Note: Descriptions are shown in the official language in which they were submitted.
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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.
)3ACKGROUND 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
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in animal models (Idanpaan-Heikkila, I. et al. (1997) J. Infect. Dis., 176:704-
712).
Particularly effective in this regard are soluble carbohydrates containing
lacto-N-
neotetraose 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
75 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. 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. etal.
(1987) Eur.
J. Biochem., 164:621-624 and McDaniel, L.S., etal. (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 N-
terminal 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.
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Choline binding proteins for anti-pneumococcal vaccines are discussed in PCT
International Application No. PCT/US97/07198. Current vaccines against S.
pneumoniae employ purified carbohydrates of the capsules of the 23 most common
serotypes of this purified bacterium, but such vaccine is only 50% protective
(Shapiro et al. NJEM 325:1453, 1991) and is mot 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.
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
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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
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set forth in Figure 2). 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
2); and C is from about amino acid 327 to amino acid 433 from the N-
terminus 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. Expression and purification of recombinant R1 and R2.
FIGURE 4. Results of passive protection in mice. Immune sera against
recombinant
R2 protected mice from lethal S. pneumoniae challenge.
FIGURE 5 Titration of anti-R2 antibody on R6x adhering to LNnT-HSA coated
plates.
FIGURE 6. Titration of anti-Cbp-A and absorbed anti-CbpA antibodies for
activity
blocking pnetunococcal adherence to LNnT-HSA coated plates.
FIGURE 7. 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
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
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polypeptide 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 KX.XE (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.
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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 (N) 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 (i.e. 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:
ENEGATQVPTSSNRANESQAEQGEQPICKLDSERDKARICEVEEYVICKIVGESY
AKSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAV
SKFEKDSSSSSSSDSSTKPEASDTAICPNKPTEPGEKVAEAKICKVEEAEKKAKD
QKEEDRRNYPTITYKTLELEIAESDVEVKKAELELVKVKANEPRDEQKIKQAE
AEVESKQAEATRLKKIKTDREEABEEAKRRADAKEQGKPKGRAKRGVPGEL
ATPDKKENDAKS SD S SVGEETLP SP SLKPEKKVAEAEKKVEEAKKKAEDQKE
EDRRNYPTNTYKTLELEIAESDVEVKKAELELVKEEAKEPRNEEKVKQAKAE
VESKKAEATRLEKIKTDRKICAEEEAKRKAAEEDKVKEKPAEQPQPAPAPKAE
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
AKSTKKRHTITVALVNELNNIKNEYLNKIVEST SESQLQILMMESRSKVDEAV
SKFEKDSSSSSSSDSSTKPEASDTAKPNKPTEPGEKVAEAKKKVEEAEKKAKD
QKEEDRRNYPTITYKTLELEIAESDVEVICKAELELVKVKANEPRDEQKIKQAE
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AEVESKQAEATRLICKIKTDREEAEEEAKRRADAKEQGKPKGRAKRGVPGEL
ATPDKKENDAKS SD S SVGEETLP SP SLKPEKKVAEAEKKVEEAKKKAEDQKE
EDRRNYPTNTYKTLELEIAESDVEVKKAELELVKEEAKEPRNEEKVKQAKAE
VESKKAEATRLEKIKTDRKKAEEEAKRKAAEEDKVKEKPA (SEQ ID NO 1).
The DNA sequence which encodes polypeptide R2 of the N-terminal truncate of
choline
binding protein A (CbpA) serotype type 4:
GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGA
AAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGA
GATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAAAATAGTGGGTG
AGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGCTCTA
GTTAACGAGTTGAACAACATTAAGAACGAGTATTTGAATAAAATAGTTGA
ATCAACCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAA
AAGTAGATGAAGCTGTGTCTAAGTTTGAAAAGGACTCATCTTCTTCGTCAA
GTTCAGACTCTTCCACTAAACCGGAAGCTTCAGATACAGCGAAGCCAAAC
AAGCCGACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTG
AAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAA
CTACCCAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCG
ATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAA
CGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAG
AGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTG
AAGAAGCAGAAGAAGAAGCTAAACGAAGAGCAGATGCTAAAGAGCAAG
GTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAAC
ACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTG
AAGAAACTCTTCCAAGCCCATCCCTGAAACCAGAAAAAAAGGTAGCAGA
AGCTGAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAA
GAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGAACT
TGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAG
TAAAAGAGGAAGCTAAGGAACCTCGAAACGAGGAAAAAGTTAAGCAAGC
AAAAGCGGAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAAA
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ATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCAG
CAGAAGAAGATAAAGTTAAAGAAAAACCAGCTG (SEQ ID NO 12).
Amino acid sequence of CbpA of serotype 4:
ENEGATQVPTS SNRANES QAEQGEQPKKLDSERDKARICEVEEYVKKIVGESY
AKSTKKRHTITVALVNELNNIKNEYLNKIVESTSESQLQILMMESRSKVDEAV
SKFEKDS S SS SS SDS STKPEASDTAKPNKPTEPGEKVAEAKKKVEEAEKKAKD
QKEEDRRNYPTITYKTLELE1AESDVEVKKAELELVKVKANEPRDEQKIKQAE
AEVESKQAEATRLKKIKTDREEAEEEAKRRADAKEQGKPKGRAKRGVPGEL
ATPDKKENDAKS SD S SVGEETLP SP SLKPEKKVAEAEKKVEEAKKKAEDQKE
EDRRNYPTNTYKTLELEIAESDVEVKKAELELVKEEAICEPRNEEKVKQAKAE
VESKKAEATRLEKIKTDRKICAEEEAKRKAAEEDKVKEKPAEQPQPAPAPItAE
KPAPAPKPENPAEQPKAEKPADQQAEEDYARRSEEEYNRLTQQQPPKTEKPA
QP S TPKTGWKQENGMWYFYNTDGSMATGWLQNNGSWYYLNSNGAMATG
WLQNNGS WYYLNANGSMATGWLQNNGS WYYLNANGSMATGVVLQYNGS
WYYLNANGSMATGWLQYNGSWYYLNANGDMATGWVKDGDTWYYLEAS
GAMKASQWFKVSDKWYYVNGSGALAVNTTVDGYGVNANGEWVN. (SEQ ID
NO2)
DNA sequence encoding the amino acid sequence of the CbpA of serotype 4:
GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGA
AAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGA
GATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAAAATAGTGGGTG
AGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGCTCTA
GTTAACGAGTTGAACAACATTAAGAACGAGTATTTGAATAAAATAGTTGA
ATCAACCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAA
AAGTAGATGAAGCTGTGTCTAAGTTTGAAAAGGACTCATCTTCTTCGTCAA
GTTCAGACTCTTCCACTAAACCGGAAGCTTCAGATACAGCGAAGCCAAAC
AAGCCGACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTG
AAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAA
CTACCCAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCG
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ATGT GGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAA
CGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAG
AGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTG
AAGAAGCAGAAGAAGAAGCTAAACGAAGAGCAGATGCTAAAGAGCAAG
GTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAAC
ACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTG
AAGAAACTCTTCC AAGCCCATCCCTGAAACCAGAAAAAAAGGTAGCAGA
AGCTGAGAAGAAGGTTGAAGAAGCTAAGAAAAAAGCCGAGGATCAAAAA
GAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGAACT
TGAAATTGCTGAGTCCGATGTGGAAGTTAAAAAAGCGGAGgCTTGAACTA
GTAAAAGAGGAAGCTAAGGAACCTCGAAACGAGGAAAAAGTTAAGCAAG
CAAAAGCGGAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAAAA
AATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAGCA
GCAGAAGAAGATAAAGTTAAAGAAAAACCAGCTGAACAACCACAACCAG
CGCCGGCTCCAAAAGCAGAAAAACCAGCTCCAGCTCCAAAACCAGAGAA
TCCAGCTGAACAACCAAAAGCAGAAAAACCAGCTGATCAAC AAGCTGAA
GAAGACTATGCTCGTAGATCAGAAGAAGAATATAATCGCTTGACTCAACA
GCAACCGCCAAAAACTGAAAAACCAGCACAACCATCTACTCCAAAAACA
GGCTGGAAACAAGAAAACGGTATGTGGTACTTCTACAATACTGATGGTTC
AATGGCGACAGGATGGCTCCAAAACAAtGGCTCAtGGTAcTACcTCAACAG
CAATGGCGCTATGGCGACAGGATGGCTCCAAAACAATGGTTCATGGTACT
ATCTAAACGCTAATGGTTCAATGGCAACAGGATGGCTCCAAAACAATGGT
TCATGGTACTACCTAAACGCTAATGGTTCAATGGCGACAGGATGGCTCCA
ATACAATGGCTCATGGTACTACCTAAACGCTAATGGTTCAATGGCGACAG
GAT GGC TC CAATAC AATGGC TCATGGTACTACCTAAAC GC TAATGGTGAT
ATGGCGACAGGTTGGGTGAAAGATGGAGATACCTGGTACTATCTTGAAGC
ATCAGGTGCTATGAAAGCAAGCCAATGGTTCAAAGTATCAGATAAATGGT
ACTATGTCAATGGCTCAGGTGCCCTTGCAGTCAACACAACTGTAGATGGC
TATGGAGTCAATGCCAATGGTGAATGGGTAAACTAA (SEQ ID NO 13).
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"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:
ENEGATQVPTS SNRANESQAEQGEQPKKLDSERDKARKEVEEYVKKIVGESY
AKSTKKRHTITVALVNELNNIKNEYLNKIVESTSES QLQILMMESRSKVDEAV
SKFEKDSSSSSSSDSSTKPEASDTAKPNKPTEPGEKVAEAKKKVEEAEKKAKD
QKEEDRRNYPTITYKTLELEIAESDVEVICKAELELVKVICANEPRDEQKIKQAE
AEVESKQAEATRLKKIKTDREEAEEEAKRRADAKEQGKPKGRAICRGVPGEL
ATPDKICENDAKSSDSSVGEETL (SEQ ID NO 3).
The DNA sequence which encodes polypeptide R1 is:
GAGAACGAGGGAGCTACCCAAGTACCCACTTCTTCTAATAGGGCAAATGA
AAGTCAGGCAGAACAAGGAGAACAACCTAAAAAACTCGATTCAGAACGA
GATAAGGCAAGGAAAGAGGTCGAGGAATATGTAAAAAAAATAGTGGGTG
AGAGCTATGCAAAATCAACTAAAAAGCGACATACAATTACTGTAGCTCTA
GTTAACGAGTTGAACAACATTAAGAACGAGTATTTGAATAAAATAGTTGA
ATCAACCTCAGAAAGCCAACTACAGATACTGATGATGGAGAGTCGATCAA
AAGTAGATGAAGCTGTGTCT'AAGTTTGAAAAGGACTCATCTTCTTCGTCAA
GTTCAGACTCTTCCACTAAACCGGAAGCTTCAGATACAGCGAAGCCAAAC
AAGCCGACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTG
AAGAAGCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTCGTAA
CTACC CAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCG
ATGTGGAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAA
CGAACCTCGAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAG
AGTAAACAAGCTGAGGCTACAAGGTTAAAAAAAATCAAGACAGATCGTG
AAGAAGCAGAAGAAGAAGCTAAACGAAGAGCAGATGCTAAAGAGCAAG
GTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAGCAAC
ACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTAGGTG
AAGAAACTCTTC (SEQ ID NO 14).
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"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:
KPEKKVAEAEKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAE SDVEVK
KAELELVKEEAKEPRNEEKVKQAICAEVESKKAEATRLEKIKTDRKKAEEEAK
RKA (SEQ ID NO 4).
The DNA sequence of polypeptide C/R2
AAACCAGAAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTA
AGAAAAAAGCCGAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAAC
CAATACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTGGAAG
TTAAAAAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCG
AAACGAGGAAAAAGTTAAGCAAGCAAAAGCGGAAGITGAGAGTAAAAAA
GCTGAGGCTACAAGGTTAGAAAAAATCAAGACAGATCGTAAAAAAGCAG
AAGAAGAAGCTAAACGAAAAGCA (SEQ ID NO 15)
"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:
TEPGEKVAEAKKKVEEAEKKAKDQKEEDRRNYPTITYKTLELEIAESDVEVK
KAELELVKVKANEPRDEQKIKQAEAEVESKQAEATRLKK1KTDREEAEEEAK
RRADA (SEQ ID NO 5). As shown in Figure 1, region A of polypeptide R2 is the
same
region A as within Rl.
The DNA sequence which encodes the polypeptide A/R2 is:
ACAGAACCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAA
GCTGAGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGTC GTAACTACC
CAACCATTACTTACAAAACGCTTGAACTTGAAATTGCTGAGTCCGATGTG
GAAGTTAAAAAAGCGGAGCTTGAACTAGTAAAAGTGAAAGCTAACGAAC
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CTC GAGACGAGCAAAAAATTAAGCAAGCAGAAGCGGAAGTTGAGAGTAA
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 2). These
molecules
include: the incorporation of codons "preferred" for expression by selected
non-
mammalian 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
_
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substituted 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:
ENEGSTQAATS SNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVAL
NIKLSAIKTKYLRELNVLEEKSKDELP S EIKAKLDAAFEICFKKDTLKPGEKVA
EAKKKVEEAKKKAEDQKEEDRRNYPINTYKTLELEIAEFDVKVKEAELELVK
EEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKRKADAICLK
EANVATSDQGKPKGRAKRGVPGELATPDKKENDAKS SDSSVGEETLPSSSLK
SGKKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTLDLEIAESDVKVKE
AELELVKEEAKEPRDEEKIKQAKAKVESKICAEATRLENIKTDRKKAEEEAKR
KAAEEDKVKEKPA (SEQ ID NO 7)
The DNA sequence which encodes polypeptide R2 serotype R6x:
GAAAACGAAGGAAGTACCCAAGCAGCCACTTCTTCTAATATGGCAAAGAC
AGAACATAGGAAAGCTGCTAAACAAGTCGTCGATGAATATATAGAAAAA
ATGTTGAGGGAGATTCAACTAGATAGAAGAAAACATACCCAAAATGTCGC
CTTAAACATAAAGTTGAGCGCAATTAAAACGAAGTATTTGCGTGAATTAA
ATGTTTTAGAAGAGAAGTCGAAAGATGAGTTGCCGTCAGAAATAAAAGCA
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AAGTTAGACGCAGCTTTTGAGAAG'TTTAAAAAAGATACATTGAAACCAGG
AGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAAA
GCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAATACTTA
CAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTAAAGAAG
CGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAAtCTCGAAACGAGGGC
ACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTGAGGCTA
CAAGGTTAGAAAACAtCAAGACAGAtCGTAAAAAAGCAGAAGAAGAAGCT
AAACGAAAAGCAGATGCTAAGTTGAAGGAAGCTAATGTAGCGACTTCAG
AtCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTA
GCAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGT
AGGTGAAGAAACTCTTCCAAGCTCATCCCTGAAATCAGGAAAAAAGGTAG
CAGAAGCTGAGAAGAAGGTTGAAGAAGCTGAGAAAAAAGCCAAGGATCA
AAAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTG
AC CTTGAAATTGCTGAGTCCGATGTGAAAGTTAAAGAAGCGGAGCTTGAA
CTAGTAAAAGAGGAAGCTAAGGAACCTCGAGACGAGGAAAAAATTAAGC
AAGCAAAAGCGAAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGA
AAACATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAA
GCAGCAGAAGAAGATAAAGTTAAAGAAAAACCAGCTG (SEQ ID NO 17)
Amino acid sequence of CbpA of serotype R6x:
ENEGSTQAATSSNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRICHTQNVAL
NIKLSATKTKYLRELNVLEEKSKDELP SEIKAKLDAAFEKFKKDTLKPGEKVA
EAKKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVICEAELELVK
EEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKRKADAKLK
EANVATSDQGKPKGRAKRGVPGELATPDKKENDAKS SD S SVGEETLP S S SLK
S GKKVAEAEKKVEEAEKKAKD QKEEDRRNYP TNTYKTLDLEIAESDVKVKE
AELELVICEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAEBEAKR
KAAEEDKVKEKPAEQPQPAPATQPEKPAPKPEKPAEQPKAEKTDDQQAEEDY
ARRSEEEYNRLTQQQPPKTEKPAQP STPKTGWKQENGMWYFYNTDGSMAT
GWLQNNGSVVYYLNANGAMATGWLQNNGSWYYLNANGSMATGWLQNNG
SWYYLNANGAMATGWLQYNGS WYYLNSNGAMATGWLQYNGSWYYLNA
....
_
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NGDMAT GWLQNNGS WYYLNANGDMATGWLQYNGS WYYLNANGDMATG
WVKDGDTWYYLEASGAMKAS QWFKVSDKWYYVNGSGALAVNTTVDGYG
VNANGEWVN (SEQ ID NO 8).
DNA sequence encoding the amino acid sequence of the CbpA of serotype R6x:
GAAAACGAAGGAAGTACCCAAGCAGCCACTTCTTCTAATATGGCAAAGAC
AGAACATAGGAAAGCTGCTAAACAAGTCGTC GAT GAATATATAGAAAAA
ATGTTGAGGGAGATTCAACTAGATAGAAGAAAACATACCCAAAATGTCGC
CTTAAACATAAAGTTGAGCGCAATTAAAACGAAGTATTTGCGTGAATTAA
ATGTTTTAGAAGAGAAGTC GAAAGATGAGTTGC CGTCAGAAATAAAAGCA
AAGTTAGACGCAGCTTTTGAGAAGTTTAAAAAAGATACATTGAAACCAGG
AGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAAA
GCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAATACTTA
CAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTAAAGAAG
CGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAAtCTCGAAACGAGGGC
ACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTGAGGCTA
CAAGGTTAGAAAACAtCAAGACAGAtCGTAAAAAAGCAGAAGAAGAAGCT
AAACGAAAAGCAGATGCTAAGTTGAAGGAAGCTAATGTAGCGACTtCAGA
tCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGCTAG
CAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGCGTA
GGTGAAGAAACTCTTCCAAGCTCATCCCTGAAATCAGGAAAAAAGGTAGC
AGAAGCTGAGAAGAAGGTTGAAGAAGCTGAGAAAAAAGCCAAGGATCAA
AAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGA
C CTTGAAATT GC TGAGT C C GATGTGAAAGTTAAAGAAGCGGAGCTTGAAC
TAGTAAAAGAGGAAGCTAAGGAACCTCGAGACGAGGAAAAAATTAAGCA
AGCAAAAGCGAAAGTTGAGAGTAAAAAAGCTGAGGCTACAAGGTTAGAA
AACATCAAGACAGATCGTAAAAAAGCAGAAGAAGAAGCTAAACGAAAAG
CAGCAGAAGAAGATAAAGTTAAAGAAAAACCAGCTGAACAACCACAACC
AGC GC CGGCTACTCAAC CAGAAAAA C CAGCTCCAAAAC CAGAGAAGC C A
GCTGAACAACCAAAAGCAGAAAAAACAGATGATCAACAAGCTGAAGAAG
ACTATGCTCGTAGATCAGAAGAAGAATATAATCGCTTGACTCAACAGCAA
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CC GCCAAAAACTGAAAAACCAGCACAAC CATCTACTC CAAAAACAGGCT
GGAAACAAGAAAACGGTATGTGGTACTTCTACAATACTGATGGTTCAATG
GCAACAGGATGGCTCCAAAACAACGGTTCATGGTACTATCTAAACGCTAA
TGGTGCTATGGCGACAGGATGGCTCCAAAACAATGGTTCATGGTACTATC
TAAACGCTAATGGTTCAATGGCAACAGGATGGCTCCAAAACAATGGTTCA
TGGTACTACCTAAACGCTAATGGTGCTATGGCGACAGGATGGCTCCAATA
CAATGGTTCATGGTACTACCTAAACAGCAATGGCGCTATGGCGACAGGAT
GGCTCCAATACAATGGCTCATGGTACTACCTCAACGCTAATGGTGATATG
GCGACAGGATGGCTCCAAAACAACGGTTCATGGTACTACCTCAACGCTAA
TGGTGATATGGCGACAGGATGGCTCCAATACAACGGTTCATGGTATTACC
TCAACGCTAATGGTGATATGGCGACAGGTTGGGTGAAAGATGGAGATACC
TGGTACTATCTTGAAGCATCAGGTGCTATGAAAGCAAGCCAATGGTTCAA
AGTATCAGATAAATGGTACTATGTCAATGGCTCAGGTGCCCTTGCAGTCA
ACACAACTGTAGATGGCTATGGAGTCAATGCCAATGGTGAATGGGTAAAC
TAA (SEQ ID NO 18).
Polypeptide R1 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:
ENEGSTQAATS SNMAKTEHRKAAKQVVDEYIEKMLREIQLDRRKHTQNVAL
NIKLSAIKTKYLRELNVLEEKSKDELP S EIKAKLDAAFEKFKKDTLKPGEKVA
EAKICKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVKEAELELVK
EEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKRKADAKLK
EANVATSDQGKPKGRAKRGVPGELATPDKKENDAKSSDSSVGEETL (SEQ ID
NO 9).
The DNA sequence which encodes polypeptide R1 is:
GAAAACGAAGGAAGTACCCAAGCAGCCACTTCTTCTAATATGGCAAAGAC
AGAACATAGGAAAGCTGCTAAACAAGTCGTCGATGAATATATAGAAAAA
ATGTTGAGGGAGATTCAACTAGATAGAAGAAAACATACCCAAAATGTCGC
CTTAAACATAAAGTTGAGCGCAATTAAAAC GAAGTATTTGCGTGAATTAA
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ATGTTTTAGAAGAGAAGTCGAAAGATGAGTTGCCGTCAGAAATAAAAGCA
AAGTTAGAC GCAGCTTTTGAGAAGTTTAAAAAAGATACATTGAAACCAGG
AGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGAAAAAA
GCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAATACTTA
CAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTAAAGAAG
CGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAATCTCGAAACGAGGG
CACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTGAGGCT
ACAAGGTTAGAAAACAtCAAGACAGATCGTAAAAAAGCAGAAGAAGAAG
CTAAACGAAAAGCAGATGCTAAGTTGAAGGAAGCTAATGTAGCGACTTCA
GATCAAGGTAAACCAAAGGGGCGGGCAAAACGAGGAGTTCCTGGAGAGC
TAGCAACACCTGATAAAAAAGAAAATGATGCGAAGTCTTCAGATTCTAGC
GTAGGTGAAGAAACTCTTC (SEQ ID NO 19).
"Polypeptide C/R2 serotype R6x" means an polypeptide comprising a repeat
region C
within R2 (see Figure 2), 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:
KS GICKVAEAEKKVEEAEKKAKDQKEEDRRNYP TNTYKTLDLEIAESDVKVK
EAELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAEEEAK
RKA (SEQ ID NO 10)
The DNA sequence of polypeptide C/R2 serotype R6x:
AAATCAGGAAAAAAGGTAGCAGAAGCTGAGAAGAAGGTTGAAGAAGCTG
AGAAAAAAGCCAAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAAC
CAATACTTACAAAACGCTTGACCTTGAAATTGCTGAGTCCGATGTGAAAG
TTAAAGAAGC GGAGCTTGAACTAGTAAAAGAGGAAGCTAAGGAACCTCG
AGACGAGGAAAAAATTAAGCAAGCAAAAGCGAAAGTTGAGAGTAAAAAA
GCTGAGGCTACAAGGTTAGAAAACATCAAGACAGATCGTAAAAAAGCAG
AAGAAGAAGCTAAACGAAAAGCA (SEQ ID NO 20).
_ _
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Polypeptide A/R2 serotype R6x" means an polypeptide comprising a repeat region
A
within R2 (see Figure 2), 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:
PGEKVAEAKKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAEFDVKVICE
AELELVKEEAKESRNEGTIKQAKEKVESKKAEATRLENIKTDRKKAEEEAKR
KADA (SEQ ID NO 11)
The DNA sequence which encodes the polypeptide A/R2 serotype R6x is:
CCAGGAGAAAAGGTAGCAGAAGCTAAGAAGAAGGTTGAAGAAGCTAAGA
AAAAAGCCGAGGATCAAAAAGAAGAAGATCGTCGTAACTACCCAACCAA
TACTTACAAAACGCTTGAACTTGAAATTGCTGAGTTCGATGTGAAAGTTA
AAGAAGCGGAGCTTGAACTAGTAAAAGAGGAAGCTAAAGAAtCTCGAAAC
GAGGGCACAATTAAGCAAGCAAAAGAGAAAGTTGAGAGTAAAAAAGCTG
AGGCTACAAGGTTAGAAAACAtCAAGACAGATCGTAAAAAAGCAGAAGA
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 ID NOS 22 or 23,
including
fragments, mutants, variants, or analogs, or derivatives, thereof.
SPSLKPEKKVAEAEKKVEEAKKKAEDQKEEDRRNYPTNTYKTLELEIAESDV
EVKKAELELVKEEAKEPRNEEKVKQAKAEVESKKAEATRLEKIKTDRKKAEE
EAICRKAAEEDKVKEKPA (SEQ ID NO 22; serotype 4; position 323-434); or
PSS SLKSGICKVAEAEKKVEEAEKKAKDQKEEDRRNYPTNTYKTLDLEIAESD
VKVKEAELELVKEEAKEPRDEEKIKQAKAKVESKKAEATRLENIKTDRKKAE
EEAKRKAAEEDKVKEKRA (SEQ ID NO 23, serotype R6x; position 322-434).
"Polypeptide B/R2" 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
-
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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 100CE (SEQ /1.13 NO 6).
This invention is directed to a polypeptide comprising an amino acid sequence
of a N-
terminal 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 defmed 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
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invention 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
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compound. 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-2-
nitrobenzene-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 "L" 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. NH2
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
amino-
terminus 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 (Na-amino protected Na-t-butyloxycarbonyl) amino acid resin with
the
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standard deprotecting, neutralization, coupling and wash protocols of the
original solid
phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the
base-
labile 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 (e.g., n-methyl amino acids, Ca-methyl
amino acids,
and Na-methyl amino acids, etc.) to convey special properties. Synthetic amino
acids
include omithine 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, 13 sheets, y-tums, and cyclic peptides can
be generated.
In one aspect of the invention, the peptides may comprise a special amino acid
at the C-
terminus which incorporates either a CO2H or CONH2 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, e.g., pyroglutamate, when
the
particular N-terminal group is present in 50% of the peptides, would readily
be
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demonstrated by comparing activity of a completely (100%) blocked peptide with
a non-
blocked (0%) peptide.
In addition, the present invention envisions preparing peptides that have more
well
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
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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 Meien.hofer, 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 cross-
links 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 at., 1991, J. Am. Chem. Soc. 113:2275-2283); (2S,3S)-methyl-
phenylalanine, (2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and
(2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, 1991, Tetrahedron Lett.);
2-
aminotetrahydronaphthalene-2-carboxylic acid (Landis, 1989, Ph.D. Thesis,
University
of Arizona); hydroxy-1,2,3,4-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); MC (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-turn inducing dipeptide analog (Kemp et
al., 1985,
J. Org. Chem. 50:5834-5838); (3-sheet inducing analogs (Kemp et al., 1988,
Tetrahedron
Lett. 29:5081-5082); (3-turn inducing analogs (Kemp et al., 1988, Tetrahedron
Lett.
29:5057-5060); ochelix inducing analogs (Kemp et al., 1988, Tetrahedron Lett.
29:4935-
4938); y-tuni 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
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turn 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 of peptides 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, e.g., myristoylated. In
another
embodiment an amino acid comprising an aliphatic side chain of the structure -
(CH2)nCH3 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 (i.e., 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 (i.e., 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
=
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protein. 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 NH2 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, e.g., Sambrook et al,
"Molecular
Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology"
Volumes [Ausubel, R. M., ed. (1994)]; "Cell Biology: A Laboratory Handbook"
Volumes I-PLI [J. E. Celis, ed. (1994))j; "Current Protocols in Immunology"
Volumes I-
.
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III [Coligan, J. E., ed. (1994)]; "Oligonucleotide Synthesis" (M.J. Gait ed.
1984);
"Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)];
"Transcription
And Translation" [B.D. Hames & S.J. Higgins, eds. (1984)]; "Animal Cell
Culture" [RI.
Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)1; 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 (0%) 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.
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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 21cDa and
about
1001cDa (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 (e.g., 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 (e.g., 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 (e.g., mono, di-, tri-, etc.), the molecular weight of the
polymer selected,
whether the polymer is branched or unbranched, and the reaction conditions.
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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, e.g., EP 0 401 384 (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
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limit it to any particular tertiary forms. Thus, this term includes double-
stranded DNA
found, inter alia, in linear DNA molecules (e.g., 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
(i.e., 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 (e.g., 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 (AV),
adeno-
associated 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
well-
known 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
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limited 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 NEH
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.g., E. coli
plasmids col El, pCR1, pBR322, pMB9 and their derivatives, plasmids such as
RP4;
phage DNAS, e.g., the numerous derivatives of phage A, e.g., NM989, and other
phage
DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such
as the
21.1 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 1, the control
regions of fd
coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic
enzymes, the
promoters of acid phosphatase (e.g., 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, R1.1, B-W and L-M cells, African Green Monkey
kidney
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cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., 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, e.g., 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.
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The term "antibody" includes, by way of example, both naturally occurring and
non-
naturally 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, e.g.,
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 (e.g.,
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, e.g., 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 B-
cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the
EBV-
hybridoma 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 germ-
free 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.
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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(ab1)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, e.g., 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 (e.g., 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.
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Antibodies can be labeled for detection in vitro, e.g., 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, e.g., 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 3H, 14C, 32P, 35S, Cl,36 "Cr, "Co, "Co, "Fe, "Y, 1251,
1311, and '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,13-glucuronidase,13-D-glucosidase, 13-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,
_
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e.g., "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) 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;
(b) other reagents; and
(c) 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,
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or 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, e.g., 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, e.g., 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
_
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treatment 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.
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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 (e.g., Tris-HC1., acetate, phosphate), pH and ionic strength,
additives such
as albumin or gelatin to prevent absorption to surfaces, detergents (e.g.,
Tween 20,
Tween 80, Pluronic F68, bile acid salts). solubilizing agents (e.g., glycerol,
polyethylene
glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),
preservatives (e.g.,
Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity
modifiers (e.g.,
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,
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a 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 (e.g., fatty acids, waxes, oils). Also comprehended by
the invention
are particulate compositions coated with polymers (e.g., 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
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emulsions, 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
lipoptiilic depots
(e.g. fatty acids, waxes, oils). Also comprehended by the invention are
particulate
compositions coated with polymers (e.g. 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
gg/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.
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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 "A" (where "A" is a single protein, DNA molecule,
vector,
etc.) is substantially free of "B" (where "B" 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 "A". Preferably, "A" comprises at least about 90% by
weight of the
A+B species in the composition, most preferably at least about 99% by weight.
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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, e.g., 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, intraderrnal, 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, mucosa' 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; i.e., 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 of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
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
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infusion, an implantable osmotic pump, a transdennal 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
(eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug
Bioavailability, Drug
Product Design and Performance, Smolen and Ball (eds.), 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, i.e., the brain,
thus requiring
only a fraction of the systemic dose (see, e.g., 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., i.e., 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 level's for treatment of various
conditions in various
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patients, 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 of ten 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; (2) soluble carbohydrate inhibitors of bacterial adhesin; (3)
other small
molecule inhibitors of bacterial adhesin; (4) inhibitors of bacterial
metabolism,
transport, or transformation; (5) stimulators of bacterial lysis, or (6) anti-
bacterial
antibodies or vaccines directed at other bacterial antigens. Other potential
active
components include anti-inflammatory agents, such as steroids and non-
steroidal anti-
inflammatory 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:
Fornngations
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Intravenous Formulation I
Ingredient mg/m1
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 ml
Intravenous Formulation II
Ingredient mg/m1
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
Ingredient ms/m1
gentamicin (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 mg/ml
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
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Intravenous Formulation V
Ingredient mg/ml
Polypeptide antagonist 5.0
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, i.e., 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 of Internal
Medicine,
Vol. I11, pp. 206-212 (1989) (al-antitrypsin); Smith et al., J. Clin. Invest.
84:1145-1146
(1989) (a- 1 -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
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addition 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 (e.g., 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
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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.,
Grit. 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, S.P., 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, e.g.,
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
_ _
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plasma 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 marmitol in amounts
which facilitate
dispersal of the powder from the device, e.g., 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.
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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. 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).
Liposonial encapsulation may be used and the liposomes may be derivatized with
various polymers (e.g., 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. 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
I material in the intestine.
Also specifically contemplated are oral dosage forms of the above derivatize,d
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 (a) inhibition of proteolysis; and (b) 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 mayle,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
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will 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 lmm. 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
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chloride. 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 (MC), ethyl cellulose (EC) and
carboxymethyl
cellulose (CMC). Polyvinyl pyrrolidone (P'VP) 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
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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
80, 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. 111,
pp. 206-212
(a!- 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 H, 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
,
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to 25 mg of biologically active protein per mL of solution. The formulation
may also
include a buffer and a simple sugar (e.g., 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 marmitol in amounts which facilitate
dispersal of the
powder from the device, e.g., 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.
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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 3'. 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 AU
GGC 3'. This primer incorporates a Sall 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 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 Sall
restriction enzymes and cloned into the pQE30 expression vector (Qiagen, Inc.)
digested
with BamHI, Xbal, and Smal restriction enzymes.
rolypeptide
The naturally occuring PvulI 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.-
_ -
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Polypeptide 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
pQE30 was digested with AatII and SmaI. 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 of R1 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 37 in 6.0 mls LB buffer with 5014/m1 lcanamycin and 1001.tg/m1 ampicillin.
This 6.0
ml culture was added to 1L LB with antibiotics at above concentrations. The
culture was
shaken at 37 C until A600 = -0.400. 1M IPTG was added to the 1L culture to a
final
concentration of 1mM. The culture was then shaken at 37 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 NaH2PO4, 10mM Tris, 6M GuC 1 ,
300mM NaC1, pH 8.0 (Buffer A). This mixture was rotated at room temperature
for 30
minutes and sonicated on a (VibraCell Sonicator (Sonics and Materials, Inc.,
Danbury,
CT) using the micro tip, two times, for 30 secs, at 50% Cuty Cycle and with
the output
setting at 7. The mixture was spun 5 min at 10K in a JA20 rotor and the
supernatant
removed and discarded. The supernatant was loaded onto a 10 ml Talon
(Clonetech, Palo
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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
NaH2PO4, 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 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 1).
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 gg/m1), then,
without
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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 1). In three separate experiments, CbpA full
length or
Polypeptide R2 competitively inhibited binding of pnetunococci 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 pnetunococci to 3' sialyl lactose
was not
inhibited by R2 (79 and 101%) compared to the full length CbpA (74 and 66%).
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 2). Given the high degree of
homology of
sequence between R1 and R2, it is further possible that both R1 and R2 are
needed for
lectin activity or that they are both lectin with slightly different
specificities ( sialic
acid).
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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) peiwell
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 (50)
Polypeptide 2461 74, 53, 65 2639 101
R2 1288 (672) 1670 (420) 79
1440 (530)
Polypeptide 3002 91, 92, 112 1052 40
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 2). Full
length
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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 90%
(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 -410 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.
_ _
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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 (64); 88 (4) 100%
Antibody to Truncate R2 56 (11); 9 (2) 28%; 10%
5 ill of rabbit antibody undiluted +5 ill 2 x 107 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 (5111) plus 5 1 of 2 x 107 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
5 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% of pneurnococcal 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
_
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assay 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. coli 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
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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(85%)
Anti CbpA antiserum 294 72 (52%)
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 Wangles.
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 (i.e.,
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 of pre-immune serum, a New
Zealand
white rabbit was immunized with 250 tig R2 containing both amino terminal
repeats
(preparation 483:58 above), in Complete Freund's Adjuvant. The rabbit was
given a
CA 02326388 2000-10-06
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PCT/US99/07669
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boost of 125 ps 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
I 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 4). 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 g in 50 Al PBS, plus 50 1 Complete Freund's Adjuvant). A
group
of 10 sham immunized mice received PBS and adjuvant. A second immunization was
administered four weeks later, 15 g 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
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PCT/US99/07669
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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 inununogen 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 1 of PBS containing 0.8 g of
polypeptide R2 or
R1 or no protein. 15 min later Type 3 pneumococci (Strain SIII) (10 pa
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 Sifi alone displayed 2200, 6500, 6900
and 8700
(mean 6075) colonies per 10 1. Animals treated with truncate R2 showed the
greatest
decrease (3600, 3500, 2500, 2100) to mean 2925 bacteria 10 1 (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.
CA 02326388 2006-10-26
-68-
.
D is cussion :
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
carbohydrates are recognized: LNnT by a peptide containing both N-terminal
repeats (A
and C) in Figure 1 and sialic acid by a peptide containing only the single
most N-
terminal repeat (A). The truncate containing the N-terminal repeat polypeptide
R1 and
R2 demonstrates lectin activity in cell culture assays as well.
Important features of polyp eptide R2 activity include: 1) complete
correlation of
bioactivity 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 (e.g. 6B and 2)
which is
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.
,
,
CA 02326388 2001-04-06
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SEQUENCE LISTING
<110> St. Jude Children's Research Hospital
Medimmune, Inc.
<120> A POLYPEPTIDE COMPRISING THE AMINO ACID OF AN N-TERMINAL CHOLINE
BINDING A TRUNCATE, VACCINE DERIVED THEREFROM AND USES THEREOF
<130> 2721-309 FC/gc
<140> 2,326,388
<141> 1999-04-07
<150> PCT/US99/07669
<151> 1999-04-07
<150> 60/080,878
<151> 1998-04-07
<150> 09/056,019
<151> 1998-04-07
<160> 39
<170> PatentIn Ver. 2.0
<210> 1
<211> 406
<212> PRT
<213> Streptococcus pneumoniae
<400> 1
Glu Asn Glu Gly Ala Thr Gin Val Pro Thr Ser Ser Asn Arg Ala Asn
1 5 10 15
Glu Ser Gin Ala Glu Gin Gly Glu Gin Pro Lys Lys Leu Asp Ser Glu
20 25 30
Arg Asp Lys Ala Arg Lys Glu Val Glu Glu Tyr Val Lys Lys Ile Val
35 40 45
Gly Glu Ser Tyr Ala Lys Ser Thr Lys Lys Arg His Thr Ile Thr Val
50 55 60
Ala Leu Val Asn Glu Leu Asn Asn Ile Lys Asn Glu Tyr Leu Asn Lys
65 70 75 80
Ile Val Glu Ser Thr Ser Glu Ser Gin Leu Gin Ile Leu Met Met Glu
85 90 95
Ser Arg Ser Lys Val Asp Glu Ala Val Ser Lys Phe Glu Lys Asp Ser
100 105 110
-
.
CA 02326388 2001-04-06
-68b-
Ser Ser Ser Ser Ser Ser Asp Ser Ser Thr Lys Pro Glu Ala Ser Asp
115 120 125
Thr Ala Lys Pro Asn Lys Pro Thr Glu Pro Gly Glu Lys Val Ala Glu
130 135 140
Ala Lys Lys Lys Val Glu Glu Ala Glu Lys Lys Ala Lys Asp Gln Lys
145 150 155 160
Glu Glu Asp Arg Arg Asn Tyr Pro Thr Ile Thr Tyr Lys Thr Leu Glu
165 170 175
Leu Glu Ile Ala Glu Ser Asp Val Glu Val Lys Lys Ala Glu Leu Glu
180 185 190
Leu Val Lys Val Lys Ala Asn Glu Pro Arg Asp Glu Gln Lys Ile Lys
195 200 205
Gln Ala Glu Ala Glu Val Glu Ser Lys Gln Ala Glu Ala Thr Arg Leu
210 215 220
Lys Lys Ile Lys Thr Asp Arg Glu Glu Ala Glu Glu Glu Ala Lys Arg
225 230 235 240
Arg Ala Asp Ala Lys Glu Gln Gly Lys Pro Lys Gly Arg Ala Lys Arg
245 250 255
Gly Val Pro Gly Glu Leu Ala Thr Pro Asp Lys Lys Glu Asn Asp Ala
260 265 270
Lys Ser Ser Asp Ser Ser Val Gly Glu Glu Thr Leu Pro Ser Pro Ser
275 280 285
Leu Lys Pro Glu Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu
290 295 300
Ala Lys Lys Lys Ala Glu Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr
305 310 315 320
Pro Thr Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala Glu Ser Asp
325 330 335
Val Glu Val Lys Lys Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys
340 345 350
Glu Pro Arg Asn Glu Glu Lys Val Lys Gln Ala Lys Ala Glu Val Glu
355 360 365
Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Lys Ile Lys Thr Asp Arg
370 375 380
Lys Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala Ala Glu Glu Asp Lys
385 390 395 400
Val Lys Glu Lys Pro Ala
405
,
CA 02326388 2001-04-06
-68c-
<210> 2
<211> 655
<212> PRT
<213> Streptococcus pneumoniae
<400> 2
Glu Asn Glu Gly Ala Thr Gin Val Pro Thr Ser Ser Asn Arg Ala Asn
1 5 10 15
Glu Ser Gin Ala Glu Gin Gly Glu Gin Pro Lys Lys Leu Asp Ser Glu
20 25 30
Arg Asp Lys Ala Arg Lys Glu Val Glu Glu Tyr Val Lys Lys Ile Val
35 40 45
Gly Glu Ser Tyr Ala Lys Ser Thr Lys Lys Arg His Thr Ile Thr Val
50 55 60
Ala Leu Val Asn Glu Leu Asn Asn Ile Lys Asn Glu Tyr Leu Asn Lys
65 70 75 80
Ile Val Glu Ser Thr Ser Glu Ser Gin Leu Gin Ile Leu Met Met Glu
85 90 95
Ser Arg Ser Lys Val Asp Glu Ala Val Ser Lys Phe Glu Lys Asp Ser
100 105 110
Ser Ser Ser Ser Ser Ser Asp Ser Ser Thr Lys Pro Glu Ala Ser Asp
115 120 125
Thr Ala Lys Pro Asn Lys Pro Thr Glu Pro Gly Glu Lys Val Ala Glu
130 135 140
Ala Lys Lys Lys Val Glu Glu Ala Glu Lys Lys Ala Lys Asp Gin Lys
145 150 155 160
Glu Glu Asp Arg Arg Asn Tyr Pro Thr Ile Thr Tyr Lys Thr Leu Glu
165 170 175
Leu Glu Ile Ala Glu Ser Asp Val Glu Val Lys Lys Ala Glu Leu Glu
180 185 190
Leu Val Lys Val Lys Ala Asn Glu Pro Arg Asp Glu Gin Lys Ile Lys
195 200 205
Gin Ala Glu Ala Glu Val Glu Ser Lys Gin Ala Glu Ala Thr Arg Leu
210 215 220
Lys Lys Ile Lys Thr Asp Arg Glu Glu 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
245 250 255
¨ --.¨
CA 02326388 2001-04-06
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Gly Val Pro Gly Glu Leu Ala Thr Pro Asp Lys Lys Glu Asn Asp Ala
260 265 270
Lys Ser Ser Asp Ser Ser Val Gly Glu Glu Thr Leu Pro Ser Pro Ser
275 280 285
Leu Lys Pro Glu Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu
290 295 300
Ala Lys Lys Lys Ala Glu Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr
305 310 315 320
Pro Thr Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala Glu Ser Asp
325 330 335
Val Glu Val Lys Lys Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys
340 345 350
Glu Pro Arg Asn Glu Glu Lys Val Lys Gln Ala Lys Ala Glu Val Glu
355 360 365
Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Lys Ile Lys Thr Asp Arg
370 375 380
Lys Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala Ala Glu Glu Asp Lys
385 390 395 400
Val Lys Glu Lys Pro Ala Glu Gln Pro Gln Pro Ala Pro Ala Pro Lys
405 410 415
Ala Glu Lys Pro Ala Pro Ala Pro Lys Pro Glu Asn Pro Ala Glu Gln
420 425 430
Pro Lys Ala Glu Lys Pro Ala Asp Gln Gln Ala Glu Glu Asp Tyr Ala
435 440 445
Arg Arg Ser Glu Glu Glu Tyr Asn Arg Leu Thr Gln Gln Gln Pro Pro
450 455 460
Lys Thr Glu Lys Pro Ala Gln Pro Ser Thr Pro Lys Thr Gly Trp Lys
465 470 475 480
Gln Glu Asn Gly Met Trp Tyr Phe Tyr Asn Thr Asp Gly Ser Met Ala
485 490 495
Thr Gly Trp Leu Gln Asn Asn Gly Ser Trp Tyr Tyr Leu Asn Ser Asn
500 505 510
Gly Ala Met Ala Thr Gly Trp Leu Gln Asn Asn Gly Ser Trp Tyr Tyr
515 520 525
Leu Asn Ala Asn Gly Ser Met Ala Thr Gly Trp Leu Gln Asn Asn Gly
530 535 540
Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Ser Met Ala Thr Gly Trp Leu
545 550 555 560
CA 02326388 2001-04-06
-68e-
Gin Tyr Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Ser Met Ala
565 570 575
Thr Gly Trp Leu Gin Tyr Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn
580 585 590
Gly Asp Met Ala Thr Gly Trp Val Lys Asp Gly Asp Thr Trp Tyr Tyr
595 600 605
Leu Glu Ala Ser Gly Ala Met Lys Ala Ser Gin Trp Phe Lys Val Ser
610 615 620
Asp Lys Trp Tyr Tyr Val Asn Gly Ser Gly Ala Leu Ala Val Asn Thr
625 630 635 640
Thr Val Asp Gly Tyr Gly Val Asn Ala Asn Gly Glu Trp Val Asn
645 650 655
<210> 3
<211> 284
<212> PRT
<213> Streptococcus pneumoniae
<400> 3
Glu Asn Glu Gly Ala Thr Gin Val Pro Thr Ser Ser Asn Arg Ala Asn
1 5 10 15
Glu Ser Gin Ala Glu Gin Gly Glu Gin Pro Lys Lys Leu Asp Ser Glu
20 25 30
Arg Asp Lys Ala Arg Lys Glu Val Glu Glu Tyr Val Lys Lys Ile Val
35 40 45
Gly Glu Ser Tyr Ala Lys Ser Thr Lys Lys Arg His Thr Ile Thr Val
50 55 60
Ala Leu Val Asn Glu Leu Asn Asn Ile Lys Asn Glu Tyr Leu Asn Lys
65 70 75 80
Ile Val Glu Ser Thr Ser Glu Ser Gin Leu Gin Ile Leu Met Met Glu
85 90 95
Ser Arg Ser Lys Val Asp Glu Ala Val Ser Lys Phe Glu Lys Asp Ser
100 105 110
Ser Ser Ser Ser Ser Ser Asp Ser Ser Thr Lys Pro Glu Ala Ser Asp
115 120 125
Thr Ala Lys Pro Asn Lys Pro Thr Glu Pro Gly Glu Lys Val Ala Glu
130 135 140
Ala Lys Lys Lys Val Glu Glu Ala Glu Lys Lys Ala Lys Asp Gin Lys
145 150 155 160
¨
CA 02326388 2001-04-06
-68f-
Glu Glu Asp Arg Arg Asn Tyr Pro Thr Ile Thr Tyr Lys Thr Leu Glu
165 170 175
Leu Glu Ile Ala Glu Ser Asp Val Glu Val Lys Lys Ala Glu Leu Glu
180 185 190
Leu Val Lys Val Lys Ala Asn Glu Pro Arg Asp Glu Gln Lys Ile Lys
195 200 205
Gln Ala Glu Ala Glu Val Glu Ser Lys Gln Ala Glu Ala Thr Arg Leu
210 215 220
Lys Lys Ile Lys Thr Asp Arg Glu Glu Ala Glu Glu Glu Ala Lys Arg
225 230 235 240
Arg Ala Asp Ala Lys Glu Gln Gly Lys Pro Lys Gly Arg Ala Lys Arg
245 250 255
Gly Val Pro Gly Glu Leu Ala Thr Pro Asp Lys Lys Glu Asn Asp Ala
260 265 270
Lys Ser Ser Asp Ser Ser Val Gly Glu Glu Thr Leu
275 280
<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
1 5 10 15
Lys Lys Lys Ala Glu Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr Pro
20 25 30
Thr Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala Glu Ser Asp Val
35 40 45
Glu Val Lys Lys Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys Glu
50 55 60
Pro Arg Asn Glu Glu Lys Val Lys Gln Ala Lys Ala Glu Val Glu Ser
65 70 75 80
Lys Lys Ala Glu Ala Thr Arg Leu Glu Lys Ile Lys Thr Asp Arg Lys
85 90 95
Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala
100 105
<210> 5
<211> 109 =
CA 02326388 2001-04-06
-68g-
<212> PRT
<213> Streptococcus pneumoniae
<400> 5
Thr Glu Pro Gly Glu Lys Val Ala Glu Ala Lys Lys Lys Val Glu Glu
1 5 10 15
Ala Glu Lys Lys Ala Lys Asp Gin Lys Glu Glu Asp Arg Arg Asn Tyr
20 25 30
Pro Thr Ile Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala Glu Ser Asp
35 40 45
Val Glu Val Lys Lys Ala Glu Leu Glu Leu Val Lys Val Lys Ala Asn
50 55 60
Glu Pro Arg Asp Glu Gin Lys Ile Lys Gin Ala Glu Ala Glu Val Glu
65 70 75 80
Ser Lys Gin. Ala Glu Ala Thr Arg Leu Lys Lys Ile Lys Thr Asp Arg
85 90 95
Glu Glu Ala Glu Glu Glu Ala Lys Arg Arg Ala Asp Ala
100 105
<210> 6
<211> 4
<212> PRT
<213> Streptococcus pneumoniae
<220>
<221> NON CONS
<222> (2)..(3)
<223> They could be any amino acid at these two
locations.
<400> 6
Lys Xaa Xaa Glu
1
<210> 7
<211> 376
<212> PRT
<213> Streptococcus pneumoniae
<400> 7
Glu Asn Glu Gly Ser Thr Gin Ala Ala Thr Ser Ser Asn Met Ala Lys
1 5 10 15
Thr Glu His Arg Lys Ala Ala Lys Gin Val Val Asp Glu Tyr Ile Glu
20 25 30
CA 02326388 2001-04-06
-68h-
Lys Met Leu Arg Glu Ile Gin Leu Asp Arg Arg Lys His Thr Gin Asn
35 40 45
Val Ala Leu Asn Ile Lys Leu Ser Ala Ile Lys Thr Lys Tyr Leu Arg
50 55 60
Glu Leu Asn Val Leu Glu Glu Lys Ser Lys Asp Glu Leu Pro Ser Glu
65 70 75 80
Ile Lys Ala Lys Leu Asp Ala Ala Phe Glu Lys Phe Lys Lys Asp Thr
85 90 95
Leu Lys Pro Gly Glu Lys Val Ala Glu Ala Lys Lys Lys Val Glu Glu
100 105 110
Ala Lys Lys Lys Ala Glu Asp Gin Lys Glu Glu Asp Arg Arg Asn Tyr
115 120 125
Pro Thr Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala Glu Phe Asp
130 135 140
Val Lys Val Lys Glu Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys
145 150 155 160
Glu Ser Arg Asn Glu Gly Thr Ile Lys Gin Ala Lys Glu Lys Val Glu
165 170 175
Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile Lys Thr Asp Arg
180 185 190
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 Asp Gin Gly Lys Pro Lys Gly Arg Ala
210 215 220
Lys Arg Gly Val Pro Gly Glu Leu Ala Thr Pro Asp Lys Lys Glu Asn
225 230 235 240
Asp Ala Lys Ser Ser Asp Ser Ser Val Gly Glu Glu Thr Leu Pro Ser
245 250 255
Ser Ser Leu Lys Ser Gly Lys Lys Val Ala Glu Ala Glu Lys Lys Val
260 265 270
Glu Glu Ala Glu Lys Lys Ala Lys Asp Gln Lys Glu Glu Asp Arg Arg
275 280 285
Asn Tyr Pro Thr Asn Thr Tyr Lys Thr Leu Asp Leu Glu Ile Ala Glu
290 295 300
Ser Asp Val Lys Val Lys Glu Ala Glu Leu Glu Leu Val Lys Glu Glu
305 310 315 320
Ala Lys Glu Pro Arg Asp Glu Glu Lys Ile Lys Gin Ala Lys Ala Lys
325 330 335
CA 02326388 2001-04-06
-68i-
Val Glu Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile Lys Thr
340 345 350
Asp Arg Lys Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala Ala Glu Glu
355 360 365
Asp Lys Val Lys Glu Lys Pro Ala
370 375
<210> 8
<211> 663
<212> PRT
<213> Streptococcus pneumoniae
<400> 8
Glu Asn Glu Gly Ser Thr Gln Ala Ala Thr Ser Ser Asn Met Ala Lys
1 5 10 15
Thr Glu His Arg Lys Ala Ala Lys Gln Val Val Asp Glu Tyr Ile Glu
20 25 30
Lys Met Leu Arg Glu Ile Gln Leu Asp Arg Arg Lys His Thr Gln Asn
35 40 45
Val Ala Leu Asn Ile Lys Leu Ser Ala Ile Lys Thr Lys Tyr Leu Arg
50 55 60
Glu Leu Asn Val Leu Glu Glu Lys Ser Lys Asp Glu Leu Pro Ser Glu
65 70 75 80
Ile Lys Ala Lys Leu Asp Ala Ala Phe Glu Lys Phe Lys Lys Asp Thr
85 90 95
Leu Lys Pro Gly Glu Lys Val Ala Glu Ala Lys Lys Lys Val Glu Glu
100 105 110
Ala Lys Lys Lys Ala Glu Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr
115 120 125
Pro Thr Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala Glu Phe Asp
130 135 140
Val Lys Val Lys Glu Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys
145 150 155 160
Glu Ser Arg Asn Glu Gly Thr Ile Lys Gln Ala Lys Glu Lys Val Glu
165 170 175
Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile Lys Thr Asp Arg
180 185 190
Lys Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala Asp Ala Lys Leu Lys
195 200 205
CA 02326388 2001-04-06
-68j-
Glu Ala Asn Val Ala Thr Ser Asp Gln Gly Lys Pro Lys Gly Arg Ala
210 215 220
Lys Arg Gly Val Pro Gly Glu Leu Ala Thr Pro Asp Lys Lys Glu Asn
225 230 235 240
Asp Ala Lys Ser Ser Asp Ser Ser Val Gly Glu Glu Thr Leu Pro Ser
245 250 255
Ser Ser Leu Lys Ser Gly Lys Lys Val Ala Glu Ala Glu Lys Lys Val
260 265 270
Glu Glu Ala Glu Lys Lys Ala Lys Asp Gln Lys Glu Glu Asp Arg Arg
275 280 285
Asn Tyr Pro Thr Asn Thr Tyr Lys Thr Leu Asp Leu Glu Ile Ala Glu
290 295 300
Ser Asp Val Lys Val Lys Glu Ala Glu Leu Glu Leu Val Lys Glu Glu
305 310 315 320
Ala Lys Glu Pro Arg Asp Glu Glu Lys Ile Lys Gln Ala Lys Ala Lys
325 330 335
Val Glu Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile Lys Thr
340 345 350
Asp Arg Lys Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala Ala Glu Glu
355 360 365
Asp Lys Val Lys Glu Lys Pro Ala Glu Gln Pro Gln Pro Ala Pro Ala
370 375 380
Thr Gln Pro Glu Lys Pro Ala Pro Lys Pro Glu Lys Pro Ala Glu Gln
385 390 395 400
Pro Lys Ala Glu Lys Thr Asp Asp Gln Gln Ala Glu Glu Asp Tyr Ala
405 410 415
Arg Arg Ser Glu Glu Glu Tyr Asn Arg Leu Thr Gln Gln Gln Pro Pro
420 425 430
Lys Thr Glu Lys Pro Ala Gln Pro Ser Thr Pro Lys Thr Gly Trp Lys
435 440 445
Gln Glu Asn Gly Met Trp Tyr Phe Tyr Asn Thr Asp Gly Ser Met Ala
450 455 460
Thr Gly Trp Leu Gln Asn Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn
465 470 475 480
Gly Ala Met Ala Thr Gly Trp Leu Gln Asn Asn Gly Ser Trp Tyr Tyr
485 490 495
Leu Asn Ala Asn Gly Ser Met Ala Thr Gly Trp Leu Gln Asn Asn Gly
500 505 510
¨T-
CA 02326388 2001-04-06
-68k-
_
Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Ala Met Ala Thr Gly Trp Leu
515 520 525
Gln Tyr Asn Gly Ser Trp Tyr Tyr Leu Asn Ser Asn Gly Ala Met Ala
530 535 540
Thr Gly Trp Leu Gln Tyr Asn Gly Ser Trp Tyr Tyr Leu Asn Ala Asn
545 550 555 560
Gly Asp Met Ala Thr Gly Trp Leu Gln Asn Asn Gly Ser Trp Tyr Tyr
565 570 575
Leu Asn Ala Asn Gly Asp Met Ala Thr Gly Trp Leu Gln Tyr Asn Gly
580 585 590
Ser Trp Tyr Tyr Leu Asn Ala Asn Gly Asp Met Ala Thr Gly Trp Val
595 600 605
Lys Asp Gly Asp Thr Trp Tyr Tyr Leu Glu Ala Ser Gly Ala Met Lys
610 615 620
Ala Ser Gln Trp Phe Lys Val Ser Asp Lys Trp Tyr Tyr Val Asn Gly
625 630 635 640
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 pneumoniae
<400> 9
Glu Asn Glu Gly Ser Thr Gln Ala Ala Thr Ser Ser Asn Met Ala Lys
1 5 10 15
Thr Glu His Arg Lys Ala Ala Lys Gln Val Val Asp Glu Tyr Ile Glu
20 25 30
Lys Met Leu Arg Glu Ile Gln Leu Asp Arg Arg Lys His Thr Gln Asn
35 40 45
Val Ala Leu Asn Ile Lys Leu Ser Ala Ile Lys Thr Lys Tyr Leu Arg
50 55 60
Glu Leu Asn Val Leu Glu Glu Lys Ser Lys Asp Glu Leu Pro Ser Glu
65 70 75 80
Ile Lys Ala Lys Leu Asp Ala Ala Phe Glu Lys Phe Lys Lys Asp Thr
85 90 95
___ ,
CA 02326388 2001-04-06
-681-
Leu Lys Pro Gly Glu Lys Val Ala G1u Ala Lys Lys Lys Val Glu Glu
100 105 110
Ala Lys Lys Lys Ala Glu Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr
115 120 125
Pro Thr Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala Glu Phe Asp
130 135 140
Val Lys Val Lys Glu Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys
145 150 155 160
Glu Ser Arg Asn Glu Gly Thr Ile Lys Gln Ala Lys Glu Lys Val Glu
165 170 175
Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile Lys Thr Asp Arg
180 185 190
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 Asp Gln Gly Lys Pro Lys Gly Arg Ala
210 215 220
Lys Arg Gly Val Pro Gly Glu Leu Ala Thr Pro Asp Lys Lys Glu Asn
225 230 235 240
Asp Ala Lys Ser Ser Asp Ser Ser Val Gly Glu Glu Thr Leu
245 250
<210> 10
<211> 106
<212> PRT
<213> Streptococcus pneumoniae
<400> 10
Lys Ser Gly Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu Ala
1 5 10 15
Glu Lys Lys Ala Lys Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr Pro
20 25 30
Thr Asn Thr Tyr Lys Thr Leu Asp Leu Glu Ile Ala Glu Ser Asp Val
35 40 45
Lys Val Lys Glu Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys Glu
50 55 60
Pro Arg Asp Glu Glu Lys Ile Lys Gln Ala Lys Ala Lys Val Glu Ser
65 70 75 80
Lys Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile Lys Thr Asp Arg Lys
85 90 95
CA 02326388 2001-04-06
-68m-
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 15
Lys Lys Ala Glu Asp Gln Lys Glu Glu Asp Arg Arg Asn Tyr Pro Thr
20 25 30
Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala Glu Phe Asp Val Lys
35 40 45
Val Lys Glu Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys Glu Ser
50 55 60
Arg Asn Glu Gly Thr Ile Lys Gln Ala Lys Glu Lys Val Glu Ser Lys
65 70 75 80
Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile Lys Thr Asp Arg Lys Lys
85 90 95
Ala Glu Glu Glu Ala Lys Arg Lys Ala Asp Ala
100 105
<210> 12
<211> 1219
<212> DNA
<213> Streptococcus pneumoniae
<400> 12
gagaacgagg gagctaccca agtacccact tcttctaata gggcaaatga aagtcaggca 60
gaacaaggag aacaacctaa aaaactcgat tcagaacgag ataaggcaag gaaagaggtc 120
gaggaatatg taaaaaaaat agtgggtgag agctatgcaa aatcaactaa aaagcgacat 180
acaattactg tagctctagt taacgagttg aacaacatta agaacgagta tttgaataaa 240
atagttgaat caacctcaga aagccaacta cagatactga tgatggagag tcgatcaaaa 300
gtagatgaag ctgtgtctaa gtttgaaaag gactcatctt cttcgtcaag ttcagactct 360
tccactaaac cggaagcttc agatacagcg aagccaaaca agccgacaga accaggagaa 420
aaggtagcag aagctaagaa gaaggttgaa gaagctgaga aaaaagccaa ggatcaaaaa 480
gaagaagatc gtcgtaacta cccaaccatt acttacaaaa cgcttgaact tgaaattgct 540
gagtccgatg tggaagttaa aaaagcggag cttgaactag taaaagtgaa agctaacgaa 600
cctcgagacg agcaaaaaat taagcaagca gaagcggaag ttgagagtaa acaagctgag 660
gctacaaggt taaaaaaaat caagacagat cgtgaagaag cagaagaaga agctaaacga 720
agagcagatg ctaaagagca aggtaaacca aaggggcggg caaaacgagg agttcctgga 780
gagctagcaa cacctgataa aaaagaaaat gatgcgaagt cttcagattc tagcgtaggt 840
gaagaaactc ttccaagccc atccctgaaa ccagaaaaaa aggtagcaga agctgagaag 900
aaggttgaag aagctaagaa aaaagccgag gatcaaaaag aagaagatcg ccgtaactac 960
CA 02326388 2001-04-06
-68n-
ccaaccaata cttacaaaac gcttgaactt gaaattgctg agtccgatgt ggaagttaaa 1020
aaagcggagc ttgaactagt aaaagaggaa gctaaggaac ctcgaaacga ggaaaaagtt 1080
aagcaagcaa aagcggaagt tgagagtaaa aaagctgagg ctacaaggtt agaaaaaatc 1140
aagacagatc gtaaaaaagc agaagaagaa gctaaacgaa aagcagcaga agaagataaa 1200
gttaaagaaa aaccagctg 1219
<210> 13
<211> 1969
<212> DNA
<213> Streptococcus pneumoniae
<400> 13
gagaacgagg gagctaccca agtacccact tcttctaata gggcaaatga aagtcaggca 60
gaacaaggag aacaacctaa aaaactcgat tcagaacgag ataaggcaag gaaagaggtc 120
gaggaatatg taaaaaaaat agtgggtgag agctatgcaa aatcaactaa aaagcgacat 180
acaattactg tagctctagt taacgagttg aacaacatta agaacgagta tttgaataaa 240
atagttgaat caacctcaga aagccaacta cagatactga tgatggagag tcgatcaaaa 300
gtagatgaag ctgtgtctaa gtttgaaaag gactcatctt cttcgtcaag ttcagactct 360
tccactaaac cggaagcttc agatacagcg aagccaaaca agccgacaga accaggagaa 420
aaggtagcag aagctaagaa gaaggttgaa gaagctgaga aaaaagccaa ggatcaaaaa 480
gaagaagatc gtcgtaacta cccaaccatt acttacaaaa cgcttgaact tgaaattgct 540
gagtccgatg tggaagttaa aaaagcggag cttgaactag taaaagtgaa agctaacgaa 600
cctcgagacg agcaaaaaat taagcaagca gaagcggaag ttgagagtaa acaagctgag 660
gctacaaggt taaaaaaaat caagacagat cgtgaagaag cagaagaaga agctaaacga 720
agagcagatg ctaaagagca aggtaaacca aaggggcggg caaaacgagg agttcctgga 780
gagctagcaa cacctgataa aaaagaaaat gatgcgaagt cttcagattc tagcgtaggt 840
gaagaaactc ttccaagccc atccctgaaa ccagaaaaaa aggtagcaga agctgagaag 900
aaggttgaag aagctaagaa aaaagccgag gatcaaaaag aagaagatcg ccgtaactac 960
ccaaccaata cttacaaaac gcttgaactt gaaattgctg agtccgatgt ggaagttaaa 1020
aaagcggagg cttgaactag taaaagagga agctaaggaa cctcgaaacg aggaaaaagt 1080
taagcaagca aaagcggaag ttgagagtaa aaaagctgag gctacaaggt tagaaaaaat 1140
caagacagat cgtaaaaaag cagaagaaga agctaaacga aaagcagcag aagaagataa 1200
agttaaagaa aaaccagctg aacaaccaca accagcgccg gctccaaaag cagaaaaacc 1260
agctccagct ccaaaaccag agaatccagc tgaacaacca aaagcagaaa aaccagctga 1320
tcaacaagct gaagaagact atgctcgtag atcagaagaa gaatataatc gcttgactca 1380
acagcaaccg ccaaaaactg aaaaaccagc acaaccatct actccaaaaa caggctggaa 1440
acaagaaaac ggtatgtggt acttctacaa tactgatggt tcaatggcga caggatggct 1500
ccaaaacaat ggctcatggt actacctcaa cagcaatggc gctatggcga caggatggct 1560
ccaaaacaat ggttcatggt actatctaaa cgctaatggt tcaatggcaa caggatggct 1620
ccaaaacaat ggttcatggt actacctaaa cgctaatggt tcaatggcga caggatggct 1680
ccaatacaat ggctcatggt actacctaaa cgctaatggt tcaatggcga caggatggct 1740
ccaatacaat ggctcatggt actacctaaa cgctaatggt gatatggcga caggttgggt 1800
gaaagatgga gatacctggt actatcttga agcatcaggt gctatgaaag caagccaatg 1860
gttcaaagta tcagataaat ggtactatgt caatggctca ggtgcccttg cagtcaacac 1920
aactgtagat ggctatggag tcaatgccaa tggtgaatgg gtaaactaa 1969
<210> 14
<211> 853
<212> DNA
<213> Streptococcus pneumoniae
og EBelpoPPBe ov6pp2oE64 vq.eugoqqol qopooSpobp upoopq6upb Bue53PP2p6
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f7T <00t>
-ogg-
90-170-TOOZ 889ZEZO VD
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0961 Teuqp63uuu qp3uqop.466 quoqq66qup ou'euvooq36 EcTe6Ecepuuo 56Teupgq66
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OzET POOPPDPO6P ODUPPPU6qo PUUUPD0600 PPO6POPPOq DP6T4060qP ezeTe-25.2.26
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09E PPoqP56vEo 06PPPPPP6P eqp6u-e5ev6 4.466-upbp.26 upqoaevbeo 5E-455-evEa6
00E E.66upouu.28 qq-eopTebep pyypq3q6up Ece6qqqqp6p 3Eoe6v4q6-2 ppo6Pegy
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081 Eovep.eqq-eP 36o6P6qq6e. ppgeovvpqq. poboq6qupp POODP4POPP PESPPE,Pqa6
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09 66uTepeu6u op6eupp66q uquu4pq-43.4 qopooftobp vooppq5e.E.6 .6.eu6oevee6
81 <00T7>
auTuownaud snopopoqdaaqs <ETZ>
VNG <Z1Z>
Z661 <TIZ>
81 <OTZ>
6Z11
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ogoT Pp6uPE.EP6e o5epeuevq6 Dqvbpoe6uv D4POPPPP5P qq66ppopqo 66y8;36uvu
ozOT puuq6P6u6q 18E-eu6D6up puo6uup&e.e. ;Teppvv.266 p6op&E,6oqo 3es66p8l36
096 PPE6P6PPPP q6.2qoppaqq. D5p.663.6-epE. pppqq.Epeu6 4EcTebooq5e
5q36qqp.euE)
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0t8 5PP336PPPP uu6p6qp6up 5pp5qq65ut. Euu6e6.4p6e v6pobeq66E. uuupP66epq
08L Erep.6.43poqu 3g36Pup34g ogouup5pub g5E.-eq636vq oqquaeoggo .46yE.636qv6
on 4PPPP5PPPP ppze6gpoup puoSpqoft,E, pE.6.4=4.46E, 88pE3uppp3 666p66E6Pu
099 PoopPeq66p PoTe6poqq3 v636E1.6.4.ep 4D6p.e.56puE. qq6euqa6me BPD6PPPP60
009 es.eq36vP6E, P6E-eSuo6P-E, pppulbolu6 P3P6PPOTe3 PPPP6P1q86 veouqobBuE.
ots 436.e.evpuug SpEce6qq6pp p8p6pypp36 uypbepqq-ey 3Eo5E6pEou vs6ogogyy6
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on, 4.4PPubqqop p64.4pEoPuu popqqopqPp pouupopeqo pugEogEoqu 5E.e&ev6P.E.E.
09E ve3qe6.6v63 OBPPPPPP&P uq36ve6E-26 .1366.ev6p.e6 puloSpuBpo Eceq6Eceuuv5
00E p86pooppp5 qquopquEcep pppplqq6ut. 6u6qqqq36p pEopEreqqft. PP36PPPPqP
OVZ PPEceoq6006 qq6p6lu6vp y6oq6E.E.Ece6 ppBuqq446.4 vevq4P-e6q6 3ErT4TeqEvee
081 60-e.eveqq.ep 3636e6446e puqaeupqq 3363z64epp POODP1PDPP PP6PP6P4P6
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vqp640.6.e.e.e.
-d99-
9O-0-TOOZ 889ZEZO VD
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otz Peuq5p5e6.4 36ppy6P6up eva6pppEcep qqppppo666 u6ovuP6oqp TeP6ppPqp6
081 Pe66u6PEPP .46pq3PPEcqq 36pE6D6pu6 epuqq&epp6 q6qp6oqq6e 66e5
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09 6v63o6p.ePP PPE,PPqD6PP 6PP6qq56PP 6PP6PP4D6P v5E,D6P-486.e. veu8PB6Poo
TZ <00V>
avTuownaud snpoopoqdaaqs <ETz>
VNG <ZIZ>
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8TE ppEPPpub
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00E eebypEcepEce PPPPPq6D4P 6PDP6PPOTP oppppSyqq6 Bpp3u-438Ece 6.4o6uev-evu
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5Ereeqp6vv.E.
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09 D36PPPPPP6 v6qp6e-e6ve Eqq6Ecee5ev 6e6 65 o6vq.6.6.e.e2P vP66poqPee
OZ <00T7>
avTuomnaud snop000qdaaqs <ETZ>
VNa <ZIZ>
8TE <TTZ>
OZ <OTZ>
E9L p.4.4
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009 PPP406PP6P PE,PPE,POSPP eeprgEoquE) POPEPPOqPD peep&eqq86 ePopqoEBR5
ots g36upevEeq 5u6P6qq6ep P6P6PPPPD6 PPOSPPqqPP Dvo6B6E,Bou v-e6pqoqu'u5
08f, evygo6p.E.66 p6Eve,e16pq ove6qqoSub 636vv5pppq q6ueeEq64P 6ogg6p6136
ozt qTeee6qqae v6T436ovvp popqqopTet. oopuppouqo ppqEoq63-4E. 8Pu6vP6Pve
09E vuo3p66u6D 3E.PPPPPP6P vqp6p.E.E.p.e6 qq66p-e6p.e6 PPq06PP8PD
6uq5.6.2PPE.E.
00E .266P33pPP8 qqeDequbep p.euvqqq&eu 6-26qq1.4D6u 363e6E-qq5v vuo6PPE-eze
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081 SOPPPPqqPP 3636e6qq6p ppqpovvpqq. opEoqaTepp PODOPT2OPP uPbev&eze6
on PqoEvoqqvE, p656uEclq6q PPPPPPE.PqP qugye6qp63 lboq6ueopp P.4364pEcePp
09 66E-IPOPPEP DP6PPP0661 Pqppqa4qpq qopopaeobv POODPq6PP6 6ee6oevvE6
61 <00T7>
auTuownaud snopopoqdazqs <ETZ>
Vtla <ZTZ>
E9L <ITZ>
61 <OTZ>
Z66T PP
qoppvq666.4
0861 pp6466Tepo o6Teepq6u6 6Teq365ge6 vq6q3ueDPo evoz6v36qq. 3336486p3q
0z61 366
35uqoPq861.e.e, vqp6poqe18 ppv3qq66Te PD36PPD5PP e6Teqp6q66
0981 voq-236-eu6q q3quqoeqE16 qoaeqp6p66 Te6eep6q66 6q466.2op6o .661eTe6q66
0081 zePgabovvo qo3pqqvq66 zeoqq66Dep 3uTev33l36 666e63 66TequEq66
-b89-
9O-0-TOOZ 889ZEZO VO
CA 02326388 2001-04-06
-68r-
gctaaacgaa aagcagatgc t 321
<210> 22
<211> 121
<212> PRT
<213> Streptococcus pneumoniae
<400> 22
Ser Pro Ser Leu Lys Pro Glu Lys Lys Val Ala Glu Ala Glu Lys Lys
1 5 10 15
Val Glu Glu Ala Lys Lys Lys Ala Glu Asp Gin Lys Glu Glu Asp Arg
20 25 30
Arg Asn Tyr Pro Thr Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala
35 40 45
Glu Ser Asp Val Glu Val Lys Lys Ala Glu Leu Glu Leu Val Lys Glu
50 55 60
Glu Ala Lys Glu Pro Arg Asn Glu Glu Lys Val Lys Gln Ala Lys Ala
65 70 75 80
Glu Val Glu Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Lys Ile Lys
85 90 95
Thr Asp Arg Lys Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala Ala Glu
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 Glu Ala Glu Lys
1 5 10 15
Lys Val Glu Glu Ala Glu Lys Lys Ala Lys Asp Gin Lys Glu Glu Asp
20 25 30
Arg Arg Asn Tyr Pro Thr Asn Thr Tyr Lys Thr Leu Asp Leu Glu Ile
35 40 45
Ala Glu Ser Asp Val Lys Val Lys Glu Ala Glu Leu Glu Leu Val Lys
50 55 60
Glu Glu Ala Lys Glu Pro Arg Asp Glu Glu Lys Ile Lys Gin Ala Lys
65 70 75 80
CA 02326388 2001-04-06
-68s-
Ala Lys Val Glu Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile
85 90 95
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> 24
<211> 428
<212> PRT
<213> Streptococcus pneumoniae
<400> 24
Glu Asn Glu Gly Ala Thr Gln Val Pro Thr Ser Ser Asn Arg Ala Asn
1 5 10 15
Glu Ser Gln Ala Glu Gln Gly Glu Gln Pro Lys Lys Leu Asp Ser Glu
20 25 30
Arg Asp Lys Ala Arg Lys Glu Val Glu Glu Tyr Val Lys Lys Ile Val
35 40 45
Gly Glu Ser Tyr Ala Lys Ser Thr Lys Lys Arg His Thr Ile Thr Val
50 55 60
Ala Leu Val Asn Glu Leu Asn Asn Ile Lys Asn Glu Tyr Leu Asn Lys
65 70 75 80
Ile Val Glu Ser Thr Ser Glu Ser Gln Leu Gln Ile Leu Met Met Glu
85 90 95
Ser Arg Ser Lys Val Asp Glu Ala Val Ser Lys Phe Glu Lys Asp Ser
100 105 110
Ser Ser Ser Ser Ser Ser Asp Ser Ser Thr Lys Pro Glu Ala Ser Asp
115 120 125
Thr Ala Lys Pro Asn Lys Pro Thr Glu Pro Gly Glu Lys Val Ala Glu
130 135 140
Ala Lys Lys Lys Val Glu Glu Ala Glu Lys Lys Ala Lys Asp Gln Lys
145 150 155 160
Glu Glu Asp Arg Arg Asn Tyr Pro Thr Ile Thr Tyr Lys Thr Leu Glu
165 170 175
Leu Glu Ile Ala Glu Ser Asp Val Glu Val Lys Lys Ala Glu Leu Glu
180 185 190
Leu Val Lys Val Lys Ala Asn Glu Pro Arg Asp Glu Gln Lys Ile Lys
195 200 205
CA 02326388 2001-04-06
-68t-
Gin Ala Glu Ala Glu Val Glu Ser Lys Gin Ala Glu Ala Thr Arg Leu
210 215 220
Lys Lys Ile Lys Thr Asp Arg Glu Glu 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
245 250 255
Gly Val Pro Gly Glu Leu Ala Thr Pro Asp Lys Lys Glu Asn Asp Ala
260 265 270
Lys Ser Ser Asp Ser Ser Val Gly Glu Glu Thr Leu Pro Ser Pro Ser
275 280 285
Leu Lys Pro Glu Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu
290 295 300
Ala Lys Lys Lys Ala Glu Asp Gin Lys Glu Glu Asp Arg Arg Asn Tyr
305 310 315 320
Pro Thr Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala Glu Ser Asp
325 330 335
Val Glu Val Lys Lys Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys
340 345 350
Glu Pro Arg Asn Glu Glu Lys Val Lys Gin Ala Lys Ala Glu Val Glu
355 360 365
Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Lys Ile Lys Thr Asp Arg
370 375 380
Lys Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala Ala Glu Glu Asp Lys
385 390 395 400
Val Lys Glu Lys Pro Ala Glu Gin Pro Gin Pro Ala Pro Ala Pro Lys
405 410 415
Ala Glu Lys Pro Ala Pro Ala Pro Lys Pro Glu Asn
420 425
<210> 25
<211> 23
<212> DNA
<213> Streptococcus pneumoniae
<400> 25
ggcggatcca tggaraayga rgg 23
<210> 26
<211> 33
<212> DNA
CA 02326388 2001-04-06
-68u-
<213> Streptococcus pneumoniae
<400> 26
gccgtcgact tagtttaccc attcaccatt ggc 33
<210> 27
<211> 5
<212> PRT
<213> Streptococcus pneumoniae
<220>
<221> VARIANT
<222> (1)
<223> It could be any amino acid.
<400> 27
Xaa Glu Asn Glu Gly
1 5
<210> 28
<211> 439
<212> PRT
<213> Streptococcus pneumoniae
<220>
<221> VARIANT
<222> (243)
<223> It could be any amino acid.
<400> 28
Ala Val Ala Ser Leu Phe Met Gly Ser Val Val His Ala Thr Glu Lys
1 5 10 15
Glu Val Thr Thr Gln Val Ala Thr Ser Ser Asn Lys Ala Asn Lys Ser
20 25 30
Gln Thr Glu His Met Lys Ala Ala Lys Gln Val Asp Glu Tyr Ile Lys
35 40 45
Lys Lys Leu Gln Leu Asp Arg Arg Lys His Thr Gln Asn Val Gly Leu
50 55 60
Leu Thr Lys Leu Gly Val Ile Lys Thr Glu Tyr Leu His Gly Leu Ser
65 70 75 80
Val Ser Lys Lys Lys Ser Glu Ala Glu Leu Pro Ser Glu Ile Lys Ala
85 90 95
Lys Leu Asp Ala Ala Phe Glu Gln Phe Lys Lys Asp Thr Leu Pro Thr
100 105 110
CA 02326388 2001-04-06
-68v-
Glu Pro Gly Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu Ala
115 120 125
Lys Lys Lys Ala Glu Asp Gin Lys Glu Lys Asp Leu Arg Asn Tyr Pro
130 135 140
Thr Asn Thr Tyr Lys Thr Leu Glu Leu Asp Ile Ala Glu Ser Asp Val
145 150 155 160
Glu Val Lys Lys Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys Glu
165 170 175
Ser Arg Asp Glu Lys Lys Ile Asn Gin Ala Lys Ala Lys Val Glu Asn
180 185 190
Lys Lys Ala Glu Ala Thr Arg Leu Lys Asn Ile Lys Thr Asp Arg Glu
195 200 205
Lys Ala Glu Glu Ala Lys Arg Arg Ala Asp Ala Lys Leu Gin Glu Ala
210 215 220
Asn Val Ala Thr Ser Glu Gin Asp Lys Ser Lys Arg Arg Ala Lys Arg
225 230 235 240
Glu Val Xaa Gly Glu Leu Ala Thr Pro Asp Lys Lys Glu Asn Asp Ala
245 250 255
Lys Ser Ser Asp Ser Ser Val Gly Glu Glu Thr Leu Thr Ser Pro Ser
260 265 270
Leu Lys Pro Glu Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu
275 280 285
Ala Lys Lys Lys Ala Glu Asp Gin Lys Glu Glu Asp Arg Arg Asn Tyr
290 295 300
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
325 330 335
Glu Ser Arg Asn Glu Glu Lys Ile Lys Gin Val Lys Ala Lys Val Glu
340 345 350
Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile Lys Thr Asp Arg
355 360 365
Lys Lys Ala Glu Glu Glu Glu Ala Lys Arg Arg Ala Ala Glu Glu Asp
370 375 380
Lys Val Lys Glu Lys Pro Ala Glu Gin Pro Gin Pro Ala Pro Ala Pro
385 390 395 400
Gin Pro Glu Lys Pro Thr Glu Glu Pro Glu Asn Pro Ala Pro Ala Pro
405 410 415
CA 02326388 2001-04-06
-68w-
Ala Pro Lys Pro Glu Asn Pro Ala Glu Lys Pro Lys Ala Glu Lys Pro
420 425 430
Ala Asp Gln Gln Ala Glu Glu
435
<210> 29
<211> 437
<212> PRT
<213> Streptococcus pneumoniae
<400> 29
Ala Val Ala Ser Leu Phe Met Gly Ser Val Val His Ala Thr Glu Lys
1 5 10 15
Glu Val Thr Thr Gln Val Ala Thr Ser Ser Asn Arg Ala Asn Lys Ser
20 25 30
Gln Thr Glu His Met Lys Ala Ala Lys Gln Val Asp Glu Tyr Ile Lys
35 40 45
Lys Lys Leu Gln Leu Asp Arg Arg Lys His Thr Gln Asn Val Gly Leu
50 55 60
Leu Thr Lys Leu Gly Val Ile Lys Thr Glu Tyr Leu His Gly Leu Ser
65 70 75 80
Val Ser Lys Lys Lys Ser Glu Ala Glu Leu Pro Ser Glu Ile Lys Ala
85 90 95
Lys Leu Asp Ala Ala Phe Glu Gln Phe Lys Lys Asp Thr Leu Pro Thr
100 105 110
Glu Pro Gly Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu Ala
115 120 125
Lys Lys Lys Ala Glu Asp Gln Lys Glu Lys Asp Leu Arg Asn Tyr Pro
130 135 140
Thr Asn Thr Tyr Lys Thr Leu Glu Leu Asp Ile Ala Glu Ser Asp Val
145 150 155 160
Glu Val Lys Lys Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys Glu
165 170 175
Ser Arg Asp Glu Lys Lys Ile Asn Gln Ala Lys Ala Lys Val Glu Asn
180 185 190
Lys Lys Ala Glu Ala Thr Arg Leu Lys Asn Ile Lys Thr Asp Arg Glu
195 200 205
Lys Ala Glu Glu Ala Lys Arg Arg Ala Asp Ala Lys Leu Gln Glu Ala
210 215 220
CA 02326388 2001-04-06
-68x-
Asn Val Ala Thr Ser Glu Gin Asp Lys Ser Lys Arg Arg Ala Lys Arg
225 230 235 240
Glu Val Leu Gly Glu Leu Ala Thr Pro Asp Lys Lys Glu Asn Asp Ala
245 250 255
Lys Ser Ser Asp Ser Ser Val Gly Glu Glu Thr Leu Thr Ser Pro Ser
260 265 270
Leu Lys Pro Glu Lys Lys Val Ala Glu Ala Glu Lys Lys Val Glu Glu
275 280 285
Ala Lys Lys Lys Ala Glu Asp Gin Lys Glu Glu Asp Arg Arg Asn Tyr
290 295 300
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
325 330 335
Glu Ser Arg Asn Glu Glu Lys Ile Lys Gin Val Lys Ala Lys Val Glu
340 345 350
Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile Lys Thr Asp Arg
355 360 365
Lys Lys Ala Glu Glu Glu Glu Ala Lys Arg Arg Ala Ala Glu Glu Asp
370 375 380
Lys Val Lys Glu Lys Pro Ala Glu Gin Pro Gin Pro Ala Pro Ala Pro
385 390 395 400
Gin Pro Glu Lys Pro Thr Glu Glu Pro Glu Asn Pro Ala Pro Ala Pro
405 410 415
Ala Pro Lys Pro Glu Asn Pro Ala Glu Lys Pro Lys Ala Glu Lys Pro
420 425 430
Ala Asp Gin Gin Ala
435
<210> 30
<211> 439
<212> PRT
<213> Streptococcus pneumoniae
<400> 30
Val Ala Val Ala Ser Leu Val Met Gly Ser Val Val His Ala Thr Glu
1 5 10 15
Lys Glu Val Thr Thr Gin Val Ala Thr Ser Ser Asn Arg Ala Asn Glu
20 25 30
/
CA 02326388 2001-04-06
-68y-
Ser Gin Ala Gly His Arg Lys Ala Ala Glu Gin Phe Asp Glu Tyr Ile
35 40 45
Lys Thr Met Ile Gin Leu Asp Arg Arg Lys His Thr Gin Asn Phe Ala
50 55 60
Leu Asn Ile Lys Leu Ser Arg Ile Lys Thr Glu Tyr Leu Arg Lys Leu
65 70 75 80
Asn Val Leu Glu Glu Lys Ser Lys Ala Glu Leu Pro Ser Glu Thr Lys
85 90 95
Lys Glu Ile Asp Ala Ala Phe Glu Gin Phe Lys Lys Asp Thr Asn Arg
100 105 110
Thr Lys Lys Thr Val Ala Glu Ala Glu Lys Lys Val Glu Glu Ala Lys
115 120 125
Lys Lys Ala Lys Ala Gin Lys Glu Glu Asp His Arg Asn Tyr Pro Thr
130 135 140
Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala Glu Ser Asp Val Glu
145 150 155 160
Val Lys Lys Ala Glu Leu Glu Leu Val Lys Glu Glu Ala Lys Glu Ser
165 170 175
Arg Asp Asp Glu Lys Ile Lys Gin Ala Glu Ala Lys Val Glu Ser Lys
180 185 190
Lys Ala Glu Ala Thr Arg Leu Glu Asn Ile Lys Thr Asp Arg Glu Lys
195 200 205
Ala Glu Glu Glu Ala Lys Arg Arg Ala Glu Ala Lys Leu Lys Glu Ala
210 215 220
Val Glu Lys Asn Val Ala Thr Ser Glu Gin Asp Lys Pro Lys Gly Arg
225 230 235 240
Arg Lys Arg Gly Val Pro Gly Glu Gin Ala Thr Pro Asp Lys Lys Glu
245 250 255
Asn Asp Ala Lys Ser Ser Asp Ser Ser Val Gly Glu Glu Ala Leu Pro
260 265 270
Ser Pro Ser Leu Lys Pro Glu Lys Lys Val Ala Glu Ala Glu Lys Lys
275 280 285
Val Ala Glu Ala Glu Lys Lys Ala Lys Ala Gin Lys Glu Glu Asp Arg
290 295 300
Arg Asn Tyr Pro Thr Asn Thr Tyr Lys Thr Leu Glu Leu Glu Ile Ala
305 310 315 320
Glu Ser Asp Val Lys Val Lys Glu Ala Glu Leu Glu Leu Val Lys Glu
325 330 335
CA 02326388 2001-04-06
-68z-
Glu Ala Lys Glu Ser Arg Asn Glu Glu Lys Val Asn Gin Ala Lys Ala
340 345 350
Lys Val Glu Ser Lys Lys Ala Glu Ala Thr Arg Leu Glu Lys Ile Lys
355 360 365
Thr Asp Arg Lys Lys Ala Glu Glu Glu Ala Lys Arg Lys Ala Ala Glu
370 375 380
Glu Asp Lys Val Lys Glu Lys Pro Ala Glu Gin Pro Gin Pro Ala Pro
385 390 395 400
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 Glu Glu
435
<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
1 5 10 15
Glu Gly Thr Thr Gin Ala Pro Thr Ser Ser Asn Arg Gly Asn Glu Ser
20 25 30
Gin Ala Glu His Met Lys Ala Ala Lys Gin Val Asp Glu Tyr Ile Glu
35 40 45
Lys Met Leu Gin Leu Asp Arg Arg Lys His Thr Gln Asn Val Gly Leu
50 55 60
Leu Thr Lys Leu Gly Ala Ile Lys Thr Glu Tyr Leu Arg Gly Leu Ser
65 70 75 80
Val Ser Lys Glu Lys Ser Thr Ala Glu Leu Pro Ser Glu Ile Lys Glu
85 90 95
Lys Leu Thr Ala Ala Phe Lys Gin Phe Lys Lys Asp Thr Leu Lys Pro
100 105 110
Glu Lys Lys Val Ala Glu Ala Glu Lys Lys Val Ala Glu Ala Lys Lys
115 120 125
Lys Ala Glu Asp Gin Lys Glu Glu Asp Arg Arg Asn Tyr Pro Thr Ile
130 135 140
