JP2001517449A - Human complement C3 degrading proteinase from Streptococcus pneumoniae - Google Patents

Abstract

(57) [Summary] The present invention relates to The present invention relates to the identification and use of a family of human complement C3 degrading proteinases expressed by Pneumonia. This proteinase has a molecular weight of about 15 kD to about 25 kD. A preferred proteinase of the present invention comprises the amino acid sequence of SEQ ID NO: 2.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Field of the Invention The present invention is Streptococcus pneumoniae (Streptococcus
pneumoniae), and is particularly capable of degrading human complement protein C3. Relevant for the identification of Pneumonia protein. BACKGROUND OF THE INVENTION Respiratory infections caused by Streptococcus pneumoniae cause an estimated 500,000 cases of pneumonia and 47,000 deaths annually. Those at high risk for bacteriopneumococcal infection are infants younger than 2 years, individuals with compromised immune systems, and the elderly. In such populations, Pneumonia causes bacteremia pneumonia and meningitis. Further, S.I. Pneumonia is the major bacterial source of ear infection in children of all ages. Both children and the elderly have common defects in the synthesis of protective antibodies against pneumococcal enveloped polysaccharides after bacterial colonies, local or systemic infection, or vaccination with purified polysaccharides. S. Pneumonia causes invasive bacterial respiratory illness in both adults and children with HIV infection and has resulted in hematogenous infections 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).

[0002] Individuals who show the greatest risk of serious infection are unable to raise antibodies against current polysaccharide vaccines. As a result, four conjugate vaccines are currently in clinical trials. Conjugate vaccines consist of a pneumococcal enveloped polysaccharide coupled to a protein carrier or adjuvant to enhance the antibody response. By the way, conjugate vaccines currently in clinical trials have another potential problem. For example, the most prevalent pneumococcal serotypes in the United States are different from the most common serotypes in regions such as Israel, Western Europe, South Africa or Scandinavia. Thus, a vaccine that may be useful in one geographic location may not be useful elsewhere. The need to improve current encapsulated polysaccharide vaccines to accommodate geographic serotype variation or to develop protein conjugates for encapsulated vaccines has prohibitive financial and technical problems. Thus, the search for immunogenic surface-exposed proteins that are conserved worldwide across a variety of toxic serotypes is one of the most important for the prevention of pneumococcal infections and the formulation of a widespread pneumococcal vaccine. One. Furthermore, the emergence of penicillin and cephalosporin resistant pneumococci on a global basis further exacerbates the need for effective vaccines (Baquero et al., J. Antimicrob. Chemother. 1991; 28S; 31-8).

Some pneumococcal proteins are conjugated to pneumococcal enveloped polysaccharides or to S. pneumoniae. It has been proposed as a sole immunogen to stimulate immunity against Pneumonia. S. PsaA, PspC / CBP112 and IgA include surface proteins that have been reported to be involved in adhesion to epithelial cells of the respiratory tract of Pneumonia.
1 proteinase (Sampson et al., Infect. Immun. 1994; 62: 319-3).
24, Sheffield et al., Microb. Pathogen. 1992; 13: 261-9; and Wani et al., Infe.
ct. Immun. 1996; 64: 3967-3974). Antibodies to such adhesion molecules can inhibit the binding of pneumococci to respiratory epithelial cells and thus suppress their colonization. Other cytosolic pneumococcal proteins, such as pneumolysin, autolysin, pneuminidase or hyaluronidase, have been proposed as vaccine antigens, since the antibody is the S.p. This is because it can potentially block toxic effects in patients infected with Pneumonia. However, these proteins are generally Rather than being on the surface of Pneumonia, it is rather secreted or released when the cells lyse and die from bacteria (Lee et al.
Vaccine 1994; 12: 875-8 and Berry et al., Infect. Immun. 1994; 62: 1101-
1108). The use of such cytosolic proteins as immunogens is described in Antibodies to such proteins do not promote pneumococcal death, nor do they prevent primary or secondary colonization of pneumococci, although they may reduce the late consequences of pneumonia infection.

The prototype surface protein being tested as a pneumococcal vaccine is pneumococcal surface protein A (PspA). PspA is a heterogeneous protein of about 70-140 kDa. The structure of PspA contains 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. There is considerable information about the structure, but Ps
pA is not structurally conserved among the various pneumococcal serotypes, and its function is not entirely known (Yother et al., J. Bacteriol. 1992; 174: 601-9 and Yo).
ther J. Bacteriol. 1994; 176: 2976-2985). Studies have confirmed the immunogenicity of PspA in animals (McDaniel et al., Microb. Pathogen. 1994; 17; 323-
337). Despite the immunogenicity of PspA, the heterogeneity of PspA, its presence in four structural groups (or clades), and its undetermined function complicate its ability to be used as a vaccine antigen.

[0005] In patients unable to produce protective antibodies against type-specific polysaccharide encapsulation, the third component of complement, C3, and another complement pathway-related protein, are expressed in S. aureus. Form the first line of host defense against Pneumonia infection. The complement protein is S. Deposition of opsonin C3b on the pneumococcal surface is a major mediator of pneumococcal clearance because it cannot penetrate the strong cell walls of Pneumonia. The interaction of pneumococci with plasma C3 has been shown to occur during pneumococcalemia when the covalent bond of C3b, an opsonic active fragment of C3, initiates phagocytosis recognition and uptake (Johnson et al., J. . Exp. Me
d 1969; 129: 1275-1290; Hasin HE, J. Immunol. 1972; 109: 26-31 and
Hostetter et al., J. Infect. Dis. 1984; 150: 653-61). C3b is deposited on the pneumococcal envelope and cell wall. S. This method for controlling Pneumonia infection is quite inefficient. S. Methods for enhancing pneumonia opsonization may improve the disease pathways induced by this organism. S. There is currently a strong need for methods and treatments that limit pneumonia infection.

[0006] The present invention is S. Related to the identification of human complement C3 degrading proteins (proteinases) expressed by Pneumonia and the use of their families. These proteins are approximately 15 kD, for example, as determined on a 10% SDS polyacrylamide gel.
It preferably has a molecular weight of 約 25 kD. The present invention relates to S.I. Includes several proteins that can be isolated from various C3 degradants of Pneumonia.

[0007] In one aspect, the invention pertains to an isolated protein having at least 80% sequence identity to SEQ ID NO: 2. In a preferred embodiment, the protein is S. aureus. It is isolated from Pneumonia or the protein is a recombinant protein. Preferably, the isolated protein degrades human complement protein C3. A preferred protein of the invention is SEQ ID N
An isolated protein having an amino acid sequence comprising O: 2, and more preferably SEQ ID NO: 2. As used herein, the term "isolated" refers to natural and synthetic materials removed from the natural environment. As used herein, the term "protein" includes one or more functional units, including one or more peptides or polypeptides.

[0008] The present invention further relates to an isolated peptide or polypeptide derived from the C3 degrading proteinase of the present invention. Preferably, the invention relates to at least 15 derived from an isolated protein having at least 80% sequence identity to SEQ ID NO: 2.
Peptides or polypeptides of contiguous amino acids, and more preferably SEQ.
Provided is a peptide or polypeptide of at least 15 contiguous amino acids of ID NO: 2. In another aspect of the invention, the peptide or polypeptide is capable of degrading human complement protein C3.

[0009] A preferred embodiment of the present invention is an isolated protein comprising from about 1 to about 58 amino acids of SEQ ID NO: 2 and from about 1 to about 174 nucleotides of SEQ ID NO: 1 or a complement thereof. Or an isolated nucleic acid fragment comprising
Preferably, the isolated nucleic acid fragment comprises about 1 of SEQ ID NO: 1.
50 to about 174 nucleotides or the complement thereof.

[0010] In another aspect, the invention relates to an isolated protein that degrades human complement protein C3, wherein the nucleic acid encoding the protein is SEQ ID N
O: hybridizes under high stringency hybridization conditions with 1 or its complementary strand.

The invention further 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 contiguous amino acids from a protein, Where the protein is SEQ ID
NO: 2 with at least 80% sequence identity and human complement protein C
3 can be decomposed.

[0012] Preferably, the protein is S. aureus. It is isolated from Pneumonia.
In one embodiment, the immune system stimulating composition or vaccine further comprises S. aureus. Comprising at least one other immune system stimulating peptide, polypeptide or protein isolated from Pneumonia.

[0013] The invention further comprises an antibody comprising at least 80% sequence identity to SEQ ID NO: 2 and capable of binding (typically specifically binding) a protein capable of degrading human complement protein C3. About. In one embodiment, the antibody is a monoclonal antibody, and in another, the antibody is a polyclonal antibody. In another embodiment, the antibody is an antibody fragment. The antibody or antibody fragment can be obtained from a mouse, rat, goat, chicken, human or rabbit.

[0014] In another embodiment, the antibody is capable of binding to at least a portion of a protein,
Here, the nucleic acid encoding the protein can hybridize to SEQ ID NO: 2 or its complement under highly stringent hybridization conditions.

[0015] The present invention further relates to isolated nucleic acid fragments (polynucleotides) that are capable of hybridizing under highly stringent hybridization conditions to SEQ ID NO: 1 or its complement. Highly stringent hybridization conditions as used herein include, for example, 6 × SSC, 5 × Den
harddt, hybridization at 65 ° C. overnight with 100 μg / ml fragmented and denatured salmon sperm DNA in 0.5% SDS, and 2 × SSC, 0
. One wash in 1% SDS at room temperature for about 10 minutes, then at 65 ° C. for about 1 minute.
One wash over 5 minutes and one wash in 0.2X SSC, 0.1% SDS at room temperature for at least 3-5 minutes. In one embodiment,
This nucleic acid fragment is Isolated from Pneumonia, and in another embodiment, the nucleic acid fragment encodes at least a portion of a protein. In one embodiment, the protein is the human complement protein C3
Decompose. In another embodiment, the nucleic acid fragment encodes a peptide or polypeptide that does not degrade human complement C3.

[0016] In another embodiment, the nucleic acid is in a nucleic acid vector, and the vector can be an expression vector capable of producing at least a portion of a protein. Cells containing the nucleic acid fragment are also contemplated in the present invention. In one aspect, the cell is a bacterial or eukaryotic cell.

[0017] The invention further relates to an isolated nucleic acid fragment comprising the nucleic acid sequence of SEQ ID NO: 1, or the complement thereof. The present invention further provides SEQ ID NO: 1.
And a RNA fragment transcribed by a double-stranded DNA sequence comprising

In another aspect of the present invention, the invention further relates to mammalian (particularly human) S. aureus. Related to a method for generating an immune response against Pneumonia, the method comprising: administering to a animal a composition comprising a therapeutically effective amount of at least a portion of a protein to generate an immune response against the protein Wherein the nucleic acid encoding the protein hybridizes to SEQ ID NO: 1 or its complement under highly stringent hybridization conditions. The immune response can be a B cell response, a T cell response, an epithelial cell response, or an endothelial cell response. In a preferred embodiment, the composition is a vaccine composition. Preferably, the protein is at least 15 amino acids in length, and more preferably, the composition is S. aureus. It comprises at least one other immune system stimulating peptide, polypeptide or protein derived from Pneumonia. In one embodiment, the protein comprises at least 15 amino acids of SEQ ID NO: 2.

The present invention further relates to an isolated protein of about 15 kDa to about 25 kDa from Streptococcus pneumoniae capable of degrading human complement C3, as well as methods of inhibiting Streptococcus pneumoniae-mediated C3 degradation. This method uses S
Contacting Streptococcus pneumoniae with an antibody capable of binding to a protein having at least 80% amino acid sequence identity to EQ ID NO: 2.

The present invention further relates to a method for inhibiting C3-mediated inflammation and rejection in a xenograft, the method comprising the steps of providing a SEQ ID ID on the surface of the organ of the animal used for the xenograft.
Expressing a protein having the amino acid sequence of NO: 2. This method is highly advantageous for, for example, expressing the proteins described herein in pig kidneys and thus inhibiting C3-mediated inflammation after xenograft.

The present invention further comprises an isolated region comprising at least 15 nucleotides that hybridizes under high stringency hybridization conditions to at least a portion of the nucleic acid sequence of SEQ ID NO: 1 or its complement. Related to nucleic acid molecules. In one embodiment, the isolated nucleic acid molecule is capable of hybridizing under high stringency hybridization conditions to at least one region of SEQ ID NO: 1 or its complement. Preferably, the at least one region includes nucleotides 1-174 or 320-492 of SEQ ID NO: 1.

In yet another aspect, the invention relates to a region of at least 15 nucleotides that hybridizes under highly stringent hybridization conditions to at least a portion of the nucleic acid sequence of SEQ ID NO: 4, or a complement thereof. Or an isolated nucleic acid molecule comprising: In one embodiment, the isolated nucleic acid molecule is SEQ.
It can hybridize to at least one region of ID NO: 4 or its complementary strand under highly stringent hybridization conditions. Preferably, the at least one region includes nucleotides 507-681 or 827-999 of SEQ ID NO: 4.

In another aspect, 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 is SEQ.
Having the deduced amino acid sequence shown in ID NO: 5, and for example, SDS-PAGE
Has a molecular weight of about 75 kDa to about 85 kDa, as determined by

The present invention further provides SEQ ID NO: 6, SEQ ID NO: 7, SEQ I
An isolated D having the nucleic acid sequence set forth in D NO: 8 and SEQ ID NO: 9
Related to NA fragments or primers.

In another aspect of the present invention, the present invention relates to an immune system stimulating composition or vaccine comprising at least a portion of a therapeutically effective amount of a protein, wherein the nucleic acid encoding the protein is SEQ. It is capable of hybridizing to ID NO: 4 or its complementary strand under highly stringent hybridization conditions. Mammalian (particularly human) S. A method for generating an immune response to Pneumonia comprises: administering to a mammal a composition comprising a therapeutically effective amount of at least a portion of a protein to obtain an immune response to the protein, And the nucleic acid encoding the protein is SEQ ID NO: 4
Alternatively, it hybridizes to its complementary strand under stringent hybridization conditions.

In yet another aspect, the present invention provides a method comprising: The present invention relates to a vaccine or immune system stimulating composition comprising at least a portion of a protein in an amount effective to immunize or treat a mammalian subject against a Pneumonia infection or colony, together with a pharmaceutically acceptable carrier. This protein hybridizes to the nucleic acid sequence shown in SEQ ID NO: 4 or its complementary strand under highly stringent conditions, and is derived from a nucleic acid molecule encoding the protein. Preferably, the protein is present in an amount effective to provide a therapeutic effect on a mammalian subject, especially a human subject.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention identifies and isolates human complement C3 degrading proteinase having a molecular weight of about 20 kDa (± 5 kDa) on a 10% SDS-PAGE gel, and encodes the C3 degrading proteinase. Related nucleic acids. It has been observed that exponentially growing cultures of pneumococci from several serotypes can first degrade the C3 β chain and then degrade the C3 α-chain without providing constant C3 cleavage fragments (Angel J. I
nfect. Dis. 170: 600-608, 1994). This cleavage pattern of the cuts may indicate other microbial products, such as Pseudomonas
aeruginosa) and cysteine proteinases of Entamoeba histolytica.

As used herein, the term “degradation” refers to the removal of amino acids from a proteinaceous molecule to produce a peptide or polypeptide. The protein of the present invention has a C-type without specific cleavage fragments as observed on polyacrylamide gels.
Disassemble 3 There is at least some preference for the C3 degrading proteinase of the present invention over C3, for example, it does not appear to degrade other proteins such as albumin.

[0029] Wild-type S. By identifying clones with reduced C3 degrading activity as compared to Pneumonia, an approximately 20 kDa C3 degrading proteinase was isolated from the intactly disrupted pneumococcal gene library. A typical assay for evaluating the C3 degrading activity of clones is introduced in Example 1. A clone with reduced C3 degrading activity was identified, and a 546 bp SmaI insert was selected based on the sequence of the clone showing reduced C3 degrading activity. This SmaI fragment was transferred to CP1
S. made from 200 strains Used to probe the Pneumonia library. S. hybridis hybridizing to this SmaI fragment. Positive clones from the Pneumonia library were isolated and the open reading frame of the gene involved in C3 degradation activity was identified. The following oligonucleotides (SEQ ID NO: 10) having sequence identity with a part of PspA were subjected to differential hybridization to confirm that the gene encoding C3 degradation proteinase was different from the gene encoding PspA. Using.

Embedded image

The complete open reading frame of a 20 kDa protein is 492 base pairs (
It extends over the region of SEQ ID NO: 1) and is estimated to have a molecular weight of about 20 kDa (± 5 kDa) or a protein of about 163 amino acids (SEQ ID NO: 2). C
A typical gene sequence encoding a three-degraded protein is shown in FIG.
: 1 and the amino acid sequence of this protein is shown in FIG.
NO: Serve as 2. FIG. 3 combines the preferred gene sequence and the corresponding preferred translated protein as SEQ ID NO: 3.

Using SEQ ID NO: 2, the amino acid sequence of the protein is GenB
Ank or the Swiss Prot database was determined to be independent of other proteins. The putative protein contains a proline-rich sequence characteristic of the membrane domain in prokaryotes, specifically amino acids 80-108, suggesting that the protein is expressed at the surface. This amino acid sequence shows no apparent choline binding repeat. Pneumococcal lysate and CP1
Electrophoresis of C3-impregnated SDS-PAGE gels of supernatants from 200 cultures identified a lysis band of approximately 20 kDa (± 5 kDa) in both supernatants and lysates, with SEQ ID NO: : 2 confirmed that the protein of the putative size had C3 degrading activity (see Example 2). As introduced in Example 3,
The gene encoding the 20 kDa C3 degrading proteinase is conserved in at least two dozen pneumococcal isolates exhibiting five serotypes (serotypes 1,3,4,1).
4 and 19F).

The full-length gene encoding the C3-degrading proteinase of the present invention is Coli (E.c
oli) was inserted into a gene expression vector for expression within. Recombinant C3 degrading proteinase was isolated as described in the examples. One skilled in the art will appreciate that for a particular gene sequence, such as that provided in SEQ ID NO: 1, there are a variety of expression vectors available to express this gene. In addition, various methods available for producing and isolating the recombinant proteins of the present invention are known, and those skilled in the art will recognize that the C3 degradation assays of the present invention may be useful for specific expression systems other than the expression systems described in the Examples. It will also be appreciated that the system can be determined to function without undue experimentation. Various molecular and immunological techniques are fundamental technical literature, such as Sambrook et al. (Molecular Cloning, A Laboratory Manual, 1989 Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY) and Harlow et al. (Antibodi
es; A Laboratory Manual. Cold Spring Harbor, NY; Cold Spring Harbor La
boratory Press, 1988).

The gene encoding the C3-degrading protein of the present invention was identified using a plasmid library prepared from a pneumococcal genomic DNA fragment derived from the CP1200 strain. Although various methods are known for obtaining plasmid libraries, in a preferred strategy, the plasmid library is obtained from the pneumococcal strain CP120.
Sau3A digested pneumococcal genomic DNA fragment (0.5-4.
0kb) (DA Morrison, University of Illinois, Champagne-Urbana, Illin
obtained from ois, and Havarstein LF. et al., Proc. Natl. Acad. Sci. (USA) 199
5; 92: 11140-11144) and integrated shuttle vector pV
Insert into A891 (erm ^r , cm ^r ; with E. coli origin of replication) at the BamHI site. This library was transformed into E. coli by electroporation. Coli DH5
Transformed into α MCR strain. Some randomly selected E. coli Plasmid extracts of E. coli transformants showed that all contained the recombinant plasmid.

The plasmid library is E. coli. E. coli transformants and can be used to transform the CP1200 parental pneumococcal strain using insertional mutagenesis by homologous recombination.

Pneumococcal strain CP1200 cells can be made competent using a pH shift procedure with HCl in CTM medium. The competent cells are aliquoted and frozen at -70 ° C until needed.

The isolated protein of the invention can be assayed for C3 degradation by incubation with human complement C3 at 37 ° C. for 4 hours in the presence of PBS. A control sample without the isolated pneumococcal protein was used as a control for comparative purposes.

The protein of the present invention has an apparent molecular weight on a SDS-polyacrylamide gel of about 20 kDa (± 5 kDa) and is preferably about 15 kDa to about 25 kD.
a. A typical protein sequence is introduced in SEQ ID NO: 2. One skilled in the art will recognize that some variation in the amino acid sequence is expected, and that this variation does not depart from the scope of the invention. For example, a conservative mutation would not depart from the present invention and if the protein was a human complement protein C3
If the protein is capable of degrading S. Variations in amino acid sequence identity of less than about 80%, and preferably less than about 90%, of amino acid sequence identity, if isolated or derived from Pseudomonas pneumoniae, do not depart from the invention. Absent. Protein fragments also fall within the scope of the invention, especially if they are able to degrade human complement protein C3.

Some nucleic acid sequence variation, as well as some amino acid variation, are expected between pneumococcal strains and serotypes. Conservative amino acid substitutions are known in the art, and include, for example, amino acid substitutions utilizing another member from the same class to which the amino acid belongs. For example, non-polar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine and tryptophan. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such changes are not expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point. Particularly suitable conservative substitutions include, without limitation, Arg with Lys to maintain a positive charge and vice versa; Glu to maintain a negative charge.
Asp and vice versa by; Thr by Ser for maintaining the free OH; substitution of Asn by Gln for maintaining and free NH ₂ and the like.

Suitable proteins of the present invention include those having the amino acid sequence of SEQ ID NO: 2. Other proteins degrade human complement protein C3 and the nucleic acids encoding that protein are highly stringent under hybridization conditions to SEQ ID NO: 1, such as 6X SSC, 5X
Denhardt, overnight hybridization at 65 ° C. with 100 μg / ml disrupted and denatured salmon sperm DNA in 0.5% SDS, followed by 2 × S
SC, one wash in 0.1% SDS at room temperature for about 10 minutes, then 65
1 wash at ℃ C for about 15 minutes and 0.2X SSC, 0.1% SDS
And one washing at room temperature for at least 3 to 5 minutes, which is considered in the present invention. Typically, the SSC solution contains sodium chloride, sodium citrate and water for the preparation of a stock solution.
Peptides or polypeptides of the protein can also be used. Preferred proteins of the invention comprise about 1 to about 50 amino acids of SEQ ID NO: 2.

[0040] The proteins of the present invention may be isolated or prepared as recombinant proteins. That is, the nucleic acid encoding the protein or a part thereof can be integrated into an expression vector or into a chromosome of a cell to express the protein in the cell. The protein can be purified from bacteria or another cell, preferably a eukaryotic cell, and more preferably from an animal cell. On the other hand, this protein can be expressed in cells expressing the protein, such as S. aureus. It can be isolated from Pneumonia cells. Peptides or polypeptides are also contemplated in the present invention. The peptide or polypeptide is preferably at least 15 amino acids in length, and suitable peptides or polypeptides are at least 15 amino acids in SEQ ID NO: 2.
Has contiguous amino acids.

[0041] Nucleic acids encoding a 20 kDa protein are also part of the present invention. SEQ ID
NO: 1 is a preferred nucleic acid fragment encoding a C3 degrading proteinase. One skilled in the art will recognize that some substitutions will not alter the C3 degrading proteinase sequence to the extent that the characteristics or properties of the C3 degrading proteinase are substantially altered. For example, SEQ ID NO: 1 and at least 8
Nucleic acids with 0% identity belong to the scope of the present invention. A method for determining that a particular nucleic acid sequence falls within the scope of the invention is to provide a method wherein the particular nucleic acid sequence encodes a C3 degrading proteinase and has at least 80% nucleic acid identity compared to SEQ ID NO: 1. Whether or not to consider. Other nucleic acid sequences encoding a C3 proteinase include those having the same sequence as SEQ ID NO: 2 or having at least 90% sequence identity but having degeneracy in relation to the nucleic acid sequence. Is included. Degenerate codons mean that separate three letter codons are used to identify the same amino acid. For example, the following R
It is well known that NA codons (and therefore the corresponding DNA codons where U is replaced by T) can be used interchangeably to code for each particular amino acid.

Phenylalanine (Phe or F) UUU, UUC, UUA or UUG Leucine (Leu or L) CUU, CUC, CUA or CUG Isoleucine (Ile or I) AAU, AUC or AUA Methionine (Met or M) AUG Valin (Val) Or V) GUU, GUC, GUA, GUG Serine (Ser or S) AGU or AGC Proline (Pro or P) CCU, CCC, CCA, CCG 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 Glutamine (Gln or Q) CAA or CAG Asparagine (Asn or Asn) 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 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 stop codon UAA, UAG or UGA

In addition, certain DNA sequences may be modified using codons that are suitable for a particular cell type. For example, E. It is known that the preferred codon usage for E. coli is the preferred codon for animals, including humans. Such alterations are known to those skilled in the art, and thus these gene sequences are considered as part of the present invention. Other nucleic acid sequences may be at least 15, and preferably at least 30 nucleic acids in length from SEQ ID NO: 1, or hybridization conditions highly stringent to SEQ ID NO: 1, such as 6X SSC, 5X Denhardt
, Overnight hybridization at 65 ° C with 100 µg / ml disrupted and denatured salmon sperm DNA in 0.5% SDS, followed by about 10 minutes in 2X SSC, 0.1% SDS at room temperature. One wash over a minute, then one wash at 65 ° C. for about 15 minutes, and at least three to five times at room temperature in 0.2 × SSC, 0.1% SDS. Other nucleic acid fragments of at least 15 and preferably at least 30 nucleic acids that hybridize under the conditions of washing.

[0044] The nucleic acid fragment of the present invention may be SEQ ID NO: 2 or SEQ ID NO.
: 5, may encode the entirety of the gene, may not encode at all (ie, a fragment that is not transcribed, a fragment containing the regulatory region of a gene, etc.), or may encode a portion thereof, and preferably SEQ ID NO: 2 or SEQ ID NO : Contiguous nucleic acid fragments encoding at least 9 amino acids from 5 Because nucleic acid fragments encoding a portion of the C3 proteinase are considered in the present invention,
It will be appreciated that the entire nucleic acid fragment does not encode a protein or peptide or polypeptide having C3 degrading activity. Further, the nucleic acids of the invention may be mutated to eliminate or inactivate the C3 degrading activity of the protein. Accordingly, fragments without C3 degrading activity that meet the above hybridization conditions are also considered. Methods for mutating or otherwise altering nucleic acid sequences have been published in the art, and the production of immunogenic but enzymatically inactive proteins can be tested for therapeutic use.

The nucleic acid fragments of the present invention can be integrated into the nucleic acid genome or stably integrated into the host genome to produce recombinant proteins, including recombinant chimeric proteins. In one 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. Such genes and gene fragments may exist as fusions of the entire gene or a portion thereof with another gene, and the C3 degrading protein may exist as a fusion protein of one or more proteins, in which case The fusion protein is expressed as a single protein. A variety of nucleic acid vectors of the invention are known in the art and include a number of commercially available expression plasmids or viral vectors. Use of such vectors is within the knowledge of the art. Although exemplary vectors are used in this example, they do not limit the scope of the invention.

The present invention further relates to Approximately 2 which is derived from Pneumonia and can degrade human complement C3
It relates to an antibody that can bind (typically, specifically bind) a protein of 0 kDa, and preferably from about 15 kDa to about 25 kDa. Polyclonal antibodies can be prepared for part or all of the protein. Similarly, monoclonal antibodies can be prepared against the entire 20 kDa C3 degrading protein of the present invention, or against a peptide or polypeptide (fragment) thereof. Methods for preparing antibodies to proteins are well known and are described, for example, in Harlow et al. (Antibo
dies; A Laboratory Manual. Cold Spring Harbor, NY; Cold Spring Harbor
Laboratory Press, 1988). In a preferred example, the antibodies may be from human, rat, mouse, goat, chicken or rabbit. Protein binding antibody fragments and chimeric fragments are also known and fall within the scope of the present invention.

The present invention further relates to the use of an immunostimulatory composition. By "immunostimulation" or "immune system stimulating" composition is meant a protein, peptide or polypeptide according to the present invention that activates at least one cell type of a subject, for example a mammalian immune system. Preferably, the immunostimulatory composition provides an immune response or prophylactic utility to normal, ie, non-infected, subjects, and is typically a vaccine.
However, any measurable immune response in a therapeutic application or protocol is beneficial to the subject. Suitable activating cells of the immune system include phagocytic cells such as neutrophils or macrophages, T cells, B cells, epithelial cells and endothelial cells. An immunostimulatory composition comprising a peptide, polypeptide or protein of the present invention can be used to produce antibodies in animals, such as rats, mice, goats, chickens, rabbits or humans, or for S. aureus. It can be used to produce animal models to study Pneumonia infection. Suitable immunostimulatory compositions comprise an immunostimulatory amount, for example a therapeutically effective amount, of at least one peptide or polypeptide comprising at least 15 amino acids derived from C3 degrading proteinase.

The term vaccine refers to a composition for immunization. The method may comprise a protein, peptide, polypeptide, antigen, nucleic acid sequence or complementary sequence, such as antisense,
Or the administration of an antibody, or a suspension thereof, such that the molecule provides active immunity, and Provides protection against Pneumonia infection or settlement. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution or suspension in liquid prior to injection may also be prepared. The vaccine preparation may optionally be emulsified or encapsulated in liposomes.

The immunostimulatory composition (eg, a vaccine) may further transfer other proteins to a pharmaceutically acceptable buffer or carrier, such as PBS (phosphate buffered saline) or a host to stimulate the immune system. It may be included among other buffers recognized in the art as being suitable and safe for introduction. The immune stimulatory composition may contain other immune system stimulating proteins, such as adjuvants or S. aureus It may include immunostimulatory proteins, peptides or polypeptides from Pneumonia or other organisms. For example, a cocktail of peptides or polypeptides is It may be most useful for controlling Pneumonia infection. Preferably, one of the proteins of the invention
Or the use of multiple fragments in a vaccine preparation, A settlement of S. pneumonia or S. It is possible to prevent or suppress the pathological result of the pneumonia community.

As used herein, “therapeutically effective amount” means an amount effective to produce the desired result. This amount will vary depending on the health and physical conditions of the subject's immune system, ie, the synthesis of the antibody, the degree of protection desired, the formulation and other relevant factors. The amount can be expected to be of a relatively wide range and can be determined through routine testing.

The active immunostimulatory ingredient is often mixed with excipients or diluents which are pharmaceutically acceptable as carriers and are compatible with the active ingredient. "Pharmaceutically acceptable carrier"
By means a carrier that is "acceptable" in that it is compatible with the other ingredients of the composition and is not deleterious to its receptors. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, and combinations thereof. Furthermore,
If desired, the immunostimulatory composition (such as a vaccine) may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants that increase the effectiveness of the immunostimulatory composition.

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 (referred to as CGP 11637, nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine- 2- (1'-2
'-Dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, designated MTP-PE) and RIBI (three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and Cell wall skeleton (MPL + TDM + CWS) is reduced to 2% squalene / Tw
een 80 emulsion).

The present invention further relates to a method for inhibiting Streptococcus pneumoniae-mediated C3 degradation, which method comprises converting a Streptococcus pneumoniae protein into a protein, such as from about 15 kDa to about 25 kDa isolated protein from S. pneumoniae. Contacting with an antibody or other protein capable of binding to the protein. The protein capable of binding to the isolated protein of about 15 kDa to about 25 kDa is an antibody or a fragment thereof, or the protein is C3
It may be a chimeric protein comprising an antibody binding domain, for example, a variable domain, derived from an antibody capable of specifically recognizing an isolated protein of about 15 kDa to about 25 kDa derived from Streptococcus pneumoniae having degrading activity.

[0054] The isolated S. The Pneumoniae protein can be isolated, and optionally purified, and the isolated protein or immunogenic fragment thereof can be used to generate an immune response, for example, a human or experimental animal antibody response. Peptides or polypeptide fragments of proteins that are not capable of degrading C3 are It can be tested for its ability to suppress the effects of Pneumonia infection.
Similarly, a protein of the invention may be modified, for example, via a mutation that disrupts or inactivates the C3 degrading activity of the protein. Antibodies that can inhibit the C3 degrading activity of the proteins of the present invention can be used as a strategy to prevent C3 degradation or via the opsonization pathway. It can be used to promote the purification of Pneumonia. The isolated protein was S. In an assay for detecting antibodies to Pneumonia,
Or S. A vaccine or multivalent or multiprotein for the treatment of Pneumonia,
It can be used as part of a peptide or polypeptide containing vaccine.

Thus, the term “treatment”, as used herein, refers to a mammal that has been diagnosed as, or is indicative of, a normal mammalian subject or a population of pneumococcal or various pneumonia infection features or symptoms. It means preventing and / or treating any of the animal subjects. The term "treatment" refers to providing a therapeutic effect to a mammalian subject so that the subject exhibits little or no symptoms of a pneumococcal infection or other related disease. Such treatment can be achieved by administering a nucleic acid molecule (sense or antisense), protein, peptide or polypeptide of the invention, or an antibody.

The proteins of the present invention can be expressed surface on vertebrate cells and degrade C3 when complement deposition (or activation) is a problem, eg, in xenografts or complement-mediated glomerulonephritis. Available to do. For example, a whole pneumococcal protein, a recombinant protein, or any portion thereof, is incorporated into a xenograft cell and expressed as a surface protein or secreted protein to inhibit complement deposition (and / or complement-mediated inflammation). Can be prevented or minimized.

Another specific aspect of the present invention relates to the use of vaccine vectors that express isolated proteins and peptides or polypeptides. Accordingly, in a further aspect, the present invention provides a method for inducing an immune response in a mammal, the method comprising in the mammal at least one isolated protein and / or peptide or polypeptide of the present invention, or a method thereof. Providing a vaccine vector expressing the mixture. The proteins and peptides or polypeptides of the invention may be used in mammals to provide a live vaccine vector comprising the genetic material required for expression of the protein and / or peptide or polypeptide as a foreign polypeptide,
In particular, it can be introduced utilizing live recombinant bacteria, viruses or other live factors.
Specifically, bacteria that colonize the gastrointestinal tract, such as Salmonella
Shigella, Yersinia, Vibrio, Escherichia and BCG have been developed as vaccine vectors, and these and other examples are described in J. Holmgren et al., Immunobiol. 184 , 157-179 (1992). ) And J. McGhee et al., Vaccine , 10 , 75-88 (
1992).

A further aspect of the present invention relates to a method of inducing an immune response in a subject, eg, a mammal, comprising administering to the subject an isolated protein of the present invention and / or a peptide or peptide derived therefrom. Administering a predetermined amount of a DNA molecule encoding the polypeptide, optionally together with a transfection facilitating agent.
Here, the protein and / or peptide or polypeptide retains immunogenicity and, when incorporated into an immunostimulatory composition, such as a vaccine, and administered to a human, the subsequent S. cerevisiae of the human. Provides protection without inducing enhanced disease due to infection by a Pneumonia pathogen. Transfection facilitating agents are known in the art.

It is further contemplated that the antisense sequence of the gene encoding the 20 kDa protein may be used in a vaccine or therapeutic treatment for pneumococcal infection. The antisense DNA is complementary to all or part of SEQ ID NO: 1;
That is, it is defined as a non-coding sequence that is a complementary strand. For example, 5'-ATGTCA
The antisense sequence of AGC-3 'is 3'-TACAGTTCG-5'. Introduction of an antisense sequence or oligonucleotide into an animal results in production of the antibody by the animal, or integration of the sequence into living bacteria or other cells, thus transcription and transcription of the entire 20 kDa gene product or a portion thereof. And / or translation is inhibited.

The introduction of the antisense nucleic acid sequence can be achieved, for example, by filling the antisense nucleic acid into a suitable carrier, such as a liposome for introduction into pneumococcus or infected cells. Typically, antisense nucleic acid sequences having eight or more nucleotides can bind to bacterial nucleic acids or bacterial messenger RNA. The antisense nucleic acid sequence typically has at least 15 nucleotides, preferably at least 30 nucleotides, or more, to provide essential stability to the hybridization products of bacterial nucleic acids or bacterial messenger RNA. Contains nucleotides. The introduction of the sequence is preferably at least one endogenous S. aureus. Inhibits the transcription or translation of a Pneumonia nucleic acid sequence. A method for filling an antisense nucleic acid is disclosed in US Pat.
No. 42,046.

The present invention further relates to a protein having a molecular weight of about 20 kDa (SEQ ID NO:
The present invention provides a nucleic acid having an open reading frame of 2163 base pairs (SEQ ID NO: 4) including an open reading frame of a nucleic acid sequence encoding SEQ ID NO: 2 (SEQ ID NO: 1). The 20 kDa protein described herein is further characterized as a C3-degraded protein. This larger open reading frame, for example, 2163 bp (SEQ ID NO: 4) is approximately 79 kD
Encodes the putative protein of a (SEQ ID NO: 5).

[0062] All documents cited herein are incorporated herein. The present invention will be further described by the following examples, but the technical scope of the present invention is not limited by these examples.

Example 1 Identification of Insertion Mutants with Reduced C3 Degrading Activity Insertion mutants were identified as Dr. Elaine Toumanen (Rockefeller Inst., New York, New York).
York). Inserted clones were tested in the assay to determine a decrease in C3 degrading activity. 137 clones were tested by growing cells in microtiter plates overnight in room temperature in Todd Hewitt broth. Cells were transformed into a synthetic medium for pneumococcus (Sicard AM, Genetics 50: 31-44, 198).
Diluted 1:10 in 4) and the remaining cells were frozen in microtiter plates. 63 ng or 83 ng of C3 per 100 μl of medium containing 1 mg / ml of 0.1% BSA in phosphate buffered saline (PBS) was added to approximately 200 μl of the diluted cells. The cells were incubated at 37 ° C for 4 hours. 100 μl of this mixture was added to the ELISA plate and incubated at 4 ° C. overnight. The plates are washed three times with wash buffer and the wells are washed with PBS during the wash.
Filled with 0.05% Tween 20 in medium and incubated for 5 minutes. 10
0 μl of antibody to C3 (polyclonal horseradish peroxidase-conjugated goat antibody specific for human C3-IgG fraction, ICN Cappel. Costa Mesa, CA)
Was diluted 1: 1200 with 3% BSA in PBS. 3 ELISA plates
Incubate in the dark at 7 ° C. for about 30 minutes to 1 hour and wash with wash buffer as described above. The assay was developed utilizing 12 mg OPD in 30 ml of 0.1 M sodium citrate buffer containing 12 μl of 30% hydrogen peroxide. Assay results were determined by measuring optical density at 490 nm on an ELISA plate reader.

Each clone was tested four times. 4 compared to the unmutated control
Nineteen clones with a C3 resolution of less than 0% were selected. These 19 clones were screened 6 times by the assay described above, resulting in the selection of 6 clones with less than 30% C3 degrading activity compared to the control. These six clones were screened eleven times and the two clones with the lowest C3 degrading activity were selected for further study.

A partial sequence of one of these clones was received and a 546 bp Sma
The I fragment was ³² P labeled by random primer labeling (a kit available from Stratagene, La Jolla, CA). SEQ ID NO: 5 from 1
The 46 bp SmaI fragment was cloned on Southern blots into a vast number of pneumococcal strains E.
Hybridized to coRI and KpnI digests. The same fragment as S.I. It was also used to screen a library of Sau3A fragments of genomic DNA from Pneumoniae strain CP1200.

A 3.5 kb insert was identified from the CP1200 library. The insert was sequenced and a 492 base pair open reading frame containing a stop codon was identified. This open reading frame encodes a protein of 163 amino acids and a predicted molecular weight of about 18,500 daltons.

[0067] PCR primers were constructed for amplification of the open reading frame:
The 5 'PCR primer was incorporated at the BamHI site; the 3' primer was
Incorporated into the site. The amplification insert was replaced with a His-tagged E. coli. Coli expression vector p
Ligations were performed in-frame on QE30 (Qiagen, San Diego, CA). The resulting plasmid was transformed into the Lac repressor plasmid pREP4 (Qiagen).
E. E. coli strain BL21 (Novagen, Madison, WI) was used to transform. E. FIG. Inducing the E. coli culture to express a His-tagged protein;
Then, this protein was column-purified using Ni-NTA resin (Qiagen). This purified protein was confirmed by SDS-PAGE gel.

Example 2 Identification of a 20 kDa C3 Degrading Proteinase To determine the ability of a 20 kDa protein to degrade C3, 0.5 mg / ml C3 (
Tack et al., Meth. Enzymol. 80: 64-101, 1984) was prepared with a 15% acrylamide-containing sodium dodecyl sulfate (SDS) gel (15% SDS-PAGE).
E-gel). S. pneumoniae supernatants were grown to logarithmic growth phase in Todd Hewitt broma. Obtained from a culture of Pneumonia strain CP1200. Pneumococcal lysate was obtained by incubating 5 × 10 ⁸ cells with 5% SDS for 30 minutes at room temperature. This lysate was purified using a Centricon filter (Amicon, Beverly, Mass.) With a cut-off value of 10,000 mw.
It was concentrated twice. These samples were not heated before electrophoresis. Supernatant and lysate samples were added to a 15% C3-containing SDS-PAGE gel, and electrophoresis was performed at 150C at 4 ° C until the die front was drained. 50 gels
2.5 ml of 2.5% Triton-X 100 aqueous solution (2 times, 10 minutes each), 2.5 ml
% Triton X-100, 50 mM Tris-HCl, pH 7.4 (twice,
(10 min each) and 50 mM Tris-HCl, pH 7.4 (twice every 10 min) to remove SDS. After washing, 50 ml of 50 mM Tris-HCl,
pH 7.4 was poured into the gel-containing dish and the dish was covered and incubated at 37 ° C. for 1.5 hours and overnight (about 16 hours). The gel was stained with Coomassie blue for 10 minutes and the whole was destained.

[0069] Two lysis bands were observed against the dark blue background in both the lysate and the supernatant, one of which was approximately 20 kDa in size. C3 proteinase activity of pneumococcal lysate was observed after 1.5 hours of incubation at 37 ° C., and C3 proteinase activity in Pn supernatant was observed after overnight incubation.
Therefore, it was revealed that the C3 proteinase activity was mainly integrated into the cells.

Example 3 Genes encoding the 20 kD protein were identified by several S. The DNA stored in the P. pneumoniae strain was converted into various S. aureus. Pneumoniae strains (type 1, type 3, L002 and L003 (3
Type), type 4 and type 14 clinical isolates, as well as laboratory isolates CP1200, WU2
R6X, 6303, 109, 110, JY1119, JY182 and JY53)
And utilized SEQ ID NO: 3 as a probe to detect the presence of a nucleic acid encoding a 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 6X SSC, 5X De
nhart, 100 μg / ml denatured split salmon sperm DNA in 0.5% SDS
Hybridization at 65 ° C. overnight with 2 × SSC, 1 wash at room temperature for 10 min, 2 × SSC, 0.1% SDS at 65 ° C. for 15 min.
Washes and hybridization with 0.2 × SSC, 0.1% SDS at room temperature for 2 × 3 min and end-labeled SE under wash conditions.
Hybridized to Q ID NO: 3.

The results show that SEQ ID NO: 3 hybridizes similarly in each of the DNA samples tested, demonstrating that this protein is conserved between strains. In some strains, the DNA encoding the 20 kDa C3-degraded protein was found to be part of a large open reading frame of 2163 bp encoding the 79 kDa protein.

Example 4 Southern blot of Pneumonia DNA / 5F1 probe A 5 μg sample of genomic DNA was Obtained from 11 strains of Pneumonia. Each sample was digested with the restriction enzyme KpnI. These samples were then loaded on an agarose gel and resolved by electrophoresis. The sample contained in the gel was then transferred by capillary transfer to a Hybond-N + membrane available from Amersham (Upsalla, Sweden). 5F1 isolate derived 540 bp SmaI fragment ^T7 QuickPrime kit (Pharmacia, Piscathaway, NJ) using a randomized ply labeled with P ^32, and Nuc Trap columns (Stratagene, La J
(olla, CA) and purified from unincorporated nucleotides and hybridized.

Hybridization conditions were 6X SSC, 5X Denhardt, 0.
65 ° C. with 100 μg / ml split and denatured salmon sperm DNA in 5% SDS
Overnight, 2x SSC, 1 wash at room temperature for about 10 minutes in 0.1% SDS, 1 wash at 65 ° C for about 15 minutes, then 0.2X At least one wash in SSC, 0.1% SDS at room temperature for at least 3-5 minutes. This blot shows that the 20 kDa gene has the S. It is present in all tested strains of Pneumonia.

Example 5 Two DNA primers were prepared from SEQ ID NO: 1 and
It was used to amplify a 20 kDa gene sequence from genomic DNA of Pneumonia (serotype 3). 5'-primer, the first primer, SEQ ID NO:
6 spans the ATG start codon of the 20 kDa gene, has an NcoI site inserted, and has an Ala residue inserted after the ATG start codon to maintain the correct reading frame. 2 'primer, 3' primer, SEQ ID
NO: 7 straddles the stop codon of the 20 kDa gene and inserts a BamHI site.

Embedded image

[0075] Two primers were synthesized on an Applied Biosystems 380A DNA synthesizer (Foster City, CA) using reagents purchased from Glen Research (Sterling, UA). Amplify perkin Elmer Therm
This was performed using an Ocycler (ABI) according to the manufacturer's instructions. TA-tailed PC obtained from Invitrogen, Carlsbad, CA using the identified PCR product
L ligated into the R cloning vector PCR2.1 and
Used to transform t Top 10F 'competent cells (Invitrogen). Kanamycin resistant transformants were screened by restriction enzyme analysis of plasmid DNA prepared by alkaline lysis. An approximately 500 bp insert fragment was identified and subsequently digested with the restriction enzymes NcoI and BamHI. The 500 bp fragment was purified from a low melting point agarose gel,
Ligation was performed at the NcoI-BamHI site of the T7 promoted expression vector pET28a obtained from ovagen (Madison, Wis.).

The ligation mixture was then transferred to Top 10F 'cells (Invitroge).
n) and kanamycin resistant transformants were screened by restriction analysis of plasmid DNA prepared by alkaline lysis. The recombinant plasmid (pLP505) was then transformed into BL21 (Novagen) cells and grown in SOB medium with 30 μg / ml kanamycin. Cells were treated with an O.D. of 0.6. D. Grown to ₆₀₀ , then 0.4 mM IPTG (Boehringer Man
nheim, 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 expressed product was detected. 28 Coomassie stained gels
It showed a band between the kDa and 18 kDa molecular weight markers and was determined to be about 20 kDa.

The DNA sequence of the insert in this recombinant pLP505 plasmid was ABI 37
Obtained using a 0A DWA sequencer. This DNA sequence was converted to Mac Vect.
or Pastell DNA Matrix Plot Feature (Oxford Mol
ecular Group, Campbell, CA) and aligned with the DNA sequence of SEQ ID NO: 1. DNA sequence obtained from pLP505 plasmid, SE
Q ID NO: 1; Alignment with the Pneumonia (serotype 4) genome is based on an open reading frame (ORF) encoding a 20 kD protein.
) Is a larger ORF, ie, a protein with an estimated MW of about 79 kDa (SEQ.
ID NO: 4), which could be part of the 2163 bp of the serotype 4 genome. DNA SEQ ID NO: 4 encodes the deduced amino acid sequence shown in SEQ ID NO: 5.

S. Numonia (serotype 4) genomic sequence was translated into The Institute for Genomic Rese
Clustal of Mac Vector (Oxford Molecular Group, Campbell CA) from arch, www.tigr.org and / or from NCBI via www.ncbi.nlm.nih.gov.
Obtained using the W feature. Amino acid sequence of 20 kDa (SEQ.
ID NO: 2) and an estimated 79 kDa amino acid sequence (SEQ ID NO: 5).
And went in. Amino acids 1-58 and 90-1 of SEQ ID NO: 2
32 is SEQ ID NO: 5 amino acids 170-227 and amino acid 258-
It was observed to have substantially sequence identity with each of the 300. Proteins and peptides or polypeptides containing such specific regions are preferred embodiments of the present invention.

Based on the available genomic DNA (serotype 4) sequence, two primers flanking the 2163 bp ORF were designed and synthesized using an ABI 380A DNA synthesizer (SEQ ID NOs: 8 and 9). SEQ ID N
O: 8 is A to maintain the inserted NcoI site and the correct reading frame.
S. with a "Glu" residue added after the TG start codon. Numonia 5 '
It is a primer. SEQ ID NO: 9 is an S having an inserted HindIII site
. Numonia 3'-primer.

Embedded image

The DNA fragment of about 2100 bp was ligated to four types of S. Amplification from Pneumonia serotypes (serotypes 3, 5, 6B and 7) resulted in four fragments. Each of the four fragments was then cloned into the PCR cloning vector PCR2.1 (Invitr
, and OneShot Top 10F 'cells (Invitrogen). Kanamycin resistant transformants were screened by restriction analysis of plasmid DNA prepared by alkaline lysis. A recombinant plasmid containing the serotype 7 PCR product, eg, pLP512, was identified. This DNA sequence was obtained from 7 serotype clones using an ABI model 370A DNA sequencer. This DNA sequence is essentially identical to SEQ ID NO: 4 and encodes a deduced amino acid sequence essentially identical to SEQ ID NO: 5.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the nucleic acid sequence (SEQ ID:
NO: 1).

FIG. 2 shows the amino acid sequence of the C3-degrading proteinase of the present invention (SEQ ID NO: 2).
.

FIG. 3 shows C arranged together with a nucleic acid sequence encoding a C3 degrading proteinase according to the invention.
Amino acid sequence of 3-degrading proteinase (SEQ ID NOs: 2-3).

FIG. 4. Nucleic acid sequence of the predicted 79 kDa amino acid sequence (SEQ ID NO: 4).

FIG. 5. Predicted 79 kDa amino acid sequence (SEQ ID NO: 5).

FIG. 6. Sequence alignment of SEQ ID NO: 1 and SEQ ID NO: 4.

FIG. 7: Amino acids 169- of SEQ ID NO: 5 corresponding to SEQ ID NO: 2
331 sequence alignment.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/52 C12N 5/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK) , ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR) , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, D, GE, GH, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW , MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW ( 71) Applicant: American Cyanamid Company AMERICA CYANMID C COMPANIES New Jersey, USA 07940-0874 Madison Huaibiril Duff Arms (no address) (72) Inventor: Hostetter, Margaret K. United States, Connecticut 06511, New Haven, Tower Parkway 19, Ezra Styles College (72) Inventor Finkel, David J. USA United States, Minnesota 55414, Minneapolis, Ace Street Southeast 319, Apartment 303 (72) Inventor Chen, Quy United States of America, Minnesota 55442, Plymouth, Forty Avenue North 11240 (72) Inventor Green, Blues A. United States of America , New York 14534, Pittsford, Northfield Gate 49 (72) Inventor Masai, Amy W. United States, New York 14423, Caledonia, Grand Circle 326 F-term (reference) 4B024 AA01 AA13 BA14 BA31 CA03 CA07 CA09 CA12 CA20 DA01 DA02 DA05 DA06 DA11 EA04 GA11 GA19 GA25 HA13 HA14 HA17 4B050 CC01 CC03 CC05 DD02 EE01 FF09E LL01 LL03 LL05 4B065 AA26X AA49X AA49Y AA57X AA58X AA72X AA88X AA90X AA88X AA90X AB01 AC14 AC16 BA02 ZA04 CA04 A04 BA04 A04 A04 ZC192 4H045 AA11 AA20 AA30 BA10 BA41 CA11 DA76 DA 86 DA89 EA29 EA52 FA71 FA74 GA21

Claims (61)

[Claims] 1. SEQ ID NO: capable of degrading human complement protein C3
An isolated protein having at least 80% sequence identity to 2. 2. The method of claim 2, 2. The protein according to claim 1, isolated from Pneumonia. 3. The protein according to claim 1, which is a recombinant protein. 4. The protein of claim 1, which has a molecular weight of about 15 kDa to about 25 kDa. 5. An isolated peptide or polypeptide comprising at least 15 contiguous amino acids from the protein of claim 1. 6. An isolated peptide or polypeptide comprising at least 15 contiguous amino acids of SEQ ID NO: 2. 7. An isolated protein comprising SEQ ID NO: 2. 8. The isolated protein of claim 7, having a molecular weight of about 15 kDa to about 25 kDa. 9. S. 9. The protein according to claim 8, isolated from Pneumonia. 10. The protein according to claim 8, which degrades human complement protein C3. 11. The protein according to claim 7, which has SEQ ID NO: 2. 12. An isolated protein comprising from about 1 to about 58 amino acids of SEQ ID NO: 2. 13. The isolated protein of claim 12, further comprising about 90 to about 132 amino acids of SEQ ID NO: 2. 14. The amino acid sequence of SEQ ID NO: 5 from about 170 to about 227.
An isolated protein comprising: 15. The amino acid of SEQ ID NO: 5 from about 258 to about 300
15. The isolated protein of claim 14, comprising: 16. An isolated protein that degrades human complement protein C3, wherein the nucleic acid encoding the protein hybridizes to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions. Soy, isolated protein. 17. An S. cerevisiae capable of degrading human complement C3. About 15 from Pneumonia
An isolated protein of from about kDa to about 25 kDa. 18. An immune system stimulating peptide comprising at least 80% sequence identity to SEQ ID NO: 2 and comprising at least 15 contiguous amino acid sequences derived from a protein capable of degrading human complement protein C3. Alternatively, an immune system stimulating composition comprising an effective amount of a polypeptide. 19. The method according to claim 19, wherein the protein is S. aureus. 19. The immune system stimulating composition according to claim 18, which is isolated from Pneumonia. 20. S. 20. The immune system stimulating composition according to claim 19, further comprising at least one other immune system stimulating protein, peptide or polypeptide isolated from Pneumonia. 21. A protein, wherein the nucleic acid encoding the protein is S
An immune system stimulating composition comprising a therapeutically effective amount of at least a portion of a protein that hybridizes under highly stringent hybridization conditions to EQ ID NO: 1 or its complement. 22. S. 18. An immune stimulus comprising at least a portion of the protein of claim 17 in an effective amount, together with a pharmaceutically acceptable carrier, effective to immunize or treat a mammalian subject against infection or colonization of Pneumonia. Composition. 23. The immune system stimulating composition of claim 22, wherein said protein is present in an amount effective to provide a therapeutic effect to a mammalian subject. 24. An antibody comprising at least 80% sequence identity to SEQ ID NO: 2 and capable of binding to a protein capable of degrading human complement protein C3. 25. The antibody according to claim 24, which is a monoclonal antibody. 26. The antibody according to claim 24, which is obtained from a mouse, rat, goat, chicken, human or rabbit. 27. A protein, wherein the nucleic acid encoding the protein is S
An antibody capable of binding to at least a portion of a protein that hybridizes under high stringency hybridization conditions to EQ ID NO: 1 or its complement. 28. An isolated nucleic acid fragment capable of hybridizing under high stringency hybridization conditions to SEQ ID NO: 1 or its complement. 29. 29. The nucleic acid fragment according to claim 28, isolated from Pneumonia. 30. The nucleic acid fragment of claim 28, wherein said nucleic acid fragment encodes at least a portion of a protein. 31. The nucleic acid fragment of claim 30, wherein said protein degrades human complement C3. 32. The nucleic acid fragment according to claim 28, which is in a nucleic acid vector. 33. The nucleic acid fragment according to claim 32, wherein said vector is an expression vector capable of producing at least a part of a protein. 34. A cell comprising the nucleic acid of claim 28. 35. The cell of claim 34, wherein said cell is a bacterium or a eukaryotic cell. 36. About 1 to about 174 nucleotides of SEQ ID NO: 1
Or an isolated nucleic acid fragment comprising the complement thereof. 37. Nucleotides from about 320 to about 4 of SEQ ID NO: 1
37. The isolated nucleic acid fragment of claim 36, further comprising 92 or its complement. 38. An isolated nucleic acid fragment comprising the nucleic acid sequence of SEQ ID NO: 1 or a complement thereof. 39. An RNA fragment transcribed by a double-stranded DNA sequence comprising SEQ ID NO: 1 or a complement thereof. 40. A method for treating S. cerevisiae in mammals. A method for generating an immune response to Pneumonia, comprising: administering to a mammal a composition comprising a therapeutically effective amount of at least a portion of a protein in a pharmaceutically acceptable carrier. Wherein the nucleic acid encoding the protein hybridizes to SEQ ID NO: 1 or its complement under highly stringent hybridization conditions. 41. The method of claim 40, wherein said immune response is a B cell response, T cell response, epithelial cell response or endothelial cell response. 42. The method of claim 40, wherein at least a portion of said protein is at least 15 amino acids in length. 43. The composition according to claim 43, wherein the composition comprises 41. The method of claim 40, further comprising at least another immune system stimulating protein, peptide or polypeptide derived from Pneumonia. 44. At least a portion of said protein has SEQ ID NO:
41. The method of claim 40, comprising two at least 15 amino acids. 45. S.C. A method for inhibiting Pneumonia-mediated C3 degradation, comprising: Contacting the Pseudomonas pneumoniae with an antibody or fragment thereof capable of binding to a protein having the amino acid sequence of SEQ ID NO: 2. 46. A method for inhibiting C3-mediated inflammation and rejection in a xenograft, comprising: expressing the amino acid sequence of SEQ ID NO: 2 or a fragment thereof on the surface of the organ of the animal used for the xenograft. A method comprising the steps of: 47. An isolated sequence comprising a region of at least 15 nucleotides that hybridizes under highly stringent hybridization conditions to at least a portion of the nucleic acid sequence set forth in SEQ ID NO: 1 or its complement. Nucleic acid molecule. 48. SEQ ID NO: 1 or a sequence complementary thereto that comprises a sequence that hybridizes under highly stringent hybridization conditions to at least one region thereof, wherein said region comprises nucleotides 1-174 and 320.
An isolated nucleic acid molecule selected from the group consisting of -492. 49. An isolated sequence comprising a region of at least 15 nucleotides that hybridizes under highly stringent hybridization conditions to at least a portion of the nucleic acid sequence set forth in SEQ ID NO: 4 or the complement thereof. Nucleic acid molecule. 50. A sequence comprising a sequence that hybridizes under highly stringent conditions to at least one region of SEQ ID NO: 4 or the complement thereof, wherein said region comprises nucleotides 507-681 and 827-999. An isolated nucleic acid molecule selected from the group consisting of: 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 said protein has the deduced amino acid sequence of SEQ ID NO: 5. 53. SEQ ID NO: 6, SEQ ID NO: 7, SE
An isolated nucleic acid fragment having a nucleic acid sequence selected from the group consisting of Q ID NO: 8 and SEQ ID NO: 9. 54. A protein, wherein the nucleic acid encoding the protein is S
An immune system stimulating composition comprising a therapeutically effective amount of at least a portion of a protein that hybridizes under highly stringent hybridization conditions to EQ ID NO: 4 or its complement. 55. A method for treating S. cerevisiae in a mammal. A method for generating an immune response against Pneumonia, comprising: administering to a mammal a composition comprising a therapeutically effective amount of at least a portion of a protein in a pharmaceutically acceptable carrier. Wherein the nucleic acid encoding the protein hybridizes to SEQ ID NO: 4 or its complement under highly stringent hybridization conditions. 56. S. 52. An immune system stimulating composition comprising at least a portion of the protein of claim 51 effective in immunizing or treating a mammalian subject against a Pneumonia infection or colony in an effective amount with a pharmaceutically acceptable carrier. object. 57. The immune system stimulating composition of claim 56, wherein said protein is present in an amount effective to provide a therapeutic effect on a mammalian subject. 58. The immune system stimulating composition according to claim 56, wherein said composition is a vaccine. 59. A polypeptide having SEQ ID NO: 5. 60. The protein encoded by the nucleic acid sequence or its complementary strand comprising at least one endogenous S. aureus. 24. The immune system stimulating composition according to claim 23, which inhibits transcription or translation of a Pneumonia nucleic acid sequence. 61. The protein encoded by the nucleic acid sequence or its complementary strand comprises at least one endogenous S. aureus. 57. The immune system stimulating composition of claim 56, which inhibits the transcription or translation of a Pneumonia nucleic acid sequence.