccaaccaagaagatottttggtactgtgggagattcac catectaatgatgcggcagagcagacaaagetctatcaaaaceca accacctatatttccgttgyggacatcaacactaaaccagagattg gtaccaagaatagctactagatccaaagtaaacgggcaaagtgga aggatagagttcttctggacaattttaaaaccgaatgatgcaate aacttcgagagtaatggaaatttcattgctccagaatatgcatac aaaattgtcaagaaaggggactcaacaattatgaaaagtgaattg gaatatggtaactgcaacaccaagtgtcaaactccaatgggggceg ataaactctagcatgccattccacaatatacacectctcaccatc ggggaatgecccaaatatgtgaaatcaaacagattagtocttgog actgggctcagaaatacccctcaaagagagagaagaggattattt ggagctatagcaggttttatagagggaggatggcagggaatggta gatggttggtatgggtaccaccatagcaacgagcagyggagtgag tacgctgcagacaaagaatccactcaaaaggcaatagatggagtc accaataaggtcaactcgattattgacaaaatgaacactcagttt gaggecgttggaagggaatttaacaacttagaaaggagaatagag aatttaaacaagaagatggaagacaggttcctagatgtctagact tataatgctgaacttctagttctcatggaaaatgagagaactcta gactttcatgactcaaatgtcaagaacctttacgacaaggtecga ctacagcttagggataatgcaaaggagctgggtaacggttgtttc gagttctatcataaatgtgataatgaatgtatggaaagtgtaaga aacggaacgtatgactacccgcagtattcagaagaagcaagacta Figure 31 Patent Application Publication May 13,2010 Sheet 38 of 151 US 2010/0120092 AI aaaagagaggaaataagtggagtaaaattggaatcaataggaatt taccaaatattgtcaatttattctacagtggegagctccctagea ctggcaatcatggtagctggtctatccttatggatgtgcteccTG A GGAGGCTAGATT GGTGGTGGATTTCTCTCAGTTCTCCAAAGGG: A AAAATGCAATGCGCTTTCCAAGATACTGGAGCCCAAATCTCTCCA (CATTACGTAGGAT CTT GCCCGTGGGGATGCCCAGGATTTCTITG! ACCTATCTCAGGCTTTTTATCATCITCCTCTTAATCCTGCTAGTA |ECAGCAGGCTTGCTGTATCTGACGGACAACGGGTCTACTATTTTA CTTCACTACTGCCCTCGGATCCGAAATCICTCCTCOCITTAAC TTGGACTTICACTIATATGGATGACTTCCTCCTCTGCCACCCAA, ACGCTCGTCACCT TAACGCAATTAGCCACGCTGTCTGCTCTTITI, ‘ACAAGAGTTAY Figure 31 continued Patent Application Publication May 13, 2010 Sheet 39 of 151 US 2010/0120092 A1 H5ecto-S pecan Poll HS HA DHBV S HSTMD-S Irmo] HE HA DHBV S Expression of H5 HA-S proteins in transfected 293T cells H6ecto-S +S HSTMD-S +S Green = antiH5 MAb 140 Figure 32 Patent Application Publication — May 13,2010 Sheet 40 of 151 US 2010/0120092 A1 Western blot - H5ecto-S + S gradient fractions A Fraction: 12 3 4 5 6 7 Pe + HAS Fraction: | 8 8 88a: 8 ig anti-S (7012) Figure 33 Patent Application Publication — May 13,2010 Sheet 41 of 151 US 2010/0120092 A1 ELISA - Gradient fractions (HSecto-S and HSTMD-S) 12345678 90032 Fraction # Soy eon aoa Fraction # Figure 34 Patent Application Publication —- May 13,2010 Sheet 42 of 151 US 2010/0120092 A1 Trypsin digestion HSTMD-S H6ecto-S Rec. +8 +S HS — anti-H5 (H5R3) Figure 35 Patent Application Publication —_ May 13,2010 Sheet 43 of 151 US 2010/0120092 A1 Co-sedimentation of HA-1 with VLPs after trypsin digestion H-containing VLPs +f typsin digestion, utraoentritugation F6 - oe protein 5 free protein ; HeocinS +9 HTD +S S+t om ae A SS SSeS bee : fer 3 HAS | 3 Westem blot 2 : od NE a Undigested - anti HS (HERS) B y +s 3s HoecOS+8 HSTMDS+S 94 es Trypsin digested - anti HO (HERS) Figure 36 Patent Application Publication — May 13,2010 Sheet 44 of 151 US 2010/0120092 A Rabbit anti-H5 HATMD-S HA-S aggregates. HA-VLPs Figure 37 Patent Application Publication May 13,2010 Sheet 45 of 151 US 2010/0120092 A HSTMD HA-VLP, 16 h sucrose gradient Patent Application Publication May 13, 2010 Sheet 46 of 151 US 2010/0120092 A1 HANLP (Fraction 8) +/- Trypsin are Fa etrypsh im Bank Figure 39 Patent Application Publication May 13,2010 Sheet 47 of 151 US 2010/0120092 AI Western: Rabbit anti-H5 Figure 40 Patent Application Publication May 13,2010 Sheet 48 of 151 US 2010/0120092 AI E1E2 E2.661 E1E2 E2.661 —a a Endo H N glycosidase F Western: Goat anti-E2 Figure 41 Patent Application Publication May 13,2010 Sheet 49 of 151 US 2010/0120092 AI Schematic of various HIV envelope (gp140, gp160) - S protein fusion proteins for VLP formation A & rons ‘optouns0 B & orion sp ttomener ptccost witoune ersouncicros aver vena Sins oesae Figure 42 Patent Application Publication May 13,2010 Sheet 50 of 151 US 2010/0120092 AI atgagagtgaaggagaagtatcagcact tgtggagatggggatag aaatggggcaccatgctecttyggatattgatgatctgtagtget acagaaaaattgtgggtcacagtctattatggggtacctgtgtag aaggaagcaaccaccactctattttgtgeatcagatgctaaagca tatgatacagaggtacataatgtttgggecacacatgectgtgta cccacagaccccaacccacaagaagtagtattggaaaatgtgaca gaaaattttaacatgtqgaaaaataacatggtagaacagatgcat gaggatataatcagtttatgggatcaaagectaaagecatgtgta aaattaaccccactctgtgttactttaaattgcactgatttgagg aatgttactaatatcaataatagtagtgagggaatgagaggagaa ataaaaaactgctctttcaatatcaccacaagcataagagataag gtgaagaaagactatgcacttttttatagacttgatgtagtacca atagataatgataatactagctataggttgataaattgtaatacc teaaccattacacaggectgtccaaaggtatectttgagccaatt cocatacattattgtaccecggetggttttgcgattctaaagtgt aaagataagaagttcaatygaacagggccatgtaaaaatgtcage acagtacaatgtacacatggaattaggccagtagigtcaactcaa ctgctgttaaatggcagtctagcagaagaagaggtagtaattaga totagtaatttcacagacaatgcaaaaaacataatagtacagttg aaagaatctgtagaaattaattgtacaagacccaacaacaataca aggaaaagtatacatataggaccaggaagageattttatacaaca ggagacataataggagatataagacaagcacattgcaacattagt agaacaaaatggaataacactttaaatcaaatagctacaaaatta aaagaacaatttgggaataataaaacaatagtctttaatcaatce tcaggaggggacccagaaattgtaatgcacagttttaattgtgga ggggaatttttctactgtaattcaacacaactgtttaatagtact tggaattttaatggtacttggaatttaacacaatcgaatggtact gaaggaaatgacactatcacactcccatgtagaataaaacaaatt ataaacatgtggcaagaagtaggaaaagcaatgtatgocectcce atcagaggacaaattagatgttcatcaaatattacagggctgata ttaacaagagatggtggaaataaccacaataatgataccgagacce tttagacctggaggaggagatatgagggacaattggagaagtgaa ttatataaatataaagtagtaaaaattgaaccattaggagtagca Figure 43 Patent Application Publication — May 13,2010 Sheet S1 of 151 US 2010/0120092 A1 cecaccaaggcaaagagaagagtggtgcagagagaalaccggt|sca gtgggaacaataggagctatgttecttgggttcttgggagcagca ggaagcactatgggcgcagcgtcaataacgctgacggtacaggce agactattattgtctggtatagtgcaacagcagaacaacttgctg agggctattgaggcgcaacagcatctgttgcaactcacagtctgg ggcatcaagcagct ccaggcaagagtcctggctgtggaaagatac ctaagggatcaacagetectagggatttggggttgctctggaaaa cteatctgcaccactgctgtgccttggaatgctagttggagtaat aaatctctggaacagatttggaataacatgacctggatggagtyg gacagagaaattaacaattacacaagcttaatacactccttaatt gaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattg gaatt agataaatgggcaagtttgtagaattggtttaacataaca aattggctgtggtatataaaaACGTCTGGTACCTICGGGGGAATA TAGCTGGCCTAATCGGATTACTGGTAAGCITITICTTGTIGATA GCCCAGGATTTCTTTGGACCTATCTCAGG TTATCATCTTC| CTCTTAATCCTGCTAGTAGCAGCAGGCTTGCTGTATCTGACGGAC| AACGGGTCTACTATTTTAGGAAAGCTCCAATGGGCGTCGGTCTCAl (CCCTITTCTCCTCCATCICTICACTACTGCCCTCGGATCCGAAA ‘CTCTCGTCGCTTTAACGTTTGGACTTTCACTTATATGGATGAC: CCTCCTCTGCCACCCAAACGCT CGTCACCTTAACGCAATTAGCC |ACGCTGTCTGCTCTTTTTTACAAGAGTTAG Figure 43 continued Patent Application Publication May 13,2010 Sheet 52 of 151 US 2010/0120092 AI atgagagt gaaggagaagtatcagcact tgtggagatgggggtag aaatggggcaccatgctccttgggatattgatgatetgtagtgct acagaaaaattgtgggtcacagtctattatggggtacctgtgtag aaggaagcaaccaccactctattttgtgcatcagatgctaaagea tatgatacagaggtacataatgtttgggecacacatgectgtgta cccacagaccccaacccacaagaagtagtattggaaaatgtgaca gaaaattttaacatgtggaaaaataacatggtagaacagatgcat gaggatataatcagtttatgggatcaaagectaaagecatgtgta aaattaaceccactctgtgttactttaaattgcactgatttgagg aatgttactaatatcaataatagtagtgagggaatgagaggagaa ataaaaaactgetctttcaatatcaccacaagcataagagataag gtgaagaaagactatgcacttttttatagacttgatgtagtacea atagataatgataatactagctataggttgataaattgtaatacce teaaccattacacaggectgtccaaaggtatcctttgagecaatt cccatacattattgtaccceggetggttttgcgattctaaagtgt aaagataagaagt tcaatggaacagggccatgtaaaaatgtcage acagtacaatgtacacatggaattaggccagtagtgtcaactcaa ctgctgttaaatggcagtctagcagaagaagaggtagtaattaga tctagtaatttcacagacaat gcaaaaaacataatagtacagttg aaagaatctgtagaaattaattgtacaagacccaacaacaataca aggaaaagtatacatataggaccaggaagagcattttatacaaca ggagacataataggagatataagacaagcacattgcaacattagt agaacaaaat ggaataacact ttaaatcaaatagctacaaaatta aaagaacaatttgggaataataaaacaatagtctttaatcaatco toaggaggggacecagaaattgtaatycacagttttaattgtgga ggggaatttttctactgtaattcaacacaactgtttaatagtact tggaattttaatggtacttggaatttaacacaatcgaatggtact gaaggaaatgacactatcacactcccatgtagaataaaacaaatt ataaacatgtggcaagaagtaggaaaagcaatgtatgccectece atcagaggacaaattagatgttcatcaaatattacagggctgata ttaacaagagatggtggaaataaccacaataatgataccgagace tttagacctggaggaggagatatgagugacaattggagaagtgaa ttatataaatataaagtagtaaaaattgaaccattaggagtagca Figure 44 Patent Application Publication May 13,2010 Sheet 53 of 151 US 2010/0120092 AI cccaccaaggcaaagagaagagtggtgcagagagaapaaagalyca gtgggaacaataggagctatgttccttgggttcttgggagcagca ggaagcactatgggcgcagegtcaataacgetgacggtacaggcee agactattattgtctggtatagtgcaacageagaacaacttgctg agggctattgaggcgcaacagcatctgttgcaactcacagtctgg ggcatcaagcagct ccaggcaagagtectggetgtggaaagatac ctaagggatcaacagctoctagggatttggggttgctctggaaaa etcatctgcaccactgctgtgecttggaatgctagttggagtaat aaatctctggaacagatttggaataacatgacctggatggagtgg gacagagaaattaacaattacacaagcttaatacactecttaatt gaagaat cgcaaaaccagcaagaaaagaatgaacaagaattattg gaattagataaatgggcaagtttgtggaattggtttaacataaca aattggctgtggtatataaaaACGPCTGCTACCTTCCGGGGAATAl {TAGCTGGCCTAATCGGATTACTGGTAAGCTTTTTCTTGITGATA AAAATTCTAGAAATACTGAGGAGGCTAGATTGGTGGTGGATTTCT TCAGTTCTCCAAAGGGAAAAATGCAATGCGCITTCCAAGATACT! eco AACGGGTCTACTATITTAGGAAAGCTCCAATGGGCCTCGGICTCA CCCTTTICTCCTCCATCTCTTCACTACTGCCCTCGGATCCGAAA] PCICTCGTCGCTTTAACGTTTGGACTTTCACTTATATGGATGACT| ‘CCTCCTCTGCCACCCAAACGCTCGTCACCTTAACGCAATTAGCKC| .CGCTGTCTGCTCTTTTTTACAAGAGTTAG Figure 44 continued Patent Application Publication May 13, 2010 Sheet 54 of 151 US 2010/0120092 A1 Mock pCLs Green = anti-HIV MAb 2612 or 7012 Red = nuclear stain Figure 45 Patent Application Publication —- May 13,2010 Sheet 55 of 151 US 2010/0120092 A1 A Fraction# 1 234667 8 9101112 gpt40unc-Ds Patent Application Publication Deco May 13,2010 Sheet 56 of 151 US 2010/0120092 A1 gpaaceps +S prorie 3 Siese seo) UH, gp140-VLPs Figure 47 Patent Application Publication May 13,2010 Sheet 57 of 151 US 2010/0120092 AI A gpl4duneDS +8 VLP profile cd Je 712 s e202 a0 ,3 i to fo) i 44 u s 4 | oo ae jo oeo123 45 6789 uD | { Fran = ‘gpl40uneDSTML +5 VLP profile | | Figure 48 Patent Application Publication May 13,2010 Sheet 58 of 151 US 2010/0120092 AI ‘gp140cDS+8 VEP profile ODeae-siom Coe. ce. 8 Figure 48 continued Patent Application Publication May 13,2010 Sheet 59 of 151 US 2010/0120092 A1 gpl6uncdCTDS +S VLP profile »E a \s eat x0 an Ne B is! | 1 | ,” cv — as 4 ota303 6735 8 0 2 Past ‘gplG@edCTDS +8 VLP profile | Hi | i | d 4 Proetin Figure 48 continued May 13, 2010 Sheet 60 of 151 US 2010/0120092 A1 140 cleaved VLP construct Fraction # 1234 56789 Figure 49 US 2010/0120092 A1 S 2 Fe a g ¢ osz ovz oce oz otz < [vl Ta@1ZI4NQ 40 NOILWISNWaE N93 YU Add A094 LH TT¥ A LIDIOLLIIOINWOOIWOYODWIDOWIIOIOOOILOLOI"OOWLOIIWLO WWOODWWIDOWILIIILIOIOLIOILIVIODIDIIVOWIILOITYOOOLYD ooz o6t ost Out 091 < {W] IGHILIEND 40 NOLLWISNWL “Ww YdA ATS SNGOGNLAHS D9OOPOOLLOVINIWOWWISOLVOWWLLLOOOOWDLOWLIWDLOWOWLOOL ODDO WYO LWLWLOLLVOOWLOLLWWWOOIDLIWLEWLOWOLOLWOOY ost Ort oct ot Ott < (¥] TGS TLIZNA dO NOLLWISNWUL os Se Oe A wo eA WWWOSIOWDYVOLVLLIIOLVOLLOWLWODILO¥ISINIVOWOOILIVOV LLLODDILILIOWINYOOOVLOWWOLVLIVOVO.LINNIIILIOOWDLOL oT 06 og oL 09 < [v] IGSILIZNA 0 NOILWISNWEL

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