(19) United States
US 20010014332A1
(12) Patent Application Publication (10) Pub. No.: US 2001/0014332 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 10154
(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, 1998.
Publication Classi?cation
(51) Int. Cl? ... .. A61K 39/02, A61K 39/00
(52) US. Cl. .... .. 424/190.1,424/192.1
(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
40 45
Lys Lys Arg Ser Leu Ser Thr Asn Thr Ser Asp Ile
5O 55 60
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
' 100 105
S_er Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn
110 115 120
Ser Ser Val Arg Gly Ala Val Asn Asp Leu Leu Ala
125 130
Lys 'I'rp His Gln Asp Tyr Gly Gln Val Asn Asn Val
135 140
Pro Ala Arg Met Gln 'I‘yr Glu Lys Ile Thr Ala His
145 150 155
Ser Met Glu Gln Leu Lys Val Lys Phe Gl-y Ser Asp
160 165
Phe Glu Lys Thr Gly Asn Ser Leu Asp Ile Asp Phe
170 175 180
Asn Ser Val His Ser Gly Glu Lys Gln Ile Gln Ile
185 190
Va-l Asn Phe Lys Gln Ile Tyr Tyr Thr Val Ser Val
195 200
Asp Ala Val Lys Asn Pro Gly Asp Val Phe Gln Asp
205 210- 215
Thr Val Thr Val Glu Asp Leu Lys Gln Arg Gly Ile
220 225
Ser Ala Glu Arg Pro Leu Val Tyr Ile Ser Ser Val
23 235 240
Ala Tyr Gly Arg Gln Val 'I‘yr Leu Lys Leu Glu Thr
245 250
Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe Glu
255 260
Ala Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr
265 270 275
Glu Trp Lys Gln Ile Leu Asp Asn Thr Glu Val Lys
280 285
Patent Application Publication Aug. 16, 2001 Sheet 6 0f 23 US 2001/0014332 A1
Ala Val Ile Leu Gly Gly Asp Pro Ser Ser Gly Ala
290 295 300
Arg Val Val Thr Gly Lys Val Asp Met Val Glu Asp
305 310
Leu Ile Gln Glu Gly Ser Arg Phe Thr Ala Asp His
315 320
Pro G‘ly Leu Pro Ile Ser Tyr Thr Thr Ser Phe Leu
325 ' 330 335
Arg Asp Asn Val Val Ala Thr Phe Gln Asn Ser Thr
340 345 ‘
Asp 'I'yr Val Glu Thr Lys Val Thr Ala 'I‘yr Arg Asn
‘ 350 355 ‘ 360
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
375 380
Asp His Gln Gly Lys Glu Val Leu Thr Pro Lys Ala
385 390 395
Trp Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe
‘ 400 405 ‘
Thr Thr Ser Ile Pro Leu Lys Gly Asn Val Arg Asn
410 415 420
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 ' 450 455
Gly Thr Thr Leu Tyr Pro Gln Val Glu Asp Lys Val
460 ' 465
Glu Asn Asp
470
40 45
Lys Lys Arg Ser Leu Ser Thr Asn 'I'hr Ser Asp Ile
5O 55 60
Xaa Val Xaa Ala Thr Xaa Asp Ser Arg Leu Tyr Pro
65 70
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
100 105
Ser Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn
110 115 120
Ser Ser Val Arg Gly Ala Xaa Xaa Asp Leu Leu Ala
125 130
Lys Trp His Gln Asp Tyr Gly Gln Val Asn Asn Val
135 140
Pro Ala Arg Xaa Gln Tyr Glu Lys Xaa Thr Ala His
145 150 155
Ser Met: Glu Gln Leu Lys Val Lys Phe Gly Ser Asp
160 165
Phe Glu Lys Xaa Gly Asn Ser Leu Asp Ile Asp Phe
170 175 180
Asn Ser Val His Ser Gly Glu Lys Xaa Ile Gln Ile
185 190
Val Asn Xaa Lys Gln Ile ‘I‘yr Tyr Thr Val Ser Val
195 200
Asp Ala Val Lys Asn Pro Gly Asp Val Phe Gln Asp
205 210‘ 215
'I'hr Val Thr Val Glu Asp Leu Lys Gln Arg Gly Ile
220 225
Ser Ala Glu Arg Pro Leu Val Tyr Ile Ser Xaa Val
230 235 240
Ala ‘I'yr Xaa Arg Gln Val 'I‘yr Leu Lys Leu Glu Thr
245 250
Thr Ser Xaa Ser Xaa Glu Val Glu Ala Ala Phe Glu
255 260
Ala Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr
265 270 275
Glu Trp Lys Gln Ile Leu Asp Asn Thr Xaa Val Lys
280 285
Patent Application Publication Aug. 16, 2001 Sheet 8 0f 23 US 2001/0014332 A1
Ala Val Ile Leu Gly Gly Asp Pro Ser Ser Gly Ala
290 295 300
Arg Val Val Thr Gly Lys Val Asp Met Val Glu Asp
305 310
Leu Ile Gln Glu Gly Ser Arg Phe Thr Ala Asp His
315 320
Pro Gly Leu Pro Ile Ser Tyr Thr Thr Ser Phe Leu
325 330 335
Arg Asp Asn Val Val Ala Thr Phe Gln Asn Ser Thr
340 345
Asp Tyr Val Glu Thr Lys Val Thr Ala 'I‘yr Arg Asn
350 355 360
Gly Asp Leu Leu Leu Asp His Ser Gly Ala ‘I‘yr Val
365 370
Ala Gln Tyr Tyr Ile Thr Trp Xaa Glu Leu Ser Tyr
375 380
Asp His Gln Gly Lys Glu Val Leu Thr Pro Lys Ala
385 390 395
Trp Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe
400 405
Thr Thr Ser Ile Pro Leu Lys Gly Asn Val Arg Asn
410 415 420
Leu Ser Val Lys Ile Arg Glu Cys Thr Gly Leu Ala
425 430
Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys Thr Asp
435 440
Leu Xaa Leu Val Arg Lys Arg Thr Ile Ser Ile Trp
445 450 455
Gly Thr Thr Leu 'I‘yr Pro- Gln Val Glu Asp Lys Val
460 465
Glu Asn Asp
470
Nde1
lao operate!’
': vletzior- .7.)
ani- ‘l: Max! Mend-and:
Ger-3.1a- IF'H: induclnj-n
1 In all?’ undurh-an
Lanz- d: 5: hr:
Patent Application Publication Aug. 16, 2001 Sheet 12 0f 23 US 2001/0014332 A1
W6. 3
ELISA lgG Ti
9253“
5
:5
m?
r
G
5223250 9% 58812 1=2=s8§_ 3.252%;
Hashes
Sb
Paco
H I I I lllllllco
I1Illlllr I llllllllco
llllllllEoxoh mscmpwh I ma mz
O.N Em>_oE:wcn_ - mam: O.N
Eoxoh mscmpmh - mm m1
m6 Em>_oE:mcn_ - mm m1
m6 I
wa>._. wtimsuummzom
mu
3
3
MG
m
3
on
‘m
2:
H
- S- 8m
W V.n
83
l
- t 6.H
D
83
.M
83:
l
.1 H... _m
a
:8
3
5863 J
W358
232 2.5a 25> 220255 “2232
22% 32 E
Patent Application Publication Aug. 16, 2001 Sheet 14 0f 23 US 2001/0014332 A1
mm
O.N
m:
mm
m6.
m1
wn>._. wwtmcuummzom
33H
mm
ma
3
MG
3
W s .mu,
83
V
G.
_ I
C!‘
.0.”
coo
3
.m
\
.._
1“
ace
2:
M"
H a m Jm ooodcq?
1
aesas 22% SE 5 saga-=8 E22:=¢=?_-@€2s~;_on_
293 8:93 swaoegcm
m9»... wBgmsuummzom
8
8.
3
m5
on
0
w
.1 2:
o
i
w
.
8m
.0
w
t
w
Pr
8o
H
s
t m1,
8o
m
A
m ..|+.In
83:
a
- u..
8o
8
m.
4
2225 2% 32 E
328.; £35
238 2:58.: Ea $28.55.:
_§8o=_=@=?_ 2222:: 3
SEE 5E2 ébowsaécao écaw?v QEESZE
Patent Application Publication Aug. 16, 2001 Sheet 16 0f 23 US 2001/0014332 A1
mm
O.N
mm
m6
I
m:
m1
ma>._. mntmsuummzom
Bang 513$ 15-8 15.2 1:3; $26
V
I u3
H
- -a
H
2;
a
I. oou
w
G - om»
,..AI
com
83
H.
D
83
|_
L i n...25
m
a
22:25 23F 82 E
333:3 E5352; 2222::
Patent Application Publication Aug. 16, 2001 Sheet 18 0f 23 US 2001/0014332 A1
100
-0— \Mld Type PLY
30 - --O~- PLYD MutantA