(21) Appl. No.: 09/120,044
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1 US A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2001/ A1 MINETTI et al. (43) Pub. Date: Aug. 16, 2001 (54) MODIFIED IMMUNOGENIC PNEUMOLYSIN COMPOSITIONS AS VACCINES (75) Inventors: CONCEICAO MINETTI, SILVER SPRING, MD (US); FRANCIS MICHON, BETHESDA, MD (US); JEFFREY K. PULLEN, COLUMBIA, MD (US); MARYELLEN POLVINO-BODNAR, ANNAPOLIS, MD (US); SHU-MEI LIANG, NANKANG (TW); JOSEPH Y. TAI, COLLEGEVILLE, PA (US) Correspondence Address: MORGAN & FINNEGAN 345 PARK AVENUE NEW YORK, NY (73) Assignee: NORTH AMERICAN VACCINE, INC. (*) Notice: This is a publication of a continued pros ecution application (CPA)?led under 37 cfr 1.53(d). (21) Appl. No.: 09/120,044 (22) Filed: Jul. 21, 1998 Related US. Application Data (63) Non-provisional of provisional application No. 60/053,306,?led on Jul. 21, 1997, Which is a non provisional of provisional application No. 60/073, 456,?led on Feb. 2, Publication Classi?cation (51) Int. Cl?..... A61K 39/02, A61K 39/00 (52) US. Cl /190.1,424/192.1 (57) ABSTRACT This invention relates to modi?ed pneumolysin polypeptides that retain the immunogenic nature of pneumolysin but have reduced or undetectable hemolytic activity compared to native pneumolysin. The invention also provides a method for generating novel pneumolysin variants With these desired characteristic properties. The invention also pro vides immunogenic compositions useful as pharmaceutical compositions including vaccines in Which non-toxic, modi?ed pneumolysin is used to stimulate protective immunity against Streptococcus pneumoniae. The vaccines may be compositions in Which the modi?ed pneumolysin is conju gated to bacterial polysaccharides or may be carried on an attenuated viral vector. In addition, the invention also pro vides a method of using the non-toxic, modi?ed pneumol ysin toxoid in order to stimulate antibodies against Strepto coccus pneumoniae in a treated individual Which are then isolated and transferred to a second individual, thereby conferring protection against Streptococcus pneumoniae in the second individual.
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6 Patent Application Publication Aug. 16, 2001 Sheet 5 0f 23 US 2001/ A1 Met: Ala Asn Lys Ala Val Asn Asp Phe Ile Leu Ala l 5 l0 Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln 15 2O Gly Glu Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu Pro Asp Glu Phe Val Val Ile Glu Arg Lys Lys Arg Ser Leu Ser Thr Asn Thr Ser Asp Ile 5O Ser Val Thr Ala Thr Asn Asp Ser Arg Leu Tyr Pro 65 7O Gly Ala Leu Leu Val Val Asp Glu Thr Leu Leu Glu '75 8O Asn Asn Pro Thr Leu Leu Ala Val Asp Arg Ala Pro 85 9O ' 95 Met Thr Tyr Ser Ile Asp Leu Pro Gly Le_u Ala Ser ' S_er Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn Ser Ser Val Arg Gly Ala Val Asn Asp Leu Leu Ala Lys 'I'rp His Gln Asp Tyr Gly Gln Val Asn Asn Val Pro Ala Arg Met Gln 'I yr Glu Lys Ile Thr Ala His Ser Met Glu Gln Leu Lys Val Lys Phe Gl-y Ser Asp Phe Glu Lys Thr Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val His Ser Gly Glu Lys Gln Ile Gln Ile Va-l Asn Phe Lys Gln Ile Tyr Tyr Thr Val Ser Val Asp Ala Val Lys Asn Pro Gly Asp Val Phe Gln Asp Thr Val Thr Val Glu Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro Leu Val Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val 'I yr Leu Lys Leu Glu Thr Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe Glu Ala Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys Gln Ile Leu Asp Asn Thr Glu Val Lys FIG. 3
7 Patent Application Publication Aug. 16, 2001 Sheet 6 0f 23 US 2001/ A1 Ala Val Ile Leu Gly Gly Asp Pro Ser Ser Gly Ala Arg Val Val Thr Gly Lys Val Asp Met Val Glu Asp Leu Ile Gln Glu Gly Ser Arg Phe Thr Ala Asp His Pro G ly Leu Pro Ile Ser Tyr Thr Thr Ser Phe Leu 325 ' Arg Asp Asn Val Val Ala Thr Phe Gln Asn Ser Thr Asp 'I'yr Val Glu Thr Lys Val Thr Ala 'I yr Arg Asn Gly Asp Leu Leu Leu Asp His Ser Gly Ala Tyr Val ' 365 ' 370 ' Ala Gln Tyr Tyr Ile Thr Trp Asn Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu Thr Pro Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr Thr Ser Ile Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val Lys Ile Arg Glu Cys Thr Gly Leu Ala 425, 430 Trp Glu Trp Trp Arg Thr Val Tyr Gl-u Lys Thr Asp 435 ' 440 Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile Trp 445 ' Gly Thr Thr Leu Tyr Pro Gln Val Glu Asp Lys Val 460 ' 465 Glu Asn Asp 470 FIG. 3
8 Patent Application Publication Aug. 16, 2001 Sheet 7 0f 23 US 2001/ A1 Met: Ala Asn Lys Ala Val Asn Asp Phe Ile Leu Ala 1 5 l0 Met Asn 'I yr Asp Xaa Xaa Lys Leu Leu Thr His Gln 15 2O Gly Glu Ser Ile Glu Asn Arg Phe Xaa Lys Glu Gly Asn Gln Leu Pro Xaa Glu Phe Val Xaa Xaa Glu Arg Lys Lys Arg Ser Leu Ser Thr Asn 'I'hr Ser Asp Ile 5O Xaa Val Xaa Ala Thr Xaa Asp Ser Arg Leu Tyr Pro Gly Ala Leu Leu Val Val Asp Glu Thr Xaa Leu Glu 75 8O Asn Asn Pro Thr Leu Leu Ala Val Asp Arg Ala Pro 85 9O 95 Met: Thr Tyr Ser Xaa Xaa Leu Pro Gly Leu Ala Ser Ser Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn Ser Ser Val Arg Gly Ala Xaa Xaa Asp Leu Leu Ala Lys Trp His Gln Asp Tyr Gly Gln Val Asn Asn Val Pro Ala Arg Xaa Gln Tyr Glu Lys Xaa Thr Ala His Ser Met: Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe Glu Lys Xaa Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val His Ser Gly Glu Lys Xaa Ile Gln Ile Val Asn Xaa Lys Gln Ile I yr Tyr Thr Val Ser Val Asp Ala Val Lys Asn Pro Gly Asp Val Phe Gln Asp 'I'hr Val Thr Val Glu Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro Leu Val Tyr Ile Ser Xaa Val Ala I'yr Xaa Arg Gln Val 'I yr Leu Lys Leu Glu Thr Thr Ser Xaa Ser Xaa Glu Val Glu Ala Ala Phe Glu Ala Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys Gln Ile Leu Asp Asn Thr Xaa Val Lys FIG. 4
9 Patent Application Publication Aug. 16, 2001 Sheet 8 0f 23 US 2001/ A1 Ala Val Ile Leu Gly Gly Asp Pro Ser Ser Gly Ala Arg Val Val Thr Gly Lys Val Asp Met Val Glu Asp Leu Ile Gln Glu Gly Ser Arg Phe Thr Ala Asp His Pro Gly Leu Pro Ile Ser Tyr Thr Thr Ser Phe Leu Arg Asp Asn Val Val Ala Thr Phe Gln Asn Ser Thr Asp Tyr Val Glu Thr Lys Val Thr Ala 'I yr Arg Asn Gly Asp Leu Leu Leu Asp His Ser Gly Ala I yr Val Ala Gln Tyr Tyr Ile Thr Trp Xaa Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu Thr Pro Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr Thr Ser Ile Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val Lys Ile Arg Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys Thr Asp Leu Xaa Leu Val Arg Lys Arg Thr Ile Ser Ile Trp Gly Thr Thr Leu 'I yr Pro- Gln Val Glu Asp Lys Val Glu Asn Asp 470 FIG. 4
10 Patent Application Publication Aug. 16, 2001 Sheet 9 0f 23 US 2001/ A1 17 termlnaior Xhol Nde1 lao operate! T7 promoter
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12 Patent Application Publication Aug. 16, 2001 Sheet 11 0f 23 US 2001/ A1 ': vletzior-.7.) ani- l: Max! Mend-and: Ger-3.1a- IF'H: induclnj-n 1 In all? undurh-an Lanz- d: 5: hr: t: hr: anal induchc-n
13 Patent Application Publication Aug. 16, 2001 Sheet 12 0f 23 W6. 3 ELISA lgg Ti H co llllllll Sb Paco I1Illlllr I llllllll co I I lllllll G r Hashes I US 2001/ A1 : m? %; 9% 1=2=s8 _
14 Patent Application Publication Aug. 16, 2001 Sheet 13 0f 23 US 2001/ A1 swig Eggs: 5 my ueuawozisari W a % E 25> mu 3 MG wa>._. wtimsuummzom Eoxoh mscmpmh Em>_oE:mcn_ mm m6 I - m1 - Eoxoh mscmpwh Em>_oE:wcn_ mam: mz O.N I J m a :8 3.1 H... _ l 83:.M D.H 83 t 6-83 l.n 8m - W V - S H 2: m m 3 on
15 Patent Application Publication Aug. 16, 2001 Sheet 14 0f 23 US 2001/ A1 1 _ G. I 182x855 w EBQEE 222%: as EVE UEUQQWEEQEZL aesas 22% SE 5 saga-=8 E22:= =?_-@ 2s~;_on_ V mu, 83 33H mm ma 3 MG wn>._. wwtmcuummzom mm O.N m6. m: m1 C!.0. coo 3.m ooodcq? m m J H a M" ace 2: 1 \.._ W s. 3
16 Patent Application Publication Aug. 16, 2001 Sheet 15 0f 23 US 2001/ A1 _ 8o=_=@=?_ 2222:: 3 SEE 5E2 ébowsaécao écaw?v QEESZE % 328.; :58.: Ea $28.55.: w t wbgmsuummzom 8. 3 m5 m9» :93 swaoegcm s Pr 8o H 4 m... 8o 83: In a m.. +. A 1, 8o m m t u -.0 8m. w i.1 o 2: 0 on w
17 Patent Application Publication Aug. 16, 2001 Sheet 16 0f 23 US 2001/ A1 w 222%: BEE 5E uizsééé 22:25 23F :3 E5352; 2222:: m»,..ai com o G - a H 2; V Bang 513$ :3; $26 ma>._. mntmsuummzom mm O.N m6 I m: m1 w I. oou 25 m a i L n... _ H. D a - - H 3 I u
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19 Patent Application Publication Aug. 16, 2001 Sheet 18 0f 23 US 2001/ A \Mld Type PLY O~- PLYD MutantA -v PLYD Mutant B i Z-é E T, 40 - C m 2 Q) a. 20 _ 0-1 1' Inhibitor Concentration (nglwell) <15 m
20 Patent Application Publication Aug. 16, 2001 Sheet 19 0f 23 US 2001/ A (nm) Wavelength HG hisuaaul aouaosamnu GAQBIGH
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