WO2002028351A2 - Recombinant mucin binding proteins from steptococcus pneumoniae - Google Patents

Abstract

The present invention provides for amino acid and nucleic acid sequences of isolated mucin-binding proteins from pneumococcal bacteria and fragments thereof. Expression vectors, transfected host cells, methods for producing recombinant mucin-binding proteins, compositions comprising the proteins, and antibodies to the proteins also are contemplated. A method of inducing an immune response is described by the present invention. Screening and diagnosing methods are also discussed.

Description

RECOMBINANT MUCIN BINDING PROTEINS FROM STREPTOCOCCUS PNEUMONIAE

PRIORITY

This application claims priority under 35 U.S.C. § 119 from US Provisional Patent

Application Serial No. 60/237,888, filed October 4, 2000 and US Provisional Patent Application Serial No. 60/267,104, filed February 7, 2001; which are each hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention provides amino acid sequences and nucleic acid sequences relating to mucin-binding proteins of Streptococcus pneumoniae having molecular

weights of 12 and 14 kilo Daltons (kDa). The present invention also pertains to compositions

for the treatment and prophylaxis of infection or inflammation associated with bacterial

infection.

BACKGROUND OF THE INVENTION

The middle ear is a sterile, air-filled cavity separated from the outer ear by the

eardrum. Attached to the eardrum are three ear bones that vibrate when sound waves strike

the eardrum. Vibrations are transmitted to the inner ear, which generates nerve impulses that are sent to the brain. Air may enter the middle ear through the Eustachian tube, which opens in the walls of the nasopharynx. The nasopharynx is located posterior to the nasal cavities. The nasopharynx is lined by the respiratory epithelium and stratified squamous epithelium. Beneath the

respiratory epithelium, the abundant mucosa-associated lymphoid tissue (MALT) forms the

nasopharyngeal tonsil (adenoids).

Bacterial infection or inflammation of the middle ear is mainly observed in children. Due to the isolation of the middle ear, it is suggested that development of middle

ear infections requires the involvement of the nasopharynx and Eustachian tube. Infections

with Streptococcus pneumoniae (S. pneumoniae) are one of the major causes of middle ear

infections, as well as bacteremia, meningitis, and fatal pneumonia worldwide (Butler, J.C, et

al, American Journal of Medicine, 1999, 107:69S-76S). The rapid emergence of multi-drug resistant pneumococcal strains throughout the world has led to increased emphasis on

prevention of pneumococcal infections by vaccination (Goldstein and Garau, Lancet, 1997, 350:233-4).

Protein antigens of S. pneumoniae have been evaluated for protective efficacy

in animal models of pneumococcal infection. Some of the most commonly studied vaccine candidates include the the PspA proteins, PsaA lipoprotein, and the CbpA protein. Numerous

studies have shown that PspA protein is a virulence factor (Grain, M.J., et al, J-nfect Immun,

1990, 58:3293-9; McDanieL L.S., et al, J Exp Med,1984, 160:386-97), but is antigenically

variable among pneumococcal strains. Additionally, a recent study has indicated that some antigenically conserved regions of a recombinant PspA variant may elicit cross-reactive

antibodies in human adults (Nabors, G.S., et al, Vaccine, 2000, 18:1743-1754). PsaA, a 37

kDa lipoprotein with similarity to other Gram-positive adhesins, is involved in manganese transport in pneumococci (Dintilhac, A., et al, Molecular Microbiology, 1997, 25(4):727-

739; Sampson, J.S., et al, Infect Immun, 1994, 62:319-24.) and has been shown to be protective in mouse models of systemic disease (Talkington, D.F., et al, Microb Pathog,

1996. 21 : 17-22). The surface exposed choline binding protein, CbpA, is antigenically

conserved and also is protective in mouse models of pneumococcal disease (Rosenow, C, et al. Molecular Microbiology, 1997, 25:819-29). Since nasopharyngeal colonization is a

prerequisite for otic disease, intranasal immunization of mice with pneumococcal proteins

and appropriate mucosal adjuvants has been used to enhance the mucosal antibody response

and thus, the effectiveness of protein vaccine candidates (Briles, D.E., et al, Infect Immun, 2000, 68:796-800; Yamamoto, M., et al, A. J Immunol, 1998, 161:4115-21).

The currently available 23-valent pneumococcal capsular polysaccharide

vaccine is not effective in children of less than 2 years of age or in immunocompromised

patients, two of the major populations at risk from pneumococcal infection (Douglas, R.M., et al, Journal of Infectious Diseases, 1983, 148:131-137). A 7-valent pneumococcal

polysaccharide-protein conjugate vaccine, was shown to be highly effective in infants and children against systemic pneumococcal disease caused by the vaccine serotypes and against

cross-reactive capsular serotypes (Shinefield and Black, Pediatr Infect Dis J, 2000, 19:394-7).

The seven capsular types cover greater than 80% of the disease isolates in the United States,

but only 57-60% of disease isolates in other areas of the world (Hausdorff, W.P., et al, Clinical Infectious Diseases, 2000, 30:100-21). Therefore, there is an immediate need for a

vaccine to cover most or all of the disease causing serotypes of pneumococci.

SUMMARY OF THE INVENTION

The present invention provides for isolated mucin-binding proteins from pneumococcal bacteria. In preferred embodiments, the mucin-binding proteins comprises (i)

an amino acid sequence as depicted in SEQ ID NO:8, SEQ ID NO: 10; (ii) which have a molecular weight of about 12 kilo Daltons (kDa) or about 14 kDa, where the molecular weight is determined using a 10-20% SDS-PAGE gel; (iii) nucleic acid sequences encoding a

mucin-binding protein, where the nucleic acid sequence has a sequence as depicted in SEQ

ID NO:7, SEQ ID NO:9; or (iv) any fragments of the disclosed nucleic acid and amino acid

sequences. Expression vectors encoding a mucin-binding protein or any fragments and host cells tranfected with the expression vectors also are contemplated. A method for producing recombinant mucin-binding proteins or any fragments also is contemplated.

Compositions are also contemplated by the present invention. The

compositions comprise (i) a mucin-binding protein, (ii) an amino acid sequence as depicted in

SEQ ID NO:8, SEQ ID NO: 10; (iii) which have a molecular weight of about 12 kilo Daltons (kDa) or about 14 kDa, where the molecular weight is determined using a 10-20% SDS- PAGE gel; (iv) nucleic acid sequences encoding a mucin-binding protein, where the nucleic

acid sequence has a sequence as depicted in SEQ ID NO: 7, SEQ ID NO:9; or (v) any

fragments of the amino acid or nucleic acid sequence; and a pharmaceutically acceptable carrier. Compositions comprising expression vectors and host cells are further contemplated.

The present invention further contemplates an immunogenic composition

comprising at least one mucin-binding protein from pneumococcal bacteria, a

pharmaceutically acceptable carrier, and optionally at least one adjuvant. In a preferred

embodiment, the mucin-binding protein comprises an amino acid sequence as depicted in SEQ ID NO: 8, SEQ ID NO: 10, or an immunogenic fragment; is encoded by a nucleic acid

sequence as depicted in SEQ ID NO:7, SEQ ID NO:9, or an immunogenic fragment; or is

polypeptide having a molecular weight of about 12 kDa or about 14 kDa, where the molecular

weight is determined using a 10-20% SDS-PAGE gel.

Immunogenic compositions comprising at least one expression vector encoding a mucin-binding protein from pneumococcal bacteria, a pharmaceutically

acceptable carrier, and optionally at least one adjuvant also are contemplated by the present

invention. In a preferred embodiment, the mucin-binding protein comprises an amino acid sequence as depicted in SEQ ID NO: 8, SEQ ID NO: 10, or an immunogenic fragment; is

encoded by a nucleic acid sequence as depicted in SEQ ID NO:7, SEQ ID NO:9, or an

immunogenic fragment; or is polypeptide having a molecular weight of about 12 kDa or

about 14 kDa, where the molecular weight is determined using a 10-20% SDS-PAGE gel.

In preferred embodiments, the immunogenic composition comprises

Streptococcus pneumoniae. In a further embodiment, the immunogenic composition elicits

protective immunity from a disease caused by Streptococcus pneumoniae. In an additional

embodiment, the disease is selected from the group consisting of otitis media, rhinosinusitis, bacteremia, meningitis, pneumonia, and lower respiratory tract infection.

A method of inducing an immune response in a mammal is contemplated by the present invention. The method comprises administering to the mammal an amount any of

the immunogenic compositions effective to induce an immune response.

A method of inducing an immune response in a mammal which is infected with pneumococcal bacteria also is contemplated by the present invention. The method

comprises administering to the mammal an amount of a compound effective to inhibit binding of an amino acid sequence as depicted in SEQ ID NO: 8 or SEQ ID NO: 10 with

mucin to induce the immune response. In a preferred embodiment, the mucin is selected

from the group consisting of human nasopharyngeal mucin and human middle ear mucin.

A method for screening for a compound which induces an immune response in a mammal having a disease caused by pneumococcal bacteria is contemplated by the present

invention. The method comprises comparing the amount of binding of an amino acid sequence as depicted in SEQ ID NO: 8 or SEQ ID NO: 10 to mucin in the presence of a compound to a second amount of binding of an amino acid sequence as depicted in SEQ ID

NO: 8 or SEQ ID NO: 10 to mucin not in the presence of the compound; where a lower first

amount of binding than the second amount binding indicates that the compound may induce

an immune response in the mammal.

The present invention also contemplates a method for diagnosing

pneumococcal bacterial infection. The method comprises comparing the level of mucin-

binding protein as depicted in SEQ ID NO: 8, SEQ ID NO: 10, or fragments, in suspect sample

to the level of mucin-binding protein as depicted in SEQ ID NO:8, SEQ ID NO:10, or

fragments, in a control sample, where a higher level of the mucin-binding protein in the suspect sample than the level of mucin-binding protein in the control sample indicates that

the suspect sample comprises pneumococcal bacterial infection.

The present invention also contemplates an antibody that binds to

Streptococcus pneumoniae mucin-binding proteins. In preferred embodiments, the antibodies selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 8, SEQ ID NO: 10, or fragments. In further embodiments, the antibody is chimeric, humanized, anti-idiotypic,

conjugated to a pharmaceutically active compound, or a monoclonal antibody. Additionally,

the monoclonal antibody may be humanized, anti-idiotypic, or conjugated to a

pharmaceutically active compound.

The present invention provides for methods for inducing an immune response in a mammal, where the method comprises administering to the mammal an amount of an

anti-idiotypic antibody effective to induce an immune response.

The present invention also provides methods for inducing an immune response in a mammal which is infected with pneumococcal bacteria, where the method comprises administering to the mammal an amount of an antibody conjugated to a pharmaceutically

active compound effective to induce an immune response.

The present invention further provides for mucin-binding protein variants and nucleic acids that encode the variant where at least one lysine residue is replaced, deleted, or

altered. In a further embodiment, the mucin-binding protein activity of the variant is

decreased when compared to mucin-binding protein activity of a wild-type mucin-binding

protein. A mucin-binding protein fragment comprised of at least 8 amino acids, where the

fragment comprises at least one lysine residue, also is contemplated. In a further embodiment, the absence of at least one of at least one lysine residue in the fragment

decreases mucin-binding protein activity when compared to a wild-type mucin binding

protein. In a further embodiment, a nucleic acid sequence encoding a mucin-binding protein

variant or fragment as discussed above, where the nucleic acid sequence is modified so that at least one lysine residue in the protein is replaced, deleted or altered; also is contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A and B depict SDS -PAGE gels of DEAE fractions from PBS

washes of S. pneumoniae strain 49136 and Mucin binding overlay assay of DEAE fractions. In each gel, lane 1, unstained standards; lane 2, fraction #18; lane 3, fraction #26; lanes 4 and

5, unstained standards. The gel in Figure A shows the distinct small bands in fraction #26

resolved by the gradient gel. The autoradiogram in Figure B shows two separate and distinct

bands in fraction #26 that bind radiolabeled mucin.

Figure 2 depicts a mucin overlay assay of whole cell lysates of recombinant

expression of pLP533 and pLP535. This shows the specific binding of radiolabeled mucin by

recombinantly expressed 12kDa and 14kDa proteins. Lanel, PBS wash of S. pneumoniae; lane 2, pLP537 (12kDa); Lane 3, Lysozyme molecular weight marker; Lane 4, unrelated

recombinantly expressed protein; Lane 5, preinduced pLP538; Lane 6, post induction pLP538

(14 Da).

Figure 3 depicts a graphical representation of mean Log 10 cfu/gram of tissue

from immunization studies in mice. Animals were treated with either 12kDa mucin binding protein (12k MBP), pneumococcal type 3 conjugate (PNC-3), KLH, or vehicle (naive)

adjuvanted with 0.1 μg of CT-E29H on weeks 0, 2, and 4. The results show that the mice

immunized with the rl2 kDa MBP had statistically significantly reduced levels of

colonization as compared to the KLH/CT-E29H control group (p<0.05 by Student's t test).

DETAILED DESCRIPTION

The proteins and nucleic acids of this invention possess diagnostic,

prophylactic and therapeutic utility for diseases caused by Streptococcus pneumoniae

infection. They can be used to design screening systems for compounds that interfere or disrupt interaction of mucin-binding proteins with mucin. The nucleic acids and proteins

also can be used in the preparation of compositions against S. pneumoniae infection and/or

other pathogens when used to express foreign genes. Fragments of the protein, variants of the

protein, and fragments of the variants can be used to assess the amino acids involved in the activity of the proteins. One can use the activity information to select fragments that contain these lysine residues or one can knockout or reduce the mucin-binding activity of a fragment

of protein of this invention.

Streptococcus Pneumoniae S. pneumoniae is a species of bacteria which is highly infectious in the human

body. There have been more than 80 serotypes identified, to date. Several of these serotypes are etiological agents in a variety of disease states including, but not limited to, pneumonia,

meningitis, endocarditis, arthritis, sinusitis, otitis, bronchitis, and laryngitis. Pneumococcal

infections have been identified as a leading cause of death in persons with

immunocompromised systems, such as those infected with HTV.

S. pneumoniae is a species of the Streptococcus genus of the Streptococcaceae

family. This family comprises Gram-positive, non-motile, spherical or oval cells that do not form endospores. S. pneumoniae have an inorganic terminal electron acceptor for oxidative-

metabolism; however, they will grow in the presence of oxygen. This allows S. pneumoniae

to grow in a variety of environments and thus it is well adapted to grow in various human

tissues. The bacteria is difficult to target with penicillin, since many strains produce a polysaccharide capsule.

The first step towards pneumococcal infection is colonization of the

nasopharynx. Disruption of binding of the pneumococci to human nasopharyngeal/otic cells

should result in reduction of colonization and a lower disease potential. Thus, isolation of the structures responsible for pneumococcal binding to human cells could lead to vaccine candidates. Pneumococci have evolved numerous mechanisms for binding to human

nasopharyngeal cells, including the PspA, PsaA, and CbpA proteins. Additionally,

pneumococci may specifically bind to human nasopharyngeal mucin as a first step in

colonization. Thus, identification of the pneumococcal structure(s) responsible for this interaction may identify potential vaccine targets. Mucins

Mucins are high molecular weight glycoproteins; that is, they are proteins

which have a large number of sugar residues attached to them. Almost 50% of their

molecular weight is comprised of sugar units. The Muc genes encode several mucin type

proteins expressed in epithelial cells. They have the functions of cellular protection and lubrication and are the major components of mucus. Specific combinations of mucin proteins

are expressed in various regions (PorchetN., et al, Journal de la Societe de Biologie, 1999,

193:85-99).

A common feature of Muc genes is that they contain tandem repeats (TR) of DNA sequence which lead to tandem repetition of amino acid motifs. There is evidence that

Muc allele distribution is different in different populations. Mucins show change in expression in inflammatory disease and cancer (Lesuffleur, T., et al. Crit Rev Oncol Hematol,

1994, 17:153-80) and short alleles in specific Muc genes appear to be associated with gastric

cancer (Carvalho, F., et al, Glycoconj J, 1997, 14:107-11).

Molecular Biology

Embodiments of this invention relate to isolated polynucleotide sequences

encoding the polypeptides or proteins, as well as variants of such sequences. Preferably, under ^'high stringency conditions, these variant sequences hybridize to polynucleotides encoding one or more mucin-binding proteins. More preferably, under high stringency

conditions, these variant sequences hybridize to polynucleotides encoding one or more

mucin-binding protein sequences, such as the polynucleotide sequences of SEQ ID NOs: 7

and 9. For the purposes of defining high stringency southern hybridization conditions, reference can conveniently be made to Sambrook et al. (1989) at pp. 387-389 which is herein incorporated by reference, where the washing step at paragraph 11 is considered high

stringency.

This invention also relates to conservative variants wherein the polynucleotide sequence differs from a reference sequence through a change to the third nucleotide of a

nucleotide triplet. Preferably these conservative variants function as biological equivalents to

the mucin-binding protein reference polynucleotide . sequences. In a preferred embodiment,

variants that function as biological equivalents are those that bind to mucins.

The present invention further comprises DNA sequences which, by virtue of

the redundancy of the genetic code, are biologically equivalent to the sequences which encode

for the mucin-binding proteins, that is, these other DNA sequences are characterized by

nucleotide sequences which differ from those set forth herein, but which encode a protein having the same amino acid sequence as that encoded by the DNA sequence in SEQ ID NO: 8 or SEQ ID NO:10.

This invention also comprises DNA sequences which encode amino acid

sequences which differ from those of the S. pneumonia mucin-binding proteins, but which are

biologically equivalent to those described for one of these proteins (SEQ ID NO: 8 or SEQ ID NO: 10). Such amino acid sequences may be said to be biologically equivalent to such mucin- binding if their sequences differ only by minor deletions from, insertions into or substitutions

to the mucin-binding sequence, such that the tertiary configurations of the sequences are

essentially unchanged from those of the wild-type protein.

For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which

result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, as well as changes based on similarities of residues in their hydropathic index, can also be

expected to produce a biologically equivalent product. Nucleotide changes which result in

alteration of the N-terminal or C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.

One can use the hydropathic index of amino acids in conferring interactive

biological function on a polypeptide, as discussed by Kyte and Doolittle (1982), wherein it

was determined that certain amino acids may be substituted for other amino acids having

similar hydropathic indices and still retain a similar biological activity. Alternatively, substitution of like amino acids may be made on the basis of hydrophilicity, particularly

where the biological function desired in the polypeptide to be generated is intended for use in immunological embodiments. See, for example, U.S. Patent 4,554,101 (which is hereby

incorporated herein by reference), which states that the greatest local average hydrophilicity of a "protein," as governed by the hydrophilicity of its adjacent amino acids, correlates with

its immunogenicity. Accordingly, it is noted that substitutions can be made based on the hydrophilicity assigned to each amino acid. In using either the hydrophilicity index or

hydropathic index, which assigns values to each amino acid, it is preferred to introduce

substitutions of amino acids where these values are ± 2, with ± 1 being particularly preferred, and those within ± 0.5 being the most preferred substitutions.

Furthermore, changes in known variable regions are biologically equivalent

where the tertiary configurations of the conserved regions are essentially unchanged from

those of the mucin-binding protein. An alternative definition of a biologically equivalent sequence is one that is still capable of generating a cross-reactive immune response. In

particular, the proteins may be modified by lengthening or shortening the corresponding insertion from the gonococcal pilin, as long as the modified protein is still capable of

generating a desired immune response.

Each of the proposed modifications is well within the routine skill in the art, as

is determination of retention of structural and biological activity of the encoded products.

Therefore, where the terms "mucin-binding protein" are used in either the specification or the

claims, it will be understood to encompass all such modifications and variations which result

in the production of a biologically equivalent protein.

Preferable characteristics of the mucin-binding proteins described herein,

encoded by the nucleotide sequences of this invention, include one or more of the following:

(a) being a membrane protein or being a protein directly associated with a membrane; (b)

capable of being separated as a protein using an SDS acrylamide gel; and (c) retaining its biological function of interacting with mucin proteins.

Variants and fragments may be attenuated, i.e. having reduced on no mucin-

binding activity when compared to wild-type mucin-binding proteins of the present invention.

One example of such variants and fragments are those amino acid sequences that have at least

one lysine residue, encompassed within the sequence, that is replaced with another amino acid, deleted from the sequence or chemically altered. Mutated lysines may include those

which play a role in mucin-binding protein activity such as, but not limited to, mucin binding.

Alternatively, these mutated lysine residues may not be involved in mucin binding but

alteration of these residues alters protein immunogenicity. In such variants and fragments, the lysine residue or lysine residues may be replaced with another amino acid of similar charge or structure. In one embodiment, the lysine residues that may be mutated are located

at positions 3, 5, 6, 42, 45, 46, 56, 57, or 70 in SEQ ID NO:8 or at positions 3, 7, 11, 12, 51,

55, 70, 78, 85, or 123 in SEQ ID NO:9. In one embodiment, all the lysine residues present may be replaced, deleted, and/or altered.

Fragments of the wild-type and variant forms of the mucin-binding proteins

may be made by one of ordinary skill in the art. These fragments may be used for

immunogenic compositions and for various chemical and biological studies including, but not

limited to, tertiary structure studies, antibody preparation, and detennining the ligand binding

domains of the proteins. The fragments may be of any size that may be needed for a specific

use. In one embodiment, the fragment is at least 8 amino acids in length. In another

embodiment, the fragment is 10-15 amino acids in length, hi a preferred embodiment, the amino acids are contiguous. In the fragment, a lysine residue that is involved in mucin-

binding protein activities (e.g. , mucin binding or immunogenicity) may be present. The lysine residue may be altered, replaced, or deleted to determine what role the residue plays in

protein activity. Alteration of the lysine residue may decrease the activity mutated fragment

when compared to a non-mutated fragment, as discussed above. Methods to produce variants

and fragments, as well as methods to assess the role of lysine in mucin-binding protein activity, are well known in the art and are discussed below.

From the present specification, including examples, and other readily available

methods, one can readily determine which lysine residue or lysine residues are responsible for

the mucin-binding activity (e.g., mucin binding and protein immunogenicity). For example,

one can compare the binding of mucin to (i) a mucin binding protein that has been mutated

and to (ii) a wild-type mucin-binding protein (e.g., SEQ ID NO: 8 or 10). Decreased or altered binding of mucin to the mutated mucin-binding protein compared to the wild-type

protein would indicate that the mutated amino acid plays a role in recognition and binding of the ligand. Accordingly, one can use this information to select fragments that contain these lysine residues or one can knockout or reduce the mucin-binding activity of a fragment of protein of this invention. Alternatively, production of antisera against a wild-type and mutant

protein can be compared. Increased antisera product for a mutant protein would indicate that the residue plays a role in immunogenicity. Therefore, one can use this information to select

fragments that contain these lysine residues that increase the immunogenicity of the present

protein.

In one embodiment, the decrease in mucin-binding activity is at least 10%. In a another embodiment, the decrease in mucin-binding activity is at least 50%. In a further embodiment, the decrease is at least 90%. The attenuated proteins of the present invention

comprise at least one epitopic region of the wild-type protein. In alternative embodiments,

the epitopic region of the protein comprises at least 20 contiguous nucleotides or 8 contiguous

amino acids.

Such variants and fragments may be encoded by nucleic acid sequences that are modified so as to encode an amino acid other than lysine, delete the amino acid, or alter

the amino acid at the specific position. Such proposed modifications to the polynucleotide

encoding the variant or fragment are well known and within the ordinary skill of one in the art (See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New

York; DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985);

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 (R.I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M.

Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.

(1994)). Preferably, the fragments and variant amino acid sequences and variant

nucleotide sequences expressing mucin-binding proteins are biological equivalents, i.e. they retain substantially the same function of the wild-type mucin-binding proteins. Such variant

amino acid sequences are encoded by polynucleotides sequences of this invention. Variant amino acid sequences may have about 70% to about 80%, and preferably about 90%, overall

similarity to the amino acid sequence of the mucin-binding proteins. The variant nucleotide

sequences may have either about 70% to about 80%, and preferably about 90%, overall

similarity to the nucleotide sequences which, when transcribed, encode the amino acid

sequence of the mucin-binding proteins or a variant amino acid sequence of the mucin- binding proteins.

The "isolated" sequences of the present invention are non-naturally occurring

sequences. For example, these sequences can be isolated from their normal state within the

genome of the bacteria; or the sequences may be synthetic, i.e. generated via recombinant

techniques, such as well-known recombinant expression systems, or generated by a machine.

The invention also provides a recombinant DNA cloning vehicle capable of

expressing a mucin-binding protein comprising an expression control sequence having

promoter and initiator sequences and a nucleic acid sequence of the present invention located

3' to the promoter and initiator sequences. Cloning vehicles can be any plasmid or expression vector known in the art, including viral vectors (see below). In a further aspect, there is

provided a host cell containing a recombinant DNA cloning vehicle and/or a recombinant

mucin-binding proteins of the present invention. Suitable expression control sequences, host

cells and expression vectors are well known in the art, and are described by way of example, in Sambrook et al. (1989).

Suitable host cells may be selected based on factors which can influence the yield of recombinantly expressed proteins. These factors include, but are not limited to,

growth and induction conditions, mRNA stability, codon usage, translational efficiency and the presence of transcriptional terminators to minimize promoter read through. Upon

selection of suitable host cells, the cell may be transfected with expression vectors comprising

nucleic acid sequences of the present invention. The cells may be transfected using any

methods known in the art (see below).

Once host cells have been transfected with expression vectors of the present

invention, cells are cultured under conditions such that polypeptides are expressed. The

polypeptide is then isolated substantially free of contaminating host cell components by

techniques that are well known to those skilled in the art.

Depending on the application of the desired recombinant proteins, a heterologous nucleotide sequence may encode a co-factor, cytokine (such as an interleukin), a

T-helper epitope, a restriction marker, adjuvant, or a protein of a different microbial pathogen

(e.g. virus, bacterium, fungus or parasite), especially proteins capable of eliciting a protective

immune response. It may be desirable to select a heterologous sequence that encodes an

immunogenic portion of a co-factor, cytokine (such as an interleukin), a T-helper epitope, a restriction marker, adjuvant, or a protein of a different microbial pathogen (e.g. virus,

bacterium or fungus). Other types of non-mucin-binding moieties include, but are not limited

to, those from cancer cells or tumor cells, allergens, amyloid peptide, protein or other

macromolecular components.

For example, in certain embodiments, the heterologous genes encode cytokines, such as interleukin- 12, which are selected to improve the prophylatic or therapeutic characteristics of the recombinant proteins.

Examples of such cancer cells or tumor cells include, but are not limited to, prostate specific antigen, carcino-embryonic antigen, MUC-1, Her2, CA-125 and MAGE-3.

Examples of such allergens include, but are not limited to, those described in

United States Patent Number 5,830,877 and published International Patent Application Number WO 99/51259, which are hereby incorporated by reference, and include pollen,

insect venoms, animal dander, fungal spores and drugs (such as penicillin). Such components interfere with the production of IgE antibodies, a known cause of allergic reactions.

Amyloid peptide protein (APP) has been implicated in diseases referred to

variously as Alzheimer's disease, amyloidosis or amyloidogenic disease. The β-amyloid

peptide (also referred to as Aβ peptide) is a 42 amino acid fragment of APP, which is

generated by processing of APP by the β and γ secretase enzymes, and has the following

sequence:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu Val

Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He Gly Leu Met Val Gly Gly Val Val lie Ala (SEQ ID NO: 11).

In some patients, the amyloid deposit takes the form of an aggregated Aβ

peptide. Surprisingly, it has now been found that administration of isolated Aβ peptide

induces an immune response against the Aβ peptide component of an amyloid deposit in a

vertebrate host (See Published International Patent Application WO 99/27944). Such Aβ

peptides have also been linked to unrelated moieties. Thus, the heterologous nucleotides

sequences of this invention include the expression of this Aβ peptide, as well as fragments of

Aβ peptide and antibodies to Aβ peptide or fragments thereof. One such fragment of Aβ

peptide is the 28 amino acid peptide having the following sequence (As disclosed in U.S. Patent 4,666,829): Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys (SEQ ID NO: 12).

The heterologous nucleotide sequence can be selected to make use of the

normal route of infection of pneumococcal bacteria, which enters the body through the

respiratory tract and can infect a variety of tissues and cells, for example, the meninges,

blood, and lung. The heterologous gene may also be used to provide agents which are used for gene therapy or for the targeting of specific cells. As an alternative to merely taking advantage of the normal cells exposed during the normal route of pneumococcal infection, the

heterologous gene, or fragment, may encode another protein or amino acid sequence from a

different pathogen which, when employed as part of the recombinant protein, directs the recombinant protein to cells or tissue which are not in the normal route of infection. In this manner, the protein becomes a targeting tool for the delivery of a wider variety of foreign

proteins.

Molecular weight of proteins may be determined by using any method known

in the art. A non-limiting list of methods includes, denaturing SDS-PAGE gel, size exclusion chromatography, and iso-electric focusing. Conditions appropriate for each method (e.g. time of separation, voltage, current, and buffers) can be determined as needed using defined

methods in the art. In a preferred embodiment, denaturing SDS-PAGE is used to determine

the molecular weight of the proteins. Additionally, the conditions used to detennine the molecular weight are preferably, 1 hour separation time at 20 milli Amps and constant current.

Detection of the proteins can be determined using various methods in the art.

These methods include, but are not limited to, Western blotting, coomassie blue staining,

silver staining, autoradiography, fluorescent and phosphorescent probing. In a preferred embodiment of this invention, the proteins were detected by Western blotting.

The term "mucin-binding protein" in describing embodiments of the invention, infra, includes embodiments that employ fragments, variants and attenuated forms

thereof as a replacement for wild-type mucin-binding protein or as addition thereto, unless specified otherwise.

Viral and Non- Viral Vectors

' Preferred vectors, particularly for cellular assays in vitro and in vivo, are viral

vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, and other recombinant viruses with desirable cellular

tropism. Thus, a gene encoding a functional or mutant protein or polypeptide domain

fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using

a tissue-specific promoter, or both. Targeted gene delivery is described in PCT Publication

No. WO 95/28494.

Viral vectors commonly used for in vivo or ex vivo targeting and therapy

procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (e.g., Miller and Rosman, BioTechniques, 1992, 7:980-990).

Preferably, the viral vectors are replication-defective, that is, they are unable to replicate

autonomously in the target cell. Preferably, the replication defective virus is a minimal virus, i. e. ,

it retains only the sequences of its genome which are necessary for encapsulating the genome to produce viral particles.

DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Ban virus (EBV), adenovirus,

adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use

of defective viral vectors allows for administration to cells in a specific, localized area, without

concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted.

Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al, Molec. Cell. Neurosci., 1991, 2:320-330), defective herpes virus vector lacking a glyco-protein L gene, or other defective herpes virus vectors (PCT Publication Nos.

WO 94/21807 and WO 92/05263); an attenuated adenovirus vector, such as the vector described

by Stratford-Perricaudet et al. (J. Clin. Invest., 1992, 90:626-630; see also La Salle et al. , Science, 1993, 259:988-990); and a defective adeno-associated virus vector (Samulski et al. , J. Virol., 1987, 61:3096-3101; Samulski et al, J. Virol., 1989, 63:3822-3828; Lebkowski et al,

Mol. Cell. Biol, 1988, 8:3988-3996).

Various companies produce viral vectors commercially, including, but not limited

to, Avigen, Inc. (Alameda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, PA; adenoviral and AAV vectors), Genvec (adenoviral vectors),

IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral,

AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United

Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).

Adenovirus vectors. Adenoviruses are eukaryotic DNA viruses that can be

modified to efficiently deliver a nucleic acid of the invention to a variety of cell types. Various

serotypes of adenovirus exist. Of these serotypes, preference is given, within the scope of the present invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses

of animal origin (see PCT Publication No. WO 94/26914). Those adenoviruses of animal origin

which can be used within the scope of the present invention include adenoviruses of canine,

bovine, murine (example: Mavl, Beard etal, Virology, 1990, 75-81), ovine, porcine, avian, and simian (example: SAV) origin. Preferably, the adenovirus of animal origin is a canine

adenovirus, more preferably a CAV2 adenovirus (e.g. , Manhattan or A26/61 strain, ATCC VR-

800, for example). Various replication defective adenovirus and minimum adenovirus vectors

have been described (PCT Publication Nos. WO 94/26914, WO 95/02697, WO 94/28938, WO 94/28152, WO 94/12649, WO 95/02697, WO 96/22378). The replication defective

recombinant adenoviruses according to the invention can be prepared by any technique known

to the person skilled in the art (Levrero et al. , Gene, 1991, 101:195; European Publication No.

EP 185 573; Graham, EMBO J., 1984, 3:2917; Graham et al, J. Gen. Virol., 1977, 36:59).

Recombinant adenoviruses are recovered and purified using standard molecular biological techniques, which are well known to one of ordinary skill in the art.

Adeno-associated viruses. The adeno-associated viruses (AAV) are DNA viruses

of relatively small size that can integrate, in a stable and site-specific manner, into the genome

of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be

involved in human pathologies. The AAV genome has been cloned, sequenced and

characterized. The use of vectors derived from the AAVs for transferring genes in vitro and in

vivo has been described (see, PCT Publication Nos. WO 91/18088 and WO 93/09239; U.S.

PatentNos. 4,797,368 and 5,139,941; European Publication No. EP 488 528). The replication defective recombinant AAVs according to the invention can be prepared by cotransfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line which is infected with a human helper virus (for example an adenovirus). The AAV recombinants which are produced are then purified by standard techniques.

Retrovirus vectors. In another embodiment the gene can be introduced in a

retroviral vector, e.g., as described in U.S. Patent No. 5,399,346; Mann et al, Cell, 1983, 33:153; U.S. Patent Nos. 4,650,764 and 4,980,289; Markowitz et al. , J. Virol., 1988, 62:1120;

U.S. Patent No. 5,124,263; European Publication Nos. EP 453 242 and EP178 220; Bernstein

etal, Genet. Eng.,1985, 7:235; McCormick, BioTechnology, 1985, 3:689; PCT Publication No.

WO 95/07358; and Kuo etal, Blood, 1993, 82:845. The retroviruses are integrating viruses that

infect dividing cells. The retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). In recombinant retroviral vectors, the gag, pol and env

genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid

sequence of interest. These vectors can be constructed from different types of retrovirus, such

as, HIV, MoMuLV ("murine Moloney leukaemia virus" MSV ("murine Moloney sarcoma

virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Suitable packaging cell lines have been described in the prior art, in

particular the cell line PA317 (U.S. Patent No. 4,861,719); the PsiCRIP cell line (PCT

Publication No. WO 90/02806) and the GP+envAm-12 cell line (PCT Publication No. WO

89/07150). In addition, the recombinant retroviral vectors can contain modifications within the

LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences which may include a part of the gag gene (Bender et al, J. Virol., 1987, 61:1639). Recombinant

retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.

Retroviral vectors can be constructed to function as infectious particles or to undergo a single round of transfection. In the former case, the virus is modified to retain all of

its genes except for those responsible for oncogenic transformation properties, and to express the

heterologous gene. Non-infectious viral vectors are manipulated to destroy the viral packaging

signal, but retain the structural genes required to package the co-introduced virus engineered to contain the heterologous gene and the packaging signals. Thus, the viral particles that are

produced are not capable of producing additional virus.

Retrovirus vectors can also be introduced by DNA viruses, which permits one

cycle of retroviral replication and amplifies tranfection efficiency (see PCT Publication Nos. WO 95/22617, WO 95/26411, WO 96/39036 and WO 97/19182).

Lentivirus vectors. In another embodiment, lentiviral vectors can be used as

agents for the direct delivery and sustained expression of a transgene in several tissue types,

including brain, retina, muscle, liver and blood. The vectors can efficiently transduce dividing and nondividing cells in these tissues, and maintain long-term expression of the gene of interest. For a review, see, Naldini, Curr. Opin. BiotechnoL, 1998, 9:457-63; see also Zufferey, et al, J.

Virol., 1998, 72:9873-80). Lentiviral packaging cell lines are available and known generally in

the art. They facilitate the production of high-titer lentivirus vectors for gene therapy. An

example is a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line that can generate virusparticles at titers greater than 106 IU/ml for at least 3 to 4 days (Kafri, et al, J. Virol., 1999, 73: 576-584). The vector produced by the inducible cell line can be concentrated

as needed for efficiently transducing non-dividing cells in vitro and in vivo.

Non-viral vectors. In another embodiment, the vector can be introduced in vivo

by lipofection, as naked DNA, or with other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene

encoding a marker (Feigner, et. al, Proc. Natl. Acad. Sci. U.S.A., 1987, 84:7413-7417; Feigner and Ringold, Science, 1989, 337:387-388; see Mackey, et al, Proc. Natl. Acad. Sci. U.S.A.,

1988, 85:8027-8031; Ulmeref a/., Science, 1993, 259:1745-1748). Useful lipid compounds and compositions for transfer of nucleic acids are described in PCT Patent Publication Nos.

WO 95/18863 and WO 96/17823, and in U.S. Patent No. 5,459,127. Lipids may be chemically

coupled to other molecules for the purpose of targeting (see Mackey, et. al, supra). Targeted

peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of a nucleic acid in

vivo, such as a cationic oligopeptide (e.g., PCT Patent Publication No. WO 95/21931), peptides

derived from DNA binding proteins (e.g., PCT Patent Publication No. WO 96/25508), or a cationic polymer (e.g., PCT Patent Publication No. WO 95/21931).

It is also possible to introduce the vector in vivo as a naked DNA plasmid. Naked

DNA vectors for gene therapy can be introduced into the desired host cells by methods known

in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate

precipitation, use of a gene gun, or use of a DNA vector transporter (e.g., Wu et al, J. Biol. Chem., 1992, 267:963-967; Wu and Wu, J. Biol. Chem., 1988, 263:14621-14624; Canadian

Patent Application No. 2,012,311; Williams etal, Proc. Natl. Acad. Sci. USA, 1991, 88:2726-

2730). Receptor-mediated DNA delivery approaches can also be used (Curiel et al. , Hum. Gene

Ther., 1992, 3:147-154; Wu and Wu, J. Biol. Chem., 1987, 262:4429-4432). U.S. PatentNos.

5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA

transfer technique, termed electrotransfer, has been described (Mir et al. , C.P. Acad. Sci., 1988,

321:893; PC Publication Nos. WO 99/01157; WO 99/01158; WO 99/01175). Assay System

Any cell assay system that allows for assessing functional activities of

immunogenic compositions and compounds that modulate binding of mucin-binding proteins to mucin is contemplated by the present invention. In a specific embodiment, the assay can be used to identify compounds that interact with mucin to decrease binding of the mucin-

binding proteins described herein to mucin. This can be evaluated by assessing the effects of

a test compound on the interaction between mucin and the proteins described herein.

Any convenient method that permits detection of the binding of mucin with the mucin-binding proteins are contemplated by the present invention. In a preferred embodiment

of the invention, protein components of S. pneumoniae can be separated on apolyacrylamide gel

and transferred to a solid support. The support then may be probed with a labeled interacting

protein (e.g. mucin). The protein may be labeled with any label known in the art including, but not limited to, radioactivity, enzyme-based, dye molecules, or a flourescent or phosphorescent

tag. In a preferred embodiment, the label is radioactive. The label may be detected by any means known in the art. For example, autoradiography, scintillation counter, or ultra-violet light. In

a preferred embodiment, the radiolabel is detected by autoradiography. Assays that amplify the

signals from the probe are also known, such as, for example, those that utilize biotin and avidin, and enzyme-labeled immunoassays, such as ELISA assays.

In Vitro Screening Methods

Candidate agents are added to assay systems, prepared by known methods in

the art, and the level of binding betwen mucin and the mucin-binding proteins is measured.

Various in vitro systems can be used to analyze the effects of a compound on mucin binding. Preferably, each experiment is performed more than once, such as, for example, in triplicate

at multiple different dilutions of compound. The screening system of the invention permits detection of binding inhibitors. An

inhibitor screen involves detecting interaction of mucin and the mucin-binding protein when contacted with a compound that regulates interaction of these proteins. If a decrease in the

binding of mucin to the mucin-binding proteins is detected, then the compound is a candidate

inhibitor. If no decrease is observed, the compound does not alter the binding of mucin to the

proteins of the present invention.

Immunogenic Compositions

In further embodiments of this invention mucin-binding proteins are employed

in immunogenic compositions comprising (i) at least one mucin-binding protein; (ii) at least one pharmaceutically acceptable buffer, diluent, or carrier; and (iii) optionally at least one adjuvant. In a preferred embodiment, the immunogenic composition is used as a vaccine.

The mucin-binding protein may be recombinantly produced or isolated from a bacterial

preparation, according to methods known in the art. Preferably, these compositions have therapeutic and prophylactic applications as immunogenic compositions in preventing, protecting and/or ameliorating pneumococcal infection. In such applications, an

immunologically effective amount of at least one mucin-binding protein of this invention is

employed in such amount to cause a reduction, preferably a substantial reduction, in the

course a normal pneumoccocal infection. The proteins may be attenuated. The term "attenuated" refers to a protein that maintains functional activity of the wild-type protein

(e.g., mucin-binding protein) while binding to mucin is decreased or deleted. The attenuated

protein maintains substantial specificity of binding to mucin epitopes.

As used herein, the term "effective amount" refers to amount of the

immunogen component (i.e. mucin-binding proteins) described herein to stimulate an immune response, i.e., to cause the production of antibodies and/or a cell-mediated response

when introduced into a subject. In a preferred embodiment, the effective amount will decrease the colonization of S. pneumoniae. The term "immunogenic component" refers to

the ability of this component to stimulate secretory antibody and/or cell-mediated response

production in local regions, e.g. nasopharynx, when administered systemically as an

immunogenic composition according to the present invention.

As used herein the term "adjuvant" refers to an agent, compound or the like, which potentiates or stimulates the immune response in a subject when administered in combination with the immunogenic composition. Thus, the immune response, elicited by the

immunogenic composition combination, as measured by any convention method known in

the art, will generally be greater than that provoked by the immunogenic composition alone.

The immunogenic compositions of the invention can include one or more adjuvants, including, but not limited to aluminum hydroxide; aluminum phosphate; STIMULON QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, MA); MPL (3-O-

deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), 529 (an amino alkyl

glucosamine phosphate compound, Corixa, Hamilton, MT), IL-12 (Genetics Institute,

Cambridge, MA); GM-CSF (Immunex Corp., Seattle, Washington); N-acetyl-muramyl~L- theronyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP

11637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-

2'-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy-ethylamine) (CGP 19835A, referred to as MTP-PE); and cholera toxin. Others which may be used are non-toxic derivatives of

cholera toxin, including its A subunit, and/or conjugates or genetically engineered fusions of the mucin-binding polypeptide with cholera toxin or its B subunit ("CTB"),

procholeragenoid, fungal polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide ("MDP") derivatives, phorbol esters, the heat labile toxin of E. coli, block

polymers or saponins.

In certain preferred embodiments, the mucin-binding proteins of this invention

are used in an immunogenic composition for oral administration which includes a mucosal

adjuvant and used for the treatment or prevention of S.pneumoniae infection in a human host.

The mucosal adjuvant can be a cholera toxin; however, preferably, mucosal adjuvants other

than cholera toxin which may be used in accordance with the present invention include non-

toxic derivatives of a cholera holotoxin, wherein the A subunit is mutagenized, chemically modified cholera toxin, or related proteins produced by modification of the cholera toxin

amino acid sequence. For a specific cholera toxin which may be particularly useful in

preparing immunogenic compositions of this invention, see the mutant cholera holotoxin Ε29H, as disclosed in Published International Application WO 00/18434, which is hereby

incorporated herein by reference in its entirety. These may be added to, or conjugated with,

the polypeptides of this invention. The same techniques can be applied to other molecules

with mucosal adjuvant or delivery properties such as Escherichia coli heat labile toxin (LT).

Other compounds with mucosal adjuvant or delivery activity may be used such as bile;

polycations such as DEAE-dextran and polyornithine; detergents such as sodium dodecyl benzene sulphate; lipid-conjugated materials; antibiotics such as streptomycin; vitamin A;

and other compounds that alter the structural or functional integrity of mucosal surfaces.

Other mucosally active compounds include derivatives of microbial structures such as MDP;

acridine and cimetidine. STIMULON, QS-21, MPL, and IL-12, as described above, may also

be used.

The immunogenic composition may be administered as a single bolus dose or

as a "series" of administrations over a defined period of time (e.g., one year). When given in later year, such series of administrations is referred to as "booster shots". These

administrations increase the antibody levels produced by the previous administration. The

immunogenic compound may be administered until sufficient antibody levels have been identified in the subject, so as to induce an immune response upon challenge from the

immunogen.

The formulation of such immunogenic compositions is well known to persons

skilled in this field. Immunogenic compositions of the invention may comprise additional antigenic components (e.g., polypeptide or fragment thereof or nucleic acid encoding an

antigen or fragment thereof) and, preferably, include a pharmaceutically acceptable carrier.

Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional

solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The term

"pharmaceutically acceptable carrier" refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered. Suitable

pharmaceutically acceptable carriers include, for example, one or more of water, saline,

phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations

thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which

enhance the shelf life or effectiveness of the antigen. The use of such media and agents for

pharmaceutically active substances is well known in the art. Except insofar as any

conventional media or agent is incompatible with the active ingredient, use thereof in immunogenic compositions of the present invention is contemplated. The immunogenic compositions of this invention maybe delivered in the form of ISCOMS (immune stimulating

complexes), ISCOMS containing CTB, liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co-glycoside) to form microspheres of a size suited to

adsorption. The isolated polypeptides of this invention may also be incorporated into oily

emulsions.

Compositions h further embodiments of this invention mucin-binding nucleic acid

sequences, amino acid sequences, expression vectors or host cells are employed in compositions comprising (i) at least one mucin-binding protein, or nucleic acid encoding an

amino acid sequence of a mucin-binding protein, or an expression vector or host cell that

expresses such nucleic acid and (ii) at least one of a pharmaceutically acceptable buffer, diluent, or carrier. The mucin-binding protein may be recombinantly produced or isolated

from a bacterial preparation, according to methods known in the art. Preferably, these

compositions have therapeutic and prophylactic applications. In such applications, a

pharmaceutically effective amount of at least one mucin-binding protein of this invention is

employed in such amount to produce a defined functional activity. As used herein, the term

"effective amount" refers to amount of the mucin-binding proteins described herein to produce a functional effect.

Administration of such compositons or immunogenic compositions maybe by

any conventional effective form, such as intranasally, parenterally, orally, or topically applied

to mucosal surface such as intranasal, oral, eye, lung, vaginal, or rectal surface, such as by aerosol spray. The preferred means of administration is parenteral or intranasal.

Oral formulations include such normally employed excipients as, for example,

pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine,

cellulose, magnesium carbonate, and the like. The polynucleotides and polypeptides of the present invention may be

administered as the sole active immunogen in an immunogenic composition. Alternatively, however, the immunogenic composition may include other active immunogens, including other immunologically active antigens against other pathogenic species. Preferably, the

present polynucleotides and polypeptides , including fragments and variants thereof, are

employed with one or more immunologically active antigens against pneumococcal infection

and/or meningococcal infections. The compositions may also be used with one or more antibiotics useful to fight infection. Additionally, they may be admimstered simultaneously

or as part of a therapeutic regimen. The other immunologically active antigens may be

replicating agents or non-replicating agents. Replicating agents include, for example,

attenuated forms of measles virus, rubella virus, variscella zoster virus (VZV), Parainfluenza virus (PIV), and Respiratory Syncytial virus (RSV).

One of the important aspects of this invention relates to a method of inducing

immune responses in a mammal comprising the step of providing to said mammal an

immunogenic composition of this invention. The immunogenic composition is a composition

which is immunogenic in the treated animal or human such that the immunologically

effective amount of the polypeptide(s) contained in such composition brings about the desired response against pneumococcal infection. Preferred embodiments relate to a method for the

treatment, including amelioration, or prevention of pneumococcal infection in a human

comprising administering to a human an immunologically effective amount of the

immunogenic composition. The dosage amount can vary depending upon specific conditions of the individual. This amount can be determined in routine trials by means known to those skilled in the art.

Certainly, the isolated amino acid sequences for the proteins of the present invention may be used in forming subunit immunogenic compositions. They also may be used as antigens for raising polyclonal or monoclonal antibodies and in immunoassays for the

detection of anti-mucin-binding protein-reactive antibodies. Immunoassays encompassed by

the present invention include, but are not limited to, those described in U.S. Patent No.

4,367,110 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901 (western blot), which U.S. Patents are incorporated herein by reference. Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.

Methods of Inducing an Immune Response

According to the present invention, colonization of S. pneumoniae involves

binding of the mucin-binding proteins to mucin present in the system. The present invention

provides for methods that prevent pneumococal infections by administering to a subject a

therapeutically effective amount of an immunogenic composition that induces an immune

response in the subject. These methods include, but are not limited to, administration of an immunogenic composition comprised of at least one mucin-binding protein, variant, fragment

or attenuated version thereof, or at least one expression vector encoding the protein variant,

fragment or attenuated version thereof.

Methods of Inhibiting Pneumococcal Infection

The present invention further provides for methods to induce an immune

response in a subject which is infected with pneumococal bacteria by administering to a

subject a therapeutically effective amount of a composition or compound that blocks the

interaction of mucin and the mucin-binding proteins. These methods include, but are not

limited to, administration of a composition comprised of at least one mucin-binding protein or fragments thereof or at least one expression vector encoding a mucin-binding protein or

administration of a compound that blocks, substantially all or at least in part, the interaction of mucin and the mucin-binding proteins.

Methods of Diagnosis

This invention also provides for a method of diagnosing a pneumococcal

infection, or identifying a pneumococcal immunogenic compositon strain that has been administered, comprising the step of determining the presence, in a sample, of an amino acid

sequence of SEQ ID NOs: 8 or 10. Any conventional diagnostic method may be used.

These diagnostic methods can easily be based on the presence of an amino acid sequence or

polypeptide. Preferably, such a diagnostic method matches for a polypeptide having at least

10, and preferably at least 20, amino acids which are common to the amino acid sequences of this invention.

The nucleic acid sequences disclosed herein also can be used for a variety of

diagnostic applications. These nucleic acids sequences can be used to prepare relatively short

DNA and RNA sequences that have the ability to specifically hybridize to the nucleic acid

sequences encoding the mucin-binding proteins. Nucleic acid probes are selected for the desired length in view of the selected parameters of specificity of the diagnostic assay. The

probes can be used in diagnostic assays for detecting the presence of pathogenic organisms, or

in identifying a pneumococcal immunogenic composition that has been administered, in a given sample. With current advanced technologies for recombinant expression, nucleic acid

sequences can be inserted into an expression construct for the purpose of screening the corresponding ohgopeptides and polypeptides for reactivity with existing antibodies or for the

ability to generate diagnostic or therapeutic reagents. Suitable expression control sequences and host cell/cloning vehicle combinations are well known in the art, and are described by way of example, in Sambrook et al. (1989).

In preferred embodiments, the nucleic acid sequences employed for hybridization studies or assays include sequences that are complementary to a nucleotide stretch of at least about 10, preferably about 15, and more preferably about 20 nucleotides. A variety of known hybridization techniques and systems can be employed for practice of the hybridization aspects of this invention, including diagnostic assays such as those described in Falkow et al, US Patent 4,358,535. Preferably, the sequences recognize or bind a nucleic acid sequence on the mucin-binding proteins that are consecutive.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridizations as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) from suspected clinical samples, such as exudates, body fluids (e.g., middle ear effusion, bronchoalveolar lavage fluid) or even tissues, is absorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.

The nucleic acid sequences which encode the mucin-binding proteins of the invention, or their variants, may be useful in conjunction with PCR* technology, as set out, e.g., in U.S. Patent 4,603,102. One may utilize various portions of any of mucin-binding protein sequences of this invention as oligonucleotide probes for the PCR* amplification of a defined portion of a mucin-binding protein gene, or nucleotide, which sequence may then be

detected by hybridization with a hybridization probe containing a complementary sequence.

In this manner, extremely small concentrations of mucin-binding protein nucleic acid sequences may be detected in a sample utilizing the nucleotide sequences of this invention.

The following examples are included to illustrate certain embodiments of the

invention. However, those of skill in the art should, in the light of the present disclosure,

appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Antibodies

The present invention describes antibodies that may be used to detect the presence of mucin-binding proteins present in samples. Additionally, the antibodies (e.g., anti-idiotypic antibodies) may be used to inhibit immune responses to pneumococcal

infections.

According to the invention, mucin-binding protein polypeptides produced recombinantly or by chemical synthesis, and fragments or other derivatives, may be used as an

immunogen to generate antibodies that recognize the polypeptide or portions thereof. The

portion of the polypeptide used as an immunogen may be specifically selected to modulate

immunogenicity of the developed antibody. Such antibodies include, but are not limited to,

polyclonal, monoclonal, humanized, chimeric, single chain, Fab fragments, and an Fab

expression library. An antibody that is specific for human mucin-binding proteins may recognize a wild-type or mutant form of the mucin-binding proteins. In a specific embodiment, the

antibody is comprised of at least 8 amino acids, preferably from 8-10 amino acids, and more preferably from 15-30 amino acids. Preferably, the antibody recognizes or binds amino acids on

the mucin-binding proteins are consecutive.

Various procedures known in the art may be used for the production of polyclonal

antibodies to polypeptides, derivatives, or analogs. For the production of antibody, various host

animals, including but not limited to rabbits, mice, rats, sheep, goats, etc, can be immunized by injection with the polypeptide or a derivative (e.g., fragment or fusion protein). The polypeptide

or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin

(BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the

immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, dinitrophenol,

and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and

Corynebacterium parvum.

Monoclonal antibodies directed toward a mucin-binding proteins, fragment, analog, or derivative thereof, may be prepared by any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not

limited to the hybridoma technique originally developed by Kohler and Milstein (Nature

256:495-497, 1975), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983; Cote et al, Proc. Natl. Acad. Sci. U.S.A.

80:2026-2030, 1983), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.

77-96, 1985). "Chimeric antibodies" may be produced (Morrison et al., J. Bacteriol. 159:870, 1984; Neuberger et al., Nature 312:604-608, 1984; Takeda et al., Nature 314:452-454, 1985) by

splicing the genes from a non-human antibody molecule specific for a polypeptide together with genes from a human antibody molecule of appropriate biological activity.

In the production and use of antibodies, screening for or testing with 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.

The foregoing antibodies can be used in methods known in the art relating to the

localization and activity of the polypeptide, e.g., for Western blotting, imaging the polypeptide in situ, measuring levels thereof in appropriate physiological samples, etc. using any of the detection techniques mentioned above or known in the art. Such antibodies can also be used in

assays for ligand binding, e.g., as described in U.S. Patent No. 5,679,582. Antibody binding

generally occurs most readily under physiological conditions, e.g., pH of between about 7 and

8, and physiological ionic strength. The presence of a carrier protein in the buffer solutions

stabilizes the assays. While there is some tolerance of perturbation of optimal conditions, e.g., increasing or decreasing ionic strength, temperature, or pH, or adding detergents or chaotropic salts, such perturbations will decrease binding stability.

In a specific embodiment, antibodies that agonize the activity of mucin-binding

proteins can be generated. In particular, intracellular single chain Fv antibodies can be used to regulate the mucin-binding proteins. Such antibodies can be tested using the assays described below for identifying ligands.

In another specific embodiment, the antibodies of the present invention are anti-idiotypic antibodies. These antibodies recognize and or bind to other antibodies present in

the system. The anti-idiotypic antibodies may be monoclonal, polyclonal, chimeric, humanized.

In another specific embodiment, antibodies of the present invention are conjugated

to a secondary component, such as, for example, a small molecule, polypeptide, or

polynucleotide. The conjugation may be produced through a chemical modification of the

antibody, which conjugates the antibody to the secondary component. The conjugated antibody will allow for targeting of the secondary component, such as, for example, an antibiotic to the

site of interest. The secondary component may be of any size or length. In a specific

embodiment, the secondary component is a pharmaceutically active compound.

A further aspect of this invention relates to the use of antibodies, as discussed supra, for targeting a pharmaceutical compound. In this embodiment, antibodies against the

mucin-binding proteins are used to present specific compounds to infected sites. The

compounds, preferably an antibiotic agent, when conjugated to the antibodies are referred to as

targeted compounds or targeted agents. Methods for generating such target compounds and agents are known in the art. Exemplary publications on target compounds and their preparation are set forth in U.S. Patent Nos. 5,053,934; 5,773,001; and 6,015,562.

EXAMPLES Materials and Methods Bacterial Strains and Plasmids S. pneumoniae CP1200, a nonencapsulated, highly

transformable derivative of R36A, a rough variant of D39, a virulent type 2 strain, (Morrison,

D.A., et al, Journal of Bacteriology, 1983, 156:281-290) were obtained from Margaret

Hostetter at Yale University, CT., and S. pneumoniae strain 49136 obtained from the ATCC. S. pneumoniae were grown to log phase (about O.D. of 0.6-0.8 at 600 nm) in Todd Hewitt media (Difco Lab., Detroit, MI) with 0.5% yeast extract (Difco) at 37° C with aeration or on

Tryptic Soy (Difco) blood agar plates. BL21(DE3), BLR(DE3) (Novagen, Madison, WI), ToplOF'(Invitrogen, San Diego, CA), and were grown in SOB media (Maniatis, T., 1982, In T. Maniatis (ed.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor

Laboratories, Cold Spring Harbor, NY) at 37 °C with aeration containing appropriate

antibiotics. The pCR2.1 TOPO (Invitrogen) and pET28a (Novagen) plasmids were used.

Where specified, chloramphenicol was used at 20 μg/ml, ampicillin at 100 μg/ml,

streptomycin at 100 μg/ml, and kanamycin at 25 μg/ml. Restriction enzymes were purchased

from New England Biolabs (Beverly, MA) and used according to manufactures directions.

Preparation and Radiolabelling of Human Nasal Mucin (HNM) and Isolation of Nasopharyngeal mucin (HNM). - Mucin was purified by a modification of a previously

described method. Nasopharyngeal secretions were collected and then lyophilized from 2 to

8 yr old children undergoing an operation for adenoidectomy for either otitis media with

effusion or for hypertrophic adenoids causing obstruction. The secretions were fractionated in

a column (1.6 x 95 cm) of Sepharose CL-2B employing Tris-guanidine buffer (0.1 M Tris- HC1, pH 7.5, with 6 M guanidine). Fractions were monitored by SDS-PAGE followed by

periodic acid Schiff reagent (PAS) staining. Fractions containing high molecular weight

glycoproteins at the interface of stacking and separating gels were pooled, dialyzed against

water and lyophilized. The sample was then solubilized in Tris-guanidine buffer, (0.1 M Tris-

HC1, pH 8.5, with 6 M guanidine), reduced for 2 h at 37° C with dithiothreitol, alkylated for 1 h at room temperature with iodoacetamide and fractionated in a column (1.6 x 95 cm) of

Sepharose CL-2B. Fractions were monitored by SDS-PAGE followed by periodic acid Schiff

reagent (PAS) staining. Mucin containing fractions were pooled and lyophilized. Radiolabeling of mucins by iodination - Iodination was performed by Chloramine T

method (Greenwood, F.C., et al, Journal of Biochemistry, 1963, 89:114-123). About (150 -

200 μg of HNM) 20 μg of protein was dissolved in 25 μl of buffer, pH 7.5 (0.5 M sodium

phosphate). Following mixing of the sample with one mCi of [¹²⁵ IJsodium iodide, 20 μl of a

1 mg/ml solution of Chloramine T in 0.1 M phosphate buffer, pH 7.5 was added and mixed

gently. After 90 sec, the reaction was terminated by the addition of 25 μl of a 2 mg/ml

solution of sodium metabisulfite 0.1 M phosphate buffer, pH 7.5. Potassium iodide (40 ml of

a 10 mg/ml in 0.1 M phosphate buffer, pH 7.5) was added to dilute the residual iodine.

Radiolabeled nasopharyngeal mucin was recovered by gel filtration in Sephadex G-75

employing a 10 ml disposable pipette as column. TBS was used as eluent. Prior to chromatography of radiolabeled reaction mixture, about 4 mg of bovine serum albumin

(BSA) dissolved in 2 ml of TBS was passed through the column and washed with TBS to

saturate any protein binding sites present on the gel and the glass disposable column.

Approximately, 0.5 ml fractions were collected and lμl was counted in a gamma counter.

Appropriate fractions were pooled and stored at 4° C. An aliquot was subjected to SDS- PAGE (Laemmli, U.K., Nature (London), 1970, 227:680-685) /autoradiography to assess the

purity of the sample.

Mucin binding overlay assay - Electrophoretic (Western) transfers of protein components of

S. pneumoniae were made to a polyvinyldifluoride (PVDF) membrane (Immobilon P

membrane, Millipore Corp) at room temperature for about 30 min at 100 n A employing 0.1

M Tris with 0.192 M glycine in 20% methanol as transfer buffer (Burnette, W.N., Analytical

Biochemistry, 1981, 112:195-203; Towbin, H, et al, Proceedings of the National Academy of Science, 1979, 76:4350-4). Following the transfer, the Immobilon P membrane was

incubated with a solution of BSA (2% w/v in TBS) for 1 h at room temperature to block the remaining binding sites. [¹²⁵I]HNM, about 600000 cpm, were added and incubated at room

temperature for about 12 h with gentle shaking. Unbound radioactivity was aspirated

followed by washing the membrane with TBS, three times for 10 min each. The membrane

was gently blotted on a filter paper, air dried, and subjected to autoradiography. For autoradiography, the membrane was exposed to X-ray (X-OMAT AR) film and developed as per the instruction of the manufacturer (Eastman Kodak Company, Rochester, NY).

Identification of Mucin Binding Proteins in Outer Membrane Fractions of S.

pneumoniae and extraction of mucin binding components - Bacteria were grown in 4

liters of Todd Hewitt broth, and harvested by centrifugation at 8000 x g for 30 minutes. The

pellet was suspended in about 175 ml of PBS with the aid of a pipette and immediately

centrifuged at 20000 x g for 30 min. The wash was filtered through a 0.45μ filter (Nalgene,

Rochester, NY), dialyzed and lyophilized.

Ion-exchange chromatography of mucin binding protein components -The PBS extract of

S. pneumoniae was dissolved in Tris-HCl, pH 7.6 (10 mM, 100 ml) and subjected to ion

exchange chromatography in a column of DEAE-Sepharose CL-6B. After washing the

column with the sample buffer, it was eluted first with 200 mM Tris-HCl, pH 7.6 followed by a linear NaCl gradient to a final NaCl concentration of 0.75 M (in 200 mM Tris-HCl, pH 7.6) over 300 ml. Column fractions were analyzed by SDS-PAGE gel and by mucin binding

overlay assay of a western blot. Fractions containing mucin binding components were pooled,

desalted by Centricon SR3 concentrator and lyophilized. The mucin binding overlay assay, utilizing radioiodinated HNM was used to

identify 2 mucin binding proteins of approximately 12kDa and 14 kDa from outer membrane components of S. pneumoniae (See figure 1A and B, lane 3). Lane 3 in figure 1 A and B

represents fraction #26 from a DEAE column. There are clearly two bands that bind mucin just above and below the 14kDa lysozyme maker, which also binds mucin. The low

molecular weight bands flanking the 14kDa lysozyme marker were resolved on a preparative

SDS-PAGE gel and transferred to a PVDF membrane. The PVDF membrane has a high

binding capacity, which increases sample recovery and sequencing performance, allowing

efficient determination of the amino terminal residues. Initial studies indicate that these

proteins are specifically bind to HNM and do not bind to other known human mucins, e.g. gastric mucin (data not shown).

N-terminal Amino Acid Sequence Analysis by PVDF Blot Excision. - The sample was

diluted to 1 mg/mL total protein and combined 1 : 1 with 2X Tris-SDS-β-ME sample loading

buffer (0.25M Tris-HCl pH6.8, 2% SDS, 10% β-mercaptoethanol, 30% glycerol, 0.01%

Bromophenol Blue) (Owl Separation, Portsmouth, NH) and heated at 100° C for 5 minutes.

Approximately lOμg of total protein (20uL of heated solution) of sample was loaded in each

often lanes on a 12 lane, 10cm x 10cm x 1mm, 10-20% gradient acrylamide/bis-acrylamide gel (Zaxis, Hudson, OH). Molecular weight markers (Novex, San Diego, CA) were loaded in

the two outermost lanes of each side of the gel. Electrophoresis was carried out on an Owl

Separations Mini-Gel rig at a constant amperage of 50 mA for 1 hour in Bio-Rad Tris-

Glycine-SDS running buffer. The gel was then rinsed with deionized water and transferred to Millipore Immobilon-P PVDF (polyvinylidene fluoride) using a semi-dry blotting system supplied by Owl Separations at constant amperage of 150 mA for 1 hour. The resulting blot

was stained with Amido Black (10% acetic acid, 0.1% amido black in deionized water) and destained in 10% acetic acid. The protein band was then excised from all ten lanes using a

methanol cleaned scalpel or mini-Exacto knife and placed in the reaction cartridge of the

Applied Biosystems 477A Protein Sequencer (Foster City, CA). The N-terminal Sequencer

was then run under optimal blot conditions for 12 or more cycles (1 cycle Blank, 1 cycle Standard, and 10 or more cycles for desired residue identification). PTH-amino acid detection was done on the Applied Biosystems 120 A PTH Analyzer. The cycles were collected both on

an analog chart recorder and digitally via the instrument software. N-terminal amino acid

assignment was perfomed by comparison of the analog and digital data to a standard set of

PTH-amino acids and their respective retention times on the analyzer (cystiene residues are

destroyed during conversion and are not detected).

Subcloning and Expression of the Recombinant Mucin Binding Proteins - N-terminal

sequence was compared against both the NCBI non redundant database located at

www.ncbi.nlm.org and the public release of the S. pneumoniae genome (serotype 4), made available by The Institute for Genomic Research (TIGR, www.tigr.org), using the BLAST algorithim developed by Altschul (Altschul S.F., et al, Journal of Molceular Biology, 1990,

215:403-10). Subsequent DNA analyses of the corresponding ORFs (open reading frames) in

S pneumoniae genomic sequence and primer designs were performed using the DNASTAR

(Madison, WI) Lasergene DNA and protein analysis software.

Primers flanking each ORF were designed and subsequently synthesized using the ABI 380A DNA synthesizer. To facilitate subcloning the PCR product into the pET28a

expression vector, restriction sites were designed into the PCR primers. An Ncol site was included in the 5' primers for both genes, which allowed both for the ligation into the Ncol

site of the expression vector and also included an ATG start codon. To maintain the correct

reading frame, two extra bases included in each 5' primer, resulting in the addition of a codon

for Isoleucine in the 12kDa MBP protein and a codon for a Lysine in the 14kDa MBP protein.

A BamHl site was included in the 3' primers for both ORFs. See SEQ ID NOs: 1-4.

PCR fragments of the expected size were generated from CP1200 and each

fragment was ligated into the pCR2.1 vector and used to transform OneShot Top 10F' cells

(Invitrogen). Ampicillin resistant transformants were screened screened by restriction

digestion of plasmid DNA prepared by alkaline lysis (Birnboim and Doly, Nucleic Acids Res, 1979, 7:1513-1523). Two recombinant plasmids, one containing the 12kDa MBP gene, and the other containing the 14kDa MBP gene were identified (pLP535 and 536). DNA sequence

was obtained from the clones using the ABI model 370A DNA sequencer. The inserts were

excised by restriction digestion with Ncol and Sphl, and separated on a 1.5% Agarose gel. The DNA fragments were cut from the gel and purified away from the agarose by a Biol 01 Spin kit (Vista, CA). The inserts were ligated with plasmid vector DNA that had also

been digested with Ncol and Sphl . The ligation mixture was subsequently transformed into

Topi OF' cells (Invitrogen) and the kanamycin resistant transformants were screened by

restriction digestion of plasmid DNA prepared by alkaline lysis (Birnboim and Doly, Nucleic Acids Res, 1979, 7:1513-1523). Recombinant plasmids were subsequently transformed into

BL21 cells (Novagen) to create pLP537(12kDa) and ρLP538(14kDa) and grown in SOB

media supplemented with 30ug/ml kanamycin. Cells were grown to an O.D.₆₀₀ of 0.6, and

were subsequently induced with 0.4mM IPTG (Boehringer Mannheim, Indianapolis, IN) for

2-4 hours. Whole cell lysates were prepared and electrophoresed on a 15% SDS-PAGE gel (Laemmli, U.K., Nature (London), 1970, 227:680-685) to confirm expression of the desired recombinant product. Whole cell material was also assayed by the Mucin binding overlay

assay as previously described.

The amino terminal sequence (SEQ ID NOs: 5-6) of these two proteins allowed the identification of two corresponding open reading frames in the S. pneumoniae

genome (SEQ ID NOs: 7 and 9). Unexpectedly, these ORFs showed similarity to ribosomal

assembly proteins in other organisms, which may indicate similar function in S. pneumoniae.

Subcloning and expression of the two ORFs provided recombinant material of the expected size that also bound mucin (Figure 2). The 2 mucin binding components in a PBS wash of S.

pneumoniae can clearly be seen in lane 1. The recombniantly expressed 12kDa protein

(pLP537) in lane 2 reveals two proteins which bind mucin, one at the expected size and one

slightly larger. Neither band is present in whole cell lysates from a non related recombinantly expressed protein, indicating this larger band does not originate from the E. coli host. Some preinduction expression and nonspecific binding to mucin is seen in lane 5, however a single

mucin binding species of the predicted size is expressed by pLP538, lane 6. When figure 2 is

examined closely is most likely that the recombinant products expressed by pLP537 (lane 2)

and pLP538 (lane 6) represent the mucin binding proteins seen in the PBS washes of S. pneumoniae (lane 1).

Intranasal and parenteral immunization of mice prior to challenge. Six-week old,

pathogen-free, male CBA CaHN xid/J (CBA/N) mice are purchased from Jackson Laboratories (Bar Harbor, Maine) and housed in cages under standard temperature, humidity, and lighting conditions. CBA/N mice, at 10 animals per group, are immunized with either of the mucin-binding proteins depicted in SEQ ID NOs: 8 and 10. For parenteral immunization,

the proteins are mixed with monophosphoryl lipid A (MPL) (Corixa, Hamilton, MT), for example about 100 μg, per dose to a final volume of 200 μl in saline and then injected

subcutaneously (s.c.) into mice. All groups receive a booster with the same dose and by the

same route 3 and 5 weeks after the primary immunization. Control mice are injected with

MPL alone. All mice are bled two weeks after the last boosting; sera are then isolated and stored at -20°C. For intranasal (i.n.) immunization, mice receive three i.n. immunizations

one week apart. On each occasion, mucin-binding proteins formulated with CT-E29H, a

genetically modified cholera toxin that is reduced in enzymatic activity and toxicity (Tebby,

et al, Vaccine, 2000, 18:2723-34), is slowly instilled into the nostril of each mouse in a 10 μl

volume. CT-E29H may be present in any amount, for example 0.1 μg. Mice immunized with

CT-E29H with KLH are used as controls. Serum samples are collected one week after the last immunization. Useful amounts of CT-E29H will be determined based upon the immune

response observed.

LD₅₀ determination. Six or 12- week old CBA/N mice (10 per group) are challenged

intranasally with 10 μl of a suspension of streptomycin resistant type 3 S. pneumoniae diluted

to 5 x 10⁹ CFU/ml in PBS. Two-fold serial dilutions of this suspension are also tested. The

actual doses of bacteria administrated are determined by plating dilutions of the inoculum on

streptomycin containing TSA plates. The LD₅₀ is calculated by the Reed-Muench method as

discussed by Lennette (Lennette, General Principles for laboratory diagnosis of viral, rickettsial, and chlamydial infections, 7th edition).

Mouse intranasal challenge model. Pneumococci were inoculated into 3 ml of Todd-Hewitt

broth containing 100 μg/ml of streptomycin. The culture was grown at 37 °C until mid-log phase, then diluted to the desired concentration with Todd-Hewitt broth and stored on ice until use. Each mouse was anesthetized with 1.2 mg of ketamine HC1 (For Dodge

Laboratory, Ft. Dodge, Iowa) by i.p. injection. The bacterial suspension was inoculated to the nostril of anesthetized mice (10 μl per mouse). Five mice per group were vaccinated IN

(10ml volume) with a 5 mg/dose of either rl2kDa MBP, pneumococcal type 3 conjugate

(PNC-3), or KLH, adjuvanted with 0.1 mg of CT-E29H on weeks 0, 2, and 4. The actual

dose of bacteria administrated was confirmed by plate count. Mice were then challenged at week 5 with 2xl0⁵ colony forming units (cfu) of type 3 S. pneumoniae. Four days after

challenge, mice were sacrificed, the noses were removed, and homogenized in 3 -ml sterile

saline with a tissue homogenizer (Ultra-Turax T25, Janke & Kunkel Ika-Labortechnik,

Staufen, Germany). The homogenate was 10-fold serially diluted in saline and plated on

streptomycin containing TSA plates. Fifty μl of blood collected 2 days post-challenge from each mouse also was plated on the same kind of plates. Plates were incubated overnight at

37 °C and then colonies were counted. Bacteria recovered from noses are shown in Figure 3

as Log₁₀ cfu per gram of tissue (± standard error of the mean).

The results show that the mice immunized with the rl2 kDa MBP had

statistically significantly reduced levels of colonization as compared to the KLH E29H control group (p<0.05 by Student's t test). The level of colonization was as low as the

positive control group immunized with the PNC-3 conjugate, which was also significantly

different from the KLH group (p<0.05). This demonstrates that mucosal immunization with

the rl2 kDa MBP protein can reduce pneumococcal colonization in the nasopharynx of mice.

Modification of lysine residues. Bacteria were grown in Todd Hewitt broth and harvested

by centrifugation at 8000 x g for 30 minutes. The pellet was suspended in about 175 ml PBS

with the aid of a pipette and immediately centrifuged at 20000 x g for 30 min. This preparation then was subjected to reductive methylation or N-acetylation.

For reductive methylation, a 0.6 ml aliquot of a crude preparation of mucin

binding proteins of Streptococcus pneumoniae 49136 (A280 = 9.6) was mixed with an equal

volume of 0.2 M sodium cyanoborohydride followed by the addition of 20 μmole of

formaldehyde. After incubation for 1 h at 37 °C, additional 20 μmole of formaldehyde were added and incubated at 37 °C for 24 h. The reaction mixture was concentrated to about 0.2 ml

by centrifugation at 3000 x g employing Centricon SR3 concentrator. The contents in the

Centricon tube was diluted with 1 ml of water and centrifuged. The process was repeated

once more and the sample diluted to 0.6 ml and stored frozen.

For N-acetylation, A 0.6 ml aliquot of a crude preparation of mucin binding proteins of Streptococcus pneumoniae 49136 (A280 = 9.6) was mixed with 0.2 ml of 0.2 M

sodium phosphate buffer, pH 7.5 and 0.8 ml of a saturated solution of sodium acetate and

placed on crushed ice. While stirring on a magnetic stirrer, 1 μl of acetic anhydride was added and left on crushed ice. At the end of 30 min additional 1 μl of acetic anhydride was

added under stirring on a magnetic stirrer. After 45 min on crushed ice, the contents were diluted with 1 ml of water and concentrated to about 0.2 ml by centrifugation at 3000 x g

employing Centricon SR3 concentrator. The contents in the Centricon tube was diluted with 1

ml of water and centrifuged. The process was repeated once more and the sample diluted to 0.6 ml and stored frozen.

Modified mucin binding proteins containing material (10 μl) was subjected

SDS-PAGE (100 volts for about 1 h at room temperature, acrylamide 12% concentration) and

electrophoretically transferred to a PVDF membrane (108 mA for about 45 min at room temperature). The nonspecific binding sites on the membrane were then blocked by incubation with bovine serum albumin (5 ml, 1% in TBS[Tris buffered saline, pH 7.2]) for 1 h at room temperature. About 0.7 x 10⁶ cpm of ¹²⁵I-labeled human nasopharyngeal mucin

were added and incubated overnight with gentle shaking. The membrane was washed three times for 10 min each with TBS, air dried and exposed to X-ray film.

Results indicated that binding of human nasopharyngeal mucin to mucin

binding proteins was abolished by N-acetylation of lysine residues of mucin binding proteins.

On the other hand, reductive methylation of mucin binding proteins, which modifies lysine residues to methyl lysines, did not affect binding. These results indicate that lysine residues play a role in mucin-bacterial interactions.

Biotinylation of the cell surface proteins oi Streptococcus pneumoniae. Todd-Hewitt broth

(120 g) and yeast extract (20 g) were solubilized in 4 liters of water, filtered through a 0.45 m filter to remove any particulate matter and autoclaved for sterilization. Sti'eptococcus

pneumoniae 49136 were grown for 16 h at 37 °C and centrifuged at 4,000 x g for 15 min.

Bacterial pellet was gently suspended in 50 ml of 0.1 M N-morpholinoethane sulfonic acid

buffer, pH 5.5 and centrifuged. The pellet re-suspended in 80 ml of the above buffer, 75 mg of EZ-link Biotin-PEO-Amine in 4 ml of buffer were added and mixed well by gentle stirring. 100 mg of EDC in 1 ml of buffer were then added and gently stirred at room temperature for

2h. The incubation mixture was centrifuged at 10,000 x g for 30 min and the supernatant

saved as first extract. The bacterial pellet extracted at room temperature by stirring with PBS

(phosphate buffered saline, pH 7.2) for 15 min. The extract was recovered by centrifugation at 10,000 x g for 30 min and saved as second extract. Both extracts were desalted and concentrated to about 15 ml by centrifugation at 3,000 x g employing Centriplus YM3 concentrator.

Both the extracts (10 micro liter aliquot) were subjected SDS-PAGE/western transfer to a PVDF membrane. The nonspecific binding sites on the membrane were then

blocked by incubation with bovine serum albumin for 1 h at room temperature. 1 ml aliquot

of Streptavidin Alkaline Phosphatase conjugate was added and incubated overnight at room

temperature with gentle shaking. Unbound conjugate was removed by washing the membrane

with TBS three times for 10 min each. The membrane was blotted dry on a filter paper and

transferred to the color development substrate solution (50 μl of NBT and 25 μl of BCIP in

7.5 ml of 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgC12). Positive bands appeared

as purple bands on the membrane.

The extracts (10 μl aliquot) were also subjected to the overlay binding assay employing¹²⁵I-labeled human nasopharyngeal mucin as described above.

The results showed that several proteins including mucin were labeled with

biotin. Overlay binding assay revealed that human nasopharyngeal mucin bound to low

molecular weight components which appeared to be 12 kDa and 14 kDa mucin binding

proteins. These results indicate that the mucin binding proteins are located on the surface of

the bacteria.

Point Mutation Studies. Point mutations are generated in a recombinant nucleotide

sequence by first generating a synthetic oligonucleotide, to be used as a primer, which flanks

the nucleotide to be modified. The sequence of the synthetic oligonucleotide should exactly match the target sequence, except for the base to be modified. Several kits are available for

the creation of single base pair changes and subsequent selection and expression of the mutant sequence. A preferable system will utilize a high copy number plasmid, which among

others will have two antibiotic resistance genes. One antibiotic gene is inactive and is made active during the mutagenesis step with a synthetic oligonucleotide supplied by the company. 'The other antibiotic gene is made inactive in a similar manner. A desirable vector also will have convenient cloning and translation start sites, as well as SP6, T7, and tac promoters for

expression in E. coli.

Briefly, the sequence of interest is cloned "in frame" into such a vector and subjected

to alkaline denaturation to separate the strands. The synthetic oligo containing the base pair

change and the synthetic oligos containing the antibiotic resistance repair/mutagenesis

sequence are annealed to the now single stranded plasmid. T4 Polymerase and T4 DNA ligase are used to synthesize the second strand and ligate the fragments together. The plasmid is

then used to transform competent E. coli cells, and mutants are selected for on plates

containing the appropriate antibiotic. Plasmid DNA are made from the transformants and then sequenced to confirm the mutagenesis. Several rounds of mutagenesis can be performed to modify any number of bases. The mutant sequence can then be directly expressed and

evaluated.

ELISA assays for the recombinant mucin binding proteins. Antibody titers against

recombinant mucin binding proteins are determined by enzyme-linked immunosόrbent assay (ΕLISA). The ΕLISA is performed using either recombinant mucin binding to coat Nunc-

ImmunoTM PolySorp Plate or r20K to coat Nunc-ImmunoTM MaxiSorp Plate (Roskilde,

Denmark). Serial dilutions of test sera are added to plates blocked with 200 μl of 5% nonfat

dry milk in PBS. Biotinylated goat anti-mouse IgG or IgA (1 :8000 or 1 :4000; Brookwood Biomedical, Birmingham, AL) are then be added, followed by streptavidin conjugated horseradish peroxidase (1:10000; Zymed Laboratory Inc., SanFrancisco, CA). Antibody

reactivity is detected by adding ABTS substrate (KPL, Gaithersburg, MD), and quantified by

absorbance values read at 405 nm.

Pneumococcal whole cell ELISAs. S. pneumoniae type 3 is grown in Todd-Hewitt broth with 100 μg/ml of streptomycin, harvested in late-log phase by centrifugation and the

cell pellet suspended in PBS to an OD₅₅₀ =1. A 50 μl aliquot of this suspension is then added

to each well of a 96-well plates (Nunc, Roskilde, Denmark). The plates are air-dried at room

temperature, and blocked with 200 μl of PBS containing 5% (w/v) dry milk (blocking buffer) for 1 hour. Following this, the plates are washed 5 times with PBS, and 100 μl of mouse sera

diluted in blocking buffer is added to each well and incubated at room temperature for 1.5

hour. The plates are then washed with PBS and incubated with 100 μl of peroxidase-labeled

goat anti-mouse IgG or IgA (1:1000 in PBS; Kirkeguard Perry Laboratories, Gaithersburg, MD) for 1.5 hour at room temperature. Finally, the plates are washed 5 times with PBS and developed with ABTS (KPL, Gaithersburg, MD) for 20 min followed by stopping reactions

with 1% SDS. The absorbance values are read at 405 nm using a VERSAmax microplate

reader.

The patents, applications, test methods, and publications mentioned herein are

hereby incorporated by reference in their entirety.

Many variations of the present invention will suggest themselves to those skilled in the art in light of the above detailed description. All such obvious variations are

within the full intended scope of the appended claims.

Claims

We Claim:

1. An isolated mucin-binding protein from pneumococcal bacteria.

2. A mucin-binding protein as defined in claim 1 which has an amino acid sequence as depicted in SEQ ID NO: 8, SEQ ID NO: 10, or a fragment thereof.

3. A mucin-binding protein as defined in claim 1 which has a molecular weight

of about 12 kilo Daltons (kDa) or about 14 kDa, wherein said molecular weight is determined

using a 10-20% SDS-PAGE gel.

4. A nucleic acid sequence encoding a mucin-binding protein, wherein said

nucleic acid sequence has a sequence as depicted in SEQ ID NO:7, SEQ ID NO:9, or a

fragment thereof.

5. A nucleic acid as defined in claim 4 which is a cDNA.

6. An expression vector comprising a nucleic acid sequence encoding a mucin-

binding protein or a fragment thereof, wherein said sequence is operatively associated with an

expression control sequence.

7. A vector as defined in claim 6, wherein said mucin-binding protein has an

amino acid sequence as depicted in SEQ ID NO:8, SEQ ID NO: 10, or a fragment thereof.

8. A vector as defined in claim 6, wherein said mucin-binding protein has a

molecular weight of about 12 kDa or about 14 kDa, wherein said molecular weight is

determined using a 10-20% SDS-PAGE gel.

9. A vector as defined in claim 6, wherein said nucleic acid sequence has a

sequence as sequence as depicted in SEQ ID NO:7, SEQ ID NO:9, or a fragment thereof.

10. A host cell transfected with the vector as defined in claim 6.

11. A method for producing recombinant mucin-binding protein, which method

comprises isolating mucin-binding protein produced by the host cells as defined in claim 10,

wherein the host cells have been cultured under conditions that provide for expression of said

mucin-binding protein by said vector.

12. A composition comprising at least one mucin-binding protein as defined in

claim 1 and a pharmaceutically acceptable carrier.

13. A composition comprising at least one mucin-binding protein as defined in claim 2 and a pharmaceutically acceptable carrier.

14. A composition comprising at least one nucleic acid sequence encoding a

mucin-binding protein as defined in claim 3 and a pharmaceutically acceptable carrier.

15. A composition comprising the expression vector as defined in claim 6 and a pharmaceutically acceptable carrier.

16. A composition comprising the host cell as defined in claim 10 and a

pharmaceutically acceptable carrier.

17. An immunogenic composition comprising at least one mucin-binding protein from pneumococcal bacteria, a pharmaceutically acceptable carrier, and optionally at least

one adjuvant.

18. A composition as defined in claim 17, wherein said pneumococcal bacteria is

Streptococcus pneumoniae.

19. A composition as defined in claim 18, wherein said composition elicits

protective immunity from a disease caused by Streptococcus pneumoniae.

20. A composition as defined in claim 19, wherein said disease is selected from the group consisting of otitis media, rhinosinusitis, bacteremia, meningitis, pneumonia, and

lower respiratory tract infection.

21. A composition as defined in claim 17, wherein said mucin-binding protein

comprises an amino acid sequence as depicted in SEQ ID NO: 8, SEQ ID NO: 10, or an immunogenic fragment thereof.

22. A composition as defined in claim 17, wherein said mucin-binding protein is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 7, SEQ ID NO: 9, or an

immunogenic fragment thereof.

23. A composition as defined in claim 17, wherein said mucin-binding protein is polypeptide having a molecular weight of about 12 kDa or about 14 kDa, wherein said

molecular weight is determined using a 10-20% SDS-PAGE gel.

24. An immunogenic composition comprising at least one expression vector encoding a mucin-binding protein from pneumococcal bacteria, a pharmaceutically

acceptable carrier, and optionally at least one adjuvant.

25. A composition as defined in claim 24, wherein said pneumococcal bacteria is

Streptococcus pneumoniae.

26. A composition as defined in claim 25, wherein said composition elicits

protective immunity from a disease caused by Streptococcus pneumoniae.

27. A composition as defined in claim 26, wherein said disease is selected from the group consisting of otitis media, rhinosinusitis, bacterenia, meningitis, pneumonia, and

lower respiratory tract infection.

28. A composition as defined in claim 24, wherein said expression vector comprises a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO:8, SEQ ID NO: 10, or an immunogenic fragment thereof.

29. A composition as defined in claim 24, wherein said expression vector comprises a nucleic acid sequence depicted in SEQ ID NO: 7, SEQ ID NO: 9, or an

immunogenic fragment thereof.

30. A composition as defined in claim 24, wherein said expression vector comprises a nucleic acid sequence encoding a polypeptide having a molecular weight of

about 12 kDa or about 14 kDa, wherein said molecular weight is determined using a 10-20%

SDS-PAGE gel.

31. A method of inducing an immune response in a mammal, said method comprising administering to said mammal an amount of a composition as defined in claim 17

effective to induce an immune response in said mammal.

32. A method of inducing an immune response in a mammal, said method comprising administering to said mammal an amount of a composition as defined in claim 24

effective to induce an immune response in said mammal.

33. A method of inducing an immune response in a mammal which is infected

with pneumococcal bacteria, said method comprising administering to said mammal an amount of a compound effective to inhibit binding of an amino acid sequence as depicted in SEQ ID NO: 8 or SEQ ID NO: 10 with mucin to induce said immune response said mammal.

34. The method of claim 33, wherein said mucin is selected from the group consisting of human nasopharyngeal mucin and human middle ear mucin.

35. A method for screening for a compound which induces an immune response in a mammal infected with pneumococcal bacteria, said method comprising comparing a first amount of binding of an amino acid sequence as depicted in SEQ ID NO: 8 or SEQ ID NO: 10

to mucin in the presence of said compound to a second amount of binding of an amino acid

sequence as depicted in SEQ ID NO: 8 or SEQ ID NO: 10 to mucin not in the presence of said

compound; whereby a lower first amount of binding than said second amount binding indicates that said compound may induce said immune response in said mammal.

36. A method for diagnosing pneumococcal bacterial infection, said method

comprising comparing the level of mucin-binding protein as depicted in SEQ ID NO: 8, SEQ ID NO: 10, or fragments thereof, in suspect sample to the level of mucin-binding protein as

depicted in SEQ ID NO: 8, SEQ ID NO: 10, or fragments thereof, in a control sample, whereby

a higher level of said mucin-binding protein in said suspect sample than the level of said

mucin-binding protein in said control sample indicates that said suspect sample comprises

pneumococcal bacterial infection.

37. An antibody which binds to Streptococcus pneumoniae mucin-binding

proteins.

38. An antibody as defined in claim 37, which selectively recognizes an amino acid sequence as depicted in SEQ ID NO:8, SEQ ID NO:10, or fragments thereof.

39. An antibody as defined in claim 37, which is chimeric.

40. An antibody as defined in claim 37, which is humanized.

41. An antibody as defined in claim 37, which is anti-idiotypic.

42. An antibody as defined in claim 37, which is conjugated to a pharmaceutically

active compound.

43. An antibody as defined in claim 37, which is a monoclonal antibody.

44. An antibody as defined in claim 43, which is humanized.

45. An antibody as defined in claim 43, which is anti-idiotypic.

46. An antibody as defined in claim 43, which is conjugated to a pharmaceutically

active compound.

47. A method for inducing an immune response in a mammal, said method comprising administering to said mammal an amount of an antibody as defined in claim 41 effective to induce an immune response in said mammal.

48. A method for inducing an immune response in a mammal, said method

comprising administering to said mammal an amount of an antibody as defined in claim 45 effective to induce an immune response in said mammal.

49. A method for inducing an immune response in a mammal infected with

pneumococcal bacteria, said method comprising administering to said mammal an amount of an antibody as defined in claim 42 effective to induce an immune response in said mammal.

50. A method for inducing an immune response in a mammal infected with

pneumococcal bacteria, said method comprising administering to said mammal an amount of an antibody as defined in claim 46 effective to induce an immune response in said mammal.

51. A mucin-binding protein variant or fragment thereof, wherein at least one

lysine residue is replaced, deleted or altered.

52. A mucin-binding protein variant of claim 51 , wherein mucin-binding protein activity of the variant is decreased when compared to mucin-binding protein activity of a

wild-type mucin-binding protein.

53. A mucin-binding protein fragment comprised of at least 8 amino acids,

wherein the fragment comprises at least one lysine residue.

54. The mucin-binding protein fragment of claim 53, wherein absence of at least one of at least one lysine residue decreases mucin-binding protein activity when compared to

a wild-type mucin binding protein.

55. A nucleic acid sequence encoding a mucin-binding protein variant or fragment

of claim 52 or 53, wherein said nucleic acid sequence is modified so that at least one lysine

residue in the protein is replaced, deleted or altered.