AU764811B2 - A polypeptide comprising the amino acid of an n-terminal choline binding protein A truncate, vaccine derived therefrom and uses thereof - Google Patents

Description

WO 99/51188 PCT/US99/07669 A POLYPEPTIDE COMPRISING THE AMINO ACID OF AN N-TERMINAL CHOLINE BINDING PROTEIN A TRUNCATE, VACCINE DERIVED THEREFROM AND USES THEREOF FIELD OF THE INVENTION The present invention relates generally to a polypeptide of a N-terminal choline binding protein A truncate. The invention also relates to vaccines which provide protection or elicit protective antibodies to bacterial infection, specifically pneumococcus, and to antibodies and antagonists against such polypeptide for use in diagnosis and passive immune therapy. The polypeptide and/or the nucleic acid encoding the polypeptide are also useful as a competitive inhibitor of bacterial adhesin of pneumococcus. Lastly, this invention is directed to therapeutics using the polypeptide.

BACKGROUND OF THE INVENTION Streptococcus pneumoniae is a gram positive bacteria which is a major cause of invasive infections such as sepsis, meningitis, otitis media and lobar pneumonia (Tuomanen et al NEJM 322:1280-1284, 1995). Pneumococci bind avidly to cells of the upper and lower respiratory tract. Like most bacteria, adherence of pneumococci to human cells is achieved by presentation of bacterial surface proteins that bind to eukaryotic carbohydrates in a lectin-like fashion (Cundell, D. Tuomanen, E. (1994) Microb Pathog 17:361-374). Pneumococci bind to non-inflamed epithelium, a process that can be viewed as asymptomatic carriage. It has been proposed that the conversion to invasive disease involves the local generation of inflammatory factors which, activating the human cell, change the number and type of receptors available on the human cells (Cundell, D. et al. (1995) Nature, 377:435-438). Presented with an opportunity in this new setting, pneumococci appear to take advantage and engage one of these unregulated receptors, the platelet activating factor (PAF) receptor (Cundell et al. (1995) Nature, 377:435-438. Within minutes of the appearance of the PAF receptor, pneumococci undergo waves of enhanced adherence and invasion. Inhibition of bacterial binding to activated cells, for instance by soluble receptor analogs, blocks the progression to disease WO 99/51188 PCT/US99/07669 -2in animal models (Idanpaan-Heikkila, I. et al. (1997) J. Infect. Dis., 176:704-712).

Particularly effective in this regard are soluble carbohydrates containing lacto-Nneotetraose with or without an additional sialic acid which prevent pneumococcal attachment to human cells in vitro and prevent colonization in the lung in vivo.

Choline binding proteins: candidate structural adhesin gene: Pneumococci produce a family of surface proteins capable of binding to the bacterial surface by non-covalent association to the cell wall teichoic acid or lipoteichoic acid.

The surface of Streptococcus pneumoniae is decorated with a family of CBPs (Choline Binding Proteins) that are non-covalently bound to the phosphorylcholine. CbpA, is an kD surface-exposed choline binding protein that shows a chimeric architecture. There is a unique N-terminal domain a proline rich region followed by a C-terminal domain comprised of 10 repeated region responsible for binding to choline.

CbpA, is an adhesin (ligand) for the glycoconjugate containing receptors present on the surface of eucaryotic cells. Mutants with defects in cbpA showed reduced virulence in the infant rat model for nasopharyngeal colonization. This binding is directed to choline determinants which decorate the teichoic acid and is mediated by a signature choline binding domain in each of the members of this family of proteins. The choline binding domain was discovered and fully characterized by Lopez et al. in his studies of the autolytic enzyme (Ronda et al. (1987) Eur. J. Biochem, 164:621-624). Other proteins containing this domain include the autolysin of the pneumococcal phage and the protective antigen, pneumococcal surface protein A (PspA) (Ronda, C. et al. (1987) Eur.

J. Biochem., 164:621-624 and McDaniel, et al. (1992) Microb Pathog, 13:261-269).

CbpA, fails to colonize the nasopharynx domain which is shared with its other family members C terminus) but its activity of binding to human cells arises from its unique Nterminal domain. Since the process of colonization and the progression to disease depend on pneumococcal attachment to human cells as a primary step, interruption of the function of the N terminal domain, either by cross reactive antibody or by competitive inhibition with a peptide mimicking this domain, may be critical to blocking disease.

WO 99/51188 PCT/US99/07669 -3- Choline binding proteins for anti-pneumococcal vaccines are discussed in PCT International Application No. PCT/US97/07198 and such PCT Application is incorporated in its entirety by reference. Current vaccines against S. pneumoniae employ purified carbohydrates of the capsules of the 23 most common serotypes of this bacterium, but such vaccine is only 50% protective (Shapiro et al. NJEM 325:1453, 1991) and is not immunogenic under the age of 2. Further, a therapeutic polypeptide would offer a therapeutic option in cases of infection with multi resistant organisms.

Therefore, the invention herein fills a long felt need by providing a protective vaccines.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in Australia as at the priority date of any of the claims.

SUMMARY OF THE INVENTION The present invention provides an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate. The polypeptide comprises the amino acid sequence as set forth in SEQ ID NOS 1, 3- 7, or 9-11, including fragments, mutants, variants, analogs, or derivatives, thereof. Also, this invention provides an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate having the amino acid as set forth in SEQ ID NO 24, wherein the polypeptide exhibits its tertiary structure and methods of preparation such a polypeptide. The isolated polypeptide are suitable for use in immunizing animals and humans against bacterial infection, preferably pneumococci.

In a still further aspect, the present invention extends to an N-terminal choline binding protein A truncate having lectin activity and no choline binding activity. Still further, this invention provides an immunogenic N-terminal choline binding protein A truncate or a fragment thereof.

The present invention also relates to isolated nucleic acids, such as recombinant

DNA

molecules or cloned genes, or degenerate variants thereof, mutants, analogs, or fragments thereof, which encode the isolated polypeptide or which competitively inhibit the activity of the polypeptide. Preferably, the isolated nucleic acids which includes degenerates, variants, mutants, analogs, or fragments thereof, has a sequence as set forth in SEQ ID WO 99/51188 PCT/US99/07669 -4- NOS: 12, 14-17, 19-22 or 23. In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present invention, and more particularly, the DNA sequences or fragments thereof determined from the sequences set forth above.

Antibodies against the isolated polypeptide include naturally raised and recombinantly prepared antibodies. These may include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for diagnostic use conjunctive with their capability of modulating bacterial adherence including but not limited to acting as competitive agents.

It is still a further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the bacteria or its subunits, so as to treat or avert the adverse consequences of invasive, spontaneous, or idiopathic pathological states. This invention provides pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the isolated polypeptides, their subunits or their binding partners.

Lastly, this invention provides pharmaceutical compositions, vaccines, and diagnostic and therapeutic methods of use thereof.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1. Schematic representation of choline binding protein A (CbpA) and recombinant truncates R1 (from about amino acid 16 to amino acid 321 from the N-terminus of CbpA as set forth in Figure 2) and R2 (from about amino acid 16 to amino acid 444 from the N-terminus of CbpA as WO 99/51188 PCT/US99/07669 set forth in Figure Domain A is from about amino acid 153 to amino acid 321 from the N-terminus of CbpA amino acid sequence as set forth in Figure 2 domain B is from about amino acid 270 to amino acid 326 from the N-terminus of CbpA amino acid sequence as set forth in Figure and C is from about amino acid 327 to amino acid 433 from the Nterminus of CbpA amino acid sequence as set forth in Figure 2.

FIGURES 2A-B Comparison of homologies of various serotypes of the nucleic acid and amino acid sequence of the N-terminal region of CbpA.

FIGURE 3.

FIGURE 4.

FIGURE 5 Expression and purification of recombinant R1 and R2.

Results of passive protection in mice. Immune sera against recombinant R2 protected mice from lethal S. pneumoniae challenge.

Titration of anti-R2 antibody on R6x adhering to LNnT-HSA coated plates.

Titration of anti-Cbp-A and absorbed anti-CbpA antibodies for activity blocking pneumococcal adherence to LNnT-HSA coated plates.

Results of active protection in mice. Immune sera against recombinant R1 protected mice from lethal S. pneumoniae challenge (challenge 560 cfu serotype 6B).

DETAILED DESCRIPTION FIGURE 6.

FIGURE 7.

The present invention is directed to an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate. The polypeptides are suitable for use in immunizing animals against pneumococcal infection. These WO 99/51188 PCT/US99/07669 -6polypeptide or peptide fragments thereof, when formulated with an appropriate adjuvant, are used in vaccines for protection against pneumococci, and against other bacteria with cross-reactive proteins.

This invention provides an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate. In one embodiment the polypeptide has the amino acid sequence as set forth in any of SEQ ID NO 1, 3-5, 7, or 9-11 including fragments, mutants, variants, analogs, or derivatives, thereof. In another embodiment the polypeptide has the amino acid KXXE (SEQ ID NO 6).

This invention provides an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate as set forth in Figure 2. In one embodiment, the polypeptide has an amino acid sequence which is a conserved region as set forth in Figure 2. For example, conserved regions include but are not limited to amino acid sequence 158 to 210; 158 to 172; 300 to 321; 331 to 339; 355 to 365; 367 to 374; 379 to 389; 409 to 427; and 430 to 447. Figure 2 sets forth homologies of various serotypes of the nucleic acid and amino acid sequence of the N-terminal region of CbpA which are contemplated by this invention.

Further, this invention provides an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate having the amino acid as set forth in SEQ ID NO 24, wherein the polypeptide exhibits its tertiary structure. In one embodiment the polypeptide is an analog, fragment, mutant, or variant thereof. Variants contemplated are set forth in Figure 2. This invention also provides an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate having the amino acid from about position 16 to about position 475 of serotype 4 as set forth in Figure 2 or a corresponding amino acid of serotype 4 as shown in Figure 2 wherein the polypeptide exhibits its tertiary structure. In one embodiment tertiary structure corresponds to that present in the native protein.

WO 99/51188 PCT1US99/07669 -7- Methods of preparation of the polypeptide are for example as follows: cleaving a full length choline binding protein A with hydroxylamine, wherein the hydroxylamine cleaves the choline binding protein A at amino acid Asparagine at position 475 of serotype R6x and serotype 4, or the corresponding amino acid of serotype R6x or serotype 4 in a different serotype as shown in Figure 2, thereby creating the N-terminal choline binding protein A truncate. Alternative methods which create a truncated choline binding protein A or fragment thereof, and retain the native tertiary structure that of the full length choline binding protein A) are contemplated and known to those skilled in the art. Because the polypeptide retains its tertiary structure, the isolated polypeptide is suitable for use as an immunogen in immunizing animals and humans against bacterial infection, preferably pneumococci.

The polypeptide comprising the amino acid sequence of choline binding protein A (CbpA) serotype type 4 is as follows:

ENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESY

AKSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAV

SKFEKDSSSSSSSDSSTKPEASDTAKPNKPTEPGEKVAEAKKKVEEAEKKAKD

QKEEDRRNYPTITYKTLELEIAESDVEVKKAELELVKVKANEPRDEQKIKQAE

AEVESKQAEATRLKKIKTDREEAEEEAKRRADAKEQGKPKGRAKRGVPGEL

ATPDKKENDAKSSDSSVGEETLPSPSLKPEKKVAEAEKKVEEAKKKAEDQKE

EDRRNYPTNTYKTLELEIAESDVEVKKAELELVKEEAKEPRNEEKVKQAKAE

VESKKAEATRLEKIKTDRKKAEEEAKRKAAEEDKVKEKPAEQPQPAPAPKAE

KPAPAPKPEN (SEQ ID NO 24).

"Polypeptide R2" means a polypeptide comprising the amino acid sequences from position 16 to position 444 of the N-terminal truncate of choline binding protein A (CbpA) serotype type 4 (see Figure 1) which has the following sequence

ENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESY

AKSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAV

SKFEKDSSSSSSSDSSTKPEASDTAKPNKPTEPGEKVAEAKKKVEEAEKKAKD

QKEEDRRNYPTITYKTLELEIAESDVEVKKAELELVKVKANEPRDEQKIKQAE

Wo 99/51188 WO 9951188PCTIUS99/07669 -8-

AEVESKQAEATRLKKIKTDREEAEEEAKRRADAKEQGKPKGRAKRGVPGEL

ATPDKKENDAKSSDSSVGEETLPSPSLKPEKKVAEAEKKTEEAKKKAEDQKE

EDRRNYPTNTYKTLELEIAESDVEVKXAELELVKEEAKEPRINEEKVKQAKAE

VESKKAEATRLEKIKTDRKKAEEEAKRKAAEEDKVKEKPA (SEQ ID NO 1).

The DNA sequence which encodes polypeptide, R2 of the N-termfinal truncate of choline binding protein A (CbpA) serotype type 4:

GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGA

AAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGA

GATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAAAATAGTGGGTG

AGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGCTCTA

GTTAACGAGTTGAACAACATTAAGAACGAGTATTTGAATAAAATAGTTGA

ATCAACCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAA

AAGTAGATGAAGCTGTGTGTAAGTTTGAAAAGGACTCATCTTCTTCGTCAA

GTTCAGACTCTTCCACTAAACCGGAAGCTTCAGATACAGCGAAGCCAAAC

AAGCCGACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTG

AAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAA

CTACCCAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCG

ATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAGTGAAAGCTAA

CGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTrGAG

AGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTG

AAGAAGCAGAAGAAGAAGCTAAACGAAGAGCAGATGCTAAAGAGCAAG

GTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAAC

ACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTG

AAGAAACTCTTCCAAGCCCATCCCTGAAACCAGAAAAAAGGTAGCAGA

AGCTGAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAA

GAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGAACT

TGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAG

TAAAAGAGGAAGCTAAGGAACCTCGAAACGAGGAAAAAGTTAAGCAAGC

AAAAGCGGAAGTTGAGAGTAAAAAAGCTGAGGCTACAJAGGT'IAGI&AAJL

WO 99/51188 WO 9951188PCT/US99/07669 -9-

ATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCAG

CAGAAGAAGATAAAGTTAAAGAAAAACCAGCTG (SEQ ID NO 12).

Amino acid sequence of CbpA of serotype 4:

ENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKE\TEEYVKKJVGESY

AKSTKKRHTITVALVNELNNIKNEYLNJVESTSESQLQILMMESRSKVD1EAV SKFEKDSSSSSSSDSSTKPEASDTAKPNKTEPGEKVAKEyE&JnK

QKEEDRRNYPTITYKTLELEESDVEVKJELELVKVKEPRDEQKKQE

AEVESKQAEATRLKKIKTDREEAEEEAXIURADAKEQGKPKGRAKRGVPGEL

ATPDKKENDAKSSDSSVGEETLPSPSLKEKKVAEAEKKVEEAKADQKE

EDRRNYPTNTYKTLELEIAESDVEVKKAELELVKEEAKEPRNEEKVKQAKAE

VESKKAEATRLEKIKTDRKKAEEEAKRKAA]EDKVKKPAQPQPAPAP1JqE

KPAPAPKPENPAEQPKAEKPADQQAEEDYARI(SEEEYNRLTQQQPPKTEKPA

QPSTPKTGWKQENGMWYFYNTDGSMATGWLQNNGSWYYLNSNGMATG

WLQNNGSWYYLNANGSMATGWLQNNGSWYYLNANGSMATGWLQYNGS

WYYLNANGSMATGWLQYNGSWYYLNANGDMATGWVKDGDTWYYLEAS

GAMASQWFKVSDKWYYVNGSGALAVNTTVDGYGVNANsGEWVN. (SEQ ID NO02) DNA sequence encoding the amino acid sequence of the CbpA of serotype 4:

GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGA

AAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGA

GATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAATAGTGGGTG

AGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGGTCTA

GTTAACGAGTTGAACAACATTAAGAACGAGTATTTGATATAGTTGA

ATCAACCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAA

AAGTAGATGAAGCTGTGTCTAAGTTTGAAAAGGACTCATCTTCTTCGTCAA

GTCGCCTCCAACGAGTCGTCGGACAA

AACGCGACGAAAGTGAAGTAAGAGT

AAGAAGCTGAGAAAAAAGCCAAGGATCAAGAAGAAGATCGTCGTA

CTACCCAACCATTACTTACAAAACGCTTGALCTTGAAATTGCTGAGTCCG

WO 99/51188 WO 99/1 188PCT/US99/07669

ATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAA

CGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAG

AGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTG

AAGAAGCAGAAGAAGAAGGTAAACGAAGAGCAGATGCTAAAGAGCAAG

GTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAAC

ACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTG

AAGAAACTCTTCCAAGCCCATCCCTGAAACCAGAAAAAAAGGTAGCAGA

AGCTGAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAA

GAAGAAGATCGCCGTAACTACGCAACCAATACTTACAAAACGCTTGAACT

TGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGgCTTGAACTA

GTAAAAGAGGAAGCTAAGGAACCTCGAAACGAGGAAAAAGTTAAGCAAG

CAAAAGCGGAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAA

AATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCA

GCAGAAGAAGATAAAGTTAAAGAAAAACCAGCTGAACAACCACAACCAG

CGCCGGCTCCAAAAGCAGAAAAACCAGCTCCAGCTCCAAAACCAGAGAA

TCCAGCTGAACAACCAAAAGCAGAAAAACCAGCTGATCAACAAGCTGAA

GAAGACTATGCTCGTAGATCAGAAGAAGAATATAATCGCTTGACTCAACA

GCAACCGCCAAAAACTGAAAAACCAGCACAACCATCTACTCCAAAAACA

GGCTGGAAACAAGAAAACGGTATGTGGTACTTCTACAATACTGATGGTTC

AATGGCGACAGGATGGCTCCAAAACAAtGGCTCAtGGTAcTACcTCAACAG

CAATGGCGCTATGGCGACAGGATGGCTCCAAAACAATGGTTCATGGTACT

ATCTAAACGCTAATGGTTCAATGGCAACAGGATGGCTCCAAAACAATGGT

TCATGGTACTACCTAAACGCTAATGGTTCAATGGCGACAGGATGGCTCCA

ATACAATGGCTCATGGTACTACCTAAACGCTAATGGTTCAATGGCGACAG

GATGGCTCCAATACAATGGCTCATGGTACTACCTAAACGCTAATGGTGAT

ATGGCGACAGGTTGGGTGAAAGATGGAGATACCTGGTACTATCYI'GAAGC

ATCAGGTGCTATGAAAGCAAGCCAATGGTTCAAAGTATCAGATAAATGGT

ACTATGTCAATGGCTCAGGTGCCCTTGCAGTCAACACAACTGTAGATGGC

TATGGAGTCAATGCCAATGGTGAATGGGTAAACTAA (SEQ ID NO 13).

WO 99/51188 WO 9951188PCT/US99/07669 -11- "Polypeptide Ri" means a polypeptide comprising the amino acid sequences from position 16 to position 321 of the N-terminal truncate! choline binding protein A (CbpA) serotype type 4 which has the following sequence:

ENEGATQVPTSSNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESY

AKSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAV

SKFEKD S SSSS SSDS STKPEASDTAKPNKPTEPGEK VAEAKKK VEEAEKKAKD

QKEEDRRNYPTITYKTLELEIAESDVEVKKAELELVKVKANEPRDEQKIKQAE

AEVESKQAEATRLKKIKTDREEAEEEAKRRADAKEQGKPKGRAKRGVPGEL

ATPDKIKENDAKSSDSSVGEETL (SEQ ID NO 3).

The DNA sequence which encodes polypeptide R1 is:

GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGA

AAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGA

GATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAAAATAGTGGGTG

AGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGCTCTA

GTTAACGAGTTGAACAACATTAAGAACGAGTATTTGAATAAAATAGTTGA

ATCAAGCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAA

AAGTAGATGAAGCTGTGTCTAAGTTTGAAAAGGACTCATCTTCTTCGTCAA

GTTCAGACTCTTCCACTAAACCGGAAGCTTCAGATACAGCGAAGCCAAAC

AAGCCGACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTG

AAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAA

CTACCCAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCG

ATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAA

CGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAG

AGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTG

AAGAAGCAGAAGAAGAAGCTAAACGAAGAGCAGATGCTAAAGAGCAAG

GTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAAC

ACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTG

AAGAAACTCTTC (SEQ ID NO 14).

Wo 99/51188 WO 9951188PCTIUS99/07669 -12- "Polypeptide C/R2" means a polypeptide comprising a repeat region C within R2, wherein the repeat region C has the amino acid sequences from position 327 to position 433 of the N-terminal choline binding protein A (CbpA) serotype type 4 which has the following sequence: KPEKKVAEAEKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAESDVEvK

KAELELVKEEAKEPRNEEKVKQAKAEVESKKAEATRLEKIKTDRKKAEEEAK

RKA (SEQ ID NO 4).

The DNA sequence of polypeptide CIR2

AAACCAGAAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTA

AGAAAAAAGCCGAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAAC

CAATACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTGGAAG

TTAAAAAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCG

AAACGAGGAAAAAGTTAAGCAAGCAAAAGCGGAAGTTGAGAGTAAAAAA

GCTGAGGCTACAAGGTTAGAAAAAATCAAGACAGATCGTAAAAAAGCAG

AAGAAGAAGCTAAACGAAAAGCA (SEQ ED NO "Polypeptide A/R2" means a polypeptide comprising a repeat region A within R2, wherein the repeat region A has the amino acid sequences from position 153 to position 269 of the N-terminal of choline binding protein A (CbpA) serotype type 4 which has the following sequence: TEPGEKVAEAKKKVEEAEKKAKDQKEEDR1RNYPTTYKLELEIAESDVEVK

KAELELVKVKANEPRDEQKIKQAEABVESKQAEATRLKKIKTDREEAEEEAK

RRADA (SEQ ID NO As shown in Figure 1, region A of polypeptide R2 is the same region A as within RI.

The DNA sequence which encodes the polypeptide A/R2 is:

ACGACGAAAGTGAGACAGAAGTGAA

GCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTALCTACC

CAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTG

GAGTAAACGGTGATGAAGGAGTAGA

WO 99/51188 PCT/US99/07669 -13-

CTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAGAGTAA

ACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTGAAGAA

GCAGAAGAAGAAGCTAAACGAAGAGCAGATGCT (SEQ ID NO 16).

The identity or location of one or more amino acid residues may be changed or modified to include variants such as, for example, deletions containing less than all of the residues specified for the protein, substitutions wherein one or more residues specified are replaced by other residues and additions wherein one or more amino acid residues are added to a terminal or medial portion of the polypeptide (see Figure These molecules include: the incorporation of codons "preferred" for expression by selected nonmammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors. Specifically, examples of the amino acid substitutions of serotype 4, included but not limited to, are as follows: E at position 154 is substituted with K; P at position 155 is substituted with L; G at position 156 is substituted with E; E at position 157 is substituted with K; K at position 181 is substituted with E; D at position 182 is substituted with A; R at position 187 is substituted with Y, H, or L; I at position 194 is substituted with N; E at position 200 is substituted with D; E at position 202 is substituted with D; E at position 209 is substituted with K; K at position 212 is substituted with E; V at position 218 is substituted with L; V at position 220 is substituted with K or E; K at position 221 is substituted with E; N at position 223 is substituted with D or K; P at position 225 is substituted with S, T, or R; D at position 227 is substituted with N; E at position 228 is substituted with K; Q at position 229 is substituted with E, G, or D; K at position 230 is substituted with T; K at position 232 is substituted with N; E at position 235 is substituted with K; A at position 236 is substituted with E; E at position 237 is substituted with K; S at position 240 is substituted with N; K at position 241 is substituted with E; Q at position 242 is substituted with K; K at position 249 is substituted with E; K at position 250 is substituted with N; E at position 257 is substituted with Q or K; A at position 263 is substituted with L; K at position 264 is substituted with E; R at position 265 is substituted with N; R at position 266 is WO 99/51188 PCT/US99/07669 -14substituted with I; A at position 267 is substituted with K or V; D at position 258 is substituted with T; A at position 269 is substituted with D; A at position 291 is substituted with T, V, P, G, or X; G at position 294 is substituted with G, A, or E; V at position 295 is substituted with D, or A; P at position 295 is substituted with L or F; L at position 2999 is substituted with P or Q; P at position 328 is substituted with S; E at position 329 is substituted with G; E at position 340 is substituted with A; K at position 343 is substituted with E or D; E at position 347 is substituted with K; D at position 349 is substituted with A; R at position 354 is substituted with H; E at position 366 is substituted with D; E at position 375 is substituted with K; K at position 378 is substituted with E; E at position 390 is substituted with G; P at position 391 is substituted with S; N at position 393 is substituted with D; V at position 397 is substituted with I; and K at position 408 is substituted with Q.

"Polypeptide R2 serotype R6x" means an polypeptide comprising the amino acid sequences from position 16 to position 444 of the N-terminal truncate of Choline Binding Protein A (CbpA) serotype R6x which has the following sequence:

ENEGSTQAATSSNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVAL

NIKLSAIKTKYLRELNVLEEKSKDELPSEIKAKLDAFEKFKKDTLKPGEKVA

EAKKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVKEAELELVK

EEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKRKADAKLK

EANVATSDQGKPKGRAKRGVPGELATPDKKENDAKSSDSSVGEETLPSSSLK

SGKKVAEAEKKVEEAEKKADQKEDRRNYPTNTYKTLDLEIAESDVKVKE

AELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAEEEAKR

KAAEEDKVKEKPA (SEQ ID NO 7) The DNA sequence which encodes polypeptide R2 serotype R6x:

GAAAACGAAGGAAGTACCCAAGCAGCCACTTCTTCTAATATGGCAAGAC

AGAACATAGGAAAGCTGCTAAACAAGTCGTCGATGAATATATAGAAAAA

ATGTTGAGGGAGATTCAACTAGATAGAAGAAAACATACCCAAAATGTCGC

CTTAAACATAAAGTTGAGCGCAATTAAAACGAAGTATTTGCGTGAATTAA

ATGTTTTAGAAGAGAAGTCGAAAGATGAGTTGCCGTCAGAAATAAAAGCA

WO 99/51188 WO 9951188PCT/US99/07669

AAGTTAGACGCAGCTTTTGAGAAGTTTAAAAAAGATACATTGAAACCAGG

AGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAAA

GCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTAGCCAACCAATACTTA

CAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTAALGAAG

CGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAAtCTCGAAACGAGGcJC

ACAATTAAGCAAGCAAAGAGAAGTTGAGAGTAAAAGCTGAGGCTA

CAAGGTTAGAAAACAtCAAGACAGAtCGTAAkAGCAGAAGAAGAAGCT

AAACGAAAAGCAGATGCTAAGTTGAAGGAAGCTAATGTAGCGACTTCAG

AtCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGcAGAGy2TA

GCAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGT

AGGTGAAGAAACTCTTCCAAGCTCATCCCTGAAATCAGGAAAAAAGGTAG

CAGAAGCTGAGAAGAAGGTTGAAGAAGCTGAGAAAAAAGCCAAGGATCA

AAAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAACGCTTG

ACCTTGAAATTGCTGAGTCCGATGTGAAAGTTAAAGAAGCGGAGCTTGAA

CTAGTAAAAGAGGAAGCTAAGGAACCTCGAGACGAGGAAAAATAAGC

AAGCAAAAGCGAAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGA

AAACATCAAGACAGATCGTAAAAJAAGCAGAAGAAGAAGCTAAACGAAAA

GCAGCAGAAGAAGATAAAGTTAAAGAAA&AACCAGCTG (SEQ ID NO 17) Amino acid sequence of CbpA of serotype R6x:

ENEGSTQAATSSNMAKTEHRAAAKQVVDEYIEKMVLREIQLDRPYJITQNVAI

NIKLSALKTKYLRELNVLEEKSKDELPSEIIkKDAAFEKFKKDTLKPGEKVA EAKKKVEEAKKKEDQKEEDRRYPTNTYKTLELEIAEFDVKVKEELL1}K

EEKSNGIQKKEKAATLNKDKAEARAAL

EANVATSDQGKPKGRAKRGVPGELATPDKKENDAKSSDSSVGEETLPSSSLK

SGKKVAEAEKKVEEAEKKIIDQKEEDRRNYPTNTYKTLDLEAESDVKVyE AELELVKEEAKEPRDEEKKQA11KVESKAEATRLENIKTDRKKEEEAYR

KAEEDKVKEKPAEQPQPAPATQPEKPAPKPEKPAEQPKAEKTDDQQAEEDY

ARRSEEEYNRLTQQQPPKTEKPAQPSTPKTGWKQENGMWYFYNTDGSMAT

GWQNGWYNNAAGLNGVYLAGMTWQN

SWYYLNANGAMATGWLQYNGSWYYLNSNGAMATGWLQYNGSWYYLNA

WO 99/51188 WO 99/1 188PCT/US99/07669 -16-

NGDMATGWLQNNGSWYYLNANGDMATGWLQYNGSWYYLNANGDMATG

WVKDGDTWYYLEASGAMKASQWFKVSDKWYYVNGSGALAVNTTVDGYG

VNANGEWVN (SEQ ID NO 8).

DNA sequence encoding the amino acid sequence of the CbpA of serotype R6x:

GAAAACGAAGGAAGTACCCAAGCAGCCACI'TCTTCTAATATGGCU&AGAC

AGAACATAGGAAAGCTGCTAAACAAGTCGTCGATGAATATATAGAAAA

ATGTTGAGGGAGATTCAACTAGATAGAAGAAAACATACCCAAAATGTCGC

CTTAAACATAAAGTTGAGCGCAATTAAACGAAGTATTTGCGTGTTA

ATGTTTTAGAAGAGAAGTCGAAAGATGAGTTGCCGTCAGT&A-A

AAGTTAGACGCAGCTTTTGAGAAGTTTAAAAAAGATACATTGAAACGAGG

AGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAA

GCCGAGGATCAAAAAGAAGAAGATCGTC'GTAACTACCCAACCAATACTTA

CAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAGTTAAAJGAAG

CGGAGCTTGAACTAGTAAAAGAGGAAGCTAAjGAAtCTCGAAACGAGGGC

ACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAALAAGCTGAGCTA

CAGTAAACtAGCGtCTAAACGAAGAC AAACGAAAAGCAGATGCTAAGTTGAAGGAAGCTAATGTAGCGACTtCAGA tCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAG

CAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTACGTA

GGTGAAGAAACTCTTCCAAGCTCATCCCTGAATCAGGAJLAAJAGGTAGC

AGAAGCTGAGAAGAAGGTTGAAGAAGCTGAGAAGCCAGGATCAA

AAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAACGCTTGA

CCTTGAAATTGCTGAGTCCGATGTGAAAGTTAAAGAAGCGGAGGTTGAAC

TAGTAAAAGAGGAAGCTAAGGAACCTCGAGACGAGGAAATAAGICA

AGCAAAAGCGAAAGTTGAGAGTAAGCTGAGGCTACAJArjGTTAGAA

AACATCAAGACAGATCGTAAAAGCAGAAGAAGAAGCTAACGAAG

CAGCAGAAGAAGATAAAGTTAAAGAACCAGCTGAACACCACAACC

AGGCGTCCACGAACACCAACAAAGC

GCTGAACAACCAAAAGCAGAAAAACAGATGATCACAAGCTGAGAG

ACTATGCTCGTAGATCAGAAGAAGATATATCGCTTGACTAACAGCAA

WO 99/51188 WO 9951188PCTIUS99/07669 -17-

CCGCCAAAAACTGAAAAACCAGCACAACCATCTACTCCAAAAACAGGCT

GGAAACAAGAAAACGGTATGTGGTACTTCTACAATACTGATGGTTCAATG

GCAACAGGATGGCTCCAAAACAACGGTTCATGGTACTATCTAAACGCTAAh

TGGTGCTATGGCGACAGGATGGCTCCAAAACAATGGTTCATGGTACTATC

TAAACGCTAATGGTTCAATGGCAACAGGATGGCTCCAAAACAATGGTTCA

TGGTACTACCTAAACGCTAATGGTGCTATGGCGACAGGATGGCTCCAATA

CAATGGTTCATGGTACTACCTAAACAGCAATGGCGCTATGGCGACAGGAT

GGCTCCAATACAATGGCTCATGGTACTACCTCAACGCTAATGGTGATATG

GCGACAGGATGGCTCCAAAACAACGGTTCATGGTACTACCTCAACGCTAA

TGGTGATATGGCGACAGGATGGCTCCAATACAACGGTTCATGGTATTACC

TCAACGCTAATGGTGATATGGCGACAGGTTGGGTGAAAGATGGAGATACC

TGGTACTATCTTGAAGCATCAGGTGCTATGAAAGCAAGCCAATGGTTCAA

AGTATCAGATAAATGGTACTATGTCAATGGCTCAGGTGCCCTTGCAGTCA

ACACAACTGTAGATGGCTATGGAGTCAATGCCAATGGTGAATGGGTPAkAC TAA (SEQ IDNO 18).

Polypeptide RI Serotype R6x" means an polypeptide comprising the amino, acid sequences from position 16 to position 321 of the N-terminal truncate/truncate of choline binding protein A (CbpA) serotype R6x which has the following sequence

ENEGSTQAATSSNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVAL

NIKLSAIKTKYLRELNVLEEKSKDELPSEIKAKLDAAFEKFKKDTLKPGEKVA

EAKKKVEEAKKKAEDQKIEEDRRNYPTNTYKTLELEIAEFDVKVKEAELELVK

EEAKESRNEGTIKQAKEKVESKKAEATRENIKTDRKKAEEEAKRKADAKcIK EANVATSDQGKPKGRAXRGVPGELATPDKKENDAJ(SSDSSvGEETL (SEQ ID NO 9).

The DNA sequence which encodes polypeptide RI is:

GAAAACGAAGGAAGTACCCAAGCAGCCACTTCTTCTAATATGGCAAAGAC

AGAACATAGGAAAGCTGCTAAACAAGTCGTCGATGAATATATAGAAAAA

ATGTTGAGGGAGATTCAACTAGATAGAAGAAAACATACCCAAAATGTCGC

CTTAAACATAAAGTTGAGCGCAATTAAAACGAAGTATTTGCGTGAATTAA

WO 99/51188 WO 99/1 188PCTIUS99/07669

ATGTTTTAGAAGAGAAGTCGAAAGATGAGTTGCCGTCAGAAATAAAAGCA

AAGTTAGACGCAGCTTTTGAGAAGTTTAAAAAAGATACATTGAAACCAGG

AGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAAA

GCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAATACTTA

CAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTAAAGAAG

GGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAATCTCGAAACGAGGG

CACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTGAGGCT

ACAAGGTTAGAAAACAtCAAGACAGATCGTAAAAAAGCAGAAGAAGAAG

CTAAACGAAAAGCAGATGCTAAGTTGAAGGAAGCTAATGTAGCGACTTCA

GATCAAGGTAAACCAAAGGGGGGGCAAAACGAGGAGTTCCTGGAGAGC

TAGCAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGC

GTAGGTGAAGAAACTCTTC (SEQ ED NO 19).

"Polypeptide C/R2 serotype R6x" means an polypeptide comprising a repeat region C within R2 (see Figure wherein the repeat region C has the amino acid sequences from position 327 to position 433 of the N-terminal of choline, binding protein A (CbpA) serotype R6x which has the following sequence:

KSGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTDLEIASDTKVK

EAELELVKEEAKEPRDEEKJKQAKAKVESKKAEATRLENIKTDRKKAEEEAK

RKA (SEQ ID NO The DNA sequence of polypeptide, C/R2 serotype R6x:

AAATCAGGAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTG

AGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAAC

CAATACTTACAAAACGCTTGACCTTGAAATTGCTGAGTCCGATGTGAAAG

TTAAAGAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCG

AGACGAGGAAAAAATTAAGCAAGCAAAAGCGAAAG'TGAGAGTAAAAAA

GCTGAGGCTACAAGGTTAGAAAACATCAAGACAGATCGTAAAAAAGCAG

AAGAAGAAGCTAAACGAAAAGCA (SEQ ID) NO WO 99/51188 PCT/US99/07669 -19- Polypeptide A/R2 serotype R6x" means an polypeptide comprising a repeat region A within R2 (see Figure wherein the repeat region A has the amino acid sequences from position 155 to position 265 of the N-terminal of choline binding protein A (CbpA) serotype R6X which has the following sequence:

PGEKVAEAKKKVEEAKKKAEDQKEEDRYPTNTYKTLELEIAEFDVKVKV

AELELVKEEAKESRNEGTIKQAKEKVESKKAEATRLENIK

KADA (SEQ ID NO 11) The DNA sequence which encodes the polypeptide A/R2 serotype R6x is:

CCAGGAGAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTGA

AAAAAGCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAA

TACTTACAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTA

AAGAAGCGGAGCTTGAACTAGTAAAAGAGGAAGTAAAGAAtCTCGAC

GAGGGCACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTG

AGGCTACAAGGTTAGAAAACAtCAAGACAGATCGTA4AGCAGAAGA AGAAGCTAAACGAAAAGCAGATGCT (SEQ ID NO 21).

This invention is directed to an isolated polypeptide, wherein the isolated polypeptide consists of the amino acid sequence as set forth in SEQ II) NOS 22 or 23, including fragments, mutants, variants, or analogs, or derivatives, thereof

SPSLKPEKKVAEAEKVEEAKKDQKEDRRNYPTNTYKTLELEIESDV

EVKKAELELVKEEAKEPRNEEKVKQAKAEVESKKAEATRL

EAKRKAAEEDKVKEKPA (SEQ ID NO 22; serotype 4; position 323-434); or

PSSSLKSGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNT

VKVKEAELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRENTDRYJ

EEAKRKAAEEDKVKEKRA (SEQ ID NO 23, serotype R6x; position 322-434).

"Polypeptide BIR2" means a polypeptide comprising the amino acid sequences from position 270 to position 326 of the N-terminal truncate of choline binding protein A (CbpA) serotype type 4 as set forth in Figure 2. "Polypeptide B/R2 serotype R6x" means an polypeptide comprising the amino acid sequences from position 264 to WO 99/51188 PCT/US99/07669 position 326 of the N-terminal truncate of Choline Binding Protein A (CbpA) serotype R6x as set forth in Figure 2. This invention contemplates a polypeptide having the amino acid sequence of regions A, B, C, A+B, B+C, A+C as shown in Figure 1.

Further, this invention provides an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate, wherein the polypeptide has the amino acid KXXE (SEQ ID NO 6).

This invention is directed to a polypeptide comprising an amino acid sequence of a Nterminal choline binding protein A truncate, wherein the amino acid sequence is set forth in Figure 2. In one embodiment, the polypeptide has an amino acid sequence which is a conserved region as set forth in Figure 2. For example, conserved regions include but are not limited to amino acid sequence 158 to 172; 300 to 321; 331 to 339; 355 to 365; 367 to 374; 379 to 389; 409 to 427; and 430 to 447 Figure 2 sets forth homologies of various serotypes of the nucleic acid and amino acid sequence of the N-terminal region of CbpA which are contemplated by this invention.

This invention provides an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate, wherein the polypeptide has lectin activity and does not bind to choline. In one embodiment the polypeptide has the amino acid sequence as set forth in any of SEQ ID NO 1, 3-5, 7, or 9-11 including fragments, mutants, variants, analogs, or derivatives, thereof.

As used herein, "a polypeptide having a lectin activity" means a polypeptide, peptide or protein which binds noncovalently to a carbohydrate. As defined herein, "adhesin" means noncovalent binding of a bacteria to a human cell or secretion that is stable enough to withstand washing. As defined herein, "binds to the LNnT" means binds to Lacto-N-neotetraose coated substrates more than albumin-control.

This invention provides an isolated immunogenic polypeptide comprising an amino acid sequence of an N-terminal choline binding protein A truncate. It is contemplated by this WO 99/51188 PCT/US99/07669 -21invention that the immunogenic polypeptide has the amino acid sequence as set forth in any of SEQ ID NOS 1, 3-7, or 9-11, including fragments, mutants, variants, analogs, or derivatives, thereof. This invention provides an isolated polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate as set forth in Figure 2. In one embodiment, the polypeptide has an amino acid sequence which is a conserved region as set forth in Figure 2.

This invention is directed to analogs of the polypeptide which comprise the amino acid sequence as set forth above. The analog polypeptide may have an N-terminal methionine or an N-terminal polyhistidine optionally attached to the N or COOH terminus of the polypeptide which comprise the amino acid sequence.

In another embodiment, this invention contemplates peptide fragments of the polypeptide which result from proteolytic digestion products of the polypeptide. In another embodiment, the derivative of the polypeptide has one or more chemical moieties attached thereto. In another embodiment the chemical moiety is a water soluble polymer.

In another embodiment the chemical moiety is polyethylene glycol. In another embodiment the chemical moiety is mon-, di-, tri- or tetrapegylated. In another embodiment the chemical moiety is N-terminal monopegylated.

Attachment of polyethylene glycol (PEG) to compounds is particularly useful because PEG has very low toxicity in mammals (Carpenter et al., 1971). For example, a PEG adduct of adenosine deaminase was approved in the United States for use in humans for the treatment of severe combined immunodeficiency syndrome. A second advantage afforded by the conjugation of PEG is that of effectively reducing the immunogenicty and antigenicity of heterologous compounds. For example, a PEG adduct of a human protein might be useful for the treatment of disease in other mammalian species without the risk of triggering a severe immune response. The compound of the present invention may be delivered in a microencapsulation device so as to reduce or prevent an host immune response against the compound or against cells which may produce the WO 99/51188 PCT/US99/07669 -22compound. The compound of the present invention may also be delivered microencapsulated in a membrane, such as a liposome.

Numerous activated forms of PEG suitable for direct reaction with proteins have been described. Useful PEG reagents for reaction with protein amino groups include active esters of carboxylic acid or carbonate derivatives, particularly those in which the leaving groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or 1-hydroxy-2nitrobenzene-4-sulfonate. PEG derivatives containing maleimido or haloacetyl groups are useful reagents for the modification of protein free sulfhydryl groups. Likewise, PEG reagents containing amino hydrazine or hydrazide groups are useful for reaction with aldehydes generated by periodate oxidation of carbohydrate groups in proteins.

In one embodiment, the amino acid residues of the polypeptide described herein are preferred to be in the isomeric form. In another embodiment, the residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of lectin activity is retained by the polypeptide. NH 2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. Abbreviations used herein are in keeping with standard polypeptide nomenclature, J. Biol. Chem., 243:3552- 59 (1969).

It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of aminoterminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.

Synthetic polypeptide, prepared using the well known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (N"-amino protected N"-t-butyloxycarbonyl) amino acid resin with the WO 99/51188 PCT/US99/07669 -23standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure ofMerrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the baselabile N"-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409). Thus, polypeptide of the invention may comprise D-amino acids, a combination of D- and L-amino acids, and various "designer" amino acids P-methyl amino acids, Ca-methyl amino acids, and Na-methyl amino acids, etc.) to convey special properties. Synthetic amino acids include ornithine for lysine, fluorophenylalanine for phenylalanine, and norleucine for leucine or isoleucine. Additionally, by assigning specific amino acids at specific coupling steps, a-helices, P turns, P sheets, y-turns, and cyclic peptides can be generated.

In one aspect of the invention, the peptides may comprise a special amino acid at the Cterminus which incorporates either a CO 2 H or CONH 2 side chain to simulate a free glycine or a glycine-amide group. Another way to consider this special residue would be as a D or L amino acid analog with a side chain consisting of the linker or bond to the bead. In one embodiment, the pseudo-free C-terminal residue may be of the D or the L optical configuration; in another embodiment, a racemic mixture of D and L-isomers may be used.

In an additional embodiment, pyroglutamate may be included as the N-terminal residue of the peptide. Although pyroglutamate is not amenable to sequence by Edman degradation, by limiting substitution to only 50% of the peptides on a given bead with N-terminal pyroglutamate, there will remain enough non-pyroglutamate peptide on the bead for sequencing. One of ordinary skill would readily recognize that this technique could be used for sequencing of any peptide that incorporates a residue resistant to Edman degradation at the N-terminus. Other methods to characterize individual peptides that demonstrate desired activity are described in detail infra. Specific activity of a peptide that comprises a blocked N-terminal group, pyroglutamate, when the particular N-terminal group is present in 50% of the peptides, would readily be WO 99/51188 PCT/US99/07669 -24demonstrated by comparing activity of a completely (100%) blocked peptide with a nonblocked peptide.

In addition, the present invention envisions preparing peptides that have more well defined structural properties, and the use ofpeptidomimetics, and peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties. In another embodiment, a peptide may be generated that incorporates a reduced peptide bond, Ri-CH 2

-NH-

R

2 where R, and R 2 are amino acid residues or sequences. A reduced peptide bond may be introduced as a dipeptide subunit. Such a molecule would be resistant to peptide bond hydrolysis, protease activity. Such peptides would provide ligands with unique function and activity, such as extended half-lives in vivo due to resistance to metabolic breakdown, or protease activity. Furthermore, it is well known that in certain systems constrained peptides show enhanced functional activity (Hruby, 1982, Life Sciences 31:189-199; Hruby et al., 1990, Biochem J. 268:249-262); the present invention provides a method to produce a constrained peptide that incorporates random sequences at all other positions.

A constrained, cyclic or rigidized peptide may be prepared synthetically, provided that in at least two positions in the sequence of the peptide an amino acid or amino acid analog is inserted that provides a chemical functional group capable of cross-linking to constrain, cyclise or rigidize the peptide after treatment to form the cross-link.

Cyclization will be favored when a turn-inducing amino acid is incorporated. Examples of amino acids capable of cross-linking a peptide are cysteine to form disulfide, aspartic acid to form a lactone or a lactase, and a chelator such as y-carboxyl-glutamic acid (Gla) (Bachem) to chelate a transition metal and form a cross-link. Protected y-carboxyl glutamic acid may be prepared by modifying the synthesis described by Zee-Cheng and Olson (1980, Biophys. Biochem. Res. Commun. 94:1128-1132). A peptide in which the peptide sequence comprises at least two amino acids capable of cross-linking may be treated, by oxidation of cysteine residues to form a disulfide or addition of a metal ion to form a chelate, so as to cross-link the peptide and form a constrained, cyclic or rigidized peptide.

WO 99/51188 PCT/US99/07669 The present invention provides strategies to systematically prepare cross-links. For example, if four cysteine residues are incorporated in the peptide sequence, different protecting groups may be used (Hiskey, 1981, in The Peptides: Analysis, Synthesis, Biology, Vol. 3, Gross and Meienhofer, eds., Academic Press: New York, pp. 137-167; Ponsanti et al., 1990, Tetrahedron 46:8255-8266). The first pair of cysteine may be deprotected and oxidized, then the second set may be deprotected and oxidized. In this way a defined set of disulfide cross-links may be formed. Alternatively, a pair of cysteine and a pair of collating amino acid analogs may be incorporated so that the crosslinks are of a different chemical nature.

The following non-classical amino acids may be incorporated in the peptide in order to introduce particular conformational motifs: 1, 2 ,3, 4 -tetrahydroisoquinoline-3-carboxylate (Kazmierski et al., 1991, J. Am. Chem. Soc. 113:2275-2283); (2S,3S)-methylphenylalanine, (2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, 1991, Tetrahedron Lett.); 2aminotetrahydronaphthalene-2-carboxylic acid (Landis, 1989, Ph.D. Thesis, University of Arizona); hydroxy-l,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al., 1989, J. Takeda Res. Labs. 43:53-76); P-carboline (D and L) (Kazmierski, 1988, Ph.D. Thesis, University of Arizona); HIC (histidine isoquinoline carboxylic acid) (Zechel et al., 1991, Int. J. Pep. Protein Res. 43); and HIC (histidine cyclic urea) (Dharanipragada).

The following amino acid analogs and peptidomimetics may be incorporated into a peptide to induce or favor specific secondary structures: LL-Acp (LL-3-amino- 2-propenidone-6-carboxylic acid), a P-tur inducing dipeptide analog (Kemp et al., 1985, J. Org. Chem. 50:5834-5838); P-sheet inducing analogs (Kemp et al., 1988, Tetrahedron Lett. 29:5081-5082); P-turn inducing analogs (Kemp et al., 1988, Tetrahedron Lett.

29:5057-5060); =-helix inducing analogs (Kemp et al., 1988, Tetrahedron Lett. 29:4935- 4938); y-turn inducing analogs (Kemp et al., 1989, J. Org. Chem. 54:109:115); and analogs provided by the following references: Nagai and Sato, 1985, Tetrahedron Lett.

26:647-650; DiMaio et al., 1989, J. Chem. Soc. Perkin Trans. p. 1687; also a Gly-Ala WO 99/51188 PCT/US99/07669 -26turn analog (Kahn et al., 1989, Tetrahedron Lett. 30:2317); amide bond isostere (Jones et al., 1988, Tetrahedron Lett. 29:3853-3856); tretrazol (Zabrocki et al., 1988, J. Am.

Chem. Soc. 110:5875-5880); DTC (Samanen et al., 1990, Int. J. Protein Pep. Res.

35:501:509); and analogs taught in Olson et al., 1990, J. Am. Chem. Sci. 112:323-333 and Garvey et al., 1990, J. Org. Chem. 56:436. Conformationally restricted mimetics of beta turns and beta bulges, and peptides containing them, are described in U.S. Patent No.

5,440,013, issued August 8, 1995 to Kahn.

The present invention further provides for modification or derivatization of the polypeptide or peptide of the invention. Modifications ofpeptides are well known to one of ordinary skill, and include phosphorylation, carboxymethylation, and acylation.

Modifications may be effected by chemical or enzymatic means. In another aspect, glycosylated or fatty acylated peptide derivatives may be prepared. Preparation of glycosylated or fatty acylated peptides is well known in the art. Fatty acyl peptide derivatives may also be prepared. For example, and not by way of limitation, a free amino group (N-terminal or lysyl) may be acylated, myristoylated. In another embodiment an amino acid comprising an aliphatic side chain of the structure

(CH

2 )nCH 3 may be incorporated in the peptide. This and other peptide-fatty acid conjugates suitable for use in the present invention are disclosed in U.K. Patent GB- 8809162.4, International Patent Application PCT/AU89/00166, and reference 5, supra.

Mutations can be made in a nucleic acid encoding the polypeptide such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting WO 99/51188 PCT/US99/07669 -27protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point.

Particularly preferred substitutions are: Lys for Arg and vice versa such that a positive charge may be maintained; Glu for Asp and vice versa such that a negative charge may be maintained; Ser for Thr such that a free -OH can be maintained; and Gin for Asn such that a free NH 2 can be maintained.

Synthetic DNA sequences allow convenient construction of genes which will express analogs or "muteins". A general method for site-specific incorporation of unnatural amino acids into proteins is described in Noren, et al. Science, 244:182-188 (April 1989).

This method may be used to create analogs with unnatural amino acids.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-II [Ausubel, R. ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes I-ni E. Celis, ed. "Current Protocols in Immunology" Volumes I- WO 99/51188 PCT/US99/07669 -28- III [Coligan, J. ed. (1994)]; "Oligonucleotide Synthesis" Gait ed. 1984); "Nucleic Acid Hybridization" Hames S.J. Higgins eds. (1985)]; "Transcription And Translation" Hames S.J. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I.

Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

In an additional embodiment, pyroglutamate may be included as the N-terminal residue of the peptide. Although pyroglutamate is not amenable to sequence by Edman degradation, by limiting substitution to only 50% of the peptides on a given bead with N-terminal pyroglutatamate, there will remain enough non-pyroglutamate peptide on the bead for sequencing. One of ordinary skill in would readily recognize that this technique could be used for sequencing of any peptide that incorporates a residue resistant to Edman degradation at the N-terminus. Other methods to characterize individual peptides that demonstrate desired activity are described in detail infra.

Specific activity of a peptide that comprises a blocked N-terminal group, e.g., pyroglutamate, when the particular N-terminal group is present in 50% of the peptides, would readily be demonstrated by comparing activity of a completely (100%) blocked peptide with a non-blocked peptide.

Chemical Moieties For Derivatization. Chemical moieties suitable for derivatization may be selected from among water soluble polymers. The polymer selected should be water soluble so that the component to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable.

One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/component conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations. For the present component or components, these may be ascertained using the assays provided herein.

WO 99/51188 PCT/US99/07669 -29- The water soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co- polymers, polyoxyethylated polyols and polyvinyl alcohol.

Polyethylene glycol propionaldenhyde may have advantages in manufacturing due to its stability in water.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 2kDa and about 100kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

The number of polymer molecules so attached may vary, and one skilled in the art will be able to ascertain the effect on function. One may mono-derivatize, or may provide for a di-, tri-, tetra- or some combination of derivatization, with the same or different chemical moieties polymers, such as different weights of polyethylene glycols).

The proportion of polymer molecules to component or components molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is no excess unreacted component or components and polymer) will be determined by factors such as the desired degree of derivatization mono, di-, tri-, etc.), the molecular weight of the polymer selected, whether the polymer is branched or unbranched, and the reaction conditions.

WO 99/51188 PCT/US99/07669 The polyethylene glycol molecules (or other chemical moieties) should be attached to the component or components with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, EP 0 401 384 herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., 1992, Exp. Hematol. 20:1028-1035 (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group include lysine residues and the terminal amino acid residues; those having a free carboxyl group include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydrl groups may also be used as a reactive group for attaching the polyethylene glycol molecule(s). Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

This invention provides an isolated nucleic acid encoding a polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate. This invention provides an isolated nucleic acid encoding a polypeptide comprising an amino acid sequence of a N-terminal choline binding protein A truncate as set forth in Figure 2. In one embodiment the nucleic acid is set forth in any of SEQ ID NOS 12, 14-17, or 19-21, including fragments, mutants, variants, analogs, or derivatives, thereof. The nucleic acid is DNA, cDNA, genomic DNA, RNA. Further, the isolated nucleic acid may be operatively linked to a promoter of RNA transcription. It is contemplated that the nucleic acid is used to competitively inhibit the lectin activity.

A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A "DNA" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not WO 99/51188 PCT/US99/07669 -31limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA the strand having a sequence homologous to the mRNA).

A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked" includes having an appropriate start signal ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

Further this invention also provides a vector which comprises the above-described nucleic acid molecule. The promoter may be, or is identical to, a bacterial, yeast, insect or mammalian promoter. Further, the vector may be a plasmid, cosmid, yeast artificial chromosome (YAC), bacteriophage or eukaryotic viral DNA.

Other numerous vector backbones known in the art as useful for expressing protein may be employed. Such vectors include, but are not limited to: adenovirus adenoassociated virus (AAV), simian virus 40 (SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Moloney murine leukemia virus, DNA delivery systems, i.e. liposomes, and expression plasmid delivery systems. Further, one class of vectors comprises DNA elements derived from viruses such as bovine papilloma virus, polyoma virus, baculovirus, retroviruses or Semliki Forest virus. Such vectors may be obtained commercially or assembled from the sequences described by methods wellknown in the art.

This invention also provides a host vector system for the production of a polypeptide which comprises the vector of a suitable host cell. Suitable host cells include, but are not WO 99/51188 PCT/US99/07669 -32limited to, prokaryotic or eukaryotic cells, e.g. bacterial cells (including gram positive cells), yeast cells, fungal cells, insect cells, and animals cells. Numerous mammalian cells may be used as hosts, including, but not limited to, the mouse fibroblast cell NIH 3T3, CHO cells, HeLa cells, Ltk- cells, Cos cells, etc.

A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.

Suitable vectors include derivatives of SV40 and known bacterial plasmids, E. coli plasmids col El, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAS, the numerous derivatives of phage X, NM989, and other phage DNA, M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2p plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Any of a wide variety of expression control sequences sequences that control the expression of a DNA sequence operatively linked to it may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage X, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase Pho5), the promoters of the yeast a-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, RI.1, B-W and L-M cells, African Green Monkey kidney WO 99/51188 PCT/US99/07669 -33cells COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.

This invention further provides a method of producing a polypeptide which comprises growing the above-described host vector system under suitable conditions permitting the production of the polypeptide and recovering the polypeptide so produced.

This invention further provides an antibody capable of specifically recognizing or binding to the isolated polypeptide. The antibody may be a monoclonal or polyclonal antibody. Further, the antibody may be labeled with a detectable marker that is either a radioactive, colorimetric, fluorescent, or a luminescent marker. The labeled antibody may be a polyclonal or monoclonal antibody. In one embodiment, the labeled antibody is a purified labeled antibody. Methods of labeling antibodies are well known in the art.

WO 99/51188 PCT/US99/07669 -34- The term "antibody" includes, by way of example, both naturally occurring and nonnaturally occurring antibodies. Specifically, the term "antibody" includes polyclonal and monoclonal antibodies, and fragments thereof. Furthermore, the term "antibody" includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.

Various procedures known in the art may be used for the production of polyclonal antibodies to polypeptide or derivatives or analogs thereof (see, Antibodies A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press: Cold Spring Harbor, Ne York, 1988). For the production of antibody, various host animals can be immunized by injection with the truncated CbpA, or a derivative fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc.

In one embodiment, the polypeptide can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvant may be used to increase the immunological response, depending on the host species.

For preparation of monoclonal antibodies, or fragment, analog, or derivative thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used (see, Antibodies A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory Press: Cold Spring Harbor, Ne York, 1988). These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human Bcell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBVhybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germfree animals utilizing recent technology (PCT/US90/02545). According to the invention, human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, J. Bacteriol.

WO 99/51188 PCT/US99/07669 159-870; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule specific for a polypeptide together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention.

Such human or humanized chimeric antibodies are preferred for use in therapy of human diseases or disorders (described infra), since the human or humanized antibodies are much less likely than xenogenic antibodies to induce an immune response, in particular an allergic response, themselves. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the polypeptide, or its derivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

WO 99/51188 PCT/US99/07669 -36- Antibodies can be labeled for detection in vitro, with labels such as enzymes, fluorophores, chromophores, radioisotopes, dyes, colloidal gold, latex particles, and chemiluminescent agents. Alternatively, the antibodies can be labeled for detection in vivo, with radioisotopes (preferably technetium or iodine); magnetic resonance shift reagents (such as gadolinium and manganese); or radio-opaque reagents.

The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. The polypeptide can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from 3 H, 14 C, 3 2 P, 35 S, 36 C1, 51 Cr, 5Co, 5 8 Co, 5 Fe, 90Y, 25 13 1 1, and 86 Re.

Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, B-glucuronidase, B-D-glucosidase, B-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.

In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of predetermined binding activity or predetermined binding activity capability to suspected target cells. In accordance with the testing techniques discussed above, one class of such kits will contain at least the labeled polypeptide or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, WO 99/51188 PCT/US99/07669 -37- "competitive," "sandwich," "DASP" and the like. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of the presence or capability of cells for predetermined bacterial binding activity, comprising: a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of the present the polypeptide or a specific binding partner thereto, to a detectable label; other reagents; and directions for use of said kit.

This invention provides antagonist or blocking agents which include but are not limited to: peptide fragments, mimetics, a nucleic acid molecule, a ribozyme, a polypeptide, a small molecule, a carbohydrate molecule, a monosaccharide, an oligosaccharide or an antibody. Also, agents which competitively block or inhibit pneumococcal bacterium are contemplmated by this invention. This invention provides an agent which comprises an inorganic compound, a nucleic acid molecule, an oligonucleotide, an organic compound, a peptide, a peptidomimetic compound, or a protein which inhibits the polypeptide.

This invention provides a vaccine which comprises the polypeptide having the amino acid sequence as set forth in any of SEQ ID NOS: 1, 3-7, 9-11, 22, and 23 and a pharmaceutically acceptable adjuvant or carrier. The polypeptide may comprise an amino acid sequence of a N-terminal choline binding protein A truncate as set forth in Figure 2. This invention provides a vaccine which comprises the polypeptide having the amino acid sequence which comprises a conserved region as set forth in Figure 2 and a pharmaceutically acceptable adjuvant or carrier. For example, conserved regions include but are not limited to amino acid sequence 158 to 172; 300 to 321; 331 to 339; 355 to 365; 367 to 374; 379 to 389; 409 to 427; and 430 to 447. This invention provides a vaccine comprising the isolated nucleic acid encoding the polypeptide and a pharmaceutically acceptable adjuvant or carrier.

Active immunity against Gram positive bacteria, particularly pneumococcus, can be induced by immunization (vaccination) with an immunogenic amount of the polypeptide, WO 99/51188 PCTIUS99/07669 -38or peptide derivative or fragment thereof, and an adjuvant, wherein the polypeptide, or antigenic derivative or fragment thereof, is the antigenic component of the vaccine.

The polypeptides of the present invention, or derivatives or fragments thereof, can be prepared in an admixture with an adjuvant to prepare a vaccine. Preferably, the derivative or fragment thereof, used as the antigenic component of the vaccine is an adhesin. More preferably, the polypeptide or peptide derivative or fragment thereof, used as the antigenic component of the vaccine is an antigen common to all or many strains of a species of Gram positive bacteria, or common to closely related species of bacteria. Most preferably, the antigenic component of the vaccine is an adhesin that is a common antigen.

Vectors containing the nucleic acid-based vaccine of the invention can be introduced into the desired host by methods known in the art, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem.

263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990).

The vaccine can be administered via any parenteral route, including but not limited to intramuscular, intraperitoneal, intravenous, and the like. Preferably, since the desired result of vaccination is to elucidate an immune response to the antigen, and thereby to the pathogenic organism, administration directly, or by targeting or choice of a viral vector, indirectly, to lymphoid tissues, lymph nodes or spleen, is desirable. Since immune cells are continually replicating, they are ideal target for retroviral vector-based nucleic acid vaccines, since retroviruses require replicating cells.

Passive immunity can be conferred to an animal subject suspected of suffering an infection with a Gram positive bacterium, preferably streptococcal, and more preferably pneumoccal, by administering antiserum, polyclonal antibodies, or a neutralizing monoclonal antibody against a polypeptide of the invention to the patient. Although passive immunity does not confer long term protection, it can be a valuable tool for the WO 99/51188 PCT/US99/07669 -39treatment of a bacterial infection of a subject who has not been vaccinated. Passive immunity is particularly important for the treatment of antibiotic resistant strains of Gram positive bacteria, since no other therapy may be available. Preferably, the antibodies administered for passive immune therapy are autologous antibodies. For example, if the subject is a human, preferably the antibodies are of human origin or have been "humanized," in order to minimize the possibility of an immune response against the antibodies. The active or passive vaccines of the invention, or the administration of an adhesin, can be used to protect an animal subject from infection of a Gram positive bacteria, preferably streptococcus, and more preferably, pneumococcus.

This invention provides a pharmaceutical composition comprising an amount of the polypeptide as described and a pharmaceutically acceptable carrier or diluent.

For example, such pharmaceutical composition for preventing pneumococcal attachment to mucosal surface may include antibody to lectin domain and/or soluble excess lectin domain proteins. Blocking adherence by either mechanism blocks the initial step in infection thereby reducing colonization. This in turn decreases person to person transmission and prevents development of symptomatic disease.

This invention provides a method of inducing an immune response in a subject which has been exposed to or infected with a pneumococcal bacterium comprising administering to the subject an amount of the pharmaceutical composition, thereby inducing an immune response.

This invention provides a method for preventing infection by a pneumococcal bacterium in a subject comprising administering to the subject an amount of the pharmaceutical composition effective to prevent pneumococcal bacterium attachment, thereby preventing infection by a pneumococcal bacterium.

This invention provides a method for preventing infection by a pneumococcal bacterium in a subject comprising administering to the subject an amount of a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier or diluent, thereby preventing infection by a pneumococcal bacterium.

WO 99/51188 PCT/US99/07669 This invention provides a method for treating a subject infected with or exposed to pneumococcal bacterium comprising administering to the subject a therapeutically effective amount of a vaccine of the invention, thereby treating the subject.

This invention provides a method of inhibiting colonization of host cells in a subject which has been exposed to or infected with a pneumococcal bacterium comprising administering to the subject an amount of the pharmaceutical composition comprising the polypeptide consisting of the amino acid sequence as set forth in any of SEQ ID NOS 1, 3-5, 7, or 9-11, thereby inducing an immune response. The therapeutic peptide that blocks colonization is delivered by the respiratory mucosa. The pharmaceutical composition comprising the polypeptide consisting of the amino acid sequence as set forth in Figure 2.

As used herein, "pharmaceutical composition" could mean therapeutically effective amounts of polypeptide products of the invention together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers useful in to confer a therapeutic effect or benefit by e.g. preventing pneumococcal colonization. A "therapeutically effective amount" as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content Tris-HC1., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents Tween Tween 80, Pluronic F68, bile acid salts). solubilizing agents glycerol, polyethylene glycerol), anti-oxidants ascorbic acid, sodium metabisulfite), preservatives Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of the therapeutic agent. The choice of compositions will depend on the physical and chemical properties of the protein having therapeutic activity. For example, WO 99/51188 PCT/US99/07669 -41a product derived from a membrane-bound form of the active may require a formulation containing detergent. Controlled or sustained release compositions include formulation in lipophilic depots fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers poloxamers or poloxamines) and actives coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors. Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral.

Further, as used herein "pharmaceutically acceptable carrier" are well known to those skilled in the art and include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al., Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, California, p. 384). Often, a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response.

Adjuvant include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon WO 99/51188 PCT/US99/07669 -42emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvant such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Preferably, the adjuvant is pharmaceutically acceptable.

Controlled or sustained release compositions include formulation in lipophilic depots fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors. Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral.

When administered, compounds are often cleared rapidly from mucosal surfaces or the circulation and may therefore elicit relatively short-lived pharmacological activity.

Consequently, frequent administrations of relatively large doses of bioactive compounds may by required to sustain therapeutic efficacy. Compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987). Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound.

As a result, the desired in vivo biological activity may be achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound.

Dosages. The sufficient amount may include but is not limited to from about 1 pg/kg to about 1000 mg/kg. The amount may be 10 mg/kg. The pharmaceutically acceptable form of the composition includes a pharmaceutically acceptable carrier.

WO 99/51188 PCT/US99/07669 -43- As noted above, the present invention provides therapeutic compositions comprising pharmaceutical compositions comprising vectors, vaccines, polypeptides, nucleic acids and antibodies, anti-antibodies, and agents, to compete with the pneumococcus bacterium for pathogenic activities, such as adherence to host cells.

The preparation of therapeutic compositions which contain an active component is well understood in the art. Typically, such compositions are prepared as an aerosol of the polypeptide delivered to the nasopharynx or as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.

In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

An active component can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

A composition comprising (where is a single protein, DNA molecule, vector, etc.) is substantially free of (where comprises one or more contaminating proteins, DNA molecules, vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is Preferably, comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight.

WO 99/51188 PCT/US99/07669 -44- The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host. In the context of the present invention, a deficit in the response of the host is evidenced by continuing or spreading bacterial infection. An improvement in a clinically significant condition in the host includes a decrease in bacterial load, clearance of bacteria from colonized host cells, reduction in fever or inflammation associated with infection, or a reduction in any symptom associated with the bacterial infection.

According to the invention, the component or components of a therapeutic composition of the invention may be introduced parenterally, transmucosally, orally, nasally, pulmonarailly, or rectally, or transdermally. Preferably, administration is parenteral, e.g., via intravenous injection, and also including, but is not limited to, intra-arterial, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Oral or pulmonary delivery may be preferred to activate mucosal immunity; since pneumococci generally colonize the nasopharyngeal and pulmonary mucosa, mucosal immunity may be a particularly effective preventive treatment. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; carrier, or vehicle.

In another embodiment, the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

In yet another embodiment, the therapeutic compound can be delivered in a controlled release system. For example, the polypeptide may be administered using intravenous WO 99/51188 PCT/US99/07669 infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, the brain, thus requiring only a fraction of the systemic dose (see, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Preferably, a controlled release device is introduced into a subject in proximity of the site of inappropriate immune activation or a tumor. Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

A subject in whom administration of an active component as set forth above is an effective therapeutic regimen for a bacterial infection is preferably a human, but can be any animal. Thus, as can be readily appreciated by one of ordinary skill in the art, the methods and pharmaceutical compositions of the present invention are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., for veterinary medical use.

In the therapeutic methods and compositions of the invention, a therapeutically effective dosage of the active component is provided. A therapeutically effective dosage can be determined by the ordinary skilled medical worker based on patient characteristics (age, weight, sex, condition, complications, other diseases, etc.), as is well known in the art.

Furthermore, as further routine studies are conducted, more specific information will emerge regarding appropriate dosage levels for treatment of various conditions in various WO 99/51188 PCT/US99/07669 -46patients, and the ordinary skilled worker, considering the therapeutic context, age and general health of the recipient, is able to ascertain proper dosing. Generally, for intravenous injection or infusion, dosage may be lower than for intraperitoneal, intramuscular, or other route of administration. The dosing schedule may vary, depending on the circulation half-life, and the formulation used. The compositions are administered in a manner compatible with the dosage formulation in the therapeutically effective amount. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations often nanomolar to ten micromolar in the blood are contemplated.

Administration with other compounds. For treatment of a bacterial infection, one may administer the present active component in conjunction with one or more pharmaceutical compositions used for treating bacterial infection, including but not limited to (1) antibiotics; soluble carbohydrate inhibitors of bacterial adhesin; other small molecule inhibitors of bacterial adhesin; inhibitors of bacterial metabolism, transport, or transformation; stimulators of bacterial lysis, or anti-bacterial antibodies or vaccines directed at other bacterial antigens. Other potential active components include anti-inflammatory agents, such as steroids and non-steroidal antiinflammatory drugs. Administration may be simultaneous (for example, administration of a mixture of the present active component and an antibiotic), or may be in seriatim.

Accordingly, in specific embodiment, the therapeutic compositions may further include an effective amount of the active component, and one or more of the following active ingredients: an antibiotic, a steroid, etc. Exemplary formulations are given below: Formulations WO 99/51188 WO 9951188PCTIUS99/07669 -47- Intravenous Formulation I Ing-redient Mg/mil cefotaxime 250.0 Polypeptide, 10.0 dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium USP 0.1 water for injection q.s.a.d. 1.0 MI Intravenous Formulation II Ingredient mg/mi ampicillin 250.0 Polypeptide 10.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml Intravenous Formulation III Ingredien mg/mi gentanicin (charged as sulfate) 40.0 Polypeptide 10.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml Intravenous Formulation IV Ingredient m~n Polypeptide 10.0 dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium. USP 0.1 water for injection q.s.a.d. 1.0 ml WO 99/51188 PCT/US99/07669 -48- Intravenous Formulation V Ingredient mg/ml Polypeptide antagonist sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml Thus, in a specific instance where it is desired to reduce or inhibit the infection resulting from a bacterium mediated binding of bacteria to a host cell, or an antibody thereto, or a ligand thereof or an antibody to that ligand, the polypeptide is introduced to block the interaction of the bacteria with the host cell.

Also contemplated herein is pulmonary delivery of the present polypeptide having lectin activity which acts as an adhesin inhibitory agent (or derivatives thereof), of the invention. The adhesin inhibitory agent (or derivative) is delivered to the lungs of a mammal, where it can interfere with bacterial, streptococcal, and preferably pneumococcal binding to host cells. Other reports of preparation of proteins for pulmonary delivery are found in the art [Adjei et al. Pharmaceutical Research, 7:565-569 (1990); Adjei et al., International Journal of Pharmaceutics, 63:135-144 (1990) (leuprolide acetate); Braquet et al., Journal of Cardiovascular Pharmacology, 13(suppl. 5):143-146 (1989) (endothelin-1); Hubbard et al., Annals oflnternal Medicine, Vol. I, pp. 206-212 (1989) (al-antitrypsin); Smith et al., J. Clin. Invest. 84:1145-1146 (1989) (a-l-proteinase); Oswein et al., "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (1990) (recombinant human growth hormone); Debs et al., J. Immunol. 140:3482-3488 (1988) (interferon-y and tumor necrosis factor alpha); Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating factor)]. A method and composition for pulmonary delivery of drugs is described in U.S. Patent No. 5,451,569, issued September 19, 1995 to Wong et al.

All such devices require the use of formulations suitable for the dispensing of adhesin inhibitory agent (or derivative). Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in WO 99/51188 PCT/US99/07669 -49addition to the usual diluents, adjuvant and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Chemically modified adhesin inhibitory agent may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise adhesin inhibitory agent (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active adhesin inhibitory agent per ml of solution.

The formulation may also include a buffer and a simple sugar for adhesin inhibitory agent stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the adhesin inhibitory agent caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the adhesin inhibitory agent (or derivative) suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin.

Oleic acid may also be useful as a surfactant.

The liquid aerosol formulations contain adhesin inhibitory agent and a dispersing agent in a physiologically acceptable diluent. The dry powder aerosol formulations of the present invention consist of a finely divided solid form of adhesin inhibitory agent and a dispersing agent. With either the liquid or dry powder aerosol formulation, the formulation must be aerosolized. That is, it must be broken down into liquid or solid particles in order to ensure that the aerosolized dose actually reaches the mucous membranes of the nasal passages or the lung. The term "aerosol particle" is used herein to describe the liquid or solid particle suitable for nasal or pulmonary administration, i.e., that will reach the mucous membranes. Other considerations, such as construction of the delivery device, additional components in the formulation, and particle characteristics are WO 99/51188 PCT/US99/07669 important. These aspects of pulmonary administration of a drug are well known in the art, and manipulation of formulations, aerosolization means and construction of a delivery device require at most routine experimentation by one of ordinary skill in the art.

In a particular embodiment, the mass median dynamic diameter will be 5 micrometers or less in order to ensure that the drug particles reach the lung alveoli [Wearley, L.L., Crit. Rev. in Ther. Drug Carrier Systems 8:333 (1991)].

Systems of aerosol delivery, such as the pressurized metered dose inhaler and the dry powder inhaler are disclosed in Newman, Aerosols and the Lung, Clarke, S.W. and Davia, D. editors, pp. 197-22 and can be used in connection with the present invention.

In a further embodiment, as discussed in detail infra, an aerosol formulation of the present invention can include other therapeutically or pharmacologically active ingredients in addition to adhesin inhibitory agent, such as but not limited to an antibiotic, a steroid, a non-steroidal anti-inflammatory drug, etc.

Liquid Aerosol Formulations. The present invention provides aerosol formulations and dosage forms for use in treating subjects suffering from bacterial, streptococcal, in particularly pneumococcal, infection. In general such dosage forms contain adhesin inhibitory agent in a pharmaceutically acceptable diluent. Pharmaceutically acceptable diluents include but are not limited to sterile water, saline, buffered saline, dextrose solution, and the like. In a specific embodiment, a diluent that may be used in the present invention or the pharmaceutical formulation of the present invention is phosphate buffered saline, or a buffered saline solution generally between the pH 7.0-8.0 range, or water.

The liquid aerosol formulation of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, surfactants and excipients. The formulation may include a carrier. The carrier is a macromolecule which is soluble in the circulatory system and which is physiologically acceptable where physiological acceptance means that those of skill in the art would accept injection of said carrier into a patient as part of a therapeutic regime.

The carrier preferably is relatively stable in the circulatory system with an acceptable WO 99/51188 PCT/US99/07669 -51plasma half life for clearance. Such macromolecules include but are not limited to Soya lecithin, oleic acid and sorbitan trioleate, with sorbitan trioleate preferred.

The formulations of the present embodiment may also include other agents useful for pH maintenance, solution stabilization, or for the regulation of osmotic pressure. Examples of the agents include but are not limited to salts, such as sodium chloride, or potassium chloride, and carbohydrates, such as glucose, galactose or mannose, and the like.

The present invention further contemplates liquid aerosol formulations comprising adhesin inhibitory agent and another therapeutically effective drug, such as an antibiotic, a steroid, a non-steroidal anti-inflammatory drug, etc.

Aerosol Dry Powder Formulations. It is also contemplated that the present aerosol formulation can be prepared as a dry powder formulation comprising a finely divided powder form of adhesin inhibitory agent and a dispersant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing adhesin inhibitory agent (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, 50 to 90% by weight of the formulation.

The adhesin inhibitory agent (or derivative) should most advantageously be prepared in particulate form with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm, for most effective delivery to the distal lung. In another embodiment, the dry powder formulation can comprise a finely divided dry powder containing adhesin inhibitory agent, a dispersing agent and also a bulking agent. Bulking agents useful in conjunction with the present formulation include such agents as lactose, sorbitol, sucrose, or mannitol, in amounts that facilitate the dispersal of the powder from the device.

The present invention further contemplates dry powder formulations comprising adhesin inhibitory agent and another therapeutically effective drug, such as an antibiotic, a steroid, a non-steroidal anti-inflammatory drug, etc.

WO 99/51188 PCT/US99/07669 -52- Contemplated for use herein are oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton PA 18042) at Chapter 89, which is herein incorporated by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers U.S. Patent No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and C.T. Rhodes Chapter 10, 1979, herein incorporated by reference. In general, the formulation will include the component or components (or chemically modified forms thereof) and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the above derivatized component or components. The component or components may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits inhibition of proteolysis; and uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981, "Soluble Polymer-Enzyme Adducts" In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp.

367-383; Newmark, et al., 1982, J. Appl. Biochem. 4:185-189. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties.

For the component (or derivative) the location of release may be the stomach, the small intestine (the duodenum, the jejunem, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release WO 99/51188 PCT/US99/07669 -53will avoid the deleterious effects of the stomach environment, either by protection of the protein (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential.

Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used.

The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.

The peptide therapeutic can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the therapeutic with an inert material. These diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextran and starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium WO 99/51188 PCT/US99/07669 -54chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants. Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium WO 99/51188 PCT/US99/07669 chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the polypeptide (or derivative) are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.

Pulmonary Delivery. Also contemplated herein is pulmonary delivery of the present polypeptide (or derivatives thereof). The polypeptide (or derivative) is delivered to the lungs of a mammal while inhaling and coats the mucosal surface of the alveoli. Other reports of this include Adjei et al., 1990, Pharmaceutical Research, 7:565-569; Adjei et al., 1990, International Journal of Pharmaceutics, 63:135-144 (leuprolide acetate); Braquet et al., 1989, Journal of Cardiovascular Pharmacology, 13(suppl. 5):143-146 (endothelin-1); Hubbard et al., 1989, Annals of Internal Medicine, Vol. III, pp. 206-212 (al- antitrypsin); Smith et al., 1989, J. Clin. Invest. 84:1145-1146 (a-1-proteinase); Oswein et al., 1990, "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J. Immunol. 140:3482-3488 (interferon-g and tumor necrosis factor alpha) and Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating factor). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Patent No. 5,451,569, issued September 19, 1995 to Wong et al.

Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise polypeptide (or derivative) dissolved in water at a concentration of about 0.1 WO 99/51188 PCT/US99/07669 -56to 25 mg of biologically active protein per mL of solution. The formulation may also include a buffer and a simple sugar for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the polypeptide (or derivative) suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin.

Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing polypeptide (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, 50 to 90% by weight of the formulation. The protein (or derivative) should most advantageously be prepared in particulate form with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm, for most effective delivery to the distal lung.

Nasal Delivery. Nasal or nasopharyngeal delivery of the polypeptide (or derivative) is also contemplated. Nasal delivery allows the passage of the polypeptide directly over the upper respiratory tract mucosal after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran.

The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

WO 99/51188 PCT/US99/07669 -57- EXPERIMENTAL DETAILS SECTION EXAMPLE 1: Peptide truncates of choline binding protein A (CbpA) A polypeptide comprising a truncated N-terminal fragment of the CbpA (serotype 4) was generated. Full length CbpA was amplified with PCR primers SJ533 and SJ537, the primers were designed based on the derived N-terminal amino acid sequence of the CbpA polypeptide. 5' forward primer SJ533 5' GGC GGA TCC ATG GA(A,G) AA(C,T) GA(A,G) GG This degenerate primer designed from the amino acid sequence XENEG, incorporates both BamHI and NcoI restriction sites and an ATG start codon.

3'reverse primer SJ537 5'GCC GTC GAC TTA GTT TAC CCA TTC ACC ATT GGC This primer incorporates a Sail restriction site for cloning purposes, and the natural stop codon from CbpA, and is based on both type 4 and R6x sequence.

PCR product was generated from genomic DNA as a template with primers SJ533 and SJ537 amplified 30 cycles with an annealing temperature of 50 0 C using High Fidelity enzyme (Boehringer Mannheim). The resulting PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Inc.) then digested with BamHI and SalI restriction enzymes and cloned into the pQE30 expression vector (Qiagen, Inc.) digested with BamHI, XbaI, and SmaI restriction enzymes.

Polypeptide R2: The naturally occuring PvuII site at the end of the second repeat region, namely the C region as shown in Figure 1, (nucleic acid 1228 of Type 4 sequence) was exploited to create a truncated version of the cbpA gene, containing only the 5' portion of the gene.

To create the truncate clone, the full length clone PMI580 (Type 4) or PMI581(R6x) was digested with PvuII and XbaI, the resulting fragment was ligated into the expression vector, PQE30 and transformed into the appropriate host. The protein was expressed and purified. In this instance the stop codon utilized by the expression vector is downstream of the insert, so the protein expressed is larger than the predicted size of the insert due to additional nucleic acids at the 5' end of the cloning site. The amino acid sequence of polypeptide R2 is set forth in SEQ ID NO 1.

WO 99/51188 PCT/US99/07669 -58- Polvpeptide R1: A similar strategy was used to express only the first repeat region within the N-terminal region of CbpA, namely the A region of polypeptide R1. Here the naturally occuring XmnI site between the two amino repeats (nucleic acid 856 of the Type 4 sequence) was utilized. cbpA full length clone PMI580 was digested with XmnI and AatII. The vector was digested with AatII and Smal. Once again the two sized fragments were ligated, transformed into E. coli and clones screened for inserts. One positive clone was selected and recombinant protein purified from this stain.

All polypeptides were expressed and purified with the Qia Expression System (Qiagen) using an E. coli the pQE30 vector. The amino terminus of the His tagged proteins are detected by host and Western analysis using both anti-histidine antibodies and protein specific antibodies.

Purification ofR1 and R2 To induce production of and purify recombinant proteins from E. coli a single colony was selected from plated bacteria containing the recombinant plasmid and grown overnight at 370 in 6.0 mls LB buffer with 50gg/ml kanamycin and 100gg/ml ampicillin. This ml culture was added to 1L LB with antibiotics at above concentrations. The culture was shaken at 37° C until Ao -0.400. 1M IPTG was added to the 1L culture to a final concentration of 1mM. The culture was then shaken at 37 0 C for 3-4 hrs. The 1L culture was spun for 15 min at 4000 rpm in a model J-6B centrifuge. The supernatant was discarded and the pellet stored at -20 C.

The 1L pellet was re-suspended in 25 ml 50 mM NaH 2

PO

4 10mM Tris, 6M GuC1, 300mM NaC1, pH 8.0 (Buffer This mixture was rotated at room temperature for minutes and sonicated on a (VibraCell Sonicator (Sonics and Materials, Inc., Danbury, CT) using the micro tip, two times, for 30 sees, at 50% Cuty Cycle and with the output setting at 7. The mixture was spun 5 min at 1 OK in a JA20 rotor and the supernatant removed and discarded. The supernatant was loaded onto a 10 ml Talon (Clonetech, Palo WO 99/51188 PCT/US99/07669 -59- Alto, CA) resin column attached to a GradiFrac System (Pharmacia Biotech, Upsala, Sweden). The column was equilibrated with 100 ml Buffer A and washed with an additional 200 ml of this buffer. A volume based pH gradient using 100% 50 mM NaH 2

PO

4 8M Urea, 20mM MES, pH6.0 (Buffer B) as the final target buffer was run over a total volume of 100 ml. Protein eluted at -30% Buffer B. Eluted peaks were collected and pooled.

For refolding, dialysis was carried out with a 2L volume of PBS at room temperature for approximately 3 hrs using dialysis tubing with a molecular weight cutoff of 14,000. The sample was then dialyzed overnight in 2L of PBS at 4 0 C. Additional buffer exchange was accomplished during the concentration of the protein using Centriprep-30 spin columns by adding PBS to the spun retenate and re-spinning. The protein concentration was determined using the BCA protein assay and the purity visualized using a Coomassie stained 4-20% SDS-PAGE gel (Figure 3).

EXAMPLE 2: Lectin activity of polypeptides R1 and R2 LNnt is a carbohydrate analog of the receptors for pneumococci present on eukaryotic cells. It has been shown that a CbpA defective pneumococcal mutant failed to adhere to either eukaryotic cells or immobilized sugar indicating that CbpA is the adhesive ligand.

CbpA is a modular protein that can be divided into two regions: the N-Terminal functional domain and the C-terminal choline binding domain (Figure Polypeptides R1 and R2 were analyzed for biological activity to determine if the activities of the entire CbpA were localized in the unique N terminus (modelled by R2) or a fragment thereof (modelled by R1). It was determined whether or not the N-terminal domain alone (R2) contained the lectin binding biological activity in the absence of the choline binding domain (CBD). This was tested using the full length CbpA and polypeptide R2 (truncate missing the CBD region beyond the Pvu II site in the proline rich region).

The assay was to coat tissue culture wells with glycoconjugates known to be recognized by CbpA: LNnT-albumin, 3' sialyl lactose-albumin, and the negative control albumin.

The plates were then blocked with the albumin, washed and either full length CbpA Polypeptide R2, or polypeptide R1 were added for 15 minutes (0.8 Rtg/ml), then, without WO 99/51188 PCT/US99/07669 washing, fluoresein labelled R6 pneumococci were added for 30 minutes, washed and adherent bacteria counted visually.

Binding of R6 to carbohydrate without any peptide addition was the positive control and was calibrated at 100% (Table In three separate experiments, CbpA full length or Polypeptide R2 competitively inhibited binding of pneumococci to LNnT coated surfaces. CbpA full length inhibited to 71. 64% and 63% of control: polypeptide R2 inhibited to 65%, 53% and 74% of control. The equivalent activity of CbpA and R2 indicates the choline binding domain is not necessary for LNnT lectin activity of CbpA, and that R2 is a candidate LNnT lectin.

In contrast to binding to LNnT, binding of pneumococci to 3' sialyl lactose was not inhibited by R2 (79 and 101%) compared to the full length CbpA (74 and This indicates that the sialic acid recognition activity is lost when the CBD is missing. In contrast R1 seems to be active in recognition of sialic acid, a property shared with CbpA but apparently masked in R2. This indicates that folding of polypeptide into functional domains is influenced by the composition and length of the polypeptide. Slight sequence variation is found in other strains (see Figure Given the high degree of homology of sequence between R1 and R2, it is further possible that both RI and R2 are needed for lectin activity or that they are both lectin with slightly different specificities sialic acid).

WO 99/51188 PCT/US99/07669 -61- Table 1 Inhibition of Binding of R6 pneumococci to purified glycoconjugate by soluble forms of CbpA LNnT 3' sialyl lactose Cbp form pneumococci control pneumococci per monolayer per monolayer control (SD) pewnell No peptide 3282 100% 2611 100% 2421 (489) 2115 (125) 2210 (350) Full length 2075 63, 71, 64 1933 74 CbpA 1740 (167) 1405 (240) 66 1415 Polypeptide 2461 74, 53, 65 2639 101 R2 1288 (672) 1670 (420) 79 1440 (530) Polypeptide 3002 91, 92, 112 1052 R1 2245 (182) 1445 (526) 68 2500 (310) N= 3 experiments LNnt each 3 wells N=2 experiments SiL each 3 wells Lectin activity correlates with cell binding activity Human cells bear surface molecules that contain carbohydrates (glycoprotein, and glycolipid) and bacteria bind to these glycoconjugates by the carbohydrate despite very different protein or lipid backbones. Thus, bacteria bearing polypeptide with lectin activity in vitro can adhere to human cell surfaces. This direct correlation between in vitro lectin activity and cell binding action is known for pneumococci. For example, LNnT competitively inhibits binding of pneumococci to TNF activated A549 human lung cells and blocks the progression of pneumonia in vivo. To establish that the lectin activity of truncates of CbpA reflects cell binding activity, CbpA and truncates were tested for inhibition of binding of pneumococci to lung cells (Table Full length WO 99/51188 PCT/US99/07669 -62- CbpA and polypeptide R2 competitively inhibited adherence of pneumococci to lung cells to 58% and 63% of controls respectively. Polypeptide R1 was not effective, indicating the LNnt binding activity of R2 is needed for and explains pneumococcal binding to lung cells.

Table 2 Binding of R6 pneumococci to TNF activated human lung cells A549 Lung Cbp form pneumococci per monolayer (mean) control No peptide 697,704,674 100% 702,722 (700) Full length CbpA 376,431 58% (403) Polypeptide R2 517,693 63% 314,342,350 (443) Polypeptide R1 696,642,552 (630) N= 2 experiments of 2 or 3 wells each LNnT Lectin activity is dependent on R2 The N-terminal region of CbpA contains two repeats of -110 amino acids each (see Figure 1, regions A and C within polypeptide R2). To study the relative contribution of the two domains to bio-activity R1, containing only domain A was compared to R2 and full length CbpA. When tested in the adherence assay, polypeptide R1 did not inhibit adherence to LNnT at all (91, 92, and 112% of wild type). However, polypeptide R1 demonstrated some inhibition of binding to Sialyl lactose (68 and 40% of control). This demonstrates that the polypeptide R2 is required for LNnT lectin activity and R2 is a candidate LNnT lectin domain. In contrast R1 seems to be active in recognition of sialic acid.

WO 99/51188 PCT/US99/07669 -63- Antibodies to N-terminal Domain of CbpA Block Cell Binding: Given that the N-terminal domain of CbpA binds cells, interference with the N-terminal domain activity will prevent or reverse bacterial binding to cells or purified glycoconjugates. One such mechanism of interference is antibody.

Table 3 Inhibition of binding of R6 pneumococci to LNnT coated surfaces by anti-CbpA R2 antibodies pneumococci per control (mean) monolayer (SD) Prelmmune Antibody 198 88 100% Antibody to Truncate R2 56 9 28%; gl of rabbit antibody undiluted 5 ul 2 x 10 7 R6x Preincubate, 6 at RT x 30 min, then add to LNnT coated wells for adherence assay. Two independent experiments are shown.

Antisera raised to the recombinant N-terminal domain of CbpA (R2) was tested for the ability to block adherence of pneumococci to LNnT. Rabbit polyclonal anti CbpA antisera (5gl) plus 5gl of 2 x 10 7 of labeled bacteria were incubated at room temperature for 30 min. This mixture was overlaid onto immobilized LNnT for 30 min., and then washed 3 times with PBS to remove unbound bacteria. Bacteria bound to the plates were enumerated microscopically and results are presented as the mean values plus the standard deviation from six wells. Results shown in Table 3 demonstrate that antisera raised against the R2 polypeptide blocked the binding of pneumococci to LNnT. Figure demonstrates a titration curve of prelmmune versus anti-CbpA R2 antibody for inhibition of binding of pneumococci R6x to the model receptor LNnT. Greater than 70% ofpneumococcal adherence was blocked by anti-R2 at dilutions of 1:100 and 1:200.

Further dilution to 1:400 eliminated activity indicating the specificity of the effect.

The CbpA used to prepare the antisera shown in Table 3 and Figure 5 was raised against CbpA from serotype 4. The R6x strain pneumococci used in the inhibition of adherence WO 99/51188 PCT/US99/07669 -64assay was derived from serotype 2. The ability of the antibody to block adherence of a heterologous serotype of bacteria indicates cross protective activity across serotypes.

Such activity is highly desired for an effective vaccine immunogen.

Activity of antibodies to native conformation of N terminus of CbpA: CbpA can be purified over a choline affinity column from its natural host, the pneumococcus, as described by Rosenow et al. Alternatively, a polyhistidine tag can be engineered onto the end of the gene such that the transcribed protein is extended by several histidine residues. These residues facilitate purification over a nickel affinity matrix Purification of full length polypeptides as opposed to shorter truncates favors retention of the native tertiary structure. CbpA purified especially from pneumococcus but also from E. col or other host bacteria by these biochemical means retains its native tertiary structure. Used as an immunogen, natively folded CbpA engenders antibodies that potentially differ from those elicited by immunization with a truncate which may fold differently. Similarly, CbpA used as a therapeutic may have tertiary structure differing from the truncate which would improve its ability to block adherence. Given these considerations, it may be advantageous to produce CbpA as full length protein allowing it to fold to its native tertiary structure and then cleave the C terminal (CBD) away biochemically. For example, treatment with hydroxylamine will cleave CbpA at amino acid position 475 of serotype R6x and of serotype 4 of choline binding protein A, separating the N and C termini. The N terminal fragment is then suitable as a therapeutic or an immunogen.

Alternatively, native CbpA can be used as an immunogen and antisera to the active structure. The bioactive anti-N terminal antibodies in this mixture can be enriched by removing antibodies to the BD by absorption. Such an antibody was prepared by incubating 200, ul serum with 1 x 108 CbpA defective bacteria for 1 hour at R1. The other choline binding proteins on this mutant absorb out anti-CBD antibodies which are then removed from the antiserum by centrifuging and removing the bacteria.

To demonstrate the bioactivity of absorbed anti CbpA antibodies, the ability of the absorbed antiserum to block pneumococcal adherence to the model receptor LNnT was WO 99/51188 PCT/US99/07669 determined. R6x pneumococci were incubated with 1:600 dilution of antiserum and then added to wells coated with LNnT albumin.

Table 4 Absorbed anti CbpA antiserum blocks adherence Antisera (1:600) Number of pneumococci per well SD of control) No antibody 563 11 (100%) Prelmmune antiserum 479 11 Anti CbpA antiserum 294 72 (52%) 0 Anti CbpA antiserum absorbed 175 38 (31%) to remove CBD antibodies These results indicate that antibodies to the N terminal domain of Cbp/A in its native conformation strongly block adherence. This activity is greater than that to the truncate of Figure 5 which was inactive at 1:600 dilution. Further demonstration of this activity of absorbed anti CbpA antiserum is shown by the titration study of Figure 5. Baseline adherence of pneumococci Type 4 to LNnT coated wells is shown by the triangles.

Pre-incubation of pneumoccoci with unabsorbed (squares) or absorbed (diamonds) antiserum at the various dilutions indicated yielded decreased adherence. The fact that both antisera showed similar decreases in adherence demonstrates that the majority of the blocking activity of antibody to CbpA resides in the N-terminus removal of antibodies to the choline binding domain by absorption does not decrease bioactivity.

EXAMPLE 3: Passive Protection With Anti-R2 Antiserum Generation of Rabbit Immune Sera: Rabbit immune sera against polypeptide R2 (CbpA truncate) and CbpA were generated at Covance (Denver, PA). Following collection ofpre-immune serum, a New Zealand white rabbit was immunized with 250 gg R2 containing both amino terminal repeats (preparation 483:58 above), in Complete Freund's Adjuvant. The rabbit was given a WO 99/51188 PCT/US99/07669 -66boost of 125 gg R2 in Incomplete Freund's Adjuvant on day 21 and bled on day 31. A second rabbit was similarly immunized with purified CbpA.

Passive Protection in Mice: C3H/HeJ mice (5/group) were passively immunized intraperitoneally by with 100 tl of a 1:2 dilution of rabbit anti R2 or preimmune sera in sterile PBS (pre-immune and day 31 immune sera). One hour after administration of serum, mice were challenged with 1600 CFU Streptococcus pneumoniae serotype 6B (strain SP317). Mice were monitored for 14 days for survival. Eighty percent of the mice immunized with rabbit immune serum raised against polypeptide R2 survived challenge (Figure All mice immunized with pre-immune rabbit serum were dead by day 7.

This data demonstrates that antibodies specific for CbpA are protective against systemic pneumococcal infection. The data further indicate that the choline-binding region is not necessary for protection, as antibody specific for the truncated protein polypeptide R2, lacking the conserved choline binding repeats, was sufficient for protection. In addition, serum directed to CbpA of serotype 4 was protective against challenge with serotype 6B.

EXAMPLE 4: Active Protection With Anti-R1 Antiserum C3H/HeJ mice (10/group) were immunized intraperitoneally with CbpA truncate protein R1 (15 pg in 50 pl PBS, plus 50 pl Complete Freund's Adjuvant). A group of 10 sham immunized mice received PBS and adjuvant. A second immunization was administered four weeks later, 15 pg protein i.p. with Incomplete Freund's Adjuvant (sham received PBS plus IFA). Blood was drawn (retro-orbital bleed) at weeks 3, 6, and 9 for analysis of immune response. The ELISA end point anti-CbpA truncate titer of pooled sera from the 10 CbpA immunized mice at 9 weeks was 4,096,000. No antibody was detected in sera from sham immunized mice. Mice were challenged at week 10 with 560 CFU Streptococcus pneumoniae serotype 6B (strain SPSJ2p, provided by P. Flynn, St. Jude Children's Research Hospital, Memphis, TN). Mice were monitored for 14 days for survival. Eighty percent of the mice immunized with WO 99/51188 PCT/US99/07669 -67- CbpA truncate protein R1 survived challenge. All sham immunized mice were dead by day 8 (Figure 7).

This data demonstrates that immunization with a recombinant fragment of CbpA elicits production of specific antibodies capable of protecting against systemic pneumococcal infection and death. The data further indicates that the choline-binding region is not necessary for protection, as the immunogen is the truncated protein R1.

Additionally, the results suggest that a single amino terminal repeat may be sufficient to elicit a protective response. Cross protection is also demonstrated as the recombinant pneumococcal protein was generated based on serotype 4 DNA sequence and protection was observed following challenge with a serotype 6B isolate.

EXAMPLE 5 Prophylaxis against nasopharyngeal colonization in the infant rat In vitro the N terminal domain of CbpA competitively inhibited pneumococcal attachment. To demonstrate the therapeutic utility of peptides with this activity, infant rats were administered truncate peptides, then challenged with pneumococci and colonization of the nasopharynx was evaluated.

Rats were treated intranasally with 10 pl of PBS containing 0.8 pg ofpolypeptide R2 or R1 or no protein. 15 min later Type 3 pneumococci (Strain SII) (10 pl containing 1 x 105 cfer) were introduced intranasally. To determine the ability of the polypeptide to competitively inhibit pneumococcal adherence and colonization, nasal washing was performed at 72 hours and the number of pneumococci recovered was quantitated in each of 4 animals per group. Rats receiving SIlI alone displayed 2200, 6500, 6900 and 8700 (mean 6075) colonies per 10l. Animals treated with truncate R2 showed the greatest decrease (3600, 3500, 2500, 2100) to mean 2925 bacteria 10tl (48% of control).

Animals treated with truncate R1 also showed decreased colonization (5000, 4800, 3500, 1600) to mean 3725 (61% of control).

This experiment demonstrates that administration of the peptide of the instant invention to animals in a therapeutic study design to animals can protect against subsequent pneumococcal challenge.

WO 99/51188 PCT/US99/07669 -68- Discussion: As demonstrated by the experiments, polypeptide R2 when: 1) administered as a vaccine antigen elicits protective antibodies and is a preferred composition for a vaccine formulation; and 2) delivered as a peptide to the respiratory tract and/or nasopharynx receptor, competitively prevents pneumococcal attachment and is a preferred composition for a prophylactic and therapeutic agent against colonization or invasive disease. Also, truncates of CbpA function as lectins without the CBD. Two 10 carbohydrates are recognized: LNnT by a peptide containing both N-tenninal repeats (A and C) in Figure 1 and sialic acid by a peptide containing only the single most Nterminal repeat The truncate containing the N-terminal repeat polypeptide R1 and R2 demonstrates lectin activity in cell culture assays as well.

15 Important features of polypeptide R2 activity include: 1) complete correlation of Sbioactivity of polypeptide R2 and full length CbpA for recognition of purified glycoconjugate receptor analogs, lung cells and animal models. Correlation is also demonstrated for antibodies to them; and 2) cross protection between type 4 derived agents and bacteria in in vitro assays using other serotype 6B and 2) which is 20 important for useful vaccine, prophylactic and therapeutic modalities.

While the invention has been described and illustrated herein by references to various specific material, procedures and examples, it is understood that the invention is not restricted to the particular material combinations of material, and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. Likewise, any references cited herein are, to the extent relevant to the disclosure of the invention, to be considered as incorporated herein by reference.

Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

EDITORIAL NOTE APPLICATION NUMBER 34797/99 The following Sequence Listing pages 1 to 75 are part of the description. The claims pages follow on pages 69 to 82.

WO 99/51188 PCTUS99/07669 1 SEQUENCE LISTING <110> Tuomanen, Elaine I.

Wizemann, Theresa Masure, H. R.

Johnson, Leslie S.

Koenig, Scott <120> POLYPEPTIDE COMPRISING THE AMINO ACID OF AN N-TERMINAL CHOLINE BINDING PROTEIN A TRUNCATE, VACCINE DERIVED THEREFROM AND USES THEREOF <130> 1340-1-017 msc <140> 09/056,019 <141> 1998-04-07 <160> 39 <170> PatentIn Ver. <210> 1 <211> 406 <212> PRT <213> Streptococcus pneumoniae <400> 1 Glu Asn Glu Gly Ala Thr Gln Val Pro Thr Ser Ser Asn Arg Ala Asn 1 5 10 WO 99/51188 WO 9951188PCTIUS99/07669 2- Giu Ser Gin Arg Asp Lys Ala Giu Gin Gly Giu Gin 25 Ala Arg Lys Giu Val Giu 40 Pro Lys Lys Leu Asp Ser Giu Lys Ile Val Giu Tyr Val Lys Gly Giu Ser Tyr Ala Lys Ser Thr 55 Lys Lys Arg His Thr Ile Thr Val Tyr Leu Asn Lys Ala Leu Val Asn Giu Leu Asn Asn Ile Lys Asn Glu 70 75 Ile Val Giu Ser Thr Ser Giu Ser Gin Leu Gin Ile Leu Met Met Giu 90 Ser Lys Phe Giu Lys Asp Ser 110 Ser Arg Ser Lys Val Asp Giu Aia 100 Ser Ser Ser Ser Ser Ser Asp Ser 115 120 Ser Thr Lys Pro Glu Aia Ser Asp 125 Thr Ala 130 Lys Pro Asn Lys Pro 135 Thr Glu Pro Gly Ala Giu Lys Lys 155 Giu 140 Lys Val Ala Glu Ala 145 Lys Lys Lys Val Giu Giu 150 Ala Lys Asp Gin Lys 160 Giu Giu Asp Arg Arg Asn Tyr Pro Thr Ile Thr Tyr Lys Thr Leu Giu WO 99/51188 WO 99/1 188PCTIUS99/07669 -3- -170 165 Leu Giu Ile Ala Giu Ser Asp Val 180 Glu Val Lys Lys Ala 185 Giu Leu Glu 190 Leu Val Lys 195 Val Lys Ala Asn Glu 200 Pro Arg Asp Glu Gin 205 Lys Ile Lys Gin Ala 210 Lys Lys 225 Glu Ala Glu Val Giu 215 Ile Lys Thr Asp Arg 230 Ser Lys Gin Ala Glu Ala Thr Arg Leu 220 Glu Giu Ala Glu Glu Glu Ala Lys Arg 235 240 Arg Ala Asp Ala Lys GlU Gin Gly Lys Pro Lys Gly Arg Ala 245 250 Lys Arg 255 Gly Val Pro Lys Ser Ser 275 Gly Giu Leu Ala Thr 260 Pro Asp Lys Lys Glu 265 Asn Asp Ala 270 Ser Pro Ser Asp Ser Ser Val Gly 280 Glu Glu Thr Leii Pro 285 Leu Lys 290 Ala Lys 305 Pro Giu Lys Lys Val 295 Lys Lys Ala Giu Asp Ala Giu Ala Giu Lys 300 Gin Lys Glu Giu Asp 315 Lys Val Giu Giu Arg Arg Asn Tyr 320 WO 99/51188 WO 9951188PCT/US99/07669 -4 Pro Thr Asn Thr Tyr.Lys Thr Leu Glu 325 Lys Ala Glu Leu Glu 345 Leu Giu Ile Ala Glu 330 Ser Asp 335 Val Giu Val Glu Pro Arg 355 Leu Val Lys Glu Gin Ala Lys Ala 365 Glu Ala Lys 350 Giu Val Glu Asn Giu Giu Lys Val Lys 360 Ser Lys 370 Lys Lys 385 Lys Ala Glu Ala Thr Arg Leu Glu Lys Ile Lys 375 380 Thr Asp Arg Ala Glu Glu Giu Ala Lys Arg Lys Ala Ala Glu Giu Asp Lys 390 395 400 Val Lys Glu Lys Pro Ala 405 <210> 2 <211> 655 <212> PRT <213> Streptococcus pneumoniae <400> 2 Giu Asn Glu Gly Ala Thr Gin Val Pro Thx Ser Ser Asn Arg Ala Asn 1 5 10 Glu Ser Gin Ala Glu Gin Gly Giu Gin Pro Lys Lys Leu Asp Ser Giu WO 99/51188 WO 9951188PCT/US99/07669 Arg Asp Lys Ala Arg Lys Giu Val Giu Glu Tyr Val 40 Gly Glu Ser Tyr Ala Lys Ser Thr Lys Lys Arg His 55 Ala Leu Val Asn Giu Leu Asn Asn Ile Lys Asn Giu 70 Lys Lys Ile Val Thr Ile Thr Val Tyr Leu Asn Ile Val Glu Ser Thr Ser Glu Ser Gin Leu Gin Ile Leu Met Met Giu 90 Ser Arg Ser Ser Ser Ser 115 Lys 100 Val Asp Giu Ala Val Ser Lys Phe Giu 105 Lys Asp Ser 110 Ala Ser Asp Ser Ser Ser Asp Ser 120 Ser Thr Lys Pro Glu 125 Thr Ala 130 Ala Lys 145 Lys Pro Asn Lys Pro 135 Lys Lys Val Giu Giu 150 Thr Giu Pro Gly Giu 140 Ala Giu Lys Lys Ala 155 Lys Val Ala Giu Lys Asp Gin Lys 160 Glu Giu Asp Arg Arg Asn Tyr Pro Thr Ile Thr Tyr Lys Thr Leu Giu 165 170 175 WO 99/51188 WO 9951188PCTIUS99/07669 -6 Leu Glu Ile Leu Val Lys 195 Ala Giu-Ser Asp Val 180 Val Lys Ala Asn Glu 200 Giu Val Lys Lys Ala 185 Pro Arg Asp Giu Gin 205 Giu Leu Giu 190 Lys Ile Lys Gin Ala 210 Glu Ala Glu Val Glu Ser 215 LYS Gin Ala Giu Ala Thr Arg Leu 220 Lys Lys Ile Lys Thr Asp Arg Giu Giu Ala Glu Glu Glu Ala Lys Arg 225 230 235 240 Arg Ala Asp Ala Lys Glu Gin Gly Lys Pro Lys Gly Arg Ala Lys Arg 245 250 O' Giy Val Pro Gly Giu Leu Ala Thr Pro 260 265 Lys Ser Ser Asp Ser Ser Val Gly Glu 275 280 Asp Lys Lys Glu Asn Asp Ala 270 Ser Pro Ser Giu Thr Le.u Pro 285 Leu Lys 290 Pro Giu Lys Lys Val 295 Ala Glu Ala Giu Lys 300 Gin Lys Giu Giu Asp 315 Lys Val Giu Glu Arg Arg Asn Tyr 320 Ala 305 Lys Lys Lys Ala Giu Asp 310 Pro Thr Asn T .hr Tyr Lys Thr Leu Glu Leu Giu Ile Ala Giu Ser Asp 325 330 335 WO 99/51188 WO 9951188PCTIUS99/07669 -7- Val Giu Val Lys Lys Ala Glu Leu Giu Leu Val Lys Glu Glu Ala Lys 340 345 350 Glu Pro Arg Asn Glu Giu Lys Val Lys Gln Ala Lys Ala Glu Val Glu 355 360 365 Ser Lys 370 Lys Ala Glu Ala Thr Arg Leu Glu Lys 375 Ala Lys Arg Lys Ala Lys Ala Giu Giu Giu 390 Ile Lys Thr Asp Arg 380 Ala Glu Giu Asp Lys 400 Ala Pro Ala Pro Lys 415 Asn Pro Ala Giu Gin 430 Val Lys Glu Lys Pro 405 Ala Glu Lys Pro Ala 420 Ala Glu Gin Pro Gin 410 Pro Ala Pro Lys Pro 425 Pro Lys Ala Glu Lys Pro Ala Asp Gin Gin Ala Giu Giu Asp Tyr Ala 435 440 445 Arg Arg 450 Ser Giu Giu Glu Tyr Asn Arg Leu Thr 455 Gin Pro Ser Thr Pro 475 Gin Gin Gin Pro Pro 460 Lys Thr Gly Trp Lys 480 Lys 465 Thr Glu Lys Pro Ala 470 Gin Glu Asn Gly Met Trp Tyr Phe Tyr Asn Thr Asp Gly Ser Met Ala WO 99/51188 WO 9951188PCT[US99/07669 8- Thr Gly Trp Leu Gin Asn Asn Gly Ser 500 505 Trp Tyr Tyr Leu Asn Ser Asn 510 Asn Asn Gly Ser Trp Tyr Tyr 525 Gly Ala Met Ala Thr Gly Trp Leu Gin 515 520 Leu Asn 530 Ser Trp 545 Ala Asn Gly Ser Met Ala Thr Gly Trp Leu Gin Asn Asn Gly 535 540 Tyr Tyr Leu Asn Ala Asn Gly Ser 550 Met Ala Thr Gly Trp Leu 555 560 Gin Tyr Asn Gly Ser Trp Tyr Tyr Leu 565 Asn 570 Ala Asn Gly Ser Met Ala 575 Thr Gly Trp Gly Asp Met 595 Leu Giu Ala 610 Leu Gin Tyr Asn Gly Ser 580 585 Ala Thr Gly Trp Val Lys 600 Ser Gly Ala Met Lys Ala 615 Trp Tyr Tyr Leu Asn Ala Asn 590 Asp Gly Asp Thr Trp Tyr Tyr 605 Ser Gin Trp Phe Lys Val Ser 620 Asp Lys 625 Trp Tyr Tyr Val Asn Gly Ser Gly Ala Leu Ala Val Asn Thr 630 635 640 WO 99/51188 WO 9951188PCT/US99/07669 9- Thr Val Asp Gly Tyr Gly Val Asn Ala Asn Gly Giu Trp Val Asn 645 650 <210> <211> <212> <213> 3 284

PRT

Streptococcus pneuxnoniae <400> 3 Giu Asn Glu Gly Ala Thr Gin Val Pro 1 5 Glu Ser Gin Ala Giu Gin Gly Glu Gin 25 Arg Asp Lys Ala Arg Lys Glu Val Glu 40 Thr Ser Ser Asn Arg 10 Ala Asn Pro Lys Lys Leu Asp Ser Glu Giu Tyr Val Lys Lys Ile Val Gly Glu Ser Tyr Ala Lys Ser Thr Lys Lys Arg His Thr Ile Thr Val 55 Ala Leu Val Asn Glu Ile Val Glu Ser Thr Leu Asn Asn Ile Lys Asn Giu Tyr Leu Asn Lys 70 75 Ser Glu Ser Gin Leu Gin Ile Leu Met Met Giu 90 Ser Arg Ser Lys Val Asp Giu Ala Val Ser Lys Phe Glu Lys Asp Ser WO 99/51188 WO 9951188PCT/US99/07669 100 Ser Ser Ser-Ser Ser Ser Asp 115 110 Ser Ser Thr Lys Pro 120 Thr Glu Pro Gly Glu 140 Giu 125 Ala Ser Asp Thr Ala Lys Pro Asn Lys 130 Pro 135 Lys Val Ala Glu Lys Asp Gin Lys 160 Ala 145 Lys Lys Lys Val Giu 150 Glu Ala Giu Lys Lys Ala 155 Giu Giu Asp Arg Arg Asn 165 Tyr Pro Thr Ile Thr Tyr Lys Thr Leu Glu 170 175 Leu Giu Ile Ala Giu Ser Asp Val Glu Val Lys Lys Ala 180 185 Leu Val Lys Val Lys Ala Asn Glu Pro Arg Asp Glu Gin 195 200 205 Giu Leu Glu 190 Lys Ile Lys Gin Aia Giu Ala Glu Val 210 Lys Lys Ile Lys Thr Asp 225 230 Arg Ala Asp Ala Lys Glu Giu Ser Lys Gin Ala Glu 215 220 Arg Giu Giu Ala Glu Giu 235 Ala Thr Arg Leu Glu Ala Lys Arg 240 Gin Gly Lys Pro Lys Gly Arg Ala Lys Arg 250 255 245 WO 99/51188 WO 9951188PCTIUS99/07669 Gly Val Pro Lys Ser Ser 275 Gly Glu *Leu Ala Thr Pro Asp Lys Lys Glu Asn Asp Ala 260 265 270 Asp Ser Ser Val Gly Glu Glu Thr Leu 280 <210> 4 <211> 106 <212> PRT <213> Streptococcus pneumoniae <400> 4 Lys Pro Glu Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu Ala 2. 5 10 Lys Lys Lys Thr Asn Thr Glu Val Lys Ala Glu Asp Gln Lys Tyr Lys Thr Leu Glu 40 Lys Ala Glu Leu Glu 55 Glu Glu Asp Arg Arg Asn Tyr Pro 25 Leu Glu Ile Ala Lep Val Lys Glu Glu Ser Asp Val Glu Ala Lys Glu Pro Arg Asn Glu Glu Lys Val Lys Gln Ala Lys Ala 70 75 Glu Val Glu Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Lys Ile Lys Thr Asp Arg Lys WO 99/51188 WO 99/1 188PCT/US99/07669 12 Lys Ala Glu Giu Giu Ala Lys Arg Lys Ala 100 105 <210> <211> 109 <212> PRT <213> Streptococcus pneumoniae <400> Thr Glu Pro Gly Giu Lys Val Ala Glu Ala Lys Lys Lys Val Giu Glu 1 5 10 Ala Giu Lys Lys Ala Lyq_ Asp Gin Lys Giu Giu Asp Arg Arg Asn Tyr 25 Pro Thr Ile Thr Tyr Lys Thr Leu Glu Leu Giu Ile Ala Giu Ser Asp Val Glu Val Lys Lys Ala Giu Leu Giu Leu Val 55 Giu Pro Arg Asp Glu Gin Lys Ile Lys Gin Ala 70 75 Ser Lys Gin Ala Giu Ala Thr Arg Leu Lys Lys 90 Lys Val Lys Ala Asn Giu Ala Glu Val Giu Ile Lys Thr Asp Arg WO 99/51188 PCTIUS99/07669 -13 Glu Glu Ala Glu Glu Glu Ala Lys Arg Arg Ala Asp Ala 100 105 <210> 6 <211> 4 <212> PRT <213> Streptococcus pneumniae <400> 6 Lys Xaa Xaa Glu 1 <210> 7 <211> 376 <212> PRT <213> Streptococcus pneumoniae <400> 7 Glu Asn Glu Gly Ser Thr Gln Ala Ala Thr Ser Ser Asn Met Ala Lys 1 5 10 Thr Glu His Arg Lys Ala Ala Lys Gln Val Val Asp Glu Tyr Ile Glu 25 Lys Met Leu Arg Glu Ile Gln Leu Asp Arg Arg Lys His Thr Gin Asn 40

A

WO 99/51188 WO 9951188PCT/US99/07669 14 Val Ala Leu Asn Ile Lys Leu Ser Ala Ile 55 Glu Leu Asn Val Leu Glu Glu Lys Ser Lys 70 Ile Lys Ala Lys Leu Asp Ala Ala Phe Glu 90 Glu Lys Val Ala Glu Ala 105 Lys Thr Lys Tyr Leu Arg Asp Giu Leu Pro Ser Glu 75 Lys Phe Lys Lys Asp Thr Lys Lys Lys Val Giu Glu 110 Glu Asp Arg Arg Asn Tyr 125 Leu Lys Pro Ala Lys Lys 115 Pro Thr Asn 130 Gly 100 LYS Ala Glu Asp Gin Lys Giu 120 Thr Tyr LYS Thr Leu Glu Leu Glu Ile Ala Giu Phe Asp 135 140 Val Lys Val Lys Giu 145 Ala Glu Leu Glu Leu 150 Val Lys Giu Glu Ala Lys 155 160 Ala Lys Glu Lys Val Glu 175 Giu Ser Arg Asn Giu Gly Thr Ile Lys Gin 165 170 Ser Lys Lys Ala 180 Glu Ala Thr Arg Leu 185 Giu Asn Ile Lys Thr Asp Arg 190 LYS Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala Asp Ala Lys Leu Lys WO 99/51188 WO 99/1 188PCT/US99/07669 15 195 Glu Ala Asn Val Ala Thr 210 Lys Arg Gly Val Pro Gly 225 230 200 Ser Asp Gin Gly Lys 21S Pro Lys Gly Arg Ala 220 Asp Ala Lys Ser Asp Glu Leu Ala Thr Ser Ser Val Gly 250 Pro 235 Asp Lys Lys Glu Asn 240 Ser 245 Glu Glu Thr Leu Pro Ser 255 Glu Ala Glu Lys Lys Val 270 Ser Ser Leu Ser Gly Lys Lys Val Ala 265 Glu Glu Ala 275 Asn Tyr Pro 290 Glu Lys Lys Ala Lys Asp Gin Lys Glu Glu Asp Arg Arg 280 285 Thr Asn Thr Tyr Lys Thr Leu Asp 295 Leu Glu Ile Ala Glu 300 Ser Asp Val Lys Val Lys 305 310 Ala Lys Glu Pro Arg Asp 325 Glu Ala Glu Leu Glu 315 Glu Glu Lys Ile Lys 330 Leu Val. Lys Glu Glu 320 Gin Ala Lys Ala Lys 335 Val Glu Ser Lys Lys 340 Ala Giu Ala Thr Arg Leu Giu Asn Ile Lys Thr 345 350 wo 99/51188 WO 9951188PCT/US99/07669 16 Asp Arg Lys 355 Lys Ala -Glu Giu Glu Ala Lys Arg Lys Ala Ala Giu Glu 360 365 Asp Lys 370 Val Lys Glu Lys Pro Ala 375 <210> 8 <211> 663 <212> PRT <213> Streptococcus pneuxnoniae, <400> 8 Glu Asn Glu Gly Ser Thr Gln Ala Ala Thr Ser Ser Asn Met Ala Lys 1 5 10 Thr Glu His Arg Lys Ala Ala Lys Gin Val Val Asp Glu Tyr Ile Glu 25 Lys Met Leu Arg Glu Ile Gin Val Ala Leu Asn Ile Lys Leu 55 Glu Leu Asn Val Leu Glu Glu 70 Leu Asp Arg Arg Lys 40 Ser Ala Ile Lys Thr Lys Ser Lys Asp Glu 75 His Thr Gin Asn Lys Tyr Leu Arg Leu Pro Ser Glu Ile Lys Ala Lys Leu Asp Ala Ala Phe Glu Lys Phe Lys Lys Asp Thr WO 99/51188 WO 9951188PCTIUS99/07669 17 Leu Lys Pro Giy Glu Lys Val Ala 100 Giu Ala Lys Lys Lys 105 Lys Glu Glu Asp Arg 125 Vai Giu Glu 110 Arg Asn Tyr Ala Lys Lys Lys Ala Giu Asp 115 Gin 120 Pro Thr 130 Asn Thr Tyr Lys Thr 135 Leu Glu Leu Glu Ile Ala 140 Giu Phe Asp Val 145 Lys Val Lys Glu Ala Glu 150 Leu-Glu Leu Val Lys Glu Glu Ala Lys 155 160 Ile Lys Gin Ala Lys Glu Lys Val Glu 170 175 Giu Ser Arg Asn Giu Gly Thr 165 Ser Lys Lys Ala Giu Ala Thr Arg Leu 180 185 Lys Lys Ala Giu Glu Giu Ala Lys Arg 195 200 Giu Asn Ile Lys Lys Ala Asp Ala 205 Gly Lys Pro Lys 220 Thr Asp Arg 190 Lys Leu Lys Gly Arg Ala Glu Ala 210 Gin Asn Val Ala Thr Ser Asp 215 Gly Val Pro Gly Glu Leu Lys Arg 225 Ala Thr Pro Asp Lys Lys Glu Asn 235 240 WO 99/51188 WO 9951188PCTIUS99/07669 18 Asp Ala Lys Ser Ser Asp Ser Ser Val 245 Ser Ser Leu Lys Ser Gly Lys Lys Val 260 265 Giu Giu Ala Glu Lys Lys Ala Lys Asp 275 280 Gly Giu Glu Thr Leu Pro Ser 250 255 Ala Giu Ala Glu Lys Lys Val 270 Gin Lys Glu Glu Asp Arg Arg 285 Asn Tyr 290 Pro Thr Asn Thr Ty~r 295 Lys Thr Leu Asp Ser Asp Val Lys Val Lys 305 310 Ala Lys Glu Pro Arg Asp.- 325 Glu Ala Giu Leu Glu 315 Giu Glu Lys Ile Lys 330 Leu Glu Ile Ala Giu 300 Leu Val Lys Glu Giu 320 Gin Ala Lys Ala Lys 335 Vai Glu Ser Lys Lys Ala Giu Ala Thr Arg Leu Glu Asn Ile Lys Thr 340 345 350 Asp Arg Lys 355 LYS Ala Glu Glu Asp Lys 370 Thr Gin 385 Val Lys Glu Lys Pro 375 Pro Glu Lys Pro Ala 390 Glu Ala Lys Arg Lys 360 Ala Giu Gin Pro Gin 380 Pro Lys Pro Giu Lys 395 Ala Ala Glu Giu 365 Pro Ala Pro Ala Pro Ala Glu Gin 400 WO 99/51188 WO 9951188PCT/US99/07669 19 Pro Lys Ala Glu Lys Thr Asp Asp Gin 405 Gin Ala Glu Glu Asp 410 Tyr Ala 415 Arg Arg Ser Lys Thr Glu 435 Glu Glu Glu Tyr Asn 420 Leu Thr Gln Gin Gin Pro Pro 430 Gly Trp Lys Lys Pro Ala Gin Pro 440 Ser Thr Pro Lys Thr 445 Gin Glu 450 Asn Gly Met Trp Tyr 455 Phe Tyr Asn Thr Asp Gly 460 Ser Met Ala Thr 465 Gly Trp Leu Gin Asn Asn 470 Gly Ser Trp Tyr Tyr Leu Asn Ala 475 Asn 480 Gly Ala Met Ala Thr Gly Trp Leu Gin 485 Gly Ser Met Ala Thr 505 Leu Asn Ala Ser Trp Tyr 515 Gin Tyr Asn 530 Asn 500 Asn Asn Gly Ser Trp Tyr Tyr 490 495 Gly Trp, Leu Gin Asn Asn Gly 510 Ala Met Ala Thr Gly Trp Leu 525 Tyr Leu Asn Ala Asn Gly 520 Gly Ser Trp, Tyr Tyr Leu Asn Ser Asn Gly Ala Met Ala 535 540 Thr Gly Trp, Leu Gin Tyr Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn wo 99/51188 WO 9951188PCT/US99/07669 20 555 560 Gly Asp Met Ala Thr Gly Trp Leu Gin Asn Asn Gly Ser Trp Tyr Tyr 565 570 575 Leu Asn Ala Asn Gly Asp Met Ala Thr Gly Trp Leu Gin Tyr Asn Gly 580 585 590 Ser Trp Tyr 595 Lys Asp Gly 610 Tyr Leu Asn Ala Asp Thr Trp Tyr 615 Asn Gly Asp Met Ala 600 Tyf Leu Glu Ala Ser 620 Ser Asp Lys Trp Tyr 635 Thr Gly Trp Val 605 Gly Ala Met Lys Ser Gin Trp Phe Lys Val 630 Tyr Val Asn Gly 640 Ser Gly Ala Leu Ala Val Asn Thr Thr Val Asp Gly Tyr Gly Val Asn 645 650 655 Ala Asn Gly Glu Trp Val Asn 660 <210> 9 <211> 254 <212> PRT <213> Streptococcus pneunioniae WO 99/51188 WO 9951188PCTIUS99/07669 21 <400> 9 Glu Asn Glu Gly Ser Thr Gin Ala Ala 1 5 Thr Glu His Arg Lys Ala Ala Lys Gin 25 Thr Ser Ser Asn Met Ala Lys 10 Val Val Asp Glu Tyr Ile Glu Lys Met Leu Arg Glu Ile Gin Leu Asp Arg Arg Lys 40 Val Ala Leu Asn Ile Lys Glu Leu Asn Val Leu Glu 70 Ile Lys Ala Lys Leu Asp Leu Ser Ala Ile Lys Thr 55 Glu Lys Ser Lys Asp. Glu 75 His Thr Gin Asn Lys Tyr Leu Arg Leu Pro Ser Glu Ala Ala Phe Glu Lys Phe Lys Lys Asp Thr 90 Leu Lys Pro Gly Glu Lys Val Ala Glu Ala Lys Lys Lys 100 105 Val Glu Glu 110 Ala Lys Lys Lys Ala Glu Asp 115 Pro Thr Asn Thr Tyr Lys Thr 130 135 Gin Lys Glu Glu Asp Arg 120 125 Leu Glu Leu Glu Ile Ala 140 Arg Asn Tyr Glu Phe Asp Val Lys Vai Lys Glu Ala Glu Leu Giu Leu Val Lys Glu Giu Ala Lys WO 99/51188 WO 9951188PCT/US99/07669 155 160 Glu Ser Arg Asn Glu 165 Ser Lys Lys Ala Glu 180 Gly Thr Ile Lys Gin 170 Ala Thr Arg Leu Glu 185 Ala Lys Glu Lys Val Glu 175 Asn Ile Lys Thr Asp Arg 190 LYS LYS Ala Glu Glu Glu Ala Lys Arg Lys Ala Asp Ala Lys Leu Lys 195 200 205 Glu Ala Asn Val Ala Thr Ser Asp5 Gin Gly Lys 210 215 Lys Arg Gly Val Pro Gly Glu Leu Ala Thr Pro 225 230 235 Pro Lys Gly Arg Ala 220 Asp Lys Lys Glu Asn 240 Asp Ala Lys Ser Ser Asp Ser Ser Val Gly Glu Glu. Thr Leu 245 250 <210> <211> 106 <212> PRT <213> Streptococcus pneumoniae <400> Lys Ser Gly Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu Ala 1 5 10 WO 99/51188 WO 99/1 188PCT/US99/07669 23 Glu Lys Lys Ala Lys Asp Gin Lys Glu Glu Asp Arg Arg 25 Thr Asn Thr Tyr Lys Thr Leu Asp Leu Glu Ile Ala Glu 40 Lys Val Lys Glu Ala Glu Leu Glu Leu Val Lys Glu Glu 55 Asn Tyr Pro Ser Asp Val Ala Lys Glu Pro Arg Asp Glu Glu Lys Ile 70 Lys Gin Ala Lys Ala Lys Val Glu Ser 75 Lys Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile Lys Thr Asp Arg Lys 90 Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala 100 105 <210> 11 <211> 107 <212> PRT <213> Streptococcus pneumoniae <400> 11 Pro Gly Glu Lys Val Ala Glu Ala Lys Lys Lys Val.Glu Glu Ala Lys 1 5 10 WO 99/51188 WO 9951188PCT1LJS99/07669 24 Lys Lys Ala Asri Thr Tyr Glu Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr Pro Thr 25 Lys Thr Leu Glu Leu Glu Ile Ala GlutPhe Asp Val Lys 40 Val Lys Glu Ala Glu Leu Giu Leu Val Lys Glu Glu Ala Lys Glu Ser so 55 Arg Asn Glu Gly Thr Lys Ala Glu Ala Thr Ala Giu Giu Glu Ala 100 Ile Lys Gin Ala Lys 70 Arg Leu Glu Asn Ile 90 Glu Lys Val Glu Ser Lys 75 Lys Thr Asp Arg Lys Lys Lys.Arg Lys Ala Asp Ala 105 <210> <211> <212> <213> 12 1219

DNA

Streptococcus pneumoniae <400> 12 gagaacgagg gagctaccca agtacccact tcttctaata. gggcaaatga, aagtcaggca gaacaaggag aacaacctaa. aaaactcgat tcagaacgag ataaggcaag gaaagaggtc 120 gaggaatatg taaaaaaaat agtgggtgag agctatgcaa aatcaactaa. aaagcgacat 180 acaattactg tagctctagt taacgagttg aacaacatta. agaacgagta tttgaataaa, 240 WO 99/51188 WO 9951188PCTIUS99/07669 25 atagttgaat gtagatgaag tccactaaac aaggtagcag gaagaaga tc gagtccgatg cctcgagacg gctacaaggt agagcagatg gagc tagcaa gaagaaac tc aaggttgaag ccaaccaata aaagcggagc aagcaagcaa aagacagatc gttaaagaaa caacc tcag a ctgtgtctaa cggaagcttc aagc taagaa gtcgtaacta tggaagttaa agcaaaaaat taaaaaaaat c taaagagca cacc tgataa ttccaagccc aagc taagaa cttacaaaac ttgaactagt aagcggaagt gtaaaaaagc aaccagctg aagccaacta gtttgaaaag agatacagcg gaaggttgaa cccaaccatt aaaagcggag taagcaagca caagacagat aggtaaacca aaaagaaaat atccc tgaaa aaaagccgeig gcttgaactt aaaagaggaa tgagagtaaa agaagaagaa cagatactga gactcatctt aagccaaaca gaagc tgaga acttacaaaa cttgaactag gaagcggaag cgtgaagaag aaggggcggg gatgcgaagt ccagaaaaaa gatcaaaaag gaaattgc tg gctaaggaac aaagctgagg gc taaacgaa tgatggagag cttcgtcaag agccgacaga aaaaagccaa cgcttgaact taaaag'tgaa t tgagagtaa cagaagaaga caaaacgagg ct tcagattc aggtagcaga aagaagatcg agtccgatgt c tcgaaacga c tacaaggtt aagcagcaga tcgatcaaaa ttcagactct accaggagaa ggatcaaaaa tgaaattgct agctaacgaa acaagctgag agctaaacga agttcctgga tagcgtaggt agctgagaag ccgtaac tac ggaagttaaa ggaaaaagt t agaaaaaatc agaagataaa 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1219 <210> 13 <211> 1969 <212> DNA <213> Streptococcus pneumoniae <400> 13 gagaacgagg gaacaaggag gaggaatatg acaattactg atagttgaat gagctaccca agtacccact tcttctaata gggcaaatga aagtcaggca aacaacctaa aaaactcgat tcagaacgag ataaggcaag gaaagaggtc 120 taaaaaaaat agtgggtgag agctatgcaa aatcaactaa aaagcgacat 180 tagctctagt taacgagttg aacaacatta agaacgagta tttgaataaa 240.

caacctcaga aagccaacta cagatactga tgatggagag tcgatcaaaa 300 wo 99/51188 WO 9951188PCTIUS99/07669 26 gtagatgaag tccactaaac aaggtagcag gaagaagat c gagtccgatg cctcgagacg gctacaaggt agagcagatg gagctagcaa gaagaaactc aaggttgaag ccaaccaat a aaagcggagg taagcaagc a caagacaga t agttaaagaa agctccagct tcaacaagc t acagcaaccg acaagaaaac ccaaaacaat ccaaaacaat ccaaaacaat ccaatacaat ccaatacaat gaaagatgga gttcaaagta aactgtagat ctgtgtctaa cggaagcttc aagctaagaa gtcgtaacta tggaagttaa agcaaaaaat taaaaaaaat ctaaagagca cacctgataa ttccaagccc aagc taagaa cttacaaaac cttgaactag aaagcggaag cgtaaaaaag aaaccagctg ccaaaaccag gaagaagac t ccaaaaac tg ggtatgtggt ggctcatggt ggttcatggt ggt tcatggt ggctcatggt ggctcatggt gatacctggt tcagataaat ggctatggag gtttgaaaag agatacagcg gaaggttgaa cccaaccatt aaaagcggag taagcaagca caagacagat aggtaaacca aaaagaaaat atccctgaaa aaaagccgag gcttgaact~t taaaagagga ttgagagtaa cagaagaaga aacaaccaca agaatccagc atgctcgtag aaaaaccagc acttctacaa actacctcaa actatctaaa actacctaaa actacctaaa actacctaaa actatcttga ggtactatgt tcaatgccaa gactcatctt aagccaaaca gaagctgaga acttacaaaa cttgaactag gaagcggaag cgtgaagaag aaggggcggg gatgcgaagt ccagaaaaaa gatcaaaaag gaaattgctg agctaaggaa aaaagctgag agc taaacga accagcgccg tgaacaacca atcagaagaa acaaccatct tactgatggt cagcaatggc cgc taatggt cgctaatggt cgctaatggt cgctaatggt agcatcaggt caatggctca tggtgaatgg c ttcgtcaag agccgacaga aaaaagccaa cgcttgaact taaaagtgaa t tgagagtaa cagaagaaga caaaacgagg cttcagattc aggtagcaga aagaagatcg agtccgatgt cctCgaaacg gc tacaaggt aaagcagcag gctccaaaag aaagcagaaa gaatataatc actccaaaaa tcaatggcga gc tatggcga tcaatggcaa tcaatggcga tcaatggcga gatatggcga gctatgaaag ggtgccc ttg gtaaactaa ttcagaCct accaggagaa g'gatcaaaaa tgaaattgct agc taacgaa acaagctgag agc taaacga agttcctgga tagcgtaggt agc tgagaag ccgtaactac ggaagttaaa aggaaaaagt tagaaaaaat aagaagataa cagaaaaacc aaccagctga gcttgac tca caggc tggaa caggatggct caggatggct caggatggct caggatggct caggatggct caggttgggt caagccaatg cagtcaacac 360 420 480 540 600 660 72.0 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1 560 1620 1680 1740 1800 1860 1920 1969 WO 99/51188 WO 9951188PCT[US99/07669 27 <210> 14 <211> 853 <212> DNA <213> Streptococcus pneurnoniae <400> 14 gagaacgagg gaacaaggag gaggaatatg acaattactg atagttgaat gtagatgaag tccactaaac aaggtagcag gaagaagatc gagtccgatg cctcgagacg gctacaaggt agagcagatg gagc tagcaa gaagaaactc gagctaccca aacaacctaa taaaaaaaat tagctctagt caacctcaga ctgtgtctaa cggaagcttc aagc taagaa gtcgtaacta tggaagttaa agcaaaaaat taaaaaaaat Ctaaagagca cacc tgataa ttc agtacccact aaaactcgat agtgggtgag taacgagttg aagccaacta gtttgaaaig agatacagcg gaaggt tgaa cccaaccatt aaaagcggag taagcaagca caagacagat aggtaaacca aaaagaaaat tcttctaata tcagaacgag agctatgcaa aacaacatta cagatactga gactcatctt aagccaaaca gaagctgaga act tacaaaa cttgaactag gaagcggaag cgtgaagaag aaggggcggg gatgcgaagt gggcaaatga a taaggcaag aatcaac taa agaacgagta tgatggagag c ttcgtcaag agccgacaga aaaaagccaa cgcttgaact taaaagtgaa ttgagagtaa cagaagaaga caaaacgagg c ttcagattc aagtcaggca gaaagaggtc aaagcgacat t ttgaataaa tcgatcaaaa ttcagactct accaggagaa ggatcaaaaa tgaaattgct agctaacgaa acaagc tgag agc taaacga agttcctgga tagcgtaggt 120 180 240 300 360 420 480 540 600 660 720 780 840 853 <210> <211> 318 <212> DNA <213> Streptococcus pneuxnoniae <400> aaaccagaaa aaaaggtagc agaagctgag aagaaggttg aagaagctaa gaaaaaagcc WO 99/51188 WO 9951188PCTIUS99/07669 2.8 gaggatcaaa cttgaaattg gaagc taagg aaaaaagc tg gaagc taaac aagaagaaga tcgccgtaac tacccaacca atacttacaa aacgcttgaa ctgagtccga tgtggaagtt aaaaaagcgg agcttgaact agtaaaagag aacctcgaaa cgaggaaaaa gttaagcaag caaaagcgga agttgagagt aggctacaag gttagaaaaa atcaagacag atcgtaaaaa agcagaagaa gaaaagca 120 180 240 300 318 <210> 16 <211> 327 <212> DNA <213> Streptococcus pneuxnoniae <400> 16 acagaaccag gccaaggatc gaacttgaaa gtgaaagc ta agtaaacaag gaagaagcta gagaaaaggt aaaaagaaga ttgctgagtc acgaacctcg ctgaggctac aacgaagagc agcagaagct aagaagaagg ttgaagaagc tgagaaaaaa agatcgtcgt aactacccaa ccattactta caaaacgctt cgatgtggaa gttaaaaaag cggagcttga actagtaaaa agacgagcaa aaaattaagc aagcagaagc ggaagttgag aaggttaaaa aaaatcaaga cagatcgtga agaagcagaa agatgc t <210> 17 <211> 1129 <212> DNA <2 13> Streptococcus pneumoniae <400> 17 gaaaacgaag aaagctgc ta gatagaagaa aagtatttgc gaagtaccca agcagccact tcttctaata tggcaaagac agaacatagg aacaagtcgt cgatgaatat atagaaaaaa tgttgaggga gattcaacta 120 aacataccca aaatgtcgcc ttaaacataa agttgagcgc aattaaaacg 180 gtgaattaaa tgttttagaa gagaagtcga aagatgagtt gccgtcagaa 240 WO 99/51188 WO 9951188PCT/US99/07669 29 a taaaagcaa gaaaaggtag aaagaagaag gctgagttcg gaatctcgaa gaggctacaa cgaaaagcag aaggggcggg gatgcgaagt tcaggaaaaa gatcaaaaag gaaattgctg gc taaggaac aaagctgagg ge taaacgaa agttagacgc cagaagc taa atcgtcgtaa atgtgaaagt acgagggcac ggt tagaaaa atgctaagtt caaaacgagg cttcagattc aggtagcaga aagaagatcg agtccgatgt ctcgagacga ctacaaggtt aagcagcaga agcttttgag gaagaaggtt c tacccaacc taaagaagcg aattaagcaa catcaagaca gaaggaagct agttcctgga tagcgtaggt agc tgagaag ccgtaactac gaaagt tzaa ggaaaaaatt agaaaacatc agaagataaa aagtttaaaa gaagaagc ta aatacttaca gagcttgaac gcaaaagaga gatcgtaaaa aatgtagcga gagc tagcaa gaagaaac tc aaggttgaag ccaaccaata gaagcggagc aagcaagcaa aagacagatc gt taaagaaa aagatacatt agaaaaaagc aaacgcttga tagtaaaaga aagt tgagag aagcagaaga cttcagatca cacctgataa t tccaagctc aagc tgagaa c ttacaaaac t tgaac tagt aagcgaaagt gtaaaaaagc aaccagctg gaaaccagga cgagga tcaa acttgaaatt ggaagc taaa taaaaaagct agaagctaaa aggtaaacca aaaagaaaat atccctgaaa aaaagccaag gcttgacctt aaaagaggaa tgagagtzaaa agaagaagaa 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1129 <210> 18 <211> 1992 <212> DNA <213> Streptococcus pneumroniae <400> 18 gaaaacgaag aaagctgcta gatagaagaa aagtatttgc ataaaagcaa gaaaaggtag aaagaagaag gaagtaccca aacaagtcgt aacataccca gtgaattaaa agttagacgc cagaagctaa atcgtcgtaa agcagccact cgatgaatat aaatgtcgcc tgttttagaa agcttttgag gaagaaggt t ctacccaacc tcttctaata atagaaaaaa ttaaacataa gagaagtcga aagtttaaaa gaagaagcta aatacttaca tggcaaagac tgttgaggga agttgagcgc aagatgagtt aagatacatt agaaaaaagc aaacgcttga agaacatagg gattcaacta aattaaaacg gccgtcagaa gaaaccagga cgaggatcaa acttgaaatt 120 180 240 300 360 420 WO 99/51188 WO 9951188PCT/US99/07669 30 gctgagttcg atgtgaaagt gaatctcgaa acgagggcac gaggctacaa ggttagaaaa cgaaaagcag atgctaagtt aaggggcggg caaaacgagg gatgcgaagt cttcagattc tcaggaaaaa aggtagcaga gatcaaaaag aagaagatcg gaaattgctg agtccgatgt gctaaggaac Ctcgagacga aaagctgagg ctacaaggtt gctaaacgaa aagcagcaga ccagcgccgg ctactcaacc ccaaaagcag aaaaaacaga gaagaatata atcgcttgac tctactccaa aaacaggctg ggttcaatgg caacaggatg ggtgctatgg cgacaggatg ggttcaatgg caacaggatg ggtgctatgg cgacaggatg ggcgctatgg cgacaggatg ggtgatatgg cgacaggatg ggtgatatgg cgacaggatg ggtgatatgg cgacaggttg ggtgctatga aagcaagcca tcaggtgccc ttgcagtcaa tgggtaaact aa taaagaagcg aattaagcaa catcaagaca gaaggaagc t agttcctgga tagcgtaggt agc tgagaag ccgtaactac gaaagttaaa ggaaaaaatt agaaaacatc agaagataaa agaaaaacca tgatcaacaa tcaacagcaa gaaacaagaa gc tccaaaac gctccaaaac gc tccaaaac gc tccaatac gctccaatac gc tccaaaac gctccaatac ggtgaaagat atggttcaaa cacaactgta gagcttgaac gcaaaagaga gatcgtaaaa aatgtagcga gagctagcaa gaagaaac tc aaggt tgaag ccaaccaata gaagcggagc aagcaagcaa aagacagatc gt taaagaaa gctccaaaac gctgaagaag ccgccaaaaa aacggtatgt aacggttcat aatggttcat aatggttca t aatggttcat aatggctcat aacggttcat aacggttcat ggagatacct gtatcagata gatggctatg tagtaaaaga aagt tgagag aagcagaaga ct tcagatca cacctgataa ttccaagctc aagctgagaa cttacaaaac ttgaactagt aagcgaaagt gtaaaaaagc aaccagc tga cagagaagcc actatgctcg c tgaaaaacc ggtacttcta ggtactatct ggtactatct ggtactacc t ggtactacct ggtactacct ggtactacct ggtattacct ggtactatct aatggtacta gagtcaatgc ggaagctaaa 480 taaaaaagct 540 agaagctaaa 600 aggtaaacca 660 aaaagaaaat 720 atccctgaaa 780 aaaagccaag 840 gcttgacctt 900 aaaagaggaa 960 tgagagtaaa 1020 agaagaagaa 1080 acaaccacaa 1140 agctgaacaa 1200 tagatcagaa 1260 agcacaacca 1320 caatactgat 1380 aaacgctaat 1440 aaacgctaat 1500 aaacgctaat 1560 aaacagcaat 1620 caacgctaat 1680 caacgctaat 1740 caacgctaat 1800 tgaagcatca 1860 tgtcaatggc 1920 caatggtgaa 1980 1992 <210> 19 WO 99/51188 WO 9951188PCT/tJS99/07669 31 <211> 763 <212> DNA <213> Streptococcus pneuxnoniae <400> 19 gaaaacgaag aaagctgcta gatagaagaa aagtatttgc a taaaagcaa gaaaaggtag aaagaagaag gctgagttcg gaatctcgaa gaggctacaa cgaaaagcag aaggggcggg gatgcgaagt gaagtaccca aacaagtcgt aacataccca gtgaat taaa agttagacgc cagaagctaa atcgtcgtaa atgtgaaagt acgagggcac ggt tagaaaa atgctaagtt caaaacgagg cttcagattc agcagccact cgatgaataz aaatgtcgcc tgttttagaa agcttttgag gaagaaggtt c tacccaac~c taaagaagcg aattaagcaa c atcaagaca gaLaggaagc t agttcctgga tagcgtaggt tcttctaata atagaaaaaa t taaacataa gagaagtcga aagtttaaaa gaagaagcta aatacttaca gagcttgaac gcaaaagaga gatcgtaaaa aatgtagcga gagc tagcaa gaagaaactc tggcaaagac tgttgaggga agi tgagcgc aagatgagtt aagatacatt agaaaaaagc aaacgcttga tagtaaaaga aagttgagag aagcagaaga cttcagatca cacctgataa tc agaacatagg gattcaac ta aattaaaacg gccgtcagaa gaaaccagga cgaggatcaa acttgaaatt ggaagctaaa taaaaaagct agaagc taaa aggtaaacca aaaagaaaat 120, 180 240 300 360 420 480 540 600 660 720 763 <210> <211> 318 <212> DNA <213> Streptococcus pneumoniae <400> aaatcaggaa aaggatcaaa cttgaaattg gaagctaagg aaaaggtagc agaagctgag aagaaggttg aagaagctga gaaaaaagcc aagaagaaga tcgccgtaac tacccaacca atacttacaa aacgcttgac 120 ctgagtccga tgtgaaagtt aaagaagcgg agcttgaact agtaaaagag 180 aacctcgaga cgaggaaaaa attaagcaag caaaagcgaa agttgagagt 240 WO 99/51188 PCTIUS99/07669 32 aaaaaagctg aggctacaag gttagaaaac atcaagacag atcgtaaaaa agcagaagaa 300 gaagctaaac gaaaagca 318 <210> 21.

<211> 322.

<212> DNA <213> Streptococcus pneumoniae <400> 21 ccaggagaaa gatcaaaaag gaaattgctg gctaaagaat aaagctgagg gc taaacgaa aggtagcaga aagaagatcg agttcgatgt c tcgaaacga c tacaaggtt aagcagatgc agctaagaag aaggttgaag aagctaagaa aaaagccgag tcgtaactac ccaaccaata cttacaaaac gcttgaactt gaaagttaaa gaagcggagc ttgaactagt aaaagaggaa gggcacaatt aagcaagcaa aagagaaagt tgagagtaaa agaaaacatc aagacagatc gtaaaaaagc agaagaagaa t <210> 22 <211> 121 <212> PRT <213> Streptococcus pneumoniae <400> 22 Ser Pro Ser Leu Lys Pro Glu Lys Ly., 1 5 Val Glu Glu Ala Lys Lys Lys Ala Glu 25 Val Ala Glu Ala Glu Lys Lys 10 is Asp Gin Lys Glu Glu Asp Arg Arg Asn Tyr Pro Thr Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala WO 99/51188 WO 9951188PCT/US99/07669 33 Giu Ser Asp Val Giu Val Giu Ala Lys Giu Pro Arg 70 Glu Val Giu Ser Lys Lys Lays Lys Ala Giu Leu Giu 55 Asn Glu Giu Lys Val Lys 75 Leu Val Lys Glu Gin Ala Lys Ala Ala Glu Ala Thr Arg Leu Giu Lys Ile Lys 90 Thr Asp Arg Lys Lys Ala Glu Gla Glu Ala Lys Arg Lys Ala Ala Giu 100 105 110 Glu Asp Lys Val Lys Glu Lys Pro Ala 115 120 <210> 23 <211> 122 <212> PRT <213> Streptococcus pneumoniae <400> 23 Pro Ser Ser Ser Leu Lys Ser Gly Lys Lys Val Ala Giu Ala Glu Lys 1 5 10 Lys Val Giu Glu Ala Glu Lys Lys Ala Lys Asp Gin Lys Glu Glu Asp 25 WO 99/51188 WO 99/1 188PCTIUS99/07669 34 Arg Arg Asn Tyr Pro Thr Asn Ala Glu Ser Asp Val Lys Val 55 Glu Glu Ala Lys Glu Pro Arg 70 Thr Tyr Lys Thr Leu Asp Leu Glu Ile 40 Lys Glu Ala Glu Leu Glu Leu Val Lys Asp Glu Glu Lys Ile Lys Gin Ala Lys 75 Ala Lys Val Glu Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile 90 Lys Thr Asp Arg Lys Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala Ala 100 105 110 Glu Glu Asp Lys Val Lys Glu Lys Arg Ala 115 120 <210> <211> <212> <213> 24 428

PRT

Streptococcus pneumoniae <400> 24 Glu Asn Glu Gly Ala Thr Gin Val Pro Thr Ser Ser Asn Arg Ala Asn 1 5 10 WO 99/51188 WO 9951188PCTIUS99/07669 35 Giu Ser Gin Ala Giu Gin Gly Giu Gin Pro Lys Lys Leu Asp Ser Giu 25 Arg Asp Lys Ala Arg Lys Giu Val Glu Glu Tyr Val 40 Lys Lys Ile Val Thr Ile Thr Val Gly Giu Ser Tyr Ala Lys Ala Leu Val Asri Giu Leu 70 Ser Thr Lys Lys Arg His 55 Asn Asn Ile Lys Ile Val Giu Ser Thr Ser Arg Ser Lys Val 100 Ser Giu Ser Gin Leu 90 Asp Giu Ala Val Ser 105 Asn Giu Tyr Leu Asn Lys 75 Gin Ile Leu Met Met Giu LYS Phe Glu Lys Asp Ser 110 Ser Ser Ser Ser Ser Ser Asp Ser Ser Thr Lys Pro Giu Ala Ser Asp 115 120 125 Thr Ala Lys Pro Asn Lys 130 Pro Thr Giu Pro Giy 135 Giu Ala Giu Lys Lys 155 Aia 145 Lys Lys Lys Val Giu 150 Giu Lys Vai Ala Giu 140 Aia Lys Asp Gin Lys 160 Tyr Lys Thr Leu Giu 175 Giu Giu Asp Arg Arg 165 Asn Tyr Pro Thr Ile Thr 170 WO 99/51188 WO 9951188PCTLJS99107669 36 Leu Glu Ile Leu Val Lys 195 Ala Giu Ser Asp Val 180 Val Lys Ala Asn Giu 200 Giu 185 Val Lys Lys Ala Giu Leu Giu 190 Lys Ile Lys Pro Arg Asp Giu Gin 205 Lys Gin Ala Gi~u Ala 220 Gin Ala 210 Giu Ala Giu Val Giu Ser 215 Thr Arg Leu Lys 225 Lys Ile Lys Thr Asp Arg Giu Giu Ala Giu Giu Giu Ala Lys Arg 230 235 240 Arg Ala Asp Ala Lys Giu Gin Gly Lys 245 Pro Lys Gly Arg Ala 250 Lys Arg 255 Gly Val Pro Lys Ser Ser 275 Gly Giu Leu Ala Thr 260 Asp Ser Ser Vai Gly 280 Pro 265 Asp Lys Lys Glu Asn Asp Ala 270 Ser Pro Ser Glu Giu Thr Leu Pro 285 Leu Lys 290 Pro Glu Lys Lys Val Ala Glu 295 Ala Giu Lys Lys 300 Val Giu Giu Ala Lys 305 Lys Lys Ala Giu Asp Gin Lys Giu Giu Asp Arg Arg Asn Tyr 310 315 320 Pro Thr Asn Thr Tyr Lys Thr Leu Giu Leu Glu Ile Ala Glu Ser Asp WO 99/51188 WO 9951188PCT/US99/07669 37 325 Val Glu Val Lys Lys Ala Glu Leu Glu 340 345 Giu Pro Arg Asn Glu Glu Lys Val Lys 355 360 Leu Val Lys Glu Glu Ala Lys 350 Gin Ala Lys Ala Glu Val Giu 365 Ser Lys Lys Ala Giu Ala 370 Thr Arg Leu Glu Lys 375 Lys Lys Ala Glu Giu 385 Val Lys Giu Lys Pro 405 Ala Giu Lys Pro Ala 420 <210> <211> 23 <212> DNA <213> Streptococcus <400> Giu 390 Ala Lyt Arg Lys Ala 395 Ile Lys Thr Asp Arg 380 Ala Giu Glu Asp Lys 400 Ala Pro Ala Pro Lys 415 Ala Giu Gin Pro Gin Pro 410 Pro Ala Pro Lys Pro Glu Asn 425 pneumoniae ggcggatcca tggaraayga rgg WO 99/51188 PCTIUS99/07669 -38 <210> 26 <211> 33 <212> DNA <213> Streptococcus pneumoniae <400> 26 gccgtcgact tagtttaccc attcaccatt ggc 33 <210> 27 <211> <212> PRT <213> Streptococcus pneuznoniae' <400> 27 Xaa Glu Asn Glu Gly 1 <210> 28 <211> 439 <212> PRT <213> Streptococcus pneuxnoniae <400> 28 Ala Val Ala Ser Leu Phe Met Gly Ser Val Val His Ala Thr Glu Lys 1 5 10 Glu Val Thr Thr Gln Val Ala Thr Ser Ser Asn Lys Ala Asn Lys Ser 25 WO 99/51188 PCT/US99/07669 -39 Gin Thr Glu His Met Lys Ala Ala Lys Gin Val Asp Glu Tyr Ile Lys 40 Lys Lys Leu Thr Leu Gin Leu Asp Arg Arg Lys His Thr Gin Asn Val Gly Leu 55 Lys Leu Gly Val Ile Lys Thr Giu Tyr Leu His Giy Leu Ser 70 75 Val Ser Lys Lys Lys Ser Giu Ala Glu Leu Pro Ser Giu Ile Lys Ala 90 Lys Leu Asp Giu Pro Gly 115 Lys Lys Lys 130 Ala Ala Phe Giu Gin 100 LYS Lys Vai Ala Giu 120 Ala Glu Asp Gin Lys 135 Phe Lys Lys Asp Thr 105 Ala Giu Lys Lys Val 125 Glu Lys Asp Leu Arg 140 Leu Pro Thr 110 Giu Giu Ala Asn Tyr Pro Thr Asn Thr Tyr Lys Thr Leu Giu Leu Asp 145 150 Giu Val Lys Lys Ala Glu Leu Glu Leu Val 165 170 Ile Ala Glu Ser Asp 155 Val 160 Lys Glu Glu Ala Lys Glu 175 Ser Arg Asp Glu Lys Lys Ile Asn Gin Ala Lys Ala Lys Val Giu Asn WO 99/51188 WO 99/1 188PCT/US99/07669 40 180 190 Lys Lys Ala 195 Lys Ala Glu 210 Glu Ala Thr Arg Leu 200 Glu Ala Lys Arg Arg 215 Lys Asn Ile Lys Thr Asp Arg Glu 205 Leu Gin Glu Ala Ala Asp Ala Asn Val Ala Thr Ser Glu Gin Asp Lys Ser 225 230 Lys Arg Arg Ala Lys Arg 235 240 Lys Lys Glu Asn Asp Ala 255 Glu Val Xaa Gly Glu Leu Ala Thi Pro 245 Asp 250 Lys Ser Ser Leu Lys Pro 275 Asp 260 Ser Ser Val Gly Glu 265 Glu Thr Leu Thr Ser Pro Ser 270 Glu Lys Lys Val Ala 280 Glu Ala Glu Lys Lys Val Glu Glu 285 Lys Glu Glu Asp Arg Arg Asn Tyr 300 Ala Lys 290 Lys Lys Ala Glu Asp Gin 295 Pro Thr Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala Glu Ser Asp 305 310 315 320 Val Glu Val Lys Lys Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys 325 330 'A r WO 99/51188 WO 9951188PCT/US99/07669 41 Glu Ser Arg Asn Glu Glu Lys Ile 340 Lys Gin Val Lys Ala Lys Val Glu 345 350 Ser Lys Lys 355 Ala Giu Ala Thr Arg 360 Leu Giu Asn Ile Lys Thr Asp Arg 365 Lys Lys Ala Glu Giu Glu Giu Ala Lys Arg Arg 370 375 Lys Val Lys Glu Lys Pro Ala Glu Gin Pro Gin 385 390 395 Gin Pro Glu Lys Pro Thr Glu Glu Pro Glu Asn 405 410 Ala 380 Ala Glu Glu Asp Pro Ala Pro Ala Pro 400 Pro Ala Pro Ala Pro 415 Ala Pro Lys Pro Glu Asn Pro Ala Giu Lys Pro Lys Ala Glu Lys Pro 420 425 430 Ala Asp Gin Gin Ala Glu Giu 435 <210> <211> <212> <213> 29 437

PRT

Streptococcus pneuxnoniae <400> 29 Ala Val Ala Ser Leu Phe Met Gly Ser Val Val. His Ala Thr Glu Lys WO 99/51188 WO 9951188PCTIUS99/07669 42 Giu Val Thr Gin Thr Giu Lys Lys Leu Leu Thr Lys Thr Gin Val Ala Thr Ser Ser Asn Arg Ala Asn Lys Ser 25 Lys Gin Val Asp Giu Tyr Ile Lys His Met Lys Ala Ala 40 Gin Leu Asp Arg 55 Leu Gly Val Ile 70 Arg Lys His Thr Gin Asn Vai Gly Leu Lys Thr Giu Tyr Leu His Gly Leu Ser 75 Val Ser Lys Lys Lys Ser Giu Ala Giu Leu Pro Ser Giu Ile Lys Ala 90 Lys Leu Asp Glu Pro Giy 115 Ala Ala Phe Giu Gin 100 Phe Lys Lys Asp Thr 105 Ala Giu Lys Lys Vai 125 Leu Pro Thr 110 Giu Giu Ala Lys Lys Val Ala Giu 120 Lys Lys Lys 130 Ala Giu Asp Gin 135 Lys Glu Lys Asp Leu Arg 140 Asn Tyr Pro Thr Asn 145 Thr Tyr Lys Thr Leu 150 Glu Leu Asp Ile Ala Glu Ser Asp Val 155 160 WO 99/51188 WO 9951188PCT/US99/07669 43 Giu Val Lys Lys Ala Glu Leu Giu Leu 165 Ser Arg Asp Giu Lys Lys Ile Asn Gin 180 185 Vai 170 Lys Glu Giu Ala Lys Giu 175 Ala Lys Ala Lys Vai Glu Asn 190 Lys Lys Ala 195 Glu Ala Thr Arg Leu Lys Asn Ile Lys Thr Asp Arg Giu 200 205 Lys Ala 210 Asn Val 225 Glu Giu Ala Lys Arg Arg Ala Asp Ala 215 Lys Leu Gin Glu Ala 220 Ala Tbhr Ser Glu Gin Asp Lys Ser 230 Lys 235 Arg Arg Ala Lys Arg 240 Giu Val Leu Gly Giu 245 Leu Ala Thr Pro Asp 250 Lys Lys Glu Asn Asp Ala 255 Lys Ser Ser Asp Ser Ser 260 Vai Gly Glu Giu 265 Thr Leu Thr Ser Pro Ser 270 Leu Lys Pro 275 Giu Lys Lys Vai Ala Glu Ala Giu Lys Lys Val Glu Glu 280 285 Ala Lys 290 Pro Thr 305 LYS LYS Ala Glu Asp Gin Lys Glu Giu Asp Arg Arg'Asn Tyr 295 300 Asn Thr Tyr Lys Thr Leu Glu Leu Giu Ile Ala Giu Ser Asp 310 315 320 WO 99/51188 WO 9951188PCTIUS99/07669 44 Val Giu Val Lys Lys Ala Giu Leu Giu 325 Leu Val Lys Glu Glu 330 Ala Lys 335 Glu Ser Arg Ser Lys Lys 355 Asn Giu Glu Lys Ile Lys 340 345 Gin Vai Lys Ala Lys Val Giu 350 Thr Asp Arg Ala Giu Ala Thr Arg 360 Leu Giu Asn Ile Lys 365 Lys Lys 370 Lys Val 385 Ala Giu Glu Giu Giu 375 Lys Giu Lys Pro Aia 390 Ala Lys Arg Arg Aia 380 Giu Gin Pro Gin Pro 395 Ala Giu Glu Asp Ala Pro Ala Pro 400 Gin Pro Giu Lys Pro Thr Giu Glu Pro Giu Asn Pro Ala Pro Ala Pro 405 410 415 Ala Pro Lys Pro Glu Asn Pro Ala Giu Lys Pro Lys Ala Giu Lys Pro 420 425 430 Ala Asp Gin Gin Ala 435 <210> <211> 439 <212> PRT WO 99/51188 PCT/US99/07669 45 <213> Streptococcus pneumoniae <400> Val Ala Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu 1 5 10 Lys Glu Val Thr Thr Gln Val Ala Thr Ser Ser Asn Arg Ala Asn Glu 25 Ser Gln Ala Gly His Arg Lys Ala Ala Glu Gln Phe Asp Glu Tyr Ile 40 Lys Thr Met Ile Gln Leu Asp Arg Arg Lys His Thr Gin Asri Phe Ala 55 Leu Asn Ile Lys Leu Ser Arg Ile Lys Thr 70 Glu Tyr Leu Arg Lys Leu 75 Asn Val Leu Glu Glu Lys Ser Lys Ala Glu 90 Leu Pro Ser Glu Thr Lys Lys Glu Ile Asp Ala Ala Phe Glu Gln 100 10.5 Thr Val Ala Glu Ala Glu 120 Phe Lys Lys Asp Thr Asn Arg 110 Thr Lys Lys 115 Lys Lys Val Glu 125 Glu Ala Lys Lys Lys 130 Ala Lys Ala Gln Lys Glu Glu Asp His Arg Asn Tyr Pro Thr 135 140 WO 99/51188 WO 9951188PCTIUS99/07669 46 Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile 145 150 Ala Glu Ser Asp Val Glu 155 160 Val Lys Lys Ala Arg Asp Asp Glu 180 Lys Ala Glu Ala 195 Glu Leu Glu Leu Val Lys 165 170 Glu Glu Ala Lys Glu Ser 175 LYS Ile Lys Gln Ala 185 Glu Ala Lys Val Glu Ser Lys 190 Thr Arg Leu Glu 200 Asn Ile Lys Thr Asp 205 Ala Glu Ala Lys Leu 220 Arg Glu Lys Ala Glu Glu Glu Ala Lys Arg Arg 210 215 Lys Glu Ala Val Glu Lys Asn Val Ala Thr Ser Giu Gin 225 230 Asp Lys Pro Lys Gly Arg 235 240 Arg Lys Arg Gly Asn Asp Ala Lys 260 Ser Pro Ser Leu 275 Val Pro Gly Glu Gln Ala 245 250 Thr Pro Asp Lys Lys Glu 255 Ser Ser Asp Ser Lys Pro Glu Lys 280 Ser 265 Val Gly Glu Glu Ala Leu Pro 270 Lys Val Ala Glu Ala Glu Lys Lys 285 Val Ala Glu Ala Glu Lys Lys Ala Lys Ala Gln Lys Glu GlU Asp Arg WO 99/51188 WO 9951188PCT/US99/07669 47 290 300 Arg Asn Tyr Pro Thr Asn Thr Thyr Lys Thr 305 310 Leu 315 Giu Leu Glu Ile Ala 320 Giu Ser Asp Val Lys Val Lys Glu Ala Glu 325 330 Ser Arg Asn Glu Glu Lys 345 Leu Glu Leu Val Lys Glu 335 Glu Ala Lys Glu 340 Val Asn Gin Ala Lys Ala 350 Lys Val Giu Ser Lys Lys Ala Glu Ala Thr Arg Leu Giu Lys Ile Lys 355 360 365 Thr Asp 370 Arg Lys Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala Ala Glu 375 380 Glu Asp Lys 385 Val Lys Glu Lys Pro Ala Giu 390 Gin Pro Gin Pro Ala Pro 395 400 Ala Pro Gin Pro Glu Lys Pro Thr Glu Glu Pro Glu Asn Pro Ala Pro 405 410 415 Ala Pro Lys Pro Glu Lys Pro Ala Glu Gin Pro Lys Ala Glu Lys Thr 420 425 430 Asp Asp Gin Gin Ala Giu Glu 435 WO 99/51188 WO 9951188PCT/US99/07669 -48- <210> 31 <211> 419 <212> PRT <213> Streptococcus pneumoniae <400> 31 Ala Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu Asn 10 Glu Gly Thr Thr Gin Ala Pro Thr Ser Ser Asn Arg Gly Asn Glu Ser 25 Gin Ala Glu His Met Lys Ala Ala Lys Gin Vai Asp Glu Tyr Ile Glu 40 Lys Met Leu Gin Leu Asp Arg Arg Lys His Thr Gin Asn Val Gly Leu 55 Leu Thr Lys Leu Gly Ala Ile Lys Thr Giu Tyr Leu Arg Gly Leu Ser 70 75 Val Ser Lys Glu Lys Ser Thr Ala Giu Leu Pro Ser Giu Ile Lys Glu 90 Lys Leu Thr Ala Ala Phe Lys Gin Phe Lys Lys Asp Thr Leu Lys Pro 100 105 110 Giu Lys Lys Vai Ala Giu Ala Glu Lys Lys Val Ala Glu Ala Lys Lys WO 99/51188 WO 99/1 188PCTIUS99/07669 49 115 Lys Ala Glu Asp Gin Lys 130 125 Giu Glu Asp Arg Arg 135 Leu Giu Ile Ala Giu 155 Asn 140 Tyr Pro Thr Ile Thr Tyr Lys Thr Leu 145 Lys Lys Ala Giu Leu 165 Asp Giu Giu Lys Ile 180 Glu 150 Ser Asp Val Giu Val 160 Ala Asn Giu Pro Arg 175 Giu Leu Val Lys Val Lys 170 Lys Gin Ala Glu Ala Giu Val Giu Ser Lys Lys 185 190 Ala Glu Ala Thr Arg Leu Lys Lys Ile Lys Thr Asp 195 200 Glu Glu Glu Ala Lys Arg 210 Ser Lys Arg Arg Lys Ser 225 230 Ala Thr Pro Asp Lys Lys 245 Arg Val Asp Ala Lys Giu 215 220 Arg Vai Lys Arg Giy Asp 235 Arg Giu Lys Ala 205 Gin Asp Giu Ser Val Gly Giu Gin 240 Giu Asn Asp Ala Lys Ser Ser Asp Ser Ser 250 255 Val Giy Giu Giu Thr Leu Pro Ser Pro Ser Leu Lys Pro Gly Lys Lys 260 265 270 WO 99/51188 WO 9951188PCT/US99/07669 50 Val Ala Giu 275 Ala Giu Lys Lys Val Glu Giu Ala AspLys Lys Ala Lys 280 285 Ala Gin 290 Lys Glu Giu Asp Arg Arg Asn Tyr Pro Thr Asn Thr Tyr Lys 295 300 Thr Leu Giu Leu Glu 305 Ile Ala Glu Ser Asp Val Glu. Val Lys Lys Ala 310 315 320 Glu Leu Giu Leu Lys Glu Glu Ala Lys Glu Pro Arg Asn 330 Glu Giu 335 Lys Val Lys Thr Arg Leu 355 Gin 340 Ala Lys Ala Giu Val 345 Giu Ser Lys Lys Ala Giu Ala 350 Glu Glu Giu Giu Lys Ile Lys Thr 360 Asp Arg Lys Lys Ala 365 Ala Lys 370 Arg Lys Ala Ala Glu 375 Giu Asp Lys Val Lys Glu Lys Pro Ala 380 Glu 385 Gin Pro Lys Pro Ala Pro 390 Ala Pro Gin Pro Giu 395 Lys Pro Ala Pro 400 Lys Pro Giu Asn Pro Ala Giu Gin Pro Lys Ala Glu Lys Pro Ala Asp 405 410 415 Gin Gin Ala WO 99/51188 WO 9951188PCT/US99/07669 51 <210> <211> <212> <213> Streptococcus pneumoniae <400> 32 Val Ala Ser Leu 1 Phe Met Gly Ser 5 Val Ala Thr Ser Val Thr Thr Thr Glu His Lys Leu Gin Gin Val Val His Ala Thr Glu Lys Glu 10 Ser Asn Lys Ala Asn Lys Ser Gin 25 Gin Val Asp Glu Tyr Ile Lys Lys His Thr Gin Asn Val Gly Leu Leu Met Lys Ala Ala Lys 40 Leu Asp Arg Arg Lys 55 Thr Lys Leu Gly Val Ile Lys Thr Giu Tyr Leu His Giy Leu Ser Val 70 75 Ser Lys Lys Lys Ser Glu Ala Glu Leu Pro Ser Glu Ile Lys Ala Lys 90 Leu Asp Ala Ala Phe Giu Gin Phe Lys Lys Asp Thr Leu Pro Thr Giu 100 105 110 WO 99/51188 PCT/US99/07669 52- Pro Gly Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu Ala Lys 115 120 125 Lys Lys 130 Ala Glu Asp Gin Lys Glu Lys Asp Leu 135 Arg Asn Tyr Pro Thr 140 Asn 145 Thr Tyr Lys Thr Leu 150 Glu Leu Asp Ile Ala 155 Glu Ser Asp Val Glu 160 Val Lys Lys Ala Glu 165 Arg Asp Glu Lys Lys 180 Leu Glu Leu Val Lys 170 Ile Asn Gin Ala Lys 185 Glu Glu Ala Lys Glu Ser 175 Ala Lys Val Glu Asn Lys 190 LYS Ala Glu Ala Thr Arg Leu 195 Lys Asn Ile Lys Thr Asp Arg Glu Lys 200 205 Ala Glu Glu Ala Lys Arg 210 Val Ala Thr Ser Glu Gin 225 230 Val Phe Gly Glu Leu Ala 245 Arg 215 Ala Asp Ala Lys Leu Gin Glu Ala Asn 220 Asp Lys Ser Lys Arg 235 Thr Pro Asp Lys Lys 250 Arg Ala Lys Arg Glu 240 Giu Asn Asp Ala Lys 255 Ser Ser Asp Ser Ser Val Gly Glu Glu Thr Leu Thr Ser Pro Ser Leu 260 265 270 WO 99/51188 WO 99/1 188PCTIUS99/07669 53 Lys Pro Giu Lys Lys Val Ala 275 Giu Ala Giu Lys Lys 280 Val Giu Giu Ala 285 Arg Asn Tyr Pro Lys Lys Lys Ala Giu Asp 290 Gln 295 Lys Giu Giu Asp Arg 300 Thr 305 Asn Thr Tyr Lys Thr 310 Leu Giu Leu Giu Ile 315 Leu Giu Leu Val Lys 330 Ala Glu Ser Asp Val 320 Glu Val Lys Lys Ala Giu 325 Glu Glu Ala Lys Glu 335 Ser Arg Asn Giu Glu Lys Ile Lys 340 Gin Val LYS Ala Lys 345 Val Glu Ser 350 ASP Arg Lys Lys Lys Ala Giu Ala Thr Arg 355 Leu 360 Glu Asn Ile Lys Thr 365 Lys Ala 370 Giu Glu Glu Giu Ala .375 Ala Glu 390 Lys Arg Arg Ala Ala Giu Glu Asp Lys 380 Val 385 Lys Giu Lys Pro Gin Prp Gin Pro Ala 395 Pro Ala Pro Gin 400 Pro Giu Lys Pro Thr 405 Giu Giu Pro Glu Asn Pro Ala Pro Ala Pro Ala 410 415 Pro Lys Pro Glu Asn Pro Ala Giu Lys Pro Lys Ala Glu Lys Pro Ala WO 99/51188 WO 99/1 188PCTfUS99/07669 54 420 Asp Gin Gin Ala Glu 435 <210> 33 <211> 433 <212> PRT <213> Streptococcus pneumoniae 425 <400> 33 Cys Thr Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu 1 5 10 Asn Glu Arg Thr Thr Gin- Val Pro Glu Arg Arg Lys Ala Ala Glu Gin 40 Ile Gin Leu Asp Lys Arg Lys His 55 Gin Leu Ser Arg Ile Lys Thr Glu 70 Thr Ser Ser Asn Arg Gly Lys Pro 25 Phe Asp Glu Tyr Thr Gin Asn Leu Tyr Leu Asn Gly 75 Ile Asn Lys Met Ala Phe Asn Ile Leu Lys Giu Lys Ser Glu Ala Glu Leu Pro Ser Lys Ile Lys Ala Glu Leu Asp Ala Ala WO 99/51188 PCTIUS99/07669 55 Phe Lys Gin Phe Lys Lys Asp Thr Leu Pro Thr Giu Pro Giu Lys Lys 100 105 110 Val Ala Giu Ala Glu Lys Lys Val Glu Glu Ala Glu 115 120 Lys Lys Val Ala 125 Giu Ala Lys Lys Lys Ala 130 Lys Ala Gin Lys Giu 135 Glu 140 Asp His Arg Asn Ile Ala Giu Phe 160 Tyr 145 Pro Thr Ile Thr 150 Lys Thr Leu Asp Leu Glu 155 Asp Val Lys Val Lys 165 Asp Glu Ser Arg Asn 180 Glu Ala Giu Leu Glu 170 Glu Gly Thr Ile Asn 185 Leu Val Lys Lys Glu Ala 175 Gin Ala Lys Ala Lys Val 190 Giu Ser Giu Lys Ala Giu Ala 195 Thr Arg Leu Lys Lys 200 Ile Lys Thr Asp 205 Ala Asp Ala Lys Arg Giu 210 Lys Ala Glu Glu Glu Ala Lys Arg Arg 220 Giu 225 Gin Asp Glu Ser Lys 230 Arg Arg Lys Ser Arg 235 Gly Lys Arg Gly Ala 240 Leu Giy Glu Gin Ala Thr Pro Asp Lys Lys Giu Asn Asp Ala Lys Ser WO 99/51188 WO 9951188PCT[US99/07669 56 255 Ser Asp Ser Ser 260 Pro Gly Lys Lys 275 Val. Gly Giu Glu Thr Leu Pro Ser Pro Ser Leu Lys 265 270 Val Ala Giu Ala Giu Lys Lys Val 280 Glu Giu Ala Asp 285 Lys Lys Ala Lys Ala Gin Lys Glu Glu Asp Arg Arg 290 295 300 Asn Tyr Pro Thr Asn Thr Tyr Lys Thr 305 Val Lys Giu Ala Glu Leu Glu Leu Glu Ile 310 Leu Glu Leu Val. Lys 330 Ile Lys Gin Ala Lys 345 Ala 315 Giu Ser Asp Val. Lys 320 Giu Glu Ala Lys Giu Ser 335 Ala Lys Va. Giu Ser Lys 350 Arg Asn Glu Glu Lys 340 Lys Ala Giu 355 Ala Thr Arg Leu Giu Lys Ile Lys Thr Asp Arg Lys Lys 360 365 Ala Giu 370 Glu Lys 385 Glu Glu Ala Lys Arg Lys Ala Ala Glu 375 Pro Ala Glu Gin Pro Gin Pro Ala Pro 390 395 Glu Asp Lys Val. Lys 380 Ala Pro Gin Pro Giu 400 WO 99/51188 WO 99/1 188PCTIUS99/07669 57 Lys Pro Ala Glu Glu Pro Glu Asn Pro Val Pro Ala Pro Lys Pro Glu 405 410 415 Asn Pro Ala Glu Gin Pro Lys Ala Glu Lys Pro Ala Asp Gin Gin Ala 420 425 430 Giu <210> <211> <212> <213> 34 427

PRT

Streptococcus pneumoniae <400> 34 Val Ala Val Ala Ser Leu Val Met Gly 1 5 Lys Glu Val Thr Thr Gin Val Pro Thr 25 Glu His Arg Lys Ala Ala Lys Gin Val.

40 Ser Val Val His Ala Thr Glu 10 Tyr Ser Asn Met Ala Lys Thr Val Asp Glu Tyr Ile Glu Lys Met Leu Arg Glu Ile Gin Leu Asp Arg Arg Lys His Thr Gin Asn Phe 55 Ala Phe Asn Met Lys Leu Ser Ala Ile Lys Thr Glu Tyr Leu Tyr Gly WO 99/51188 WO 99/1 188PCTIUS99/07669 58 Leu Lys Glu Lys Ser Glu Ala Giu Leu Pro Ser Giu Val Lys Ala Lys 90 Leu Asp Ala Ala Phe Glu Gin Phe Lys Lys Asp Thr Leu Lys Leu Gly 100 105 110 Glu Lys Val Ala Glu Ala 115 Ala Lys Ala Gin Lys Glu 130 Giu Lys Lys Val Ala Glu 120 Glu Asp Arg Arg Asn Tyr 135 140 Glu Ile Ala Glu Ser Asp 155 Ala Glu Lys Lys 125 Pro Thr Asn Thr Vai Glu Val Lys 160 Tyr Lys Thr Leu Glu 145 Leu 150 Lys Ala Glu Leu Glu 165 Leu Leu Lys Glu Glu Ala Lys Thr Arg Asn Glu .170 175 Asp Thr Ile Asn Gin Ala Lys Ala Lys Val Giu Ser Lys 180 185 Ala Thr Lys Leu Giu Glu Ile Lys Thr Asp Arg Lys Lys 195 200 205 Lys Ala Glu 190 Ala Glu Glu Glu Ala Lys Arg Lys Ala Glu 210 215 Ala Glu Giu Asp Lys Val Lys Asp Lys 220 WO 99/51188 WO 9951188PCT/US99/07669 59 Leu Lys Arg Arg Thr Lys Arg Ala Val Pro 225 230 Asp Lys Lys Glu Asn Asp Ala Lys Ser Ser 245 250 Giu Thr Leu Pro Ser Pro Ser Leu Lys Ser 260 265 Gly Giu Pro Ala Thr Pro 235 240 Asp Ser Ser Val Gly Glu 255 Gly Lys Lys Val Ala Giu 270 Ala Glu Lys Lys Val Ala Glu 275 Ala Giu Lys Lys Ala Lys Asp Gin Lys 280 285 Glu Glu Asp Arg Arg Asn 290 Tyr 295 Pro Thr Asn Thr Tyr Lys Thr Leu Asp 300 Leu 305 Glu Ile Ala Glu Ser 310 Asp Val Lys Val Lys 315 Giu Ala Glu Leu Giu 320 Leu Val Lys Giu Glu Aia 325 Lys Gly Ser Arg 330 Asn Glu Glu Lys Ile Asn 335 Gin Ala Lys Ala Giu Val Giu Ser Lys 340 34.5 Glu Lys Ile Lys Thr Asp Arg Lys Lys 355 360 Lys Ala Glu Ala Thr Arg Leu 350 Ala Glu Glu Giu Ala Lys Arg 365 Lys Ala Ala Giu Giu Asp Lys Val Lys Giu Lys Pro Ala Glu Gin Pro 370 375 380 WO 99/51188 WO 9951188PCTIUS99/07669 Gin Pro Ala Pro Ala 385 Asn Pro Ala Pro Ala 405 Ala Glu Lys Thr Asp 420 Pro Gin Pro Glu Lys Pro Thr Giu Glu Pro Glu 390 395 400 Pro Lys Pro Glu Lys Pro Ala Glu Gin Pro Lys 410 415 Asp Gin Gin Ala Glu Giu 425 <210> <211> 413 <212> PRT <213> Streptococcus pneumoniae <400> Giu Asn Giu Gly Ser Thr Gin Ala Ala Thr Ser Ser Asn Met Ala Lys 1 5 1.0 Thr Glu His Arg Lys Ala Ala Lys Gin Val Val Asp Giu Tyr Ile Glu 25 Lys Met Leu Arg Glu Ile Gin Leu Asp Arg Arg Lys His Thr Gin Asn 40 Val Ala Leu Asn Ile Lys Leu Ser Ala Ile Lys Thr Lys Tyr Leu Arg 55 WO 99/51188 WO 99/1 188PCT/US99/07669 61 Giu Leu Asn Val Leu Glu Glu Lys Ser Lys 70 Ile Lys Ala Lys Leu Asp Ala Ala Phe Giu 90 Asp Giu Leu Pro Ser Glu 75 Lys Phe Lys Lys Asp Thr Leu Lys Pro Gly Glu Lys Val Ala 100 Giu Ala Lys Lys Lys Val Glu Glu 105 110 Ala Lys Lys 115 Lys Ala Giu Asp Gin 120 Lys Giu Glu Asp Arg Arg Asn Tyr 125 Pro Thr 130 Asn Thr Tyr Lys Thr 135 Leu Giu Leu Glu Ile.Ala Giu Phe Asp 140 Lys Val Lys Glu Ala Glu 150 Leu Giu Leu Val 155 Lys Glu Giu Ala Lys 160 Lys.Giu Lys Val Giu 175 Glu Ser Arg Asn Glu Gly Thr Ile Lys Gin Ala 165 170 Ser Lys Lys Ala Giu Ala Thr Arg Leu Glu Asn Ile Lys Thr Asp Arg 180 18,5 190 Lys Lys Ala Giu Giu Glu Ala Lys Arg Lys Ala Asp Ala Lys Leu Lys 195 200 205 Giu Ala Asn Val Ala Thr Ser Asp Gin Giy Lys Pro Lys Gly Arg Ala 210 215 220 WO 99/51188 WO 99/1 188PCT/US99/07669 62 Lys Arg Gly Val Pro Giy Giu Leu Ala Thr 225 230 Asp Ala Lys Ser Ser Asp Ser Ser Val Gly 245 250 Pro Asp Lys Lys Glu 235 Asn 240 Giu Giu Thr Leu Pro Ser 255 Ser Ser Leu Lys Ser Gly Lys Lys Val 260 265 Glu Glu Ala Glu Lys Lys Ala Lys Asp 275 280 Ala Giu Ala Gin Lys Glu Glu Lys Lys Val 270 Glu Asp Arg Arg 285 Glu Ile Ala Glu Val Lys Giu Glu 320 Asn Tyr 290 Pro Thr Asn Thr Tyr Lys Thr Leu Asp Leu 295 300 Glu Ala Glu Leu Glu Leu 315 Ser 305 Asp Val Lys Val Ala Lys Giu Pro Arg 325 Vai Glu Ser Lys Lys 340 Asp Giu Glu Lys Ile Lys Gin Ala Lys 3 30 Ala Lys 335 Ala Glu Ala Thr Arg Leu Glu Asn 345 Ile Lys Thr 350 Asp Arg Lys Lys Ala Giu Glu Glu Ala Lys Arg Lys Ala 355 360 365 Ala Glu Glu Asp Lys Val Lys Glu Lys Pro Ala Glu Gin Pro Gin Pro Ala Pro Ala WO 99/51188 WO 9951188PCT/US99/07669 63 380 Thr Gin Pro Giu Lys Pro Ala Pro Lys Pro Giu Lys Pro Ala Giu Gin 385 390 395 400 Pro Lys Ala Giu Lys Thr Asp Asp Gin Gin Ala Glu Glu 405 410 <210> <211> <212> <213> 36 425

PRT

Streptococcus pneuxnoniae <400> 36 Tyr Ile Ala Ser Leu Phe- Leu Giy Gly Val Val His Ala Giu Gly Val 1 5 10 Arg Ser Giu Asn Asn Pro Thr Val Lys Lys Tyr Ala Asp Giu Val Lys 40 Thr Ser Ser Gly Gin Asp Ile Ser 25 Ser His Leu Giu Lys Ile Leu Ser Glu Ile Gin Thr Asn Leu Asp Arg Ser Lys His Ile Lys Thr Val Asn 55 Leu Ile Asn Lys Leu Gin Asp Ile Lys Arg Thr Tyr Leu Tyr Giu Leu 70 75 WO 99/51188 WO 9951188PCT/US99/07669 64 Asn Val Leu Giu Asp Lys Ser Lys Ala Ala Giu Leu Asp Ala Ala Phe Giu Gin 100 105 Thr Giu Pro Gly Lys Lys Val Ala Giu Glu Leu Pro Ser Lys Ile Lys 90 Phe Lys Lys Asp Thr Leu Pro 110 Ala Lys Lys Lys Val Giu Giu 125 Giu Glu Asp Tyr Arg Asn Tyr 140 115 Ala Giu Lys Lys Ala Lys Ala Gin Lys 130 135 Pro Thr Ile Thr Tyr 145 Lys Thr Leu Giu Leu Giu 150 155 Ile Ala Giu Ser Val Lys Val Lys Glu Ser Arg Asn 180 Giu 165 Ala Glu Leu Giu Leu Vai 170 LYS Lys Giu Ala Asp 175 Giu Gly Thr Ile Asn 185 Gin Ala Lys Ala Lys Val Giu 190 Ser Glu Gin Ala Giu Ala Thr Arg Le~u Lys Lys Ile Lys Thr Asp Arg 205 195 Giu Lys Ala Giu Giu Giu Ala Lys Arg Arg Ala Asp Ala Lys Giu Gin 210 215 220 Asp Giu Ser Lys Arg Arg Lys Ser Arg Val Lys Arg Gly Asp Phe Gly WO 99/51188 WO 99/1 188PCT/US99/07669 65 240 Giu Pro Ala Thr Pro Asp Lys Lys Giu Asn Asp Ala Lys Ser Ser Asp 245 250 255 Ser Ser Val Giy Giu Giu Thr Leu 260 Pro Ser Pro Ser Leu Lys Pro Gly 265 270 Lys Lys Val Ala Giu Ala Giu Lys Lys Val Glu Glu 275 280 Ala Lys Asp Gin Lys Glu Glu Asp His Arg Asn Tyr 290 295 300 Ala Giu Lys Lys 285 Pro Thr Ile Thr Tyr Lys Thr Leu Glu 305 Leu Giu Ile 310 Ala Giu Ser Asp 315 Val Giu Val Lys Ala Giu Leu Giu 325 Glu Giu Lys Val. Lys 340 Leu Vai Lys Giu Glu Ala Lys Gly Ser Arg Asn 330 335 Gin Ala Lys Ala Glu Vai Glu Ser Lys Lys Ala 345 350 Giu Ala Thr Arg Leu Giu Lys 355 Giu Giu Ala Lys Arg Lys Ala 370 375 Ile Lys Thr Asp Arg 360 Ala Giu Giu ASP Lys 380 Lys Lys Ala Glu 365 Val Lys Glu Lys WO 99/51188 WO 9951188PCTIUS99/07669 66 Pro Ala Glu Gin Pro 385 Ala Pro Ala Pro Lys 405 Lys Pro Ala Asp Gin 420 Gin Pro Ala Pro Ala 390 Pro Giu Asn Pro Ala 410 Pro Gin Pro Giu Lys Pro 395 400 Glu Gin Pro Lys Ala Giu 415 Gin Ala Glu Glu 425 <210> 37 <211> 439 <212> PRT <213> Streptococcus pneumoniae <400> 37 Ala Ser Leu Phe Leu Gly Gly Val Vai 1 5 His Ala Giu Gly Val 10 Arg Ser Giy Asn Asn Ser Thr Val Thr Ser Ser Gly Gin Asp Ile 25 Tyr Ala Asp Glu Val Giu Ser His Leu Gin Ser Ile Leu 40 Asn Lys Asn Leu Lys Lys Val Gin His Thr Gin Asn Ala 55 Ser Lys Lys Lys Asp Val Asp Phe Asn Lys Lys Leu Ser Lys Ile Lys Thr Lys Tyr Leu Tyr Glu Leu Asn Val WO 99/51188 WO 99/1 188PCTIUS99/07669 67 Leu Giu Giu Lys Ser Glu Ala Glu Leu Thr 90 Thr Ala Ala Phe Giu Gin 105 Ser Lys Thr Lys Giu Thr Lys Glu Giu Leu 100 Phe Lys Lys Asp Thr Leu 110 Ser Thr Glu 115 Glu Ala Lys 130 Pro Giu Lys Lys Val Ala Glu Ala Lys Lys Lys Val Glu 120 125 Lys Lys Ala Glu Asp Gln Lys Giu 135 Lys Asp Arg Arg Asn 140 Tyr Pro Thr Ile Thr 145 Tyr Lys Thr Leu Glu 150 Leu Glu Ile Ala Glu Ser 155 160 Leu Val Lys Val Lys Ala 175 Asp Val Glu Val Lys Ala Glu Leu Glu 170 Asn Glu Pro Glu Ser Lys 195 Arg 180 Asp Giu Giu Lys Ile 185 Lys Gin Ala Glu Ala Lys Val 190 Leu Lys Lys Ile Lys Thr Asp 205 Gin Ala Glu Ala Thr Arg 200 Arg Glu Gin 210 Ala Giu Ala Thr Arg Leu Glu Asn Ile Lys Thr Asp Arg 215 220 WO 99/51188 WO 9951188PCT/US99/07669 68 Glu Gin Ala Glu Glu Glu Ala Lys Val Lys 225 230 Asp Glu Pro Lys Lys Arg 235 240 Thr Pro Asp Lys Lys Glu 255 Thr Lys Arg Gly Val Leu Gly Giu Pro Ala 245 250 Asn Asp Ala Ser Pro Ser 275 Ser Ser Asp Ser Ser Val Gly Giu Glu 265 Thr Leu'Pro 270 Giu Lys Lys Leu Lys Pro Glu Lys 280 Lys Val Ala Glu Ala 285 Val Giu Giu Ala Lys Lys Lys Ala 290 295 Asn Tyr Pro Thr Asn Thr Tyr 310 Giu ASP Gin Lys 300 Lys Thr Leu Glu 315 Glu Giu AspArg Leu Giu Ile Ala 320 Giu Ser Asp Val Glu Val Lys Lys Ala Giu Leu Giu Leu Val Lys Glu 325 330 335 Glu Ala Lys Glu Pro Arg Asn Glu Giu Lys Val Lys Gin Ala Lys Ala 340 34.5 350 Giu Val Glu Ser Lys Gin Ala Giu Ala Thr Arg Leu 355 360 Thr Asp Arg Lys Lys Ala Glu Glu Glu Ala Lys Arg 370 375 380 Glu Asn Ile Lys 365 Lys Ala Ala Giu WO 99/51188 WO 9951188PCTIUS99/07669 69 Glu Asp Lys Val Lys Glu Lys Pro Ala Glu 385 390 Ala Pro Gin Pro Glu Lys Pro Ala Pro Lys 405 410 Gin Pro Gin Pro Ala 395 Pro 400 Pro Glu Lys Pro Ala Pro 415 Ala Pro Lys Pro Glu Asn Pro Ala Glu 420 425 Gln Pro Lys Ala Glu Lys Pro 430 Ala Asp. Gin 435 Gin Ala Glu Glu <210> 38 <211> 460 <212> PRT <213> Streptococcus pneuxnoniae <400> 38 Cys Ile Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu 1 5 10 Asn Glu Gly Ala Thr Gin Val Pro Thr Ser Ser Asn Arg Ala Asn Glu 25 Ser Gin Ala Glu Gin Gly Glu Gin Pro Lys Lys Leu Asp Ser Glu Arg 40 WO 99/51188 WO 9951188PCTIUS99/07669 70 Asp Lys Ala Arg Lys Giu Giu Ser Tyr Ala Lys Ser 70 Leu Val Asn Glu Leu Asn Val Giu Ser Thr Ser Giu 100 Val Glu Giu Tyr Val 55 Thr Lys Lys Arg His Lys Lys Ile Vai Gly Thr Ile Thr Val Ala Asn Ile Lys Asn Glu 90 Ser Gin Leu Gin Ile 105 Tyr Leu Asn Lys Ile Leu Met Met Glu Ser 110 Arg Ser Lys Vai Asp Giu Ala Val Ser Lys Phe Glu Lys Asp Ser Ser 115 120 125 Ser Ser Ser Ser Ser Asp- Ser Ser Thr Lys Pro 130 135 Glu Ala Ser Asp Thr 140 Ala Lys Pro Asn Lys 145 LYS Lys Lys Val Glu 165 Glu Asp Arg Arg Asn 180 Pro Thr Glu Pro Gly 150 Giu 155 Lys Val Ala Glu Ala 160 Glu Ala Giu Lys Lys 170 Ala LYS Asp Gin Lys Glu 175 Tyr Pro Thr Ile 185 Thr Tyr Lys Thr Leu Glu Leu 190 Lys Lys Ala Glu Leu Glu Leu 205 Giu Ile Ala Glu Ser Asp Val Glu Val 195 200 WO 99/51188 WO 9951188PCT/US99/07669 71 Vai Lys Val Lys Ala Asn 210 Giu Pro Arg Asp 215 Ser Lys Gin Ala Giu Gin Lys Ile Lys Gin 220 Glu Ala Thr Arg Leu Lys 235 240 Ala Glu Ala Giu Val 225 Lys Ile Lys Thr Asp 245 Ala Asp Ala Lys Glu 260 Glu 230 Arg Glu Giu Ala Glu 250 Glu Glu Ala Lys Arg Arg 255 Arg Gly Gin Gly Lys Pro Lys Gly Arg Ala Lys 265 270 Val Pro Gly Glu Leu Ala Thr Pro Asp Lys Lys Glu Asn 275 280 .285 Asp Ala Lys Ser Ser Asp Ser Ser Val 290 Gly Glu Glu Thr Leu 295 Pro Ser Pro Ser Leu 300 Lys Val Glu Glu Ala 320 LYS Pro Glu Lys Lys 305 Val1 310 Ala Giu Ala Glu Lys 315 Lys Lys Lys Ala Thr Asn Thr Tyr 340 Glu Asp Gin Lys Gu Glu 325 330 Lys Thr Leu Giu Leu Glu 345 Asp Arg Arg Asn Tyr Pro 335 Ile Ala Giu Ser Asp Val 350 Glu Val Lys Lys Ala Glu Leu Giu Leu Val Lys Glu Glu Ala Lys Glu WO 99/51188 WO 99/1 188PCT/US99/07669 72 Pro Arg Asn Glu Glu Lys Val 370 375 Lys Lys Ala Glu Ala Thr Arg 385 390 Lys Gin Ala Lys Ala Glu Val Glu Ser 380 Leu Glu Lys Ile 395 Lys Thr Asp Arg Lys 400 Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala 405 410 Lys Glu Lys Pro Ala Glu Gln Pro Gin Pro 420 425 Ala Glu Glu Asp Lys Val 415 Ala Pro Ala Pro Lys Ala 430 Glu Lys Pro Ala Pro Ala Pro Lys Pro Glu Asn Pro Ala Glu Gin Pro 435 440 445 Lys Ala Glu Lys Pro Ala Asp Gin Gin Ala Glu Glu 450 455 460 <210> 39 <211> 459 <212> PRT <213> Streptococcus pneumoniae <400> 39 Ile Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu Asn 1 5 10 WO 99/51188 WO 9951188PCT/US99/07669 73 Giu Gly Ala Gin Ala Giu Lys Ala Arg Thr Gin Val Pro Thr Gin Gly Giu Gin Pro 40 Lys Giu Val Giu Giu 55 Ser Ser Asn Arg Ala Asn Giu Ser 25 Lys Lys Leu Asp Ser Glu Arg Asp Tyr Val Lys Lys Ile Val Giy Giu Ser Tyr Ala Lys Ser Thr Lys Lys Arg His Thr Ile Thr Vai Ala Leu 70 75 Val Asn Giu Leu Asn Asn Ile Lys Asn Giu Tyr Leu Asn Lys Ile Val 90 Ile Leu Met Met Giu Ser Arg 110 Giu Ser Thr Ser Lys Val 115 Ser Ser Ser 130 Ser 100 Giu Ser Gin Leu Gin 105 Asp Giu Ala Val Ser 120 Ser Asp Ser Ser Thr 135 Lys Phe Giu Lys Asp Ser Ser Ser 125 Lys Pro Giu Ala Ser Asp Thr Ala 140 Lys Pro Asn Lys Pro Thr Giu Pro Gly Giu Lys Val Ala Giu Ala Lys 145 150 155 160 Lys Lys Val Giu Glu Val Giu Lys Lys Ala Lys Asp Gin Lys Giu Giu WO 99/51188 WO 99/1 188PCT/US99/07669 74 170 Asp Arg Arg Asn Tyr Pro Thr Ile Thr Tyr Lys Thr Leu Giu Leu Glu 180 185 190 Ile Ala Glu Ser Asp Val Glu 195 Lys Val Lys Ala Asn Glu Pro 210 215 Glu Ala Giu Val Giu Ser Lys 225 230 Ile Lys Thr Asp Arg Glu Giu 245 Val Lys Lys Ala Giu Leu Glu Leu Val 200 205 Arg Asp Lys Gin Lys Ile Lys Gin Ala 220 Gin Aia Glu Ala 235 Ala Glu Glu Giu 250 Thr Arg Leu Lys Lys 240 Ala Lys Arg Arg Ala 255 Asp Ala Lys Giu Gin Gly Lys Pro Lys Gly Arg Pro Lys Arg Gly Val 260 265 270 Pro Gly Glu Leu Ala Thr. Pro Asp Lys Lys Giu Asn Asp Ala Lys Ser 275 280 285 Ser Asp Ser Ser Val Gly Glu 290 295 Pro Glu Lys Lys Val Ala Glu 305 310 Glu Thr Leu Pro Ser Pro Ser Leu Lys 300 Ala Giu Lys Lys 315 Val Giu Glu Ala Lys 320 WO 99/51188 WO 9951188PCT[US99/07669 75 Lys Lys Ala Glu ASP *Gin Lys Glu Glu 325 Asn Thr Tyr Lys Thr Leu Glu Leu Glu 340 345 Asp Arg Arg Asn Tyr Pro Thr 330 335 le Ala Glu Ser Asp Val Glu 350 Val Lys Lys Ala Glu Leu Glu 355 Leu Val Lys Glu Glu Ala Lys Glu Pro 360 365 Arg Lys 385 Asn Glu Glu Lys Val 370 Ala Glu Ala Thr Arg 390 Lys 375 Gin Ala Lys Ala Leu Glu Lys Ile Lys 395 Arg Lys Ala Ala Glu 410 Glu Val Glu Ser Lys 380 Thr Asp Arg Lys Lys 400 Glu Asp Lys Val Lys 415 Ala Glu Glu Glu Ala Lys 405 Glu Lys Pro Ala Glu Gin Pro Gin Pro Ala Pro Ala Pro Lys Thr Glu 420 425 430 Lys Pro Ala Pro Ala Pro Lys Pro Glu Asn Pro Ala Glu Gin Pro Lys 435 440 445 Ala Glu Lys Pro Ala Asp Gin Gin Ala Glu Glu 450 455

Claims (108)

1. An isolated polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1, 3, or 24, wherein said polypeptide does not bind to choline.

2. The isolated polypeptide of claim 1, wherein said amino acid sequence comprises up to 475 amino acids.

3. The isolated polypeptide of claim 1, wherein said amino acid sequence comprises up to 460 amino acids.

4. An isolated polypeptide comprising a variant of an amino acid sequence set forth in SEQ ID NO: 1, 3, or 24, wherein said amino acid sequence comprises at least one to 57 amino acid substitutions of SEQ ID NO: 1, 3, or 24, does not bind to choline and is immunogenic. An isolated polypeptide comprising an analog or a derivative of an amino acid sequence set forth in SEQ ID NO: 1, 3, or 24, wherein said polypeptide does not bind to choline, and is immunogenic.

6. An isolated polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 5, wherein said polypeptide does not bind to choline.

7. The isolated polypeptide of claim 6, wherein said amino acid sequence comprises up to 475 amino acids.

8. The isolated polypeptide of claim 6, wherein said amino acid sequence comprises up to 460 amino acids. S 30 9. An isolated polypeptide comprising a variant of an amino acid sequence set forth in SEQ ID NO: 5, wherein said variant comprises at least one to 57 **amino acid substitutions of SEQ ID NO: 5, comprises up to 398 amino acids, does not bind to choline, and is immunogenic. W:\ciska\nki\speces\34797c.doc An isolated polypeptide comprising an analog or a derivative of the amino acid sequence set forth in SEQ ID NO: 5, wherein said polypeptide does not bind to choline and is immunogenic.

11. An isolated polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4 or 22, wherein said polypeptide does not bind to choline.

12. The isolated polypeptide of claim 11, wherein said amino acid sequence comprises up to 475 amino acids.

13. The isolated polypeptide of claim 11, wherein said amino acid sequence comprises up to 460 amino acids.

14. An isolated polypeptide comprising a variant of an amino acid sequence set forth in SEQ ID NO: 4 or 22, wherein said variant comprises at least one to 57 amino acid substitutions of SEQ ID NO: 4 or 22, comprises up to 398 amino acids, does not bind to choline, and is immunogenic. An isolated polypeptide comprising an analog or derivative of an amino acid sequence set forth in SEQ ID NO: 4 or 22, wherein said polypeptide does not bind to choline and is immunogenic. 00 16. The isolated polypeptide of claim 1, 2, 3, 4, or 5, wherein said polypeptide interacts with an antibody, wherein said antibody is capable of interacting with a full-length CbpA polypeptide.

17. The isolated polypeptide of claim 6, 7, 8, 9, or 10, wherein said polypeptide interacts with an antibody, wherein said antibody is capable of interacting with a full-length CpbA polypeptide.

18. The isolated polypeptide of claim 11, 12, 13, 14, or 15, wherein said polypeptide interacts with an antibody, wherein said antibody is capable of interacting with a full-length CpbA polypeptide. W:\ciska\nki\species34797c.doc 71

19. An isolated polypeptide comprising a fragment of SEQ ID NO: 24, wherein said fragment comprises at least 138 consecutive amino acids of SEQ ID NO: 24, wherein said polypeptide does not bind to choline.

20. An isolated polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 1,3, 4, 5, 22, or 24.

21. An isolated polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 7 or 9, wherein said polypeptide does not bind to choline, and comprises up to 376 amino acids.

22. An isolated polypeptide comprising a variant of an amino acid sequence set forth in SEQ ID NO:7 or 9, wherein said variant comprises at least one to 57 amino acid substitutions of SEQ ID NO: 7or 9, does not bind to choline and is immunogenic.

23. An isolated polypeptide comprising an analog or a derivative of an amino acid sequence set forth in SEQ ID NO: 7 or 9, wherein said polypeptide does not bind to choline, comprises up to 376 amino acids, and is immunogenic.

24. An isolated polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 10, 11, or 23, wherein said polypeptide does not bind to choline, and comprises up to 328 amino acids.

25. An isolated polypeptide comprising a variant of an amino acid sequence set forth in SEQ ID NO: 10, 11, or 23, wherein said variant comprises at least one to 57 amino acid substitutions of SEQ ID NO: 10, 11, or 23, does not bind to choline, and is immunogenic. S 30 26. An isolated polypeptide comprising an analog or a derivative of an amino acid sequence set forth in SEQ ID NO: 10, 11, or 23, wherein said polypeptide does not bind to choline, comprises up to 328 amino acids, and is immunogenic. W:\ciska\nki\speces\34797c.doc 72

27. An isolated polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 7, 9, 10, 11, or 23.

28. The isolated polypeptide of claim 21, 22, 23, 24, 25, or 26, wherein said polypeptide interacts with an antibody, wherein said antibody is capable of interacting with a full-length CbpA polypeptide.

29. The isolated polypeptide of claim 1, 2, 3, 6, 7, 8, 11, 12, 13, 21, or 24, wherein said polypeptide is immunogenic. The isolated polypeptide of claim 4, 9, 14, or 22, wherein said amino acid substitutions comprise conservative amino acid substitutions.

31. The isolated polypeptide of any of claims 1-15, and 19-27, wherein said polypeptide has lectin activity.

32. The isolated polypeptide of any of claims 1-5, wherein the polypeptide is made by cleaving a full length choline binding protein A with hydroxylamine wherein the hydroxylamine cleaves the choline binding protein A at amino acid 475 thereby creating the N-terminal choline binding protein A truncate.

33. The isolated polypeptide of claim 5, 10, 15, 23, or 26, wherein the analog comprises an N-terminal methionine or an N-terminal polyhistidine. 0

34. A pharmaceutical composition comprising an isolated antibody that specifically binds to a polypeptide comprising an amino acid sequence of an N- terminal choline binding protein A truncate, wherein said isolated antibody does not bind to a choline binding domain and said isolated antibody is substantially free of antibodies that bind to the choline binding domain.

35. The isolated antibody of claim 34, wherein said amino acid sequence D NO: 7, 9, 10, 11, or 23. comprises SEQ ID NO: 7, 9, 10, 11, or23. W:\dskanki\species\34797c.doc 73

36. The isolated antibody of claim 34, wherein said amino acid sequence comprises SEQ ID NO: 1, 3, 4, 5, 22, or 24.

37. The isolated antibody of claim 34, 35, or 36, wherein the antibody is a monoclonal antibody.

38. The isolated antibody of claim 34, 35, or 36, wherein the antibody is a polyclonal antibody.

39. The isolated antibody of claim 34, 35, or 36, wherein the antibody is a chimeric (bispecific) antibody. A pharmaceutical composition comprising the polypeptide of any of claims 1-15 or 19-27, and a pharmaceutically acceptable carrier or diluent.

41. A method of inducing an immune response in a subject which has been exposed to or infected with a pneumococcal bacterium comprising administering to the subject an amount of a pharmaceutical composition comprising a polypeptide having an amino acid sequence of an N-terminal choline binding protein A truncate which does not bind to choline thereby inducing an immune response.

42. The method of claim 41, wherein said amino acid sequence comprises •SEQ ID NO: 7, 9, 10, 11, or23.

43. The method of claim 41, wherein said amino acid sequence comprises the polypeptide of claim 21 or 24.

44. The method of claim 41, wherein said amino acid sequence comprises 30 the polypeptide of claim 22 or

45. The method of claim 41, wherein said amino acid sequence comprises the polypeptide of claim 23 or 26. W:\ciska\nki\speces\34797c.doc

46. The method of claim 41, wherein said amino acid sequence comprises SEQ ID NO: 1,3, 4, 5, 22, or 24.

47. The method of claim 41, wherein said'amino acid sequence comprises a polypeptide of claim 2, 3, 7, 8, 12, 13, or 19.

48. The method of claim 41, wherein said amino acid sequence comprises a polypeptide of claim 4, 9, or 14.

49. The method of claim 41, wherein said amino acid sequence comprises a polypeptide of claim 5, 10, or The method of claim 41, 42, or 46, wherein a protective immune response is induced.

51. A method for preventing infection by a pneumococcal bacterium in a subject comprising administering to the subject an amount of a pharmaceutical composition effective to prevent pneumococcal bacterium attachment, thereby preventing infection by the pneumococcal bacterium, wherein said pharmaceutical composition comprises a polypeptide having an amino acid sequence of an N-terminal choline binding protein A truncate which does not bind to choline.

52. The method of claim 51, wherein said amino acid sequence comprises SEQ ID NO: 7, 9, 10, 11, or 23.

53. The method of claim 51, wherein said amino acid sequence comprises the polypeptide of claim 21 or 24. S 30 54. The method of claim 51, wherein said amino acid sequence comprises the polypeptide of claim 22 or 0 W:\ciska\nki\spedes\34797c.doc The method of claim 51, wherein said amino acid sequence comprises the polypeptide of claim 23 or 26.

56. The method of claim 51, wherein said amino acid sequence comprises SEQ ID NO: 1,3, 4, 5, 22, or 24.

57. The method of claim 51, wherein said amino acid sequence comprises the polypeptide of claim 2, 3, 7, 8, 12, 13, or 19.

58. The method of claim 51, wherein said amino acid sequence comprises the polypeptide of claim 4, 9 or 14.

59. The method of claim 51, wherein said amino acid sequence comprises the polypeptide of claim 5, 10, or A method for preventing infection by a pneumococcal bacterium in a subject comprising administering to the subject an amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and an antibody capable of binding to a polypeptide comprising an amino acid sequence of an N-terminal choline binding protein A truncate wherein said antibody does not bind to a choline binding domain and said pharmaceutical composition is substantially free of antibodies that bind the choline binding domain, thereby preventing infection by a pneumococcal bacterium.

61. The method of claim 60, wherein said pharmaceutical composition is delivered to the respiratory tract or the nasopharynx.

62. The method of claim 60, wherein said antibody is capable of binding to the polypeptide having the amino acid sequence comprising SEQ ID NO: 7, 9, S 30 10, 11, or 23.

63. The method of claim 60, wherein said antibody is capable of binding to the polypeptide of claim 21 or 24. W:\ciska\nki\species\34797c.doc 76

64. The method of claim 60, wherein said antibody is capable of binding to the polypeptide of claim 22 or The method of claim 60, wherein said antibody is capable of binding to the polypeptide of claim 23 or 26.

66. The method of claim 60, wherein said antibody is capable of binding to the polypeptide having the amino acid sequence comprising SEQ ID NO: 1, 3, 4, 5, 22, or 24.

67. The method of claim 60, wherein said antibody is capable of binding to the polypeptide of claim 2, 3, 7, 8, 12, 13, or 19.

68. The method of claim 60, wherein said antibody is capable of binding to the polypeptide of claim 4, 9 or 14.

69. The method of claim 60, wherein said antibody is capable of binding to the polypeptide of claim 5, 10, or

70. A vaccine comprising a polypeptide and a pharmaceutically acceptable adjuvant or carrier, wherein said polypeptide comprises an amino acid sequence of an N-terminal choline binding protein A truncate which does not bind to choline.

71. The vaccine of claim 70, wherein the polypeptide comprises the amino acid sequence as set forth in any of SEQ ID NO: 7, 9, 10, 11, or 23.

72. The vaccine of claim 70, wherein the polypeptide comprises the amino acid sequence of claim 21 or 24.

73. The vaccine of claim 70, wherein the polypeptide comprises the amino acid sequence claim 22 or W:\ciska\nki\species\34797c.doc 77

74. The vaccine of claim 70, wherein the polypeptide comprises the amino acid sequence of claim 23 or 26. The vaccine of claim 70, wherein the polypeptide comprises the amino acid sequence as set forth in any of SEQ ID NO: 1, 3, 4, 5, 22, or 24.

76. The vaccine of claim 70, wherein the polypeptide comprises the amino acid sequence as set forth in claim 2, 3, 7, 8, 12, 13, or 19.

77. The vaccine of claim 70, wherein the polypeptide comprises the amino acid sequence as set forth in claim 4, 9 or 14.

78. The vaccine of claim 70, wherein the polypeptide comprises the amino acid sequence as set forth in claim 5, 10 or

79. The vaccine of claim 70, wherein the polypeptide comprises the amino acid sequence of a N-terminal choline binding protein A truncate as set forth in Figure 2.

80. A vaccine comprising a polypeptide having an amino acid sequence which comprises a conserved region as set forth in Figure 2 and a pharmaceutically acceptable adjuvant or carrier wherein said polypeptide does not bind to choline.

81. The vaccine of claim 80, wherein the conserved region is selected from the group consisting of amino acid sequence 158 to 172; 300 to 321; 331 to 339; 355 to 365; 367 to 374; 379 to 389; 409 to 427; and 430 to 447.

82. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 1, 3, or 24, wherein said polypeptide does not bind to choline. 00o0 W:\ciskanklspedes\34797c.doc 78

83. The isolated nucleic acid molecule of claim 82, wherein said nucleotide sequence comprises SEQ ID NO: 12 or 14.

84. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a variant of SEQ ID NO: 1, 3, or 24, wherein said variant comprises at least one to 57 amino acid substitutions of SEQ ID NO: 1, 3, or 24, does not bind choline, and is immunogenic. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 22 or 4, wherein said polypeptide does not bind to choline.

86. The isolated nucleic acid molecule of claim 85, wherein said nucleotide sequence comprises SEQ ID NO:

87. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a variant of SEQ ID NO: 22 or 4, wherein said variant comprises at least one to 57 amino acid substitutions of SEQ ID NO: 22 or 4, does not bind to choline and is immunogenic.

88. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 5, wherein said polypeptide does not bind to choline.

89. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a variant of SEQ ID NO: 5, wherein said variant comprises at least one to 57 amino acid substitutions of SEQ ID NO: does not bind to choline, is immunogenic, and comprises up to 398 amino acids. The isolated nucleic acid molecule of claim 89, wherein said nucleotide sequence comprises SEQ ID NO: 16. W:\ciska\nki\species\34797c.doc 79

91. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 138 consecutive amino acids of SEQ ID NO: 24, wherein said polypeptide does not bind to choline.

92. A nucleic acid molecule comprising a nucleotide sequence having at least 318 consecutive nucleotides of SEQ ID NO: 12, wherein said nucleotide sequence encodes a polypeptide that does not bind choline.

93. An isolated nucleic acid molecule consisting of SEQ ID NO: 12, 14, 15, or 16.

94. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 7 or 9, wherein said polypeptide comprises up to 376 amino acids and does not bind to choline. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a variant of SEQ ID NO: 7 or 9, wherein said variant comprises at least one to 57 amino acid substitutions of SEQ ID NO: 7 or 9, comprises up to 376 amino acids, does not bind choline, and is immunogenic.

96. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide set forth in SEQ ID NO: 10, 11, or 23, wherein said polypeptide comprises up to 328 amino acids and does not bind to choline.

97. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a variant of SEQ ID NO: 10, 11, or 23, wherein said variant comprises at least one to 57 amino acid substitutions of SEQ ID NO: 10, 11, or 23, comprises up to 328 amino acids, does not bind S" 30 choline, and is immunogenic. o W:\dska\nki\spedes34797c.doc

98. An isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 17 or 19, wherein said nucleotide sequence encodes a polypeptide that does not bind to choline.

99. 21. An isolated nucleic acid molecule consisting of SEQ ID NO: 17, 19, 20, or

100. An isolated nucleic acid molecule comprising a nucleotide sequence set forth in SEQ ID NO: 20 or 21, wherein said nucleotide sequence encodes a polypeptide having up to 328 amino acids and wherein said polypeptide does not bind to choline.

101. The isolated nucleic acid molecule of any of claims 82-100, wherein the nucleic acid molecule is DNA.

102. The isolated nucleic acid molecule of any of claims 82-100, wherein the nucleic acid molecule is cDNA. 9. 9O 9**9 9.. 99 9

103. The isolated nucleic acid molecule of any of claims 82-100, 20 nucleic acid molecule is genomic DNA.

104. The isolated nucleic acid molecule of any of claims 82-100, nucleic acid molecule is RNA. wherein the wherein the 9* 99 9 9 999* 9 *999 99 9 99*9 999*

105. An isolated nucleic acid molecule of any of claims 82-100, wherein said nucleic acid molecule is operatively linked to a promoter.

106. The isolated nucleic acid molecule of claim 105, wherein said promoter is a promoter of RNA transcription.

107. A vector comprising the nucleic acid molecule of any of claims 82-100. W:\cska\nki\speces\34797c.doc 81

108. The vector of claim 107, wherein said vector further comprises a promoter.

109. The vector of claim 107, wherein the vector is a plasmid, a cosmid, a yeast artificial chromosome (YAC), a bacteriophage or a eukaryotic viral DNA.

110. A host vector system for the production of a polypeptide which comprises the vector of claim 107 in a suitable host cell.

111. The host vector system of claim 110, wherein the suitable host cell comprises a prokaryotic or a eukaryotic cell.

112. A cell comprising the vector of claim 107.

113. A method of obtaining a polypeptide in purified form comprising: a) introducing the vector of claim 107 into a suitable host cell; b) culturing the resulting host cell so as to produce the polypeptide; c) recovering the polypeptide produced in step and, d) purifying the polypeptide so recovered in step

114. A vaccine comprising an isolated nucleic acid molecule encoding a polypeptide and a pharmaceutically acceptable adjuvant or carrier, wherein said polypeptide comprises an amino acid sequence of an N-terminal choline binding protein A truncate that does not bind to choline.

115. The vaccine of claim 114, wherein said nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 12, 14, 15, or 16.

116. The vaccine of claim 114, wherein said nucleic acid molecule comprises the nucleotide sequence of claim 82, 85, 88, or 92.

117. The vaccine of claim 114, wherein said nucleic acid molecule comprises the nucleotide sequence of claim 84, 87, or 89. W:\iska\nkl\species\34797c.doc 82

118. The vaccine of claim 114, wherein said nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 17, 19, 20, or 21.

119. The vaccine of claim 114, wherein said nucleic acid molecule comprises the nucleotide sequence set forth in any of claim 94, 96, or 99.

120. The vaccine of claim 114, wherein said nucleic acid molecule comprises the nucleotide sequence of claim 95 or 97.

121. A vaccine comprising the vector of claim 107 and a pharmaceutically acceptable adjuvant or carrier.

122. A method for treating a subject infected with or exposed to pneumococcal bacterium comprising administering to the subject a therapeutically effective amount of the vaccine of any of claims 70-81 or 114- 121, thereby treating the subject.

123. An isolated polypeptide according to claim 1, 4, 5, 6, 9, 10, 11, 14, 19, 20, 21, 22, 23, 24, 25, 26 or 27, substantially as hereinbefore described.

124. A method according to claim 41, 51 or 60, substantially as hereinbefore described.

125. An isolated nucleic acid according to claim 82, 84, 85, 87, 88, 89, 91, 92, 95, 96, 97, 98 or 100, substantially as hereinbefore described. *0e* 0.0 0 00 :0.0 0 @000 0 *S S. egos 0* Sees .5 50 0 0 DATED: 4 July, 2003 PHILLIPS ORMONDE FITZPATRICK Attorneys for: ST JUDE CHILDREN'S RESEARCH HOSPITAL and MEDIMMUNE, INC. W:\ciskanki\species34797c.doc