AU9510598A - Human complement c3-degrading proteinase from (streptococcus pneumoniae) - Google Patents

Description

WO 99/15675 PCT/US98/20186 1 HUMAN COMPLEMENT C3-DEGRADING PROTEINASE FROM STREPTOCOCCUS PNEUMON4IAE Field of the Invention 5 This invention relates to Streptococcus pneumoniae and in particular this invention relates to the identification of an S. pneumoniae protein that is capable of degrading human complement protein, C3. 10 Background of the Invention Respiratory infection with the bacterium Streptococcus pneumoniae (S. pneumoniae) leads to an estimated 500,000 cases of pneumonia and 47,000 deaths annually. Those persons at highest risk of bacteremic pneumococcal infection are infants under two years of age, individuals with a compromised 15 immune system and the elderly. In these populations, S. pneumoniae is the leading cause of bacterial pneumonia and meningitis. Moreover, S. pneumoniae is the major bacterial cause of ear infections in children of all ages. Both children and the elderly share defects in the synthesis of protective antibodies to pneumococcal capsular polysaccharide after either bacterial colonization, local 20 or systemic infection, or vaccination with purified polysaccharides. S. pneumoniae is the leading cause of invasive bacterial respiratory disease in both adults and children with HIV infection and produces hematogenous infection in these patients (Connor et al. Current Topics in AIDS 1987; 1:185-209 and Janoff et al. Ann. Intern. Med. 1992;117(4):314-324). 25 Individuals who demonstrate the greatest risk for severe infection are not able to make antibodies to the current capsular polysaccharide vaccines. As a result, there are now four conjugate vaccines in clinical trial. Conjugate vaccines consist of pneumococcal capsular polysaccharides coupled to protein carriers or adjuvants in an attempt to boost the antibody response. However, 30 there are other potential problems with conjugate vaccines currently in clinical trials. For example, pneumococcal serotypes that are most prevalent in the United States are different from the serotypes that are most common in places WO 99/15675 PCT/US98/20186 2 such as Israel, Western Europe, South Africa, or Scandinavia. Therefore, vaccines that may be useful in one geographic locale may not be useful in another. The potential need to modify currently available capsular polysaccharide vaccines or to develop protein conjugates for capsular vaccines 5 to suit geographic serotype variability entails prohibitive financial and technical complications. Thus, the search for immunogenic, surface-exposed proteins that are conserved worldwide among a variety of virulent serotypes is of prime importance to the prevention of pneumococcal infection and to the formulation of broadly protective pneumococcal vaccines. Moreover, the emergence of 10 penicillin and cephalosporin-resistant pneumococci on a worldwide basis makes the need for effective vaccines even more exigent (Baquero et al. J. Antimicrob. Chemother. 1991;28S;31-8). Several pneumococcal proteins have been proposed for conjugation to pneumococcal capsular polysaccharide or as single immunogens to stimulate 15 immunity against S. pneumoniae. Surface proteins that are reported to be involved in adhesion of S. pneumoniae to epithelial cells of the respiratory tract include PsaA, PspC/CBPl 12, and IgAl proteinase (Sampson et al. Infect. Immun. 1994;62:319-324, Sheffield et al. Microb. Pathogen. 1992; 13: 261-9, and Wani, et al. Infect. Immun. 1996; 64:3967-3974). Antibodies to these 20 adhesins could inhibit binding of pneumococci to respiratory epithelial cells and thereby reduce colonization. Other cytosolic pneumococcal proteins such as pneumolysin, autolysin, neuraminidase, or hyaluronidase are proposed as vaccine antigens because antibodies could potentially block the toxic effects of these proteins in patients infected with S. pneumoniae. However, these proteins 25 are typically not located on the surface of S. pneumoniae, rather they are secreted or released from the bacterium as the cells lyse and die (Lee et al. Vaccine 1994; 12:875-8 and Berry et al. Infect. Immun. 1994; 62:1101-1108). While use of these cytosolic proteins as immunogens might ameliorate late consequences of S. pneumoniae infection, antibodies to these proteins would neither promote 30 pneumococcal death nor prevent initial or subsequent pneumococcal colonization. A prototypic surface protein that is being tested as a pneumococcal WO 99/15675 PCT/US98/20186 3 vaccine is the pneumococcal surface protein A (PspA). PspA is a heterogeneous protein of about 70-140 kDa. The PspA structure includes an alpha helix at the amino terminus, followed by a proline-rich sequence, and terminates in a series of 11 choline-binding repeats at the carboxy-terminus. Although much 5 information regarding its structure is available, PspA is not structurally conserved among a variety of pneumococcal serotypes, and its function is entirely unknown (Yother et al. J. Bacteriol. 1992; 174:601-9 and Yother J. Bacteriol. 1994;176:2976-2985). Studies have confirmed the immunogenicity of PspA in animals (McDaniel et al. Microb. Pathogen. 1994; 17;323-337). 10 Despite the immunogenicity of PspA, the heterogeneity of PspA, its existence in four structural groups (or clades), and its uncharacterized function complicate its ability to be used as a vaccine antigen. In patients who cannot make protective antibodies to the type-specific polysaccharide capsule, the third component of complement, C3, and the 15 associated proteins of the alternative complement pathway constitute the first line of host defense against S. pneumoniae infection. Because complement proteins cannot penetrate the rigid cell wall of S. pneumoniae, deposition of opsonic C3b on the pneumococcal surface is the principal mediator of pneumococcal clearance. Interactions of pneumococci with plasma C3 are 20 known to occur during pneumococcal bacteremia, when the covalent binding of C3b, the opsonically active fragment of C3, initiates phagocytic recognition and ingestion (Johnston et al. J. Exp. Med 1969;129:1275-1290, Hasin HE, J. Immunol. 1972; 109:26-31 and Hostetter et al. J. Infect. Dis. 1984; 150:653-61). C3b deposits on the pneumococcal capsule, as well as on the cell wall. This 25 method for controlling S. pneumoniae infection is fairly inefficient. Methods for augmenting S. pneumoniae opsonization could improve the disease course induced by this organism. There currently exists a strong need for methods and therapies to limit S. pneumoniae infection. 30 Summary of the Invention This invention relates to the identification and use of a family of human complement C3-degrading proteins (proteinases) expressed by S. pneumoniae.

WO 99/15675 PCT/US98/20186 4 The proteins preferably have a molecular weight of about 15 kD to about 25 kD, as determined, for example, on a 10% SDS polyacrylamide gel. The invention includes a number of proteins isolatable from different C3-degrading strains of S. pneumoniae. 5 In one aspect, the invention relates to an isolated protein having at least 80% sequence identity with SEQ ID NO:2. In a preferred embodiment, the protein is isolated from S. pneumoniae or alternatively the protein is a recombinant protein. Preferably, the isolated protein degrades human complement protein C3. A preferred protein of this invention is an isolated 10 protein having an amino acid sequence that includes SEQ ID NO:2, and more preferably, is SEQ ID NO:2. The term "isolated" as used herein refers to a naturally occurring species that has been removed from its natural environment, as well as to synthetic species. The term "protein" as used herein includes one or more functional units, which encompasses one or more peptides or polypeptides. 15 The invention also relates to isolated peptides or polypeptides from the C3-degrading proteinase of this invention. Preferably, the invention provides peptides or polypeptides of at least 15 sequential amino acids from an isolated protein that has at least 80% sequence identity with SEQ ID NO:2, and more preferably, peptides or polypeptides of at least 15 sequential amino acids of SEQ 20 ID NO:2. In another aspect of this invention, the peptides or polypeptides are capable of degrading human complement protein C3. Preferred embodiments of the invention include an isolated protein comprising amino acids of about 1 to about 58 of SEQ ID NO:2 and an isolated nucleic acid fragment comprising nucleotides of about 1 to about 174 of SEQ ID 25 NO: 1 or its complementary strand. Preferably, the isolated nucleic acid fragment comprises nucleotides of about 150 to about 174 of SEQ ID NO: 1 or its complementary strand. In another aspect, the invention relates to an isolated protein that degrades human complement protein C3, wherein nucleic acid encoding the 30 protein hybridizes to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions.

WO 99/15675 PCT/US98/20186 5 The invention also relates to an immune system stimulating composition (preferably, a vaccine) comprising an effective amount of an immune system stimulating peptide or polypeptide comprising at least 15 sequential amino acids derived from a protein, wherein the protein has at least 80% sequence identity 5 with SEQ ID NO:2 and is capable of degrading human complement protein C3. Preferably the protein is isolated from S. pneumoniae. In one embodiment, the immune system stimulating composition or vaccine further comprises at least one other immune system stimulating peptide, polypeptide or protein isolated from S. pneumoniae. 10 The invention further relates to an antibody capable of binding (typically, specifically binding) to a protein comprising at least 80% sequence identity with SEQ ID NO:2 and capable of degrading human complement protein C3. In one embodiment, the antibody is a monoclonal antibody and in another embodiment, the antibody is a polyclonal antibody. In another embodiment the antibody is an 15 antibody fragment. The antibody or antibody fragments can be obtained from a mouse, a rat, a goat, a chicken, a human, or a rabbit. In another embodiment, the antibody is capable of binding to at least a portion of a protein, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization 20 conditions. The invention also relates to an isolated nucleic acid fragment (polynucleotide) capable of hybridizing to SEQ ID NO:1 or its complimentary strand under highly stringent hybridization conditions. As used herein, highly stringent hybridization conditions include, for example, 6XSSC, 5X Denhardt, 25 0.5% SDS, and 100 pg/ml fragmented and denatured salmon sperm DNA hybridized overnight at 65 0 C and washed in 2X SSC, 0.1% SDS one time at room temperature for about 10 minutes followed by one time at 65 0 C for about 15 minutes followed by at least one wash in 0.2XSSC, 0.1% SDS at room temperature for at least 3-5 minutes. In one embodiment, the nucleic acid 30 fragment is isolated from S. pneumoniae and in another embodiment, the nucleic acid fragment encodes at least a portion of a protein. In one embodiment, the protein degrades human complement protein C3. In another embodiment, the WO 99/15675 PCT/US98/20186 6 nucleic acid fragment encodes a peptide or polypeptide that does not degrade human complement C3. In another embodiment, the nucleic acid fragment is in a nucleic acid vector and the vector can be an expression vector capable of producing at least a 5 portion of a protein. Cells containing the nucleic acid fragment are also contemplated in this invention. In one embodiment, the cell is a bacterium or a eukaryotic cell. The invention further relates to an isolated nucleic acid fragment comprising the nucleic acid sequence of SEQ ID NO: 1, or its complementary 10 strand. The invention further relates to an RNA fragment transcribed by a double-stranded DNA sequence comprising SEQ ID NO: 1. In another aspect of this invention, the invention relates to a method for producing an immune response to S. pneumoniae in a mammal (particularly a human) including the steps of: administering a composition comprising a 15 therapeutically effective amount of at least a portion of a protein to an animal, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO: 1, or its complementary strand, under highly stringent hybridization conditions to produce an immune response to the protein. The immune response can be a B cell response, a T cell response, an epithelial response or an endothelial 20 response. In a preferred embodiment, the composition is a vaccine composition. Preferably the protein is at least 15 amino acids in length and also preferably the composition further comprises at least one other immune system stimulating peptide, polypeptide or protein from S. pneumoniae. In one embodiment, the protein comprises at least 15 amino acids of SEQ ID NO:2. 25 The invention further relates to an isolated protein of about 15 kDa to about 25 kDa from Streptococcus pneumoniae that is capable of degrading human complement C3 and to a method for inhibiting Streptococcus pneumoniae-mediated C3 degradation comprising the step of: contacting a Streptococcus pneumoniae bacterium with antibody capable of binding to a 30 protein with at least 80% amino acid sequence identity to SEQ ID NO:2. The invention also relates to a method for inhibiting C3-mediated inflammation and rejection in xenotransplantation comprising the step of WO 99/15675 PCT/US98/20186 7 expressing on the surface of an organ of an animal used in xenotransplantation a protein with the amino acid sequence of SEQ ID NO:2. This method is particularly advantageous for causing, for example, the kidneys of pigs to express the protein described herein and thereby to inhibit C3 mediated 5 inflammation after xenotransplantation. The invention also relates to an isolated nucleic acid molecule that contains a region of at least 15 nucleotides which hybridize under highly stringent hybridization conditions to at least a portion of a nucleic acid sequence of SEQ ID NO.1 or its complementary strand. In one embodiment, an isolated 10 nucleic acid molecule is capable of hybridizing under highly stringent hybridization conditions to at least one region of SEQ ID NO.1 or its complementary strand. Preferably, the at least one region includes nucleotides 1-174 or 320-492 of SEQ ID NO:1. In yet another embodiment, the invention relates to an isolated nucleic 15 acid molecule that contains a region of at least 15 nucleotides which hybridize under highly stringent hybridization conditions to at least a portion of a nucleic acid sequence of SEQ ID NO.4 or its complementary strand. In one embodiment, an isolated nucleic acid molecule is capable of hybridizing under highly stringent conditions to at least one region of SEQ ID NO.4 or its complementary strand. 20 Preferably, the at least one region includes nucleotides 507-681 or 827-999 of SEQ ID NO:4. In another embodiment, at least a portion of the nucleic acid molecule of SEQ ID NO:4 encodes at least a portion of a protein. Preferably, the protein has a predicted amino acid sequence as shown in SEQ ID NO:5, and has a molecular 25 weight of about 75 kDa to about 85 kDa as determined, for example, by SDS PAGE. The invention also relates to isolated DNA fragments or primers having the nucleic acid sequences as shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 AND SEQ ID NO:9. 30 In another aspect of the invention, the invention relates to an immune system-stimulating composition or vaccine containing a therapeutically effective amount of at least a portion of a protein, wherein nucleic acid encoding the WO 99/15675 PCT/US98/20186 8 protein is capable of hybridizing to the sequence of SEQ ID NO:4, or its complementary strand, under highly stringent hybridization conditions. A method for producing an immune response to S. pneumoniae in a mammal (particularly a human) includes the steps of: administering a composition 5 comprising a therapeutically effective amount of at least a portion of a protein to a mammal, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO:4, or its complementary strand, under stringent hybridization conditions to obtain an immune response to the protein. In yet another embodiment, the invention relates to a vaccine or immune 10 system stimulating composition containing an amount of at least a portion of a protein that is effective to immunize or treat a mammalian subject against S. pneumoniae infection or colonization and a pharmaceutically acceptable carrier. The protein is derived from a nucleic acid molecule that hybridizes under highly stringent conditions to a nucleic acid sequence as shown in SEQ ID NO:4, or its 15 complementary strand, coding for the protein. Preferably, the protein is provided in an amount effective to provide a therapeutic effect to the mammalian subject, especially a human subject. Brief Description of the Figures 20 Figure 1 provides the nucleic acid sequence of the translated portion of a C3-degrading proteinase gene of this invention (SEQ ID NO:1). Figure 2 provides the amino acid sequence of a C3-degrading proteinase of this invention (SEQ ID NO:2). Figure 3 diagrams the amino acid sequence of a C3-degrading proteinase 25 positioned with the nucleic acid sequence encoding a C3-degrading proteinase according to this invention (SEQ ID NOS:2-3). Figure 4 provides the nucleic acid sequence for a predicted 79 kDa amino acid sequence (SEQ ID NO:4). Figure 5 provides the predicted 79 kDa amino acid sequence (SEQ ID 30 NO:5). Figure 6 shows sequence alignments of SEQ ID NO: 1 and SEQ ID NO:4.

WO 99/15675 PCT/US98/20186 9 Figure 7 shows sequence alignment of SEQ ID NO:2 with corresponding amino acids 169-331of SEQ ID NO:5. Detailed Description of the Preferred Embodiments 5 The present invention relates to the identification and isolation of a human complement C3 degrading proteinase with a molecular weight of about 20 kDa (± 5 kDa) on a 10% SDS-PAGE gel and nucleic acid encoding a C3 degrading proteinase. It has been observed that exponentially growing cultures of pneumococci from several serotypes were able to first degrade the 3-chain 10 then degrade the a chain of C3 without producing defined C3 cleavage fragments (Angel, et al. J Infect. Dis. 170:600-608, 1994). This pattern of degradation without cleavage differs substantially from that of other microbial products such as the elastase enzyme of Pseudomonas aeruginosa and the cysteine proteinase of Entamoeba histolytica. 15 The term "degrade" is used herein to refer to the removal of amino acids from proteinaceous molecules, generating peptides or polypeptides. The proteins of this invention degrade C3 without producing specific cleavage fragments as observed on a polyacrylamide gel. There is at least some preference of the C3-degrading proteinases of this invention for C3 in that, for 20 example, the C3-degrading proteinase does not appear to degrade other proteins, such as albumin. A C3-degrading proteinase of about 20 kDa was isolated from a library of insertionally interrupted pneumococcal genes by identifying those clones that had decreased C3 degrading activity as compared to wild type S. pneumoniae. 25 An exemplary assay for assessing C3-degrading activity of clones is provided in Example 1. Clones with decreased C3-degrading activity were identified and a 546 bp SmaI insert was selected, based on the sequence of the clones that had demonstrated decreased C3-degrading activity. This SmaI fragment was used to probe a S. pneumoniae library made from strain CP 1200. Positive clones from 30 the S. pneumnoniae library that hybridized to the SmaI fragment were isolated and the open reading frame of the gene associated with C3-degrading activity was identified. The following oligonucleotide (SEQ ID NO: 10), which has sequence WO 99/15675 PCT/US98/20186 10 identify with a portion of PspA, was used to confirm, by differential hybridization, that the gene encoding the C3-degrading protinease was distinct from the gene encoding PspA. 5 SEQ ID NO:10 GAAAACAATAATGTAGAAGACTACTTTAAAGAAGGTTAGA A complete open reading frame of a 20 kDa protein spans an area of 492 base pairs (SEQ ID NO:1) predicting a protein of molecular weight of about 20 10 kDa (+/- 5 kDa) or about 163 amino acids (SEQ ID NO:2). An exemplary gene sequence encoding a C3-degrading protein is provided in Figure 1 as SEQ ID NO:1 and an amino acid sequence of the protein is provided in Figure 2 as SEQ ID NO:2. Figure 3 combines a preferred gene sequence with a corresponding preferred translated protein as SEQ ID NO:3. 15 Using SEQ ID NO:2, the amino acid sequence of the protein was determined to be unrelated to other proteins in the GenBank or Swiss Prot databases. The predicted protein encompasses a proline-rich sequence characteristic of membrane domains in prokaryotes, particularly between amino acids 80-108 suggesting that the protein is expressed at the surface. The amino 20 acid sequence exhibits no apparent choline-binding repeats. Electrophoresis of pneumococcal lysates and supernatants from cultures of CP 1200 on SDS-PAGE gels impregnated with C3 identified a lytic band at about 20 kDa (± 5 kDa) in both supernatants and lysates, confirming that a protein of a size predicted by SEQ ID NO:2 had C3-degrading activity (see Example 2). As provided in 25 Example 3, the gene encoding the 20 kDa C3-degrading proteinase is conserved in at least two dozen pneumococcal isolates representing five serotypes (serotypes 1, 3, 4, 14, and 19F). The full length gene encoding a C3-degrading proteinase of this invention was inserted into a gene expression vector for expression in E. coli. 30 Recombinant C3-degrading proteinase was isolated as described in the examples. Those of ordinary skill in the art recognize that, given a particular gene sequence such as that provided in SEQ ID NO: 1, there are a variety of WO 99/15675 PCT/US98/20186 11 expression vectors that could be used to express the gene. Further, there are a variety of methods known in the art that could be used to produce and isolate the recombinant protein of this invention and those of ordinary skill in the art also recognize that the C3 degrading assay of this invention will determine whether 5 or not a particular expression system, in addition to those expression systems provided in the examples, is functioning, without requiring undue experimentation. A variety of molecular and immunological techniques can be found in basic technical texts such as those of Sambrook et al. (Molecular Cloning, A Laboratory Manual, 1989 Cold Spring Harbor Laboratory Press, 10 Cold Spring Harbor, NY) and Harlow et al. (Antibodies; A Laboratory Manual. Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press, 1988). The gene encoding the C3 degrading protein of this invention was identified using a plasmid library made with pneumococcal genomic DNA fragments from strain CP 1200. Although there are a variety of methods known 15 for obtaining a plasmid library, in a preferred strategy, a plasmid library was constructed with Sau 3A digested pneumococcal genomic DNA fragments (0.5 -4.0 kb) from pneumococcal strain CP 1200 (obtained from D.A. Morrison, University of Illinois, Champagne-Urbana, Illinois and described in Havarstein LF, et al. Proc. Natl. Acad. Sci. (USA) 1995;92:11140-11144) and inserted into 20 the Bamrn HI site of the integrative shuttle vector pVA 891 (ermr, cmr; has origin of replication for E. coli). This library was transformed into an E. coli DH5a MCR strain by electroporation. Plasmid extractions of some randomly selected E. coli transformants revealed that all of them contained recombinant plasmids. Plasmid library DNA can be extracted from the E. coli transformants and 25 used to transform the CP 1200 parent pneumococcal strain using insertional mutagenesis by homologous recombination. The pneumococcal strain CP 1200 cells can be made competent using a pH shift with HCI procedure in CTM medium. The competent cells are frozen at -70' C in small aliquots until needed. 30 The isolated protein of this invention can be incubated with human complement C3 for 4 hours at 37 0 C in the presence of PBS to detect C3 degradation. Control samples without the isolated pneumococcal protein are WO 99/15675 PCT/US98/20186 12 used as controls for comparative purposes. The protein of this invention has an apparent molecular weight on a 10% SDS-polyacrylamide gel of about 20 kDa (±5 kDa) and preferably has a molecular weight of about 15 kDa to about 25 kDa. An exemplary protein 5 sequence is provided by SEQ ID NO: 2. Those of ordinary skill in the art will recognize that some variability in amino acid sequence is expected and that this variability should not detract from the scope of this invention. For example, conserved mutations do not detract from this invention nor do variations in amino acid sequence identity of less than about 80 % amino acid sequence 10 identity and preferably less than about 90% amino acid sequence identity where the protein is capable of degrading human complement protein C3, and particularly where the protein is isolated or originally obtained from an S. pneumoniae bacterium. Fragments of the protein are also within the scope of the present invention, particularly if they are capable of degrading human 15 complement protein C3. Some nucleic acid sequence variability is expected among pneumococcal strains and serotypes as is some amino acid variability. Conserved amino acid substitutions are known in the art and include, for example, amino acid substitutions using other members from the same class to which the amino acid 20 belongs. For example, the nonpolar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, and tryptophan. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid 25 and glutamic acid. Such alterations are not expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point. Particularly preferred conservative substitutions include, but are not limited to, Lys for Arg and vice verse to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free 30 OH is maintained; and Gln for Asn to maintain a free NH 2 . A preferred protein of this invention includes a protein with the amino acid sequence of SEQ ID NO:2. Other proteins include those degrading human WO 99/15675 PCT/US98/20186 13 complement protein C3 and having nucleic acid encoding the protein that hybridizes to SEQ ID NO: 1 under highly stringent hybridization conditions such as 6XSSC, 5X Denhardt, 0.5% SDS (sodium dodecyl sulfate), and 100 pg/ml fragmented and denatured salmon sperm DNA hybridized overnight at 65 0 C and 5 washed in 2X SSC, 0.1% SDS one time at room temperature for about 10 minutes followed by one time at, 65 0 C for about 15 minutes followed by at least one wash in 0.2XSSC, 0.1% SDS at room temperature for at least 3-5 minutes are also contemplated in this invention. Typically, an SSC solution contains sodium chloride, sodium citrate, and water to prepare a stock solution. Peptides 10 or polypeptides of the protein can also be used. A preferred protein of this invention comprises amino acids of about 1 to about 50 of SEQ ID NO:2. The proteins of this invention can be isolated or prepared as recombinant proteins. That is, nucleic acid encoding the protein, or a portion of the protein, can be incorporated into an expression vector or incorporated into a chromosome 15 of a cell to express the protein in the cell. The protein can be purified from a bacterium or another cell, preferably a eukaryotic cell and more preferably an animal cell. Alternatively, the protein can be isolated from a cell expressing the protein, such as a S. pneumoniae cell. Peptides or polypeptides are also considered in this invention. The peptides or polypeptides are preferably at least 20 15 amino acids in length and preferred peptides or polypeptides have at least 15 sequential amino acids from SEQ ID NO:2. Nucleic acid encoding the 20 kDa protein is also part of this invention. SEQ ID NO: 1 is a preferred nucleic acid fragment encoding a C3-degrading proteinase. Those of ordinary skill in the art will recognize that some 25 substitution will not alter the C3-degrading proteinase sequence to an extent that the character or nature of the C3-degrading proteinase is substantially altered. For example, nucleic acid with an identity of at least 80% to SEQ ID NO: 1 is contemplated in this invention. A method for determining whether a particular nucleic acid sequence falls within the scope of this invention is to consider 30 whether or not a particular nucleic acid sequence encodes a C3-degrading proteinase and has a nucleic acid identity of at least 80% as compared with SEQ ID NO: 1. Other nucleic acid sequences encoding the C3 proteinase include WO 99/15675 PCT/US98/20186 14 nucleic acid encoding the C3 proteinase where the C3 proteinase has the same sequence or at least a 90% sequence identity with SEQ ID NO:2 but which includes degeneracy with respect to the nucleic acid sequence. A degenerate codon means that a different three letter codon is used to specify the same amino 5 acid. For example, it is well known in the art that the following RNA codons (and therefore, the corresponding DNA codons, with a T substituted for a U) can be used interchangeably to code for each specific amino acid: Phenylalanine (Phe or F) UUU, UUC, UUA or UUG Leucine (Leu or L) CUU, CUC, CUA or CUG 10 Isoleucine (Ile or I) AUU, AUC or AUA Methionine (Met or M) AUG Valine (Val or V) GUU, GUC, GUA, GUG Serine (Ser or S) AGU or AGC Proline (Pro or P) CCU, CCC, CCA, CCG 15 Threonine (Thr or T) ACU, ACC, ACA, ACG Alanine (Ala or A) GCU, GCG, GCA, GCC Tryptophan (Trp) UGG Tyrosine (Tyr or Y) UAU or UAC Histidine (His or H) CAU or CAC 20 Glutamine (Gln or Q) CAA or CAG Asparagine (Asn or N) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAU or GAC Glutamic Acid (Glu or E) GAA or GAG 25 Cysteine (Cys or C) UGU or UGC Arginine (Arg or R) AGA or AGG Glycine (Gly or G) GGU or GGC or GGA or GGG Termination codon UAA, UAG or UGA Further, a particular DNA sequence can be modified to employ the 30 codons preferred for a particular cell type. For example, the preferred codon usage for E. coli is known, as are preferred codons for animals including humans. These changes are known to those of ordinary skill in the art and WO 99/15675 PCT/US98/20186 15 therefore these gene sequences are considered part of this invention. Other nucleic acid sequences include nucleic acid fragments of at least 15, and preferably, at least 30 nucleic acids in length from SEQ ID NO: 1 or other nucleic acid fragments of at least 15, and preferably at least 30 nucleic acids in length 5 where these fragments hybridize to SEQ ID NO: 1 under highly stringent hybridization conditions such as 6XSSC, 5X Denhardt, 0.5% SDS, and 100 pg/ml fragmented and denatured salmon sperm DNA hybridized overnight at 65 0 C and washed in 2X SSC, 0.1% SDS one time at room temperature for about 10 minutes followed by one time at, 65 0 C for about 15 minutes followed by at 10 least one wash in 0.2XSSC, 0.1% SDS at room temperature for at least 3-5 minutes. The nucleic acid fragments of this invention can encode all, none (i.e., fragments that cannot be transcribed, fragments that include regulatory portions of the gene, or the like) or a portion of SEQ ID NO:2 or SEQ ID NO:5 and 15 preferably containing a contiguous nucleic acid fragment that encodes at least nine amino acids from SEQ ID NO:2 or SEQ ID NO:5. Because nucleic acid fragments encoding a portion of the C3 proteinase are contemplated in this invention, it will be understood that not all of the nucleic acid fragments will encode a protein or peptide or polypeptide with C3 degrading activity. Further, 20 the nucleic acid of this invention can be mutated to remove or otherwise inactivate the C3 degrading activity of this protein. Therefore, fragments without C3 degrading activity that meet the hybridization requirements described above are also contemplated. Methods for mutating or otherwise altering nucleic acid sequences are well described in the art and the production of an 25 immunogenic, but enzymatically inactive protein can be tested for therapeutic utility. The nucleic acid fragments of this invention can be incorporated into nucleic acid vectors or stably incorporated into host genomes to produce recombinant protein including recombinant chimeric protein. In one 30 embodiment, the C3-degrading protein is encoded by a gene in a vector and the vector is in a cell. Preferably, the cell is a prokaryotic cell such as a bacterium. The genes and gene fragments can exist as the fusion of all or a portion of the WO 99/15675 PCT/US98/20186 16 gene with another gene and the C3-degrading protein can exist as a fusion protein of one or more proteins where the fusion protein is expressed as a single protein. A variety of nucleic acid vectors of this invention are known in the art and include a number of commercially available expression plasmids or viral 5 vectors. The use of these vectors is well within the scope of what is ordinary skill in the art. Exemplary vectors are employed in the examples, but should not be construed as limiting on the scope of this invention. This invention also relates to antibodies capable of binding (typically specifically binding) to a protein of about 20 kDa, and preferably a protein of 10 about 15 kDa to about 25 kDa, from S. pneumoniae and preferably where the protein is capable of degrading human complement C3. Polyclonal antibody can be prepared to a portion of the protein or to all of the protein. Similarly, monoclonal antibodies can be prepared to all or to a peptide or polypeptide (fragment) of the about 20 kDa C3 degrading protein of this invention. Methods 15 for preparing antibodies to protein are well known and well described, for example, by Harlow et al., (Antibodies; A Laboratory Manual. Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press, 1988). In a preferred example, the antibodies can be human derived, rat derived, mouse derived, goat derived, chicken derived, or rabbit derived. Protein-binding antibody fragments 20 and chimeric fragments are also known and are within the scope of this invention. The invention also relates to the use of immune stimulating compositions. The term "immune stimulating" or "immune system stimulating" composition refers to protein, peptide or polypeptide compositions according to 25 the invention that activate at least one cell type of the immune system in a subject, such as a mammal. Preferably, the immune stimulating composition provides an immunizing response or prophylactic benefit in a normal, i.e., uninfected subject, typically a vaccine. However, any measurable immune response is beneficial to the subject in a therapy application or protocol. 30 Preferred activated cells of the immune system include phagocytic cells such as neutrophils or macrophages, T cells, B cells, epithelial cells and endothelial cells. Immune stimulating compositions comprising the peptides, polypeptides WO 99/15675 PCT/US98/20186 17 or proteins of the invention can be used to produce antibody in an animal such as a rat, mouse, goat, chicken, rabbit, or a human or an animal model for studying S. pneumoniae infection. Preferred immune stimulating compositions include an immune stimulating amount, e.g, a therapeutically effective amount, of at least 5 one peptide or polypeptide including at least 15 amino acids from the C3 degrading proteinase. The term "vaccine" refers to a composition for immunization. This process can include the administration of a protein, peptide, polypeptide, antigen, nucleic acid sequence or complementary sequence, e.g., anti-sense, or 10 antibody, or suspensions thereof, wherein upon administration, the molecule will produce active immunity and provide protection against an S. pneumoniae infection or colonization. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The vaccine 15 preparation may optionally be emulsified, or encapsulated in liposomes. The immune stimulating composition (such as a vaccine) can further include other proteins in a pharmaceutically acceptable buffer or carrier, such as PBS (phosphate buffer saline) or another buffer recognized in the art as suitable and safe for introduction of proteins into a host to stimulate the immune system. 20 The immune stimulating compositions can also include other immune system stimulating proteins such as adjuvants or immune stimulating proteins, peptides or polypeptides from S. pneumoniae or other organisms. For example, a cocktail of peptides or polypeptides may be most useful for controlling S. pneumoniae infection. Preferably one or more fragments of the proteins of this invention are 25 used in a vaccine preparation to protect against or limit S. pneumoniae colonization or the pathogenic consequences of S. pneumoniae colonization. By a "therapeutically effective amount," as used herein, refers to that amount that is effective for production of a desired result. This amount varies depending upon the health and physical condition of a subject's immune system, 30 i.e., to synthesize antibodies, the degree of protection desired, the formulation prepared and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

WO 99/15675 PCT/US98/20186 18 The active immune stimulating ingredients are often mixed with excipients or diluents that are pharmaceutically acceptable as carriers and compatible with the active ingredient. The term "pharmaceutically acceptable carrier" refers to a carrier(s) that is "acceptable" in the sense of being compatible 5 with the other ingredients of a composition and not deleterious to the recipient thereof. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the immune stimulating composition (including vaccine) may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, 10 and/or adjuvants which enhance the effectiveness of the immune stimulating compostion. Examples of adjuvants or carriers that may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 15 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in a 2% 20 squalene/Tween 80 emulsion. This invention also relates to a method for inhibiting Streptococcus pneumoniae-mediated C3 degradation comprising contacting a Streptococcus pneumonie bacterium with a protein, such as an antibody or another protein that is capable of binding to an isolated protein of about 15 kDa to about 25 kDa 25 from Streptococcuspneumoniae. The protein capable of binding to an isolated protein of about 15 kDa to about 25 kDa can be an antibody or a fragment thereof or the protein can be a chimeric protein that includes the antibody binding domain, such as a variable domain, from antibody that is capable of specifically recognizing an isolated protein of about 15 kDa to about 25 kDa 30 from Streptococcuspneumoniae having C3 degrading activity. The isolated S. pneumoniae protein of this invention can be isolated, and optionally purified, and the isolated protein or immunogenic fragments thereof WO 99/15675 PCT/US98/20186 19 can be used to produce an immunologic response, including, in one example, an antibody response in a human or an experimental animal. Peptide or polypeptide fragments of the protein without C3 degrading ability can be tested for their ability to limit the effects of S. pneumoniae infection. Similarly, the protein of 5 this invention can be modified, such as through mutation to interrupt or inactivate the C3 degrading activity of the protein. Antibody capable of inhibiting the C3-degrading activity of the protein of this invention may be used as a strategy for preventing C3 degradation and for promoting clearance of S. pneumoniae through the opsonic pathway. Isolated protein can be used in assays 10 to detect antibody to S. pneumoniae or as part of a vaccine or a multi-valent or multiple protein, peptide or polypeptide-containing vaccine for S. pneumoniae therapy. Thus, the term "treatment," as used herein, refers to prophylaxis and/or therapy of either normal mammalian subjects or mammalian subjects colonized 15 with, diagnosed with, or exhibiting characteristics or symptoms of various S. pneumoniae infections. The term "therapy" refers to providing a therapeutic effect to a mammalian subject such that the subject exhibits few or no symptoms of a pneumococcal infection or other related disease. Such treatment can be accomplished by administration of nucleic acid molecules (sense or antisense), 20 proteins, peptides or polypeptides or antibodies of the instant invention. It is further contemplated that the proteins of this invention can be surface expressed on vertebrate cells and used to degrade C3, for example, where complement deposition (or activation) becomes a problem, such as in xenotransplantation or in complement-mediated glomerulonephritis. For 25 example, the entire pneumococcal protein, a recombinant protein, or a portion of either, can be incorporated into xenotransplant cells and expressed as a surface protein or as a secreted protein to prevent or minimize complement deposition (and/or complement-mediated inflammation). Another specific aspect of the present invention relates to using a vaccine 30 vector expressing an isolated protein and peptides or polypeptides therefrom. Accordingly, in a further aspect this invention provides a method of inducing an immune response in a mammal, which comprises providing to a mammal a WO 99/15675 PCT/US98/20186 20 vaccine vector expressing at least one, or a mixture of an isolated protein and/or peptide or polypeptide of the invention. The protein and peptides or polypeptides of the present invention can be delivered to the mammal using a live vaccine vector, in particular using live recombinant bacteria, viruses or other 5 live agents, containing the genetic material necessary for the expression of the protein and/or peptides or polypeptides as a foreign polypeptide. Particularly, bacteria that colonizes the gastrointestinal tract, such as Salmonella, Shigella, Yersinia, Vibrio, Escherichia and BCG have been developed as vaccine vectors, and these and other examples are discuessed by J. Holmgren et al., Immunobiol., 10 184, 157-179 (1992) and J. McGhee et al., Vaccine, 10, 75-88 (1992). An additional embodiment of the present invention relates to a method of inducing an immune response in a subject, e.g,. mammal, comprising administering to the subject an amount of a DNA molecule encoding an isolated protein and/or peptide or polypeptide therefrom of this invention, optionally with 15 a transfection-facilitating agent, where the protein and/or peptides or polypeptides retain immunogenicity and, when incorporated into an immune stimulating composition, e.g, vaccine, and administered to a human, provides protection without inducing enhanced disease upon subsequent infection of the human with S. pneumoniae pathogen. Transfection-facilitating agents are known 20 in the art. It is further contemplated that the antisense sequence of the gene encoding the 20kDa protein may be used as a vaccine or as a therapeutic treatment for pneumococcal infection. Antisense DNA is defined as a non coding sequence that is complementary, i.e., a complementary strand, to all or a 25 portion of SEQ ID NO: 1. For example, the antisense sequence for 5' ATGTCAAGC-3' is 3'-TACAGTTCG-5'. Delivery of antisense sequence or oligonuclotides into an animal may result in the production of antibody by the animal or in the incorporation of the sequence into living bacteria or other cells whereby transcription and/or translation of all or a portion of the 20 kDa gene 30 product is inhibited. Introduction of an antisense nucleic acid sequence can be accomplished, for example, by loading the antisense nucleic acid into a suitable carrier, such as WO 99/15675 PCT/US98/20186 21 a liposome, for introduction into pneumococci or infected cells. Typically, an antisense nucleic acid sequence having eight or more nucleotides is capable of binding to the bacterial nucleic acid or bacterial messenger RNA. The antisense nucleic acid sequence, typically contains at least about 15 nucleotides, preferably 5 at least about 30 nucleotides or more nucleotides to provide necessary stability of a hybridization product of bacterial nucleic acid or bacterial messenger RNA. Introduction of the sequences preferably inhibit the transcription or translation of at least one endongenous S. pneumoniae nucleic acid sequence. Methods for loading antisense nucleic acid is known in the art as exemplified by U.S. Patent 10 4,242,046. The present invention also provides nucleic acid having an open reading frame of 2163 bases (SEQ ID NO:4) that encompasses the open reading frame of a nucleic acid sequence (SEQ ID NO: 1) that encodes a protein that has a molecular weight of about 20 kDa (SEQ ID NO:2). The 20 kDa protein, 15 described herein, is further characterized as a C3-degrading protein. The larger open reading frame, e.g., 2163 bp (SEQ ID NO:4), encodes for a putative protein of about 79 kDa (SEQ ID NO:5). All references and publications cited herein are expressly incorporated by reference into this disclosure. There are a variety of alternative techniques and 20 procedures available to those of skill in the art which would similarly permit one to successfully perform the intended invention in view of the present disclosure. It will be appreciated by those skilled in the art that while the invention has been described above in connection with particular embodiments and examples, the invention is not necessarily so limited and that numerous other embodiments, 25 examples, uses, modifications and departures from the embodiments, examples and uses may be made without departing from the inventive scope of this application.

WO 99/15675 PCT/US98/20186 22 Example 1 Identification of Insertional Mutants with Reduced C3-Degrading Activity 5 Insertional mutants were received from Dr. Elaine Tuomanen, (Rockefeller Inst., New York, New York). The clones with insertions were tested in an assay to detect reduced C3-degrading activity. 137 clones were tested by growing the cells in Todd Hewitt broth overnight at room temperature in microtitre plates. The cells were diluted 1:10 in synthetic medium for 10 pneumococci (see Sicard A. M., Genetics 50:31-44, 1984) and the remainder of the cells were frozen in the microtiter plate. Either 63 ng or 83 ng of C3 per 100 [l of medium containing 1 mg/ml of 0.1% BSA in phosphate buffered saline (PBS) was added to about 200 pl of diluted cells. The cells were incubated at 37 0 C for 4 hrs. One hundred pl of the mixture was added to ELISA plates and 15 incubated overnight at 4 0 C. The plates were washed three times with wash buffer and the wells were filled with 0.05% Tween 20 in PBS with five minute incubations between the washes. One hundred il of antibody to C3 (polyclonal horse-radish peroxidase-conjugated goat antibody specific to human C3-IgG fraction, ICN Cappel, Costa Mesa, CA) was diluted 1:1200 with 3% BSA in 20 PBS. The ELISA plate was incubated at 37 0 C for about 30 minutes to 1 hr in the dark and washed with wash buffer as above. The assay was developed using 12 mg of OPD in 30 ml of 0.1M sodium citrate buffer with 12 gl of 30% hydrogen peroxide. Assay results were determined by optical density readings at 490 nm on an ELISA plate reader. 25 Each clone was tested four times. Nineteen clones were selected that had less than 40% C3 degradation as compared to nonmutated controls. These 19 clones were screened 6 times by the assay described above and from these results 6 clones were selected with less than 30% C3-degrading activity as compared to controls. These 6 clones were screened eleven times each and the 30 two clones with the lowest C3-degrading activity were selected for further study. A partial sequence of one of the clones was received and a SmaI fragment of 546bp was labeled with 32 P by random primer labeling (kit available WO 99/15675 PCT/US98/20186 23 from Stratagene, La Jolla, CA). The 546bp Sinal fragment from SEQ ID NO: 1 was hybridized to EcoR1 and KpnI digests of numerous pneumococcal strains on Southern blots. This same fragment was also used to screen a library of Sau3A fragments of genomic DNA from S. pneumoniae strain CP1200. 5 A 3.5 kb insert was identified from the CPl200 library. The insert was sequenced and an open reading frame of 492 base pairs, including the stop codon, was identified. The open reading frame coded for a protein of 163 amino acids and a predicted molecular weight of about 18,500 daltons. PCR primers were constructed to amplify the open reading frame; the 5' 10 PCR primer incorporated a BamHI site; the 3' primer incorporated a PstI site. The amplified insert was ligated in frame to a His-Tagged E. coli expression vector pQE30 (Qiagen, San Diego, CA). The resulting plasmid was used to transform E. coli strain BL21 (Novagen, Madison, WI) containing the lac repressor plasmid pREP4 (Qiagen). E. coli cultures were induced to express the 15 His-Tagged protein and the protein was column purified with Ni-NTA resin (Qiagen). The purified protein was confirmed by SDS-PAGE gel. Example 2 Identification of a 20kDa C3-Degrading Proteinase 20 To determine the C3-degrading capability of the 20 kDa protein, 0.5 mg/ml of C3 (prepared according to Tack et al., Meth. Enzymol. 80:64-101, 1984) was copolymerized in a sodium dodecyl sulfate (SDS) gel-containing 15% acrylamide (15% SDS-PAGE gel). Pneumococcal supernatants were obtained from cultures of S. pneumoniae strain CP 1200 grown to exponential phase in 25 Todd Hewitt broth; pneumococcal lysates were obtained by incubating 5 x 108 cells with 5% SDS for 30 minutes at room temperature. The lysate was concentrated 10 fold using a Centricon filtration device with a 10,000 mw cutoff (Amicon, Beverly, MA). The samples were not heated before electrophoresis. Samples of supernatants and lysates were added to the 15% C3-containing SDS 30 PAGE gels and electrophoresis was carried out at 4oC at 150 V until the dye front ran out. The gel was washed successively with 50 ml of 2.5% Triton X 100 in water (2 times, 10 minutes), 2.5 % Triton X-100 in 50 mM Tris-HC1, pH WO 99/15675 PCT/US98/20186 24 7.4 (2 times, 10 minutes), and 50 mm Tris-HC1, pH 7.4 (2 times, 10 minutes) to remove SDS. After washes, 50 ml of 50 mM Tris-HC1, pH 7.4, was poured into dishes containing the gels, and the dishes were covered and incubated at 37 0 C for 1.5 hour and overnight (about 16 hours). The gels were stained with 5 Coomassie blue for 10 minutes and destained totally. Two lytic bands were visualized, one of which was about 20 kDa in size, against the dark blue background in both lysates and supernatant. C3 proteinase activities in the pneumococcal lysates were observed after a 1.5 hour incubation at 37'C, while C3 proteinase activities in the Pn supernatant were observed after 10 an overnight incubation. Therefore, C3 proteinase activities appeared to be mainly cell associated. Example 3 The gene encoding the 20 kD protein is conserved in a number of S. 15 pneumoniae strains. DNA was obtained from a variety of S. pneumoniae strains (Clinical isolates of Type 1, Type 3, LOO2 and LOO3 (type 3), Type 4, Type 14 and Laboratory isolates CP1200, WU2, R6X, 6303,109,110, JY1119, JY1 82, and JY53) and SEQ ID NO:3 was used as a probe to detect the presence of nucleic 20 acid encoding the 20 kD protein in DNA from these strains. Isolated chromosomal DNA was digested with EcoRI and separated by electrophoresis. The DNA was transferred to a solid support and hybridized to end-labeled SEQ ID NO:3 under the hybridization and washing conditions of 6X SSC, 5X Denhart's, 0.5% SDS, 100 tg/ml denatured fragmented salmon sperm DNA 25 hybridized at 65 oC overnight and washed in 2X SSC, 1 time at room temperature for 10 minutes and in 2X SSC, 0.1% SDS 1 time at 65 0 C for 15 minutes followed by two washes in 0.2X SSC, 0.1% SDS for 3 minutes each at room temperature. Results indicated that SEQ ID NO:3 hybridized identically in each of the 30 DNA samples tested indicating that the protein appears to be conserved among strains. In some strains, the DNA encoding the 20 kDa C3-degrading protein WO 99/15675 PCT/US98/20186 25 appears to be part of a larger open reading frame of 2163 bp that putatively encodes a 79 kDa protein. Example 4 5 Southern blot of S. pneumoniae DNA/5F1 probe Five ug samples of genomic DNA were obtained from 11 strains ofS. pneumoniae. Each sample was digested with the restriction enzyme Kpnl. The samples were subsequently loaded onto an agarose gel and resolved by electrophoresis. The samples contained in the gel were subsequently transferred 10 to a Hybond-N+ membrane available from Amersham (Upsalla, Sweden) by capillary transfer. A 540bp SmaI fragment from an 5F1 isolate was random prime labeled with P 32 using a T 7 QuickPrime kit (Pharmacia, Piscathaway, NJ) and purified from non-incorporated nucleotides using NucTrap column (Stragene, La Jolla, CA) and hybridized. 15 The hybridization conditions were 6XSSC, 5X Denhardt, 0.5% SDS, and 100 pg/ml fragmented and denatured salmon sperm DNA hybridized overnight at 65 0 C and washed in 2X SSC, 0.1% SDS once at room temperature for about 10 minutes followed by 1 time at, 65 0 C for about 15 minutes followed by at least one wash in 0.2XSSC, 0.1% SDS at room temperature for at least 3-5 minutes. 20 The blot demonstrated that the 20kDa gene was present in all tested strains of S. pneumoniae.

WO 99/15675 PCT/US98/20186 26 Example 5 Two DNA primers were prepared from SEQ ID NO: 1 and utilized to amplify the 20Kda gene sequence from S. pneumoniae (serotype 3) genomic DNA. The first primer, a 5'-primer, SEQ ID NO:6, spans the ATG start codon 5 of the 20Kda gene, inserts a Ncol site, and had an Ala residue inserted after the ATG start codon to maintain a correct reading frame. The second primer, a 3' primer, SEQ ID NO:7, spans the termination codon of the 20 Kda gene and inserts a BamH 1 site. 10 5'-GGGGG CCA TGG CC TCA AGC CTT TTA CGT GAA TTG-3'; (SEQ ID NO:6) 5'-GGGGG GGA TCC CTA GCT ATA TGA GAT AAA CTT TCC TGC T-3'; (SEQ ID NO:7) 15 The two primers were synthesized on an Applied Biosystems 380A DNA synthesizer (Foster City, CA) using reagents purchased from Glen Research (Sterling, VA). Amplifications were performed utilizing a Perkin Elmer Thermocycler (ABI) according to the manufacturer's directions. The identified 20 PCR product was ligated into the TA tailed PCR cloning vector PCR2.1, available from Invitrogen, Carlsbad, CA, and used to transform OneShot Top 1 OF' competent cells (Invitrogen). Kanamycin resistant transformants were screened by restriction enzyme analysis of plasmid DNA prepared by alkaline lysis. An approximately 500bp insert fragment was identified and subsequently 25 excised with restriction enzymes Ncol and BamH1. The 500 bp fragment was purified from a low melting agarose gel, and subsequently ligated into the Neol BamH1 sites of the T7 promoted expression vector pET 28a, available from Novagen ( Madison, WI). The ligation mixture was subsequently transformed into Top 10F'cells 30 (Invitrogen), and the kanamycin resistant transformants were screened by restriction enzyme analysis of plasmid DNA prepared by alkaline lysis. A recombinant plasmid (pLP505) was subsequently tranformed into BL21 (Novagen) cells and grown in SOB media supplemented with 30ug/ml kanamycin. Cells were grown to an O.D.

0 0 of 0.6, and were subsequently WO 99/15675 PCT/US98/20186 27 induced with 0.4mM IPTG (Boehringer Mannheim, Indianapolis, Indiana) for 2 4 hours. Whole cell lysates were prepared and electrophoresed on a 14% SDS PAGE gel. The gel was stained with Coomassie and the expression product was detected. The coomassie stained gel revealed a band between the 28 kDa and the 5 18 kDa molecular weight markers, and was determined to be approximately 20 kDa. The DNA sequence of the insert in the recombinant pLP505 plasmid was obtained using the ABI 370A DNA sequencer. The DNA sequence was aligned with the DNA sequence of SEQ ID NO: 1, using the Pustell DNA matrix plot 10 feature of MacVector (Oxford Molecular Group, Campbell, CA). Alignment of the DNA sequence obtained from the pLP505 plasmid, SEQ ID NO:1, and the S. pneumoniae (serotype 4) genome, revealed that the open reading frame (ORF) that codes for the 20Kd protein may be part of a larger ORF, i.e., a 2163 bp in the serotype 4 genome, that codes for a protein with a predicted MW of 15 approximately 79 kDa (SEQ ID NO: 4). DNA SEQ ID NO:4 encodes for a predicted amino acid sequence as shown in SEQ ID NO:5. The S. pneumoniae (serotype 4) genome sequence was obtained from The Institute for Genomic Research at www.tigr.org and/or through NCBI at www.ncbi.nlm.nih.gov, using the ClustalW feature of MacVector, (Oxford 20 Molecular Group, Campbell CA). A sequence comparison was made between the 20 kDa amino acid sequence (SEQ ID NO:2) and the predicted 79 kDa amino acid sequence (SEQ ID NO:5). It was observed that amino acids 1-58 and amino acids 90-132 of SEQ ID NO:2 have substantial sequence identity with amino acids 170-227 and amino acids 258-300 of SEQ ID NO:5, respectively. 25 Proteins and peptides or polypeptides, containing these particular regions are preferred embodiments of the invention. Based upon the available genomic DNA (serotype 4) sequence, two primers flanking the 2163bp ORF were designed and subsequently synthesized using the ABI 380A DNA synthesizer (SEQ ID NOS:8 and 9). SEQ ID NO:8 30 was an S. pneumoniae 5'-primer having an inserted Ncol site and a "Glu" residue added after the ATG start codon to maintain a correct reading frame. SEQ ID NO:9, was an S. pneumoniae 3'-primer having an inserted HindIII site.

WO 99/15675 PCT/US98/20186 28 5'-AGA GCT CCT CCC ATG GAA GAT CCG AAT TAT CAG-3'; (SEQ ID NO:8) 5 5'-CCG GGC AAG CTT TTA CTT ACT CTC CT-3'-; (SEQ ID NO:9) An approximately 2100 bp DNA fragment was then amplified from the 4 different S. pneumoniae serotypes (serotype 3, 5, 6B and 7) resulting in 4 fragments. Each of the 4 fragments were subsequently ligated into the PCR 10 cloning vector PCR2.1 (Invitrogen), and used to transform OneShot Top 1 OF' cells (Invitrogen). Kanamycin resistant transformants were screened by restriction analysis of the plasmid DNA prepared by alkaline lysis. A recombinant plasmid containing the serotype 7 PCR product was identified, e.g., pLP512. The DNA sequence was obtained from the serotype 7 clone using the 15 ABI model 370A DNA sequencer. The DNA sequence was essentially identical to SEQ ID NO:4 and encoded a predicted amino acid sequence essentially identical to SEQ ID NO:5. It will be appreciated by those skilled in the art that while the invention has been described above in connection with particular embodiments and 20 examples, the invention is not necessarily so limited and that numerous other embodiments, examples, uses, modifications and departures from the embodiments, examples and uses may be made without departing from the inventive scope of this application.

Claims (61)

1. An isolated protein having at least 80% sequence identity with SEQ ID NO:2 which is capable of degrading human complement protein C3. 5

2. The protein of claim 1 which is isolated from S. pneumoniae.

3. The protein of claim 1 which is a recombinant protein. 10

4. The protein of claim 1 having a molecular weight of about 15 kDa to about 25 kDa. 15

5. An isolated peptide or polypeptide comprising at least 15 sequential amino acids from the protein of claim 1.

6. An isolated peptide or polypeptide comprising at least 15 sequential amino acids of SEQ ID NO:2. 20

7. An isolated protein comprising SEQ ID NO:2.

8. The isolated protein of claim 7 which has a molecular weight of about 15 kDa to about 25 kDa. 25

9. The protein of claim 8 which is isolated from S. pneumoniae.

10. The protein of claim 8 which degrades human complement protein C3. 30

11. The protein of claim 7 which is SEQ ID NO:2.

12. An isolated protein comprising amino acids from about 1 to about 58 of SEQ ID NO:2. WO 99/15675 PCT/US98/20186 30

13. The isolated protein of claim 12 further comprising amino acids from about 90 to about 132 of SEQ ID NO:2.

14. An isolated protein comprising amino acids from about 170 to about 227 5 of SEQ ID NO:5.

15. The isolated protein of claim 14 further comprising amino acids from about 258 to about 300 of SEQ ID NO:5. 10

16. An isolated protein that degrades human complement protein C3, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions.

17. An isolated protein of about 15 kDa to about 25 kDa from S. pneumoniae 15 that is capable of degrading human complement C3.

18. An immune system stimulating composition comprising an effective amount of an immune system stimulating peptide or polypeptide comprising at least 15 sequential amino acids derived from a protein, 20 wherein the protein has at least 80% sequence identity with SEQ ID NO:2 and is capable of degrading human complement protein C3.

19. The immune system stimulating composition of claim 18 wherein the protein is isolated from S. pneumoniae. 25

20. The immune system stimulating composition of claim 19 further comprising at least one other immune system stimulating protein, peptide or polypeptide isolated from S. pneumoniae. 30

21. An immune system stimulating composition comprising a therapeutically effective amount of at least a portion of a protein, wherein nucleic acid WO 99/15675 PCT/US98/20186 31 encoding the protein hybridizes to SEQ ID NO:1 or its complementary strand under highly stringent hybridization conditions.

22. An immune system stimulating composition comprising an effective 5 amount of at least a portion of the protein of claim 17 effective to immunize or treat a mammalian subject against S. pneumoniae infection or colonization, and a pharmaceutically acceptable carrier.

23. The immune system stimulating composition of claim 22 wherein the 10 protein is provided in an amount effective to provide a therapeutic effect to the mammalian subject.

24. An antibody capable of binding to a protein comprising at least 80% sequence identity with SEQ ID NO:2 and capable of degrading human 15 complement protein C3.

25. The antibody of claim 24 which is a monoclonal antibody.

26. The antibody of claim 24 which is obtained from a mouse, a rat, a goat, a 20 chicken, a human, or a rabbit.

27. An antibody capable of binding to at least a portion of a protein, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions. 25

28. An isolated nucleic acid fragment capable of hybridizing to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions. 30

29. The nucleic acid fragment of claim 28 isolated from S. pneumoniae. WO 99/15675 PCT/US98/20186 32

30. The nucleic acid fragment of claim 28 wherein the nucleic acid fragment encodes at least a portion of a protein.

31. The nucleic acid fragment of claim 30 wherein the protein degrades 5 human complement C3.

32. The nucleic acid fragment of claim 28 in a nucleic acid vector.

33. The nucleic acid fragment of claim 32 wherein the vector is an 10 expression vector capable of producing at least a portion of a protein.

34. A cell comprising the nucleic acid of claim 28.

35. The cell of claim 34 wherein the cell is a bacterium or a eukaryotic cell. 15

36. An isolated nucleic acid fragment comprising nucleotides of about 1 to about 174 of SEQ ID NO: 1 or its complementary strand.

37. The isolated nucleic acid fragment of claim 36 further comprising 20 nucleotides of about 320 to about 492 of SEQ ID NO: 1 or its complementary strand.

38. An isolated nucleic acid fragment comprising the nucleic acid sequence of SEQ ID NO: 1 or its complementary strand. 25

39. An RNA fragment transcribed by a double-stranded DNA sequence comprising SEQ ID NO: 1 or its complementary strand.

40. A method for producing an immune response to S. pneumoniae in a 30 mammal comprising the steps of: administering a composition comprising a therapeutically effective amount of at least a portion of a protein to a mammal, WO 99/15675 PCT/US98/20186 33 wherein nucleic acid encoding the protein hybridizes to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions in a pharmaceutically acceptable carrier to yield an immune response. 5

41. The method of claim 40 wherein the immune response is a B cell response, a T cell response, an epithelial cell response, or an endothelial cell response. 10

42. The method of claim 40 wherein the at least a portion of a protein is at least 15 amino acids in length.

43. The method of claim 40 wherein the composition further comprises at least one other immune system stimulating protein, peptide or 15 polypeptide from S. pneumoniae.

44. The method of claim 40 wherein the at least a portion of a protein comprises at least 15 amino acids of SEQ ID NO:2. 20

45. A method for inhibiting S. pneumoniae-mediated C3 degradation comprising the step of: contacting a S. pneumoniae bacterium with an antibody capable of binding to a protein with the amino acid sequence of SEQ ID NO:2 or a fragment thereof. 25

46. A method for inhibiting C3-mediated inflammation and rejection in xenotransplantation comprising the step of: expressing on the surface of an organ of an animal used in xenotransplantation a protein with the amino acid sequence of SEQ ID 30 NO:2 or a fragment thereof.

47. An isolated nucleic acid molecule comprising a region of at least 15 WO 99/15675 PCT/US98/20186 34 nucleotides which hybridize under highly stringent hybridization conditions to at least a portion of a nucleic acid sequence as shown in SEQ ID NO:1, or its complementary strand. 5

48. An isolated nucleic acid molecule comprising a sequence that hybridizes under highly stringent hybridization conditions to at least one region of SEQ ID NO: 1 or its complementary strand wherein the region is selected from the group consisting of nucleotides 1-174 and 320-492. 10

49. An isolated nucleic acid molecule comprising a region of at least 15 nucleotides which hybridize under highly stringent hybridization conditions to at least a portion of a nucleic acid sequence as shown in SEQ ID NO:4, or its complementary strand. 15

50. An isolated nucleic acid molecule comprising a sequence that hybridizes under highly stringent hybridization conditions to at least one region of SEQ ID NO:4 or its complementary strand, wherein the region is selected from the group consisting of nucleotides 507-681 and 827-999. 20

51. The nucleic acid molecule of claim 49 which encodes at least a portion of a protein.

52. The nucleic acid molecule of claim 51 wherein the protein has a predicted amino acid sequence of SEQ ID NO:5. 25

53. An isolated nucleic acid fragment having a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, AND SEQ ID NO:9. 30

54. An immune system stimulating composition comprising a therapeutically effective amount of at least a portion of a protein, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO:4 or its complementary WO 99/15675 PCT/US98/20186 35 strand under highly stringent hybridization conditions.

55. A method for producing an immune response to S. pneumoniae in a mammal comprising the steps of: 5 administering a composition comprising a therapeutically effective amount of at least a portion of a protein to a mammal, wherein nucleic acid encoding the protein hybridizes to SEQ ID NO:4, or its complementary strand under highly stringent hybridization conditions in a pharmaceutically acceptable carrier 10 to yield an immune response.

56. An immune system stimulating composition comprising an effective amount of at least a portion of the protein of claim 51 effective to immunize or treat a mammalian subject against S. pneumoniae infection 15 or colonization, and a pharmaceutically acceptable carrier.

57. The immune system stimulating composition of claim 56 wherein the protein is provided in an amount effective to provide a therapeutic effect to the mammalian subject. 20

58. The immune system stimulating composition of claim 56 wherein the composition is a vaccine.

59. A polypeptide having SEQ ID NO:5. 25

60. The immune system stimulating composition of claim 23 where the protein encoded by the nucleic acid sequence or its complementary strand inhibits the transcription or translation of at least one endogenous S. pneumoniae nucleic acid sequence. 30

61. The immune system stimulating composition of claim 56 where the protein encoded by the nucleic acid sequence or its complementary WO 99/15675 PCT/US98/20186 36 strand inhibits the transcription or translation of at least one endogenous S. pneumoniae nucleic acid sequence.