GB2345288A

GB2345288A - Streptococcal polypeptides

Info

Publication number: GB2345288A
Application number: GB9821362A
Authority: GB
Inventors: Mario Altieri; Enrico Domenici; Frederico Faggioni; Livia Ferrari; Harald Motti; Laura Piccoli; Alessandra Polissi; Andrea Pontiggia; Emiliangelo Ratti; Daniel Simon
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 1998-10-02
Filing date: 1998-10-02
Publication date: 2000-07-05
Also published as: GB9821362D0

Abstract

This invention relates to newly identified polynucleotides and polypeptides derived from Streptococcus pneumoniae, processes for making these polynucleotides and polypeptides and uses thereof in screening for antimicrobial compounds.

Description

POLYNUCLEOTIDES AND POLYPEPTIDES This invention relates, in part, to newly identified polynucleotides and polypeptides ; variants and derivatives of these polynucleotides and polypeptides ; processes for making these polynucleotides and these polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides ; and uses of these polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of Streptocuccus pneumoniae (hereinafter referred to as"S. pneumoniae") BACKGROUND OF THE INVENTION Streptococcus pneumoniae is the major cause of bacterial pneumonia and it is also responsible for otitis media and meningitis in children.

Capsular polysaccharides were the first virulence factors to be identified. In addition a small number of proteins are considered to be virulence factors (DeVelasco et al., Microbiol Rev 59: 591-603 1995). Pneumolysin, autolysin, hyaluronidase, PspA (8), neuraminidase, IgA1 protease, pyruvate oxidase and the PsaA protein (Barry and Paton Infect Immun 64: 52555262 1996) are all been considered to be virulence factors, even though for some of them their involvement in virulence is only hypothetical. S. pneumoniae has been studied for many years, yet its virulence mechanisms are not fully understood. Furthermore very little is known conceming the temporal expression of such genes during infection and disease progression in mammalian host. Discovering the sets of genes during infection provides critical information for the screening and characterisation of novel antibacterials which can interrupt pathogenesis.

Clearly there is a need for polypeptide and polynucleotide sequences that may be used to screen for antimicrobial compound and which may also play a role in preventing, ameliorating or correcting infection, dysfunction and disease.

SUMMARY OF THE INVENTION Toward these ends, and others, it is an object of the present invention to provide novel polypeptides particularly those from S. pneumoniae G54 that have the amino acid sequences set out in Figure 2 [SEQ ID Nos 5,6,7,8], or a fragment, analogue or derivative thereof.

It is a further object of the invention, moreover, to provide novel polynucleotides that encode the polypeptides having the amino acid sequence set out in Figure 2.

In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises the sequences set out in Figure 1 [SEQ ID Nos 1, 2,3,4], or a fragment, analogue or derivative thereof.

In accordance with this aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide of S. pneumoniae G54 which is described in Pozi et al., J Bacteriol 178: 6087 6090 1996 and is available on request to the authors.

In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding polypeptides of S. penumoniae, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives, and compositions comprising same.

In accordance with another aspect of the present invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.

The invention also relates to novel oligonucleotides derived from the sequences given herein which can act as PCR primers in the process herein described to determine whether or not the S. pneumoniae gene identified herein in whole or in part is expressed in infected tissue. It is recognised that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained. The proteins so identified are also useful as targets in screens designed to identify antimicrobial compounds.

Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of the polynucleotides of the present invention and polypeptides encoded thereby.

In accordance with this aspect of the invention there are provided novel polypeptides of S. pneumoniae as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing, and compositions comprising same.

Among the particularly preferred embodiments of this aspect of the invention are variants of polypeptides encoded by naturally occurring alleles of the polynucleotides of the present invention.

In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned polypeptides.

In accordance with yet another aspect of the present invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.

In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia : assessing expression of the polynucleotides of the present invention; to treat diseases; assaying genetic variation; and administering a polypeptide or polynucleotide of the present invention to an organism to raise an immunological response against Streptococci.

In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybridize to the polynucleotide sequences of the present invention.

In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against the polypeptides of the present invention.

In accordance with another aspect of the present invention, there are provided agonists of the function of the polypeptides of the present invention which are also preferably bacteriostatic or bacteriocidal.

In accordance with yet another aspect of the present invention, there are provided antagonists of the function of the polypeptides of the present invention which are also preferably bacteriostatic or bacteriocidal.

In a further aspect of the invention there are provided compositions comprising a polynucleotide or a polypeptide of the present invention for administration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings depict certain embodiments of the invention. They are illustrative only and do not limit the invention otherwise disclosed herein.

Figure 1 shows the polynucleotide sequences of the present invention of S. pneumoniae [SEQ ID NO : 1], [SEQ ID NO : 2], [SEQ ID NO : 3], [SEQ ID NO : 4].

Figure 2 shows the amino acid sequences of the present invention of S. pneumoniae [SEQ ID NO : 5] [SEQ ID NO : 6], [SEQ ID NO : 7], [SEQ ID NO : 8] deduced from the polynucleotide sequences of Figure 1.

Particularly amino acid sequence S. pneumoniae [SEQ ID N0 : 5] is deduced from the polynucleotide sequence [SEQ ID NO : 1], amino acid sequence S. pneumoniae [SEQ ID NO : 6] is deduced from the polynucleotide sequence [SEQ ID NO : 2], amino acid sequence S. pneumoniae [SEQ ID NO : 7] is deduced from the polynucleotide sequence [SEQ ID NO : 3] and amino acid sequence S. pneumoniae [SEQ ID NO : 8] is deduced from the polynucleotide sequence [SEQ ID NO : 4].

TABLE 1 shows the closest homologue of each polypeptide encoded by each ORF of the invention based on a comparison of the sequences in Figure 2 with sequences available in the public domain.

GLOSSARY The following illustrative explanations are provided to facilitate understanding of certain terms used frequently herein, particularly in the Examples. The explanations are provided as a convenience and are not limitative of the invention.

BINDING MOLECULE refers to a molecule or ion which binds or interacts specifically with polypeptides or polynucleotides of the present invention, including, for example enzyme substrates, cell membrane components and classical receptors. Binding between polypeptides of the invention and such molecules may be exclusive to polypeptides of the invention, which is preferred, or it may be highly specific for polypeptides of the invention, which is also preferred, or it may be highly specific to a group of proteins that includes polypeptides of the invention, which is preferred, or it may be specific to several groups of proteins at least one of which includes a polypeptide of the invention. Binding molecules also include antibodies and antibody-derived reagents that bind specifically to polypeptides of the invention.

GENETIC ELEMENT generally means a polynucleotide comprising a region that encodes a polypeptide or a polynucleotide region that regulates replication, transcription or translation or other processes important to expression of the polypeptide in a host cell, or a polynucleotide comprising both a region that encodes a polypeptide and a region operably linked thereto that regulates expression. Genetic elements may be comprised within a vector that replicates as an episomal element; that is, as a molecule physically independent of the host cell genome. They may be comprised within plasmids. Plasmids generally are designated herein by a lower case p preceded and/or followed by capital letters and/or numbers, in accordance with standard naming conventions that are familiar to those of skill in the art. Genetic elements also may be comprised within a host cell genome; not in their natural state but, rather, following manipulation such as isolation, cloning and introduction into a host cell in the form of purified DNA or in a vector, among others.

HOST CELL is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.

IDENTITY, as known in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Identity can be readily calculated (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or two polypeptide sequences, the term is well known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J.

Applied Math., 48: 1073 (1988). Methods commonly employed to determine identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1) : 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403 (1990)).

ISOLATED means separated"by the hand of man"from its natural state; i. e., that, if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a naturally occurring polynucleotide or a polypeptide naturally present in a living organism in its natural state is not"isolated,"but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is"isolated", as the term is employed herein. As part of or following isolation, such polynucleotides can be joined to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance. The isolated polynucleotides, alone or joined to other polynucleotides such as vectors, can be introduced into host cells, in culture or in whole organisms. Introduced into host cells in culture or in whole organisms, such DNAs still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment. Similarly, the polynucleofides and polypeptides may occur in a composition, such as a media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein.

POLYNUCLEOTIDE (S) generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions or single-, double-and triple-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded, or a mixture of single-and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are"polynucleotides"as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.

The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. Polynucleotides embraces short polynucleotides often referred to as oligonucleotide (s).

POLYPEPTIDES, as used herein, includes all polypeptides as described below. The basic structure of polypeptides is well known and has been described in innumerable textbooks and other publications in the art. In this context, the term is used herein to refer to any peptide or protein comprising two or more amino acids joined to each other in a linear chain by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. It will be appreciated that polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques which are well known to the art. Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.

Among the known modifications which may be present in polypeptides of the present are, to name an illustrative few, acetylation, acylation, ADP ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP ribosylation, for instance, are described in most basic texts, such as, for instance PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993).

Many detailed reviews are available on this subject, such as, for example, those provided by Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182: 626646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N. Y. Acad. Sci. 663: 48-62 (1992). It will be appreciated, as is well known and as noted above, that polypeptides are not always entirely linear. For instance, polypeptides may be generally as a result of posttranslational events, including natural processing event and events brought about by human manipulation which do not occur naturally.

Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well. For instance, the amino terminal residue of polypeptides made in E. coli or other cells, prior to proteolytic processing, almost invariably will be N-formylmethionine. During post-translational modification of the peptide, a methionine residue at the NH2-terminus may be deleted. Accordingly, this invention contemplates the use of both the methionine-containing and the methionineless amino terminal variants of the protein of the invention. The modifications that occur in a polypeptide often will be a function of how it is made. For polypeptides made by expressing a cloned gene in a host, for instance, the nature and extent of the modifications in large part will be determined by the host cell posttranslational modification capacity and the modification signals present in the polypeptide amino acid sequence. For instance, as is well known, glycosylation often does not occur in bacteria hosts such as, for example, E. coli. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Insect cell often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to express efficiently mammalian proteins having native patterns of glycosylation, inter alia. Similar considerations apply to other modifications. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. In general, as used herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized recombinantly by expressing a polynucleotide in a host cell.

VARIANT (S) of polynucleotides or polypeptides, as the term is used herein, are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. Variants in this sense are described below and elsewhere in the present disclosure in greater detail.

(1) A polynucleotide that differs in nucleotide sequence from another, reference polynucleotide. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical. As noted below, changes in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide with the same amino acid sequence as the reference. Also as noted below, changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. (2) A polypeptide that differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference and the variant are closely similar overall and, in many region, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel polypeptides and polynucleotides of S. pneumoniae, among other things, as described in greater detail below.

In particular, the invention relates to novel polypeptides and polynucleotides of S. pneumoniae. The invention relates especially to the nucleotide and amino acid sequences set out in Figure 1 and Figure 2 respectively, and to the corresponding nucleotide and amino acid sequences of the G54 strain of S. pneumoniae.

Polynucleotides In accordance with one aspect of the present invention, there are provided isolated polynucleotides which encode the polypeptide having the deduced amino acid sequences of Figure 2.

Using the information provided herein, such polynucleotide sequences set out in Figure 1 [SEQ ID NO : 1, 2,3,4], a polynucleotide of the present invention may be obtained using a modification of the STM technique described by Hensel et al., Science 269: 400-403 (1995).. The following two major changes were introduced in the original STM protocol (a) A library of tagged mutants was constructed by insertion-duplication mutagenesis (Spellerberg et al., Mol Microbiol 19: 803-813 1996). Short (400-600 bp) random genomic DNA fragments inserted in the pR326 suicide plasmid vector with the molecular tag placed on it. The pR326 vectors carries the chloramphenicol resistance gene. The original transposon mutagenesis was not applied to S. pneumoniae, since we did not expect transposons to insert randomly as it was shown in Streptococcus mutans (Hallet et al., Mol Microbiol 14: 131-139 1994) and Lactococcus lactis (Renault et al., Dev Biol Stand 85: 535-541 1995). S. pneumoniae G54 was transformed with the tagged genomic library and chloramphenicol resistant clones were selected.

(b) While in the original method the filters corresponding to each pool had spots of genomic DNA from each mutant obtained by transfering bacterial colonies to the filters (colony hybridisation), we used filters containing amplified tags from each individual mutant. This modification was necessary since in our hands colony hybridisation generated a high background giving rise to false positives.

Inverse PCR was used to amplify the genomic DNA segment flanking the chloramphenicol resistance gene in the S. pneumoniae mutants.. Two micrograms of genomic DNA was digested with suitable enzymes and self ligated. The ligation mixture was amplified by PCR using oligonucleotides pairing to the chloramphenicol resistance gene. The average size of the amplified DNA product was 500 bp. The PCR products were sequenced with an automated sequencer ABI Prism 377. Predicted amino acid sequences from all six reading frames of the DNA sequences obtained above were subjected to similarity search against public databases.

The DNA sequences thus obtained is set out in Figure 1 [SEQ ID NO : 1, 2, -3, 4]. They contain an open reading frame encoding a protein of having about the number of amino acid Oresidues set forth in Figure 2 with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known in the art.

Polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The DNA may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.

The coding sequence which encodes the polypeptide may be identical to the coding sequence of the polynucleotides shown in Figure 1. It also may be a polynucleotide with a different sequence, which, as a result of the redundancy (degeneracy) of the genetic code, encodes the polypeptides of Figure 2.

Polynucleotides of the present invention which encode the polypeptide of Figure 2 [SEQ ID NO : 2] may include, but are not limited to the coding sequence for the mature polypeptide, by itself ; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro-or preproprotein sequence; th by integrated phage or insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences.

The present invention further relates to variants of the herein above described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequences of Figure 2. A variant of the polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.

Among variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.

Among the particularly preferred embodiments of the invention in this regard are polynucleotides encoding polypeptides having the amino acid sequences set out in Figure 2; variants, analogs, derivatives and fragments thereof, and fragments of the variants, analogs and derivatives.

Further particularly preferred in this regard are polynucleotides encoding variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments, which have the amino acid sequence of polypeptides of Figure 2 in which several, a few, 5 to 10,1 to 5,1 to 3,2,1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the polypeptides of Figure 2. Also especially preferred in this regard are conservative substitutions. Most highly preferred are polynucleotides encoding polypeptides having the amino acid sequences of Figure 2, without substitutions.

Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding polypeptides having the amino acid sequence set out in Figure 2, and polynucleotides which are complementary to such polynucleotides.

Altematively, most highly preferred are polynucleotides that comprise a region that is at least 80% identical over their entire length to a polynucleotide encoding a polypeptide of the present invention and polynucleotides complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.

Preferred embodiments in this respect, moreover, are polynucleotides which encode polypeptides which retain substantially the same biological function or activity as the mature polypeptides encoded by the DNA of Figure 1.

The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term"stringent conditions"means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.

As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the polynucleotides of the present invention. Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 30 bases and may have at least 50 bases. Particularly preferred probes will have at least 30 bases and will have 50 bases or less.

For example, the coding region of the polynucleotide of the present invention may be isolated by screening using the known DNA sequence to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays, inter alia.

The polynucleotides of the invention that are oligonucleotides are derived from the sequences of [SEQ ID NO : 1], [SEQ ID NO : 2], [SEQ ID NO : 3], [SEQ ID NO : 4]. They may be used in the processes herein as described, but preferably for PCR, to determine whether or not the S. pneumoniae genes identified herein in whole or in part are transcribed in infected tissue.

It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.

The polynucleotides may encode a polypeptide which is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.

Polypeptides The present invention further relates to polypeptides which have a deduced amino acid sequence of amino acids in length, as set forth in Figure 2 [SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8].

The invention also relates to fragments, analogs and derivatives of these polypeptides. The terms"fragment,""derivative"and"analog"when referring to the polypeptides of Figure 2, means a polypeptide which retains essentially the same biological function or activity as such polypeptide.

Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The fragment, derivative or analog of the polypeptides of Figure 2 may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

Among the particularly preferred embodiments of the invention in this regard are polypeptides having the amino acid sequences of set out in Figure 2, variants, analogs, derivatives and fragments thereof, and variants, analogs and derivatives of the fragments.

Among preferred variants are those that vary from a reference by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile ; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.

Further particularly preferred in this regard are variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments, having the amino acid sequence of the potypeptides of Figure 2, in which several, a few, 5 to 10,1 to 5,1 to 3,2,1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the polypeptide of the present invention. Also especially preferred in this regard are conservative substitutions. Most highly preferred are polypeptides having the amino acid sequences of Figure 2 without substitutions.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The polypeptides of the present invention include the polypeptides of Figure 2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% identity to the polypeptides of Figure 2, preferably at least 80% identity to the polypeptides of Figure 2, and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptides of Figure 2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptides of Figure 2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

Fragments Also among preferred embodiments of this aspect of the present invention are polypeptides comprising fragments of the polypeptides having the amino acid sequences set out in Figure 2, and fragments of variants and derivatives of the polypeptides of Figure 2.

In this regard a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned S. pneumoniae polypeptides and variants or derivatives thereof.

Such fragments may be"free-standing,"i. e., not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the presently discussed fragments most preferably form a single continuous region. However, several fragments may be comprised within a single larger polypeptide. For instance, certain preferred embodiments relate to a fragment of a polypeptide of the present invention comprised within a precursor polypeptide designed for expression in a host and having heterologous pre and pro-polypeptide regions fused to the amino terminus of the fragment and an additional region fused to the carboxyl terminus of the fragment. Therefore, fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein derived froma polypeptide of the present invention.

In this context"about"herein includes the particularly recited ranges larger or smaller by several, a few, 5,4,3,2 or 1 amino acid at either extreme or at both extremes.

Preferred fragments of the invention include, for example, truncation polypeptides including polypeptides having the amino acid sequences of Figure 2, or of variants or derivatives thereof, except for deletion of a continuous series of residues (that is, a continuous region, part or portion) that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or, as in double truncation mutants, deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Fragments having the size ranges set out about also are preferred embodiments of truncation fragments, which are especially preferred among fragments generally. Degradation forms of the polypeptides of the invention in a host cell are also preferred.

Also preferred in this aspect of the invention are fragments characterized by structural or functional attributes of the polypeptide of the present invention.

Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, tum and tum-forming regions, coil and coilforming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surfaceforming regions, substrate binding region, and high antigenic index regions of the polypeptide of the present invention, and combinations of such fragments.

Preferred regions are those that mediate activities of the polypeptide of the present invention. Most highly preferred in this regard are fragments that have a chemical, biological or other activity of the polypeptide of the present invention, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of S. pneumoniae or the ability to cause disease in man. Further preferred polypeptide fragments are those that comprise or contain antigenic or immunogenic determinants in an animal, especially in a human.

It will be appreciated that the invention also relates to, among others, polynucleotides encoding the aforementioned fragments, polynucleotides that hybridize to polynucleotides encoding the fragments, particularly those that hybridize under stringent conditions, and polynucleotides, such as PCR primers, for amplifying polynucleotides that encode the fragments. In these regards, preferred polynucleotides are those that correspond to the preferred fragments, as discussed above.

Vectors, host cells, expression The present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate polynucleotides and express polypeptides of the present invention. Introduction of a polynucleotides into the host cell can be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING : A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989).

Polynucelotide constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Altematively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING : A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989).

In accordance with this aspect of the invention the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector. Starting plasmids disclosed herein are either commercially available, publicly available, or can be constructed from available plasmids by routine application of well known, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art.

Preferred among vectors, in certain respects, are those for expression of polynucleotides and polypeptides of the present invention. Generally, such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate transacting factors either are supplie by the host, supplie by a complementing vector or supplie by the vector itself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide for specific expression. Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell- specific. Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives. A variety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routine by those of skill in the art.

A great variety of expression vectors can be used to express a polypeptide of the invention. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e. g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used for expression in this regard.

The appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

The DNA sequence in the expression vector is operatively linked to appropriate expression control sequence (s), including, for instance, a promoter to direct mRNA transcription. Representatives of such promoters include, but are not limited to, the phage lambda PL promoter, the E. coli lac, trp, tac and ara promoters, the SV40 early and late promoters and promoters of retroviral LTRs.

In general, expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate as well as engender expression. Generally, in accordance with many commonly practiced procedures, such regions will operate by controlling transcription, such as transcription factors, repressor binding sites and termination, among others.

Vectors for propagation and expression generally will include selectable markers and amplification regions, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, streptomyces and Bacillus subtils cells ; fungal cells, such as yeast cells and Aspergillus cells ; insect cells such as Drosophila S2 and Spodoptera Sf9 cells ; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells ; and plant cells.

The following vectors, which are commercially available, are provided by way of example. Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia, and pBR322 (ATCC 37017), and pBAD18, pBAD24, pBAD28 (Guzman et al., J Bacteriol 177: 4121-4130 1995).

Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention.

Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("CAT) transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i. e., a fragment that may contain a promoter. As is well known, introduction into the vector of a promoter-containing fragment at the restriction site upstream of the cat gene engenders production of CAT activity, which can be detected by standard CAT assays. Vectors suitable to this end are well known and readily available, such as pKK232-8 and pCM7. Promoters for expression of polynucleotides of the present invention include not only well known and readily available promoters, but also promoters that readily may be obtained by the foregoing technique, using a reporter gene.

Among known prokaryotic promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E. coli lacl and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter.

Among known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein-I promoter.

Recombinant expression vectors will include, for example, origins of replication, a promoter preferably derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.

Polynucleotides of the invention, encoding the heterologous structural sequence of a polypeptide of the invention generally will be inserted into the vector using standard techniques so that it is operably linked to the promoter for expression. The polynucleotide will be positioned so that the transcription start site is located appropriately 5'to a ribosome binding site.

The ribosome binding site will be 5'to the AUG that initiates translation of the polypeptide to be expressed. Generally, there will be no other open reading frames that begin with an initiation codon, usually AUG, and lie between the ribosome binding site and the initiation codon. Also, generally, there will be a translation stop codon at the end of the polypeptide and there will be a polyadenylation signal in constructs for use in eukaryotic hosts. Transcription termination signal appropriately disposed at the 3'end of the transcribed region may also be included in the polynucleotide construct.

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-or C-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.

Also, region also may be added to the polypeptide to facilitate purification.

Such regions may be removed prior to final preparation of the polypeptide.

The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability or to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from immunolglobulin that is useful to solubilize or purify polypeptides. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another protein or part thereof. In drug discovery, for example, proteins have been fused with antibody Fc portions for the purpose of high-throughput screening assays to identify antagonists. See, D. Bennett et al., Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471 (1995).

Cells typically then are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Mammalian expression vectors may comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation regions, splice donor and acceptor sites, transcriptional termination sequences, and 5'flanking non-transcribed sequences that are necessary for expression.

The polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

Polynucleotide assays This invention is also related to the use of the polynucleotides of the present invention to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of complementary nucleotides in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (herein also "individual (s)"), particularly mammals, and especially humans, infected with S. pneumoniae may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324: 163-166 (1986) prior to analysis. RNA or cDNA may also be used in the same ways. As an example, PCR primers complementary to the nucleic acid forming part of the polynucleotide of the present invention can be used to identify and analyze for its presence and/or expression. Using PCR, characterization of the strain of S. pneumoniae present in a mammal, and especially a human, may be made by an analysis of the genotype of the prokaryote gene. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or altematively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method. For example, a sequencing primer is used with double-stranded PCR product or a single stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.

Genetic characterization based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e. g., Myers et al., Science, 230: 1242 (1985)).

Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e. g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 43974401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, e. g., restriction fragment length polymorphisms (RFLP) and Southern blotting of genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing, mutations also can be detected by in situ analysis.

Cells carrying mutations or polymorphisms in the gene of the present invention may also be detected at the DNA level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR can be used to detect mutations. It is particularly preferred to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA or cDNA may also be used for the same purpose, PCR or RT-PCR. As an example, PCR primers complementary to the nucleic acid encoding the polypeptide of the present invention can be used to identify and analyze mutations. The primers may be used to amplify the gene isolated from the individual such that the gene may then be subject to various techniques for elucidation of the DNA sequence. In this way, mutations in the DNA sequence may be diagnosed.

The invention provides a process for diagnosing disease, arising from infection with S. pneumoniae, comprising determining from a sample derived from an individual an increased level of expression of polynucleotides having the sequences of Figure 1. Increased expression of polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northem blotting and other hybridization methods.

Polypeptide assays The present invention also relates to a diagnostic assays such as quantitative and diagnostic assays for detecting levels of the polypeptide of the present invention in cells and tissues, including determination of normal and anormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of the polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example. Assay techniques that can be used to determine levels of a the polypeptide, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Among these ELISAs frequently are preferred. An ELISA assay initially comprises preparing an antibody specific to the polypeptide, preferably a monoclonal antibody. In addition a reporter antibody generally is prepared which binds to the monoclonal antibody. The reporter antibody is attached a detectable reagent such as radioactive, fluorescent or enzymatic reagent, in this example horseradish peroxidase enzyme.

Antibodies The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. The present invention includes, for examples monoclonal and polyclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library.

Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique known in the art which provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques described for the production of single chain antibodies (U. S.

Patent No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies to immunogenic polypeptide products of this invention.

Altematively phage display technology could be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-Fbp or from naive libraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10,779-783).

The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directed against a different epitope-termed'bispecific'antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or purify the polypeptide of the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography.

Thus, among others, antibodies against the polypeptide of the present invention may be employed to inhibit and/or treat infections, particularly bacterial infections and especially infections arising from S. pneumoniae.

Polypeptide derivatives include antigenically, epitopically or immunologically equivalent derivatives which form a particular aspect of this invention. The term"antigenically equivalent derivative"as used herein encompasses a polypeptide or its equivalent which will be specifically recognised by certain antibodies which, when raised to the protein or polypeptide according to the present invention, interfere with the immediate physical interaction between pathogen and mammalian host. The term"immunologically equivalent derivative"as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stability to the polypeptide. The antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).

Alternatively a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.

Preferably the antibody or derivative thereof is modified to make it less immunogenic in the individual. For example, if the individual is human the antibody may most preferably be"humanized" ; where the complimentarity determining region (s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones, P. et al. (1986), Nature 321,522-525 or Tempest et al., (1991) Biotechnology 9,266-273.

The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1: 363, Manthorpe et al., Hum. Gene Ther. 1963: 4, 419), delivery of DNA complexe with specific protein carriers (Wu et al., J Biol Chem 1989: 264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS, 1986: 83, 9551), encapsulation of DNA in various forms of liposomes (Kaneda et al., Science 1989: 243,375), particle bombardment (Tang et al., Nature 1992,356: 152, Eisenbraun et al., DNA Cell Biol 1993,12: 791) and in vivo infection using cloned retroviral vectors (Seeger et al., PNAS 1984: 81,5849).

Binding molecules and assays This invention also provides a method for identification of molecules, such as binding molecules, that bind to the polypeptide of the present invention.

Genes encoding proteins that bind to the polypeptide, can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Such methods are described in many laboratory manuals such as, for instance, Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991). Also, a labeled ligand can be photoaffinity linked to a cell extract. Polypeptides of the invention also can be used to assess the binding capacity of a binding molecule, in cells or in cell-free preparations.

Polypeptides of the invention may also be used to assess the binding or small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics.

The invention further provides a complex of a polypeptide and a binding molecule which comprises a polypeptide as described herein and a binding molecule capable of modulating the activity of the polypeptide. A complex of this kind will arise in vivo upon administration to a patient of a binding molecule as described herein.

Antagonists and agonists-assays and molecules The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the function of polypeptides or polynucleotides of the present invention, such as its interaction with a binding molecule. The method of screening may involve high-throughput.

For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, may be prepared from a cell that expresses a molecule that binds to the polypeptide of the present invention. The preparation is incubated with labeled polypeptide in the absence or the presence of a candidate molecule which may be an agonist or antagonist.

The ability of the candidate molecule to bind the binding molecule is reflected in decreased binding of the labeled ligand. Molecules which bind gratuitously, i. e., without inducing the functional effects of the polypeptide, are most likely to be good antagonists. Molecules that bind well and elicit functional effects that are the same as or closely related to the polypeptide are agonist s.

The functional effects of potential agonists and antagonists may by measured, for instance, by determining activity of a reporter system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of the polypeptide of the present invention or molecules that elicit the same effects as the polypeptide. Reporter systems that may be useful in this regard include but are not limited to colorimetric labeled substrate converted into product, a reporter gene that is responsive to changes in the functional activity of the polypeptide, and binding assays known in the art.

Another example of an assay for antagonists is a competitive assay that combines the polypeptide of the present invention and a potential antagonist with membrane-bound binding molecules, recombinant binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. The polypeptide can be labeled, such as by radioactivity or a colorimetric compound, such that the number of polypeptide molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds to the same sites on a binding molecule without inducing functional activity of the polypeptide of the invention.

Potential antagonists include a small molecule which binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented.

Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules.

Other potential antagonists include antisense molecules (see Okano, J.

Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules).

Preferred potential antagonists include derivatives of the polypeptide of the invention.

In a particular aspect the invention provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequelae of infection. In particular the molecules of the invention may be used: i) in the prevention of adhesion of S. pneumoniae to mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; ii) to block protein mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases (Rosenshine et al., Infect. Immun. 60: 2211 (1992); iii) to block bacterial adhesion between mammalian extracellular matrix proteins and bacterial proteins which mediate tissue damage; iv) to block the normal progression of pathogenesis in infections initiated other than by the implantation of indwelling devices or by other surgical techniques.

Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds. The encoded protein upon expression can be used as a target for the screening of antibacterial drugs.

Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgamo or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.

The antagonists and agonist may be employed for instance to inhibit diseases arising from infection with Streptococci, especially S. pneumoniae.

Vaccines Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with the polypeptide of the invention, or a fragment or variant thereof, adequate to produce antibody to protect said individual from infection, particularly bacterial infection and most particularly Streptococci infections. Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises, through gene therapy or otherwise, delivering gene encoding the polypeptide, or a fragment or a variant thereof, for expressing the polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response to produce antibodies or a cell mediated T cell response, either cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not. One way of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise.

A further aspect of the invention relates to an immunological composition which, when introduced into a host capable or having induced within it an immunological response, induces an immunological response in such host, wherein the composition comprises recombinant DNA which codes for and expresses an antigen of the polypeptide of the present invention. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells.

The polypeptide of the invention or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as Glutathione- S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilise the protein and facilitate production and purification thereof.

Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein.

Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bacteria cell surface proteins in DNA constructs used in such genetic immunization experiments in animal models of infection with S. pneumoniae will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or clearing infection for the development of prophylactic agents or therapeutic treatments of S. pneumoniae infection in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue. Examples of tissue damage include wounds in skin or connective tissue caused e. g. by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina.

The present invention also includes a vaccine formulation which comprises the immunogenic recombinant protein together with a suitable carrier.

Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the bodily fluid, preferably the blood, of the individual ; and aqueous and nonaqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

While the invention has been described with reference to certain polypeptides, it is to be understood that this covers fragments of the naturally occurring protein and similar proteins with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant protein.

Compositions The invention also relates to compositions comprising the polynucleotide or the polypeptides discussed above or the agonists or antagonists. Thus, the polypeptides of the present invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration.

Kits The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.

Associated with such container (s) can be a notice in the form prescribed by a govemmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.

Administration Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as th anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amount effective for treatment or prophylaxis of a specific indication or indications.

In general, the compositions are administered in an amount of at least about 10 pg/kg body weight. In most cases they will be administered in an amount not in excess of about 8 mg/kg body weight per day. Preferably, in most cases, dose is from about 10 ug/kg to about 1 mg/kg body weight, daily. It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, its severity, route of administration, complicating conditions and the like.

In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.

Altematively the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation ; more usually they will constitute up to about 80% by weight of the formulation.

For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

In-dwelling devices include surgical implants, prosthetic devices and catheters, i. e., devices that are introduced to the body of an individual and remain in position for an extended time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters, etc.

The composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of the device. In addition, the composition could also be used to broaden perioperative cover for any surgical technique to prevent Streptococci wound infections.

Many orthopaedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia. Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality. It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis. Altematively, the composition of the invention may be used to bathe an indwelling device immediately before insertion.

The active agent will preferably be present at a concentration of 1pg/ml to 1 Omg/ml for bathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response.

A suitable unit dose for vaccination is 0.5-5g/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks.

With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.

The antibodies described above may also be used as diagnostic reagents to detect the presence of bacteria containing the protein.

In order to facilitate understanding of the following example certain frequently occurring methods and/or terms will be described.

The present invention is further described by the following examples. While illustrating certain specific aspects of the invention, the examples do not portray the limitations or circumscribe the scope of the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples were carried out using a modification of STM mutagenesis described by Hensel et al., Science 269: 400-403 (1995).

*Example 1 Library Production and Sequencing The polynucleotide having the DNA sequence given in SEQ ID NO : 1 was obtained from a library of clones of chromosomal DNA of S. pneumoniae.

Libraries may be prepared according to standard procedure. Thus, for example Method 1 Streptococcus pneumoniae chromosomal DNA was isolated from the G54 strain.

Chromosomal DNA (40 g) was sheared with a French Cell Press (SLM Aminco), as indicated by the manufacturer. The DNA ends were repaired using 25 units of Klenow polymerase, 15 units of T4 DNA polymerase and 50 uM dNTPs. The blunt ended DNA was then size fractionated by centrifugation in a 10% to 40% sucrose gradient (26,000 rpm for 18 hours in a SW28 rotor (Beckman) (Current Protocols in Molecular Biology on CD ROM, Wiley eds.). The size of the fragments from the fractions were determined by agarose gel electrophoresis. Fractions from 400 bp to 600 bp were pooled, dialyse and ethanol precipitated. The fractions were resuspended in 200 pi of 1x ligase buffer and ligated for 16 hours at 4 C with 20 pg of BamHi phosphorylated linkers (New England Biolabs) and 1000 units of T4 DNA ligase. This mixture was then digested with BamHl and ligated to the BamHl digested tagged plasmid library.

The ligation mixture was used directly to transform the S. pneumoniae strain G54 as reported by Pozzi et al. J Bacteriol 178: 6087-6090 (1996) and chloramphenicol resistant colonies were selected.

The DNA sequence given in [SEQ ID NO : 1] was obtained as follows.

Inverse PCR (IPCR) was used to amplify the genomic DNA segment flanking the chloramphenicol (cat) resistance gene in the S. pneumoniae mutants. Two micrograms of genomic DNA were digested separately with each of these enzymes : Kpnl, Hincll, Clal and Hindlll for 8 hours at 37 C.

Digested genomic DNA was diluted 100 fold and self-ligated. One microliter of ligation mixture was used as a template for PCR using primers cat 0 (5' ACAGCTTCCAGATCCATATCC-3') and cat 770 (5' TATCCCACTTTATCCAATTTT-3'). The PCR mixture (50 ut) contained primers (lpM), DATP, DTTP, DGTP and dCTP (0.2 mM each), Taq DNA polymerase (1 unit) and 5 pi Taq polymerase 10x buffer (Perkin-Elmer Cetus). Twenty cycles of denaturation (30 s at 92 C), annealing (60 s at 50 C) and polymerization (1 min and 30 s at 72 C) were performed. One microliter of the PCR reaction was used as template in a nested reaction performed under the same conditions as described above, using primers cat 1 (5'-TGACGTTGAGCCTCGGAACCCATCG-3') and cat 720 (5' GTTGAACCATTATATCACATT-3') (see Fig. 1). The average size of the amplified DNA product was 500 bp. The PCR products were sequenced using the primer cat 1 with an automated sequencer ABI Prism 377 following the manufacturer's instructions. Predicted amino acid sequences from all six reading frames of the DNA sequences obtained above were subjected to similarity search of the SWISS-PROT TREMBL database using the BLAST programs.

The polynucleotides having DNA sequences given in [SEQ ID No: 2], [SEQ ID No: 3] [SEQ ID No: 4] were obtained using analogue methods to those described for example 1.

FIGURE 1.

Polynucleotide sequence of S. pneumoniae [SEQ ID NO : 1] Sequence characteristic : lenght : 3462 bases start codon: 1 stop codon: 3459 ATGAGTAATATCAGTTTAACAACACTTGGTGGTGTGCGTGAGAATGGAAAAAATATGTACA TTGCTGAAATTGGAGAGTCCATTTTTGTTTTGAATGTAGGGTTAAAATATCCTGAAAATGA ACAATTAGGGGTCGATGTGGTGATTCCAAACATGGATTACCTTTTTGAAAATAGCGACCGT ATTGCTGGGGTTTTCTTGACCCACGGGCATGCGGATGCTATTGGTGCTCTACCTTATCTCT TGGCAGAGGCTAAAGTTCCTGTATTTGGGTCTGARTTGACCATTGAGTTGGCAAAGCTCTT TGTCAAAGGAAATGATGCCGTTAAGAAATTTAATGATTTCCATGTCATTGATGAGAATACG GAGATTGATTTTGGTGGGACAGTGGTTTCCTTCTTCCCTACGACTTACTCCGTTCCAGAGA GTCTGGGAATTGTCTTGAAGACATCGGAAGGAAGTATCGTTTATACAGGTGACTTCAAATT TGACCAAACGGCTAGTGAATCTTATGCAACTGATTTTGCTCGTTTGGCAGAGATTGGTCGT GACGGNGTCNTGGCTCTCCTCAGTGANTCGGCCACTTNNTTTGGTATAATGGTAAGCAATA ATGGACTAGAAAGAACAAAGATGCAAGATAAAATTGTCATTCATGGGGCGCGTGCCCATAA TTTAAAAAATATTGATGTGGAGATTCCGCGAGACAAGTTGGTTGTCGTGACTGGCTTGTCA GGTTCAGGGAAGTCCAGTCTGGCTTTTGATACTCTCTATGCGGAGGGACAACGTCGCTATG TAGAGAGTTTGTCTGCCTACGCTCGTCAGTTCTTGGGAAATATGGAAAAACCAGATGTAGA TGCCATTGATGGTCTCAGCCCAGCTATTTCCATCGACCAGAAAACGACTAGTAAAAATCCT CGCTCGACGGTGGGAACGACGACTGAAATCAATGACTATCTGCGTCTCCTCTACGCACGTG TGGGGACGCCTTACTGTATCAACGGACATGGAGCTATCAATGCTTCTTCTGTGGAGCAAAT CGTTGATAAGGTTTTGGAGTTACCTGAACGCCAGCGCTTGCAGATCTTGGCTCCTGTCATC CGCAAGAAAAAAGGCCAACATAAGAGTGTTATCGAGAAGGTTCAGAAAGACGGGTATGTTC GTGTTCGTGTGGATGGGGAAGTCTATGATGTGACCGAAGTGCCAGAGTTGTCTAAGAGCAA GCAACACAATATCGATGTCGTGGTTGACCGTATTGTTATCAAGGAGGGCATTCGTAGCCGT CTCTTTGATTCCATTGAAGCTGCCCTTCGTATCGCAGAAGGTTATGTGATTATCGACACCA TGGACGACTCTGAGTTGTTGTTCTCTGAGCATTATGCCTGTCCAGTTTGTGGATTTACTGT CCCAGAGTTAGAACCTCGTCTCTTCTCTTTCAATGCTCCCTTTGGCTCTTGTAGTGAGTGT GACGGCTTGGGGATTAAGCTGGAGGTGGATACTGATTTGGTAGTGCCAGATGCCAGCAAAA CCTTACATGAGGGAGCGCTGGCACCTTGGAATCCTATCTCATCCAACTACTATCCAAACAT GTTAGAGCAGGCCATGAAAGTCTTTGGAGTGGCTATGGATAAGCCTTTTGAGGACCTGTCA GAAGAAGATAAGAACTTGATTCTCTACGGCTCAGATGGTAAGGAATTCCATTTCCATTATG AAAATGAATTTGGTGGTGTGCGCGATATCGACATTCCGTTTGAGGGAGTTATCAATAATAT CAAACGTCGTTACCATGAAACCAATAGCGATTACACTCGCACTCAGATGCGTCTCTATATG AATGAGCTGACCTGCGGAACCTGTCAAGGCTATCGTCTCAATGACCAGGCCTTGTCTGTCC GTGTGGGCGGCCAGCAAGGGCCACATATTGGAGAAATCTCAGACCTGTCTATCGCTGACCA CTTGGACTTGGTGAGCCAGTTAACTTTGTCTGAAAATGAAGCCATCATTGCTCGTCCCATT CTCAAGGAAATCAAGGATCGTTTGACCTTCCTTAATAATGTGGGTCTTAACTATCTAACCC TGTCACGTTCAGCAGGAACCCTTTCAGGTGGGGAAAGTCAGCGCATTCGTTTGGCAACCCA GATTGGTTCCAACCTATCAGGTGTCCTCTATATCCTAGACGAGCCGTCAATCGGTCTTCAC CAGAGGGACAATGACCGCCTGATTGCCAGTCTGAAAAAGATGCGTGACTTGGGCAATACTC TTATCGTGGTGGAACACGACGAAGATACCATGCGTGAAGCTGATTATCTGATTGACGTTGG TCCTGGTGCCGGTGTTTTTGGTGGTGAGATTGTTGCAGCAGGTACACCTAAGCAAGTGGCT CGCAACAGCAAGTCTATCACAGGTCAGTACTTGTCAGGCAAACGTGTCATCCCAGTACCAG AAGAGCGCCGTGTCGGAAATGGTCGTTTTATTGAAGTGACAGGAGCGCGTGAGAACAACTT GCAAAATGTCACGGCTCGCTTTCCACTAGGAAAATTTATCGCAGTGACAGGTGTGTCGGGT TCAGGGAAATCGACTCTAATCAACAGCATTCTCAAAAAAGCCATTGCCCAGAAGCTCAACC GTAATTCAGACAAACCTGGTAAGTTTAAGACTATTACAGGGATTGAGCATGTCGACCGTTT GATTGATATTGACCAGAGCCCTATCGGACGGACGCCGAGGTCTAACCCAGCYACCTATACA GGAGTTTTTGACGATATACGTGACCTTTTTGCTCAGACAAATGAAGCCAAGATTCGAGGCT ACAAGAAGGGGCGCTTCAGTTTCAACGTCAAGGGAGGTCGCTGTGAAGCTTGCTCAGGTGA CGGGATTATCAAGATTGAGATGCACTTCTTGCCAGATGTTTATGTGGCTTGTGAAGTTTGC CACGGAACCCGCTATAACAGTGAAACCCTAGAAGTTCACTACAAGGAAAAGAATATTTCGC AGGTCTTGGATATGACGGTCAACGATGCGGTAGAATTTTTCCAACACATTCCGAAAATTCA ACGCAAACTCCAGACCATCAAGGATGTAGGTCTAGGCTATGTGACCTTGGGACAGCCAGCT ACCACCCTTTCTGGGGGAGAAGCCCAACGTATGAAGTTAGCTAGCGAACTCCACAAACGCT CGACAGGAAAATCTTTCTACATTCTGGATGAGCCGACGACAGGGCTTCATACAGAGGACAT TGCTCGCCTGCTCAAGGTTTTAGCTCGCTTTGTAGACGACGGCAATACAGTCCTCGTCATC GAGCACAATCTGGATGTTATCAAGACGGCAGACCATATCATTGACTTGGGACCTGAGGGCG GTGTCGGTGGTGGAACCATCATCGTAACAGGAACTCCAGAAGAAGTAGCTGCCAACGAAGC CAGCTATACAGGACACTATTTGAAAGGAAAGTTACATCATGAATAA Polynucleotide sequence of S. pneumoniae [SEQ ID NO : 2] Sequence characteristic: lenght : bases 2214 start codon: 1 stop codon : 2212 ATGGCAAAACAAGTGTTTCAAACGACTTTTGCGGGTCGTGAGTTAATTGTAGAGACTGGTC AGGTTGCTAAGCAAGCAAATGGCTCTGTTGTCGTACGTTACGGTGAGTCAACTGTCTTGAC TGCTGCCGTTATGTCTAAGAAAATGGCAACTGGGGATTTCTTCCCCCTCCAAGTCAACTAC GAAGAAAAAATGTATGCGGCTGGGAAGTTTCCTGGTGGCTTTATGAAACGTGAAGGACGTC CTTCAACAGATGCGACCTTGACAGCGCGTTTGATTGACCGTCCGATTCGTCCTATGTTTGC GGAAGGTTTCCGTAATGAAGTCCAAGTCATCAATACAGTGCTTTCTTATGATGAAAATGCA TCTGCACCAATGGCTGCTATGTTTGGTTCATCTTTGGCACTGTCTATTTCAGATATTCCAT TTGACGGACCAATTGCTGGGGTACAAGTGGGATATGTAGATGGCCAAATCATCATCAACCC AAGTCAAGAACAAGCAGAGCAATCTCTTCTTGAATTGACAGTAGCTGGAACCAAGCACGCT ATCAACATGGTAGAGTCTGGTGCCAAAGAATTGTCAGAAGAAATCATGTTGGAAGCGCTCC TTAAAGGGCACGAAGCTGTCAAAGAATTGATTGCCTTCCAAGAAGAAATCGTTGCTGCTGT CGGTAAAGAAAAAGCAGAAGTGGAATTGCTTCACGTGGATGCTGAATTGCAAGCTGAAATC ATTGCAGCCTACAACAGTGACCTCCAAAAGGCAGTTCAAGTAGAAGAGAAATTGGCCCGTG AAGCTGCGACTCAAGTAGTGAAAGACCAAGTGACTGCCGTTTACGAAGAAAAATATGCGGA CCACGAAGAATTTGACCGTATTATGCGTGATGTGGCTGAAATCTTGGAACAAATGGAACAC GCAGAAGTGCGACGTTTAATTACAGAAGACAAGGTGCGTCCTGATGGTCGTAAGGTCGATG AAATCCGTCCTTTGGATGCGGTTGTCGACTTCCTTCCTCGTGTACATGGTTCAGGTCTCTT TACTCGTGGGCAAACTCAAGCTCTTTCAGTCTTGACCTTGGCTCCGATGGGAGAAACTCAA ATCATTGATGGTTTGGATCCAGAGTACAAGAAACGCTTTATGCACCACTATAACTTCCCTC AATATTCTGTAGGGGAAACAGGTCGTTACGGTGCGCCAGGTCGTCGTGAAATCGGTCACGG TGCCCTTGGTGAGCGTGCTCTTGCTCAAGTCTTGCCAAGCTTGGAAGAATTCCCATACGCT ATCCGTCTAGTAGCAGAAGTTTTGGAATCAAACGGTTCTTCATCTCAAGCTTCTATCTGTG CGGGAACTCTTGCCCTTATGGCTGGTGGTGTGCCAATCAAGGCGCCAGTAGCTGGTATTGC TATGGGACTTATCTCAGATGGAAATAACTACACAGTATTGACAGATATCCAAGGTTTGGAA GATCACTTTGGAGATATGGACTTCAAGGTTGCAGGTACTCGTGATGGGATTACAGCCCTTC AAATGGATATCAAGATTCAAGGGATTACTGCAGAAATCTTGACGGAGGCTCTTGCTCAAGC CAAGAAAGCGCGTTTTGAAATCCTTGATGTCATTGAAGCAACCATTCCAGAAGTTCGTCTA GAATTGGCTCCAACTGCTCCGAAAATTGATACGATCAAGATTGATGTAGACAAGATTAAGA TTGTCATCGGTAAGGGTGGAGAAACCATCGACAAGATTATCGCTGAAACAGGTGTTAAGAT TGATATAGACGAAGAAGGAAATGTGTCTATCTACTCTAGTGACCAAGATGCTATTAACCGT GCCAAAGAAATTATTGCTGGTTTGGTTCGTGAAGCCAAAGTGGATGAAGTTTACCGTGCTA AAGTCGTTCGTATCGAGAAATTTGGTGCCTTTGTTAACCTCTTTGATAAGACAGATGCCCT TGTTCATATCTCTGAGATGGCTTGGACTCGTACCAATCGTGTAGAGGATTTGGTAGAAATC GGGGATGAAGTTGATGTTAAGGTTATCAAGATTGATGAAAAAGGCCGTATCGATGCCTCTA TGAAGGCTCTTCTACCTCGTCCGCCAAAACCTGAGCATGATGAAAAAGGTGAAAAGTCTGA GCGCCCTCACCGCCCACGTCATCACAAGGATCACAAACCTAAGAAAGAATTTACAGAAACA CCAAAAGATTCAGAGTAA Polynucleotide sequence of S. pneumoniae [SEQ ID NO : 3] Sequence characteristics lenght : 693 bases start codon: 1 stop codon: 691 ATGGAATTTACAGATATTGCGATGGAATTATCCAAGAAAGCTTGGCAGGCTTCCTTTCATC ATCCCTTTATTTTACAATTACAAGAGGGGAATTTAGAACCTGCCATTTTCCGCTATTACCT GATTCAGGATGCCTACTATCTGAAGGCCTTCTCAGAAATCTATCATCTTTTGGCTGATAAG ACTTCAAACCAAGAGATGAAAAGACTCTTGAAACAAAATGCTCAGGGTTTAGTGGAGGGTG AGTTATTTATTCGCCAACAATTTTTCAAGGAATTGGAAATCAGCGACCAGGAAATGGAGCA ACATCCAATCGCTCCAACCTGTTATCATTACATTTCTCATATTTATAGGCAATTTGCAGAG CCGAACTTAGCCATCGCTTTTGCAAGCTTGCTTCCTTGTCCTTGGTTATACCATGATATAG GCAAATCACTTAATCTTAAACCATCACCAAATCCTCTCTATCAACAATGGATTGAAACTTA TATTACGGATGAGTTAGAGCAACAGATCAGAGAGGAGGGAGCACTGGTCAATCAGCTCTAT CGAGAAAGTGATGAGACAGATAAGCAAAAAATGCTAGATGCCTTCCACATCAGTGTTCATA TGGAAGCCAAGTTTTGGGAGATGGCTTACCAACACCAGACATGGAAGAGCGATTTACAGTC TTTAGAAAAAGGAGAAGAATAG Polynucleotide sequence of S. pneumoniae [SEQ ID NO : 4] Sequence characteristics lenght : 747 bases start codon: 1 stop codon: 745 ATGAAAAGATTGGAACAAATTATTAAATTAGTATCAGAATATGAAAAGATCGACGTTAATA CATTATCGGAAAAATTAAATGTATCGAAAGTAACGATTAGAAAAGATTTAGATAAATTAGA GTCAAAAGGTTTATTACACAGAGAGCATGGATATGCTGTATTAAATAGTGGAGATGACTTA AATGTACGTTTGTCAATTAACTATGAAATTAAGAGAAAAATTGTTCAGGAAGCAGTAAAAT TGGTGTCAGATAATGAAACAATAATGATAGAATCTGGATCGACCTGTGCTTTACTTGCTGA GGAAATTTGCAAGCAAAAAAGAAATGTTACGATTGTAACAAATTCATTTTTTATAGCAAAT TTTGTGAGAGCTTATGATTCATGTCGTATTATTGTTCTTGGTGGAGAGTTTCAGAAGGATT CACAGGTTACTGTGGGACCTTTATTAAAAGAAATGATACAGACTTTTCATGTGCATCAAGC TTTTGTTGGGACAGATGGTTACGATAAAGAGATGGGCTTTACAGGAAAAGATTTAATGCGC AGTGAGGTAGTTCAATATATTTCAGCAGCGTCGGATAAAGTCATTGTACTAACTGACTCAA GTAAATTTGATAAAAGGGGTACAGTAAGAAGATTTGCTTTAAGTCAAGTCTATGAAGTAAT AACAGACGAAAAACTTTCTAAACAAAATATAGCTACATTAGAAAATGCTGGGATAATGGTT AAGGTAGTTTCGTAA FIGURE 2.

S. pneumoniae amino acid sequence [SEQ ID NO : 5] deduced from the polynucleotide sequence of [SEQ ID NO : 1] Sequence characteristics lenght : 1153 amino acids MYIAEIGESIFVLNVGLKYPENEQLGVDWIPNMDYLFENSDRIAGVFLTHGHADAIGALP YLLAEAKVPVFGSELTIELAKLFVKGNDAVKKFNDFHVIDENTEIDFGGTWSFFPTTYSV PESLGIVLKTSEGSIVYTGDFKFDQTASESYATDFARLAEIGRDGVALLSSATFGIMVSNN GLERTKMQDKIVIHGARAHNLKNIDVEIPRDKLVWTGLSGSGKSSLAFDTLYAEGQRRYV ESLSAYARQFLGNMEKPDVDAIDGLSPAISIDQKTTSKNPRSTVGTTTEINDYLRLLYARV GTPYCINGHGAINASSVEQIVDKVLELPERQRLQILAPVIRKKKGQHKSVIEKVQKDGYVR VRVDGEVYDVTEVPELSKSKQHNIDVWDRIVIKEGIRSRLFDSIEAALRIAEGYVIIDTM DDSELLFSEHYACPVCGFTVPELEPRLFSFNAPFGSCSECDGLGIKLEVDTDLWPDASKT LHEGALAPWNPISSNYYPNMLEQAMKVFGVAMDKPFEDLSEEDKNLILYGSDGKEFHFHYE NEFGGVRDIDIPFEGVINNIKRRYHETNSDYTRTQMRLYMNELTCGTCQGYRLNDQALSVR VGGQQGPHIGEISDLSIADHLDLVSQLTLSENEAIIARPILKEIKDRLTFLNNVGLNYLTL SRSAGTLSGGESQRIRLATQIGSNLSGVLYILDEPSIGLHQRDNDRLIASLKKMRDLGNTL IWEHDEDTMREADYLIDVGPGAGVFGGEIVAAGTPKQVARNSKSITGQYLSGKRVIPVPE ERRVGNGRFIEVTGARENNLQNVTARFPLGKFIAVTGVSGSGKSTLINSILKKAIAQKLNR NSDKPGKFKTITGIEHVDRLIDIDQSPIGRTPRSNPATYTGVFDDIRDLFAQTNEAKIRGY KKGRFSFNVKGGRCEACSGDGIIKIEMHFLPDVYVACEVCHGTRYNSETLEVHYKEKNISQ VLDMTVNDAVEFFQHIPKIQRKLQTIKDVGLGYVTLGQPATTLSGGEAQRMKLASELHKRS TGKSFYILDEPTTGLHTEDIARLLKVLARFVDDGNTVLVIEHNLDVIKTADHIIDLGPEGG VGGGTIIVTGTPEEVAANEASYTGHYLKGKLHHEMYIAEIGE S. pneumoniae amino acid sequence [SEQ ID NO : 6] deduced from the polynucleotide sequence of [SEQ ID NO : 2] Sequence characteristics lenght : 737 amino acids MAKQVFQTTFAGRELIVETGQVAKQANGSVVVRYGESTVLTAAVMSKKMATGDFFPLQVNY EEKMYAAGKFPGGFMKREGRPSTDATLTARLIDRPIRPMFAEGFRNEVQVINTVLSYDENA SAPMAAMFGSSLALSISDIPFDGPIAGVQVGYVDGQIIINPSQEQAEQSLLELTVAGTKHA INMVESGAKELSEEIMLEALLKGHEAVKELIAFQEEIVAAVGKEKAEVELLHVDAELQAEI IAAYNSDLQKAVQVEEKLAREAATQWKDQVTAVYEEKYADHEEFDRIMRDVAEILEQMEH AEVRRLITEDKVRPDGRKVDEIRPLDAWDFLPRVHGSGLFTRGQTQALSVLTLAPMGETQ IIDGLDPEYKKRFMHHYNFPQYSVGETGRYGAPGRREIGHGALGERALAQVLPSLEEFPYA IRLVAEVLESNGSSSQASICAGTLALMAGGVPIKAPVAGIAMGLISDGNNYTVLTDIQGLE DHFGDMDFKVAGTRDGITALQMDIKIQGITAEILTEALAQAKKARFEILDVIEATIPEVRL ELAPTAPKIDTIKIDVDKIKIVIGKGGETIDKIIAETGVKIDIDEEGNVSIYSSDQDAINR AKEIIAGLVREAKVDEVYRAKWRIEKFGAFVNLFDKTDALVHISEMAWTRTNRVEDLVEI GDEVDVKVIKIDEKGRIDASMKALLPRPPKPEHDEKGEKSERPHRPRHHKDHKPKKEFTET PKDSE S. pneumoniae amino acid sequence [SEQ ID NO : 7] deduced from the polynucleotide sequence of [SEQ ID NO : 3] Sequence characteristics lenght : 230 amino acids MEFTDIAMELSKKAWQASFHHPFILQLQEGNLEPAIFRYYLIQDAYYLKAFSEIYHLLADK TSNQEMKRLLKQNAQGLVEGELFIRQQFFKELEISDQEMEQHPIAPTCYHYISHIYRQFAE PNLAIAFASLLPCPWLYHDIGKSLNLKPSPNPLYQQWIETYITDELEQQIREEGALVNQLY RESDETDKQKMLDAFHISVHMEAKFWEMAYQHQTWKSDLQSLEKGEE S. pneumoniae amino acid sequence SEQ ID NO : 8] deduced from the polynucleotide sequence of [SEQ ID NO : 4] Sequence charachteristics: lenght : 248 amino acids MKRLEQIIKLVSEYEKIDVNTLSEKLNVSKVTIRKDLDKLESKGLLHREHGYAVLNSGDDL NVRLSINYEIKRKIVQEAVKLVSDNETIMIESGSTCALLAEEICKQKRNVTIVTNSFFIAN FVRAYDSCRIIVLGGEFQKDSQVTVGPLLKEMIQTFHVHQAFVGTDGYDKEMGFTGKDLMR SEWQYISAASDKVIVLTDSSKFDKRGTVRRFALSQVYEVITDEKLSKQNIATLENAGIMV KWS TABLE 1 Provided in this Table is the closest homologue of eache polypeptide encoded by each ORF of the invention based on a comparison of the sequences in Figure 2 with sequences available in the public domain (see column entitled description).

Polynucleotides Polypeptides Description [SEQ ID NO : 1] [SEQ ID NO : 5] Excinuclease ABC subunit A (UvrA) B. subtils [SEQ ID NO : 21 [SEQ ID NO : 6] Polynucleotide phosphorilase (pnp) Y. enterolitica [SEQ ID NO : 3] [SEQ ID NO : 7] Transcriptional regulator (TenA) H. Pylori [SEQ ID NO : 4] [SEQ ID NO : 8] Hypothetical transcriptional repressor E. amylovora

Claims

What is claimed is: 1. An isolated polynucleotide comprising a member selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having at least a 70% identity to the polypeptide comprising amino acids sequences of [SEQ ID Nos 5,6, 7] and/or [SEQ ID NO. 8] (b) a polynucleotide which is complementary to the polynucleotide of (a); and (c) a polynucleotide comprising at least 15 sequential bases of the polynucleotide of (a) or (b).
2. A polynucleotide of Claim 1 having at least 80% identity to a polypeptide comprising amino acids sequences of [SEQ ID Nos 5, 6,7] and/or [SEQ ID NO. 8].
3. A polypeptide of Claim 2 having at least 90% identity to a polypeptide comprising amino acids sequences of [SEQ ID Nos 5,6,7] and/or [SEQ ID NO. 8].
4. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.
5. The polynucleotide of Claim 1 wherein the polynucleotide is RNA.
6. The polynucleotide of Claim 4 wherein the polynucleotide has at least 80% identity to the polynucleotide comprising nucleotide sequences of [SEQ ID Nos 1, 2,3] and/or [SEQ ID NO. 4].
7. The polynucleotide of Claim 6 wherein the polynucleotide has at least 90% identity to the polynucleotide comprising nucleotide sequences of [SEQ ID Nos 1,2,3] and/or [SEQ ID NO. 4].
8. The polynucleotide of Claim 4 comprising nucleotide sequences of [SEQ ID Nos 1, 2,3] and/or [SEQ ID NO. 4].
9. A vector comprising the DNA of Claim 4.
10. A host cell comprising the vector of Claim 9.
11. A process for producing a polypeptide comprising: expressing from the host cell of Claim 10 a polypeptide encoded by said DNA.
12. A process for producing a cell which expresses a polypeptide comprising transforming or transfecting the cell with the vector of Claim 9 such that the cell expresses the polypeptide encoded by the DNA contained in the vector.
13. A polypeptide comprising an amino acid sequence which is at least 70% identical to the polypeptide represented by amino acid of [SEQ ID Nos 5,6,7] and/or [SEQ ID NO. 8].
14. A polypeptide comprising an amino acid sequence which is at least 80% identical to the polypeptide represented by amino acid of [SEQ ID Nos 5,6,7] and/or [SEQ ID NO. 8].
15. A polypeptide comprising an amino acid sequences which is at least 90% identical to the polypeptide represented by amino acid of [SEQ ID Nos 5,6,7] and/or [SEQ ID NO. 8].
16. A polypeptide comprising amino acid sequences of [SEQ ID Nos 5,6, 7] and/or [SEQ ID NO. 8].
17. An antibody against the polypeptide of daim 16.
18. An antagonist which inhibits the activity of the polypeptide of claim 16.
19. A method for the treatment of an individual having need to inhibit the activity of the polypeptide of Claim 16 comprising: administering to the individual a therapeutically effective amount of the antagonist of Claim 18.
20. A complex of a polypeptide and a binding molecule which comprises the polypeptide of Claim 16 and a binding molecule that is capable of antagonising the activity of the polypeptide.
21. A process for diagnosing a disease related to expression of the polypeptide of claim 17 comprising: determining a nucleic acid sequence encoding said polypeptide.
22. A diagnostic process comprising: analyzing for the presence of the polypeptide of claim 16 in a sample derived from a host.
23. A method for identifying compounds which inhibit the activity of the polypeptide of claim 16 comprising: contacting a cell expressing the polypeptide on the surface thereof, with a compound under conditions to permit binding to the polypeptide in the presence of a component capable of providing a detectable signal in response to the binding of the compound to said polypeptide ; and determining whether the compound inhibits the binding by detecting the presence or absence of a signal generated from the interaction of the compound with the binding.
24. A method for inducing an immunological response in a mammal which comprises inoculating the mammal with the polypeptide of Claim 13, or a fragment or variant thereof, adequate to to protect said animal against infection from S. pneumoniae.
25. A method of inducing an immunological response in a mammal which comprises delivering a gene encoding the polypeptide of Claim 14, or a fragment or variant thereof, and obtaining expression of the gene in vivo in order to induce an immunological response to produce antibody to protect said animal against infection from S. Pneumoniae.
26. An immunological composition comprising a DNA capable of expressing a polypeptide of Claim 13 which, when introduced into a mammal, induces an immunological response in the mammal.