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(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

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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

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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

(7)

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

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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

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Leu Ser Val Lys Ile Arg Glu Cys Thr Gly Leu Ala

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Trp Glu Trp Trp Arg Thr Val Tyr Gl-u Lys Thr Asp

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Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile Trp

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Gly Thr Thr Leu Tyr Pro Gln Val Glu Asp Lys Val

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Glu Asn Asp

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Lys Lys Arg Ser Leu Ser Thr Asn 'I'hr Ser Asp Ile

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Xaa Val Xaa Ala Thr Xaa Asp Ser Arg Leu Tyr Pro

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Gly Ala Leu Leu Val Val Asp Glu Thr Xaa Leu Glu

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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

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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

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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

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Arg Val Val Thr Gly Lys Val Asp Met Val Glu Asp

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Arg Asp Asn Val Val Ala Thr Phe Gln Asn Ser Thr

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Asp Tyr Val Glu Thr Lys Val Thr Ala 'I‘yr Arg Asn

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Gly Asp Leu Leu Leu Asp His Ser Gly Ala ‘I‘yr Val

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Ala Gln Tyr Tyr Ile Thr Trp Xaa Glu Leu Ser Tyr

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Asp His Gln Gly Lys Glu Val Leu Thr Pro Lys Ala

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Trp Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe

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Thr Thr Ser Ile Pro Leu Lys Gly Asn Val Arg Asn

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Leu Ser Val Lys Ile Arg Glu Cys Thr Gly Leu Ala

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435 440

Leu Xaa Leu Val Arg Lys Arg Thr Ile Ser Ile Trp

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Gly Thr Thr Leu 'I‘yr Pro- Gln Val Glu Asp Lys Val

460 465

Glu Asn Asp

470

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Patent Application Publication Aug. 16, 2001 Sheet 12 0f 23 US 2001/0014332 A1

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Patent Application Publication Aug. 16, 2001 Sheet 18 0f 23 US 2001/0014332 A1

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