Patent Grant
7588762
The present invention relates to a new protein, protein SIC, that can be derived from Streptococcus pyogenes strains of serotypes M1 and M57. Methods for analysis and purification and pharmaceutical preparations including vaccine compositions related to the protein as well as specific antibodies are also claimed.
Streptococcus pyogenes is an important human pathogen causing a number of acute suppurative infections such as erysipelas, necrotizing fasciitis, and pharyngitis. These Gram-positive bacteria also cause a serious toxic shock-like syndrome, whereas glomerulonephritis and rheumatic fever are serious poststreptococcal sequelae.
To elude the host defense and establish an infection, S. pyogenes has developed multiple molecular mechanisms. Some of these are dependent on genes located in a chromosomal region designated the mega locus according to a recent agreement, which is under the control of the positive regulator gene mga, previously called mry (Caparon and Scott, 1987: Proc. Natl. Acad. Sci. U.S.A. 84, 8677-8681) or virR (Simpson et al., 1990: J. Bacteriol. 172, 696-700).
Since the late 1980's unusually severe S. pyogenes infections have been reported world-wide. These hyperacute and often lethal infections have frequently been associated with the M1 serotype (Musser et al., 1993: J. Infect. Dis. 167, 337-346). This serotype is also connected with glomerulonephritis and rheumatic fever.
Based on structural variations in the antiphagocytic M protein (Fischetti, 1989: Clin. Microbiol. Rev. 2, 285-314; and Kehoe, 1994: New Compr. Biochem. 27, 217-261) S. pyogenes can be divided into more than 80 different serotypes. Most of these serotypes are rather harmless. However, during the last years, a lot of lethal streptococcal infections have been caused by strain belonging to the M1 serotype. Presently, because of the large amount of serotypes and the great similarity between these types, it is time-consuming and labourious to determine the serotype of a sample of Streptococcus pyogenes. Consequently, there is a need for simpler methods for determining whether a particular sample of Streptococcus pyogenes is virulent. There is also a need for better vaccines against virulent Streptococcus pyogenes strains.
A new protein from Streptococcus pyogenes, serotype M1, fulfilling the above mentioned needs has now been discovered and characterized. The protein and its corresponding gene has been obtained from strain AP1, which is of the M1 serotype.
In the mga locus of AP1, the regulatory gene mga is followed by emm1, the gene encoding the M1 protein. Immediately downstream of emm1 is sph ({dot over (A)}kesson et al., 1994: Biochem. J. 300, 877-886), the gene encoding an IgGFc-binding M protein-related molecule called protein H ({dot over (A)}kesson et al., 1990: Mol. Immunol. 27, 523-531; Gomi et al., 1990: J. Immunol. 144, 4046-4052; WO 91/19740; EP 0 371 199). Located adjacent to sph is a previously uncharacterized gene. This gene constitutes one of the objects of the present invention.
The protein shows some variability and M1 serotype strains of Streptococcus pyogenes producing variants of protein SIC have also been isolated. When comparing the amino acid sequences of three different protein SIC variants, some conserved regions were found.
The protein encoded by the uncharacterized gene as well as variants, subfragments and multiples of the protein having essentially the same antigenic and/or binding characteristics also constitutes an object of the present invention. The data obtained implicate that this extracellular protein plays a role in S. pyogenes pathogenicity and virulence through previously unknown molecular mechanisms. The new protein is referred to as protein SIC, Streptococcal Inhibitor of Complement-mediated lysis.
“SIC proteins” as utilized herein refers to streptococcal proteins which inhibit hemolysis by interacting with the plasma proteins clusterin and members of the cystatin protein superfamily, such as HRG, as demonstrated by affinity chromatography on a protein SIC Sepharose column or by indirect ELISA. SIC proteins may be distinguished from other proteins based upon criteria such as specific binding to the above mentioned plasma proteins and sequence homology. For example, SIC proteins of the present invention should comprise at least one of the following partial amino acid sequences:
a) glu thr tyr thr ser arg asn phe (SEQ ID NO:7);
b) asp trp ser gly asp asp trp pro glu asp asp trp (SEQ ID NO: 8);
c) arg ser gly val gly leu ser gln tyr gly trp ser (SEQ ID NO:9);
d) trp ser ser asp lys lys asp glu thr glu asp lys thr (SEQ ID NO:10);
e) gly thr gly tyr glu lys arg asp asp trp gly gly pro gly (SEQ ID NO:11);
f) lys arg asp asp trp arg gly pro gly his ile pro lys pro (SEQ ID NO:12);
preferably the amino acid sequence of the SIC proteins should be at least 70% homologous, more preferably at least 85% homologous, still more preferably at least 90% homologous and most preferably at least 95% homologous to anyone of the amino acid sequences disclosed in SEQ. ID. NOS 2, 3 and 4 .
As already mentioned and suggested above, different variants of protein SIC have been isolated. Most isolates produced a variant showing close resemblance to protein SIC isolated from strain AP1, whose amino acid sequence is disclosed in SEQ. ID. NO. 2. However, two more variants having the amino acid sequences according to SEQ. ID. NO. 3 and SEQ. ID. NO. 4, respectively, have also been discovered. After aligning the sequences of the different variants the conserved partial sequences a)-f) above were found. Regions a)-e) are present in all known protein SIC variants. Protein SIC from one of the isolates only comprised a part of the f) region. Consequently the above regions are considered to have important functions in the protein.
By “subfragment” is meant a part-fragment of the given protein having essentially the same antigenic and/or binding characteristics. By “variants” is meant proteins or peptides in which the original amino acid sequence has been modified or changed by insertion, addition, substitution, inversion or exclusion of one or more amino acids. By “multiples” is meant those proteins containing multiples of the whole original protein or those protein containing multiples of subfragments and/or variants thereof.
The present invention also relates to nucleic acid sequences encoding protein SIC. As utilized within the context of the present invention, nucleic acid sequences which encode protein SIC are deemed to be substantially similar to those disclosed herein if: (a) the nucleic acid sequence is derived from the coding region of a native protein SIC gene (including, for example, variations of the sequences disclosed herein); (b) the nucleic acid sequence is capable of hybridization to nucleic acid sequences of the present invention under conditions of either moderate or high stringency (hybridization in 5×SSPE containing 0.1% SDS and 0.1 mg/ml ssDNA, at 50-65° C. dependent on the probe length, or 10-20° C. below the Tm of the probe; washing in 1×SSPE, 0.1% SDS at 15-20° C. below the Tm of the probe for moderate stringency, and in 0.1×SSPE, 0.1% at 10° C. below the Tm of the probe for high stringency conditions) (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY, 1989); or (c) nucleic acid sequences are degenerate as a result of the genetic code to the nucleic acid sequences defined in (a) or (b). Furthermore, although nucleic acid molecules are primarily referred to herein, as should be evident to one of skill in the art given the disclosure provided herein, a wide variety of related nucleic acid molecules may also be utilized in various embodiments described herein, including for example, RNA, nucleic acid analogues, as well as chimeric nucleic acid molecules which may be composed of more than one type of nucleic acid.
Within another aspect of the present invention, probes and primers are provided for detecting nucleic acids sequences which encode protein SIC. Within one embodiment of the invention, probes are provided which are capable of hybridizing to protein SIC nucleic acids (DNA or RNA). For purposes of the present invention, probes are “capable of hybridizing” to protein SIC nucleic acids if they hybridize to Sequence I.D. No 1, 5 or 6 under conditions of moderate or high stringency (see the section above concerning nucleic acid molecules, and Sambrook et al., supra); Preferably, the probe may be utilized to hybridize to suitable nucleotide sequences in the presence of 5×SSPE, 0.1% SDS, and 0.1 mg/ml ssDNA at 10-20° C. below the Tm of the probe. Subsequent washes may be performed in 1×SSPE, 0.1% SDS at 15-20° C. for conditions of moderate stringency, and in 0.1×SSPE, 0.1% SDS at 10° C. below the Tm of the probe for conditions of high stringency.
Probes of the present invention may be composed of ether deoxyribonucleic acids (DNA) ribonucleic acids (RNA), nucleic acid analogues, or any combination of these, and may be as few as about 12 nucleotides in length, usually about 14 to 18 nucleotides in length, and possibly as large as the entire sequence which encodes protein SIC. Selection of probe size is somewhat dependent upon the use of the probe. For example, a long probe used under high stringency conditions is more specific, whereas a oligonucleotide carefully selected from the sequence can detect a structure of special interest.
Probes may be constructed and labeled using techniques which are well known in the art. Shorter probes of, for example, 12 or 14 bases may be generated synthetically. Longer probes of about 75 bases to less than 1,5 kb are preferably generated by, for example, PCR amplification in the presence of labeled precursors such as 32P-dCTP, digoxigenin-dUTP, or biotin-dATP. Probes of more than 1.5 kb are generally most easily amplified by transfecting a cell with a plasmid containing the relevant probe, growing the transfected cell into large quantities, and purifying the relevant sequence from the transfected cells (see Sambrook et al., supra).
Probes may be labeled by a variety of markers, including, for example, radioactive markers, fluorescent markers, enzymatic markers, and chromogenic markers. The use of 32P is particularly preferred for marking or labeling a particular probe.
As noted above, nucleic acid probes of the present invention may be utilized to detect the presence of protein SIC nucleic acid molecules within a sample. However, if such nucleic acids molecules are present in only a limited number, then it may be beneficial to amplify the relevant sequence such that it may be more readily detected or obtained.
A variety of methods may be utilized in order to amplify a selected sequence, including, for example, RNA amplification (see Lizardi et al., Bio/Technology 6:1197-1202, 1988; Kramer et al., Nature 339:401-402, 1989; Lomeli et al., Clinical Chem. 35(91) 1826-1831, 1989; U.S. Pat. No. 4,786,600), and nucleic acid amplification utilizing Polymerase Chan Reaction (“PCR”) (see U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159) (see also U.S. Pat. Nos. 4,876,187, and 5,011,769, which describe an alternative detection/amplification system comprising the use of scissile linkages). Within a particularly preferred embodiment, PCR amplification is utilized to detect or obtain protein SIC nucleic acids. Briefly, as described in greater detail below, a nucleic acid sample is denatured at 95° C. in order to generate single stranded nucleic acid. Specific primers, as discussed below, are then annealed at 37° C. to 70° C., depending on the proportion of AT/GC in the primers. The primers are extended at 72° C. with Taq polymerase in order to generate the opposite strand to the template. These steps constitute one cycle, which may be repeated in order to amplify the selected sequence.
Primers for the amplification of a selected sequence should be selected from sequences which are highly specific and form stable duplexes with the target sequence. The primers should also be non-complementary, especially at the 3′ end, should not form dimers with themselves or other primers, and should not form secondary structures or duplexes with other regions of nucleic acid. In general, primers of about 18 to 20 nucleotides are preferred, and may be easily synthesized using techniques well known in the art.
In another aspect, the present invention relates to vectors and host cells comprising the above mentioned nucleic acid sequences. The above described nucleic acid molecules which encode protein SIC (or portions thereof) may be readily introduced into a wide variety of host cells. Representative examples of such host cells include plant cells, eukaryotic cells, and prokaryotic cells Within preferred embodiments, the nucleic acid molecules are introduced into cells from a vertebrate or warm-blooded animal, such as a human, macaque, dog, cow, horse, pig, sheep, rat, hamster, mouse, or a fish, or any hybrid thereof.
The nucleic acid molecules (or vectors) may be introduced into host cells by a wide variety of mechanisms, including for example calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978), lipofection; gene gun (Corsaro and Pearson, Somatic Cell Gen. 1:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), retroviral, adenoviral, protoplast fusion-mediated transfection or DEAE-dextran mediated transfection (Ausubel et al., (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, N.Y., 1987).
The nucleic acid molecules, antibodies, and proteins of the present invention may be labeled or conjugated (either through covalent or non-covalent means) to a variety of labels or other molecules, including for example, fluorescent markers, enzyme markers, toxic molecules, molecules which are nontoxic but which become toxic upon exposure to a second compound, and radionuclides.
Representative examples of fluorescent labels suitable for use within the present invention include, for example, Fluorescein Isothiocyanate (FITC), Rodamine, Texas Red, Luciferase and Phycoerythrin (PE). Particularly preferred for use in flow cytometry is FITC which may be conjugated to purified antibody according to the method of Keltkamp in “Conjugation of Fluorescein Isothiocyanate to Antibodies. I. Experiments on the Conditions of Conjugation,” Immunology 18:865-873, 1970. (See also Keltkamp, “Conjugation of Fluoresscein Isothiocyanate to Antibodies. II. A Reproducible Method,” Immunology 18:875-881, 1970; and Goding, “Conjugation of Antibodies with Fluorochromes: Modification to the Standard Methods,” J. Immunol. Methods 13:215-226, 1970). For histochemical staining, HRP, which is preferred, may be conjugated to the purified antibody according to the method of Nakane and Kawaoi {“Peroxidase-Labeled Antibody: A New Method of Conjugation,” J. Histochem. Cytochem. 22:1084-1091, 1974; see also, Tijssen and Kurstak, “Highly Efficient and Simple Methods for Preparation of Peroxidase and Active Peroxidase Antibody Conjugates for Enzyme Immunoassays,” Anal. Biochem. 136:451-457, 1984).
Representative examples of enzyme markers or labels include alkaline phosphatase, horse radish peroxidase, and β-galactosidase. Representative examples of toxic molecules include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A. Representative examples of molecules which are nontoxic, but which become toxic upon exposure to a second compound include thymidine kinases such as HSVTK and VZVTK. Representative examples of radionuclides include Cu-64, Ga-67, CTa-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125, I-I31, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212.
As will be evident to one of skill in the art given the disclosure provided herein, the above described nucleic acid molecules, antibodies, proteins and peptides may also be labeled with other molecules such as colloidal gold, as well either member of a high affinity binding pair (e.g., avidin-biotin).
As noted above, the present invention also provides a variety of pharmaceutical compositions, such as vaccine compositions against certain streptococcal infections, comprising one of the above described anti-protein SIC antibodies, or protein SIC (or a peptide portion thereof), along with a pharmaceutically or physiologically acceptable carrier, excipients or diluents. Generally, such carriers should be nontoxic, to recipients at the dosages and concentrations employed. ordinarily, the preparation of such compositions entails combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents.
In addition, the pharmaceutical compositions of the present invention may be prepared for administration by a variety of different routes, including for example intraarticularly, intracranially, intradermally, intramuscularly, intraocularly, intraperitoneally, intrathecally, intravenously, subcutaneously or even directly into a tumor (for example, by stereotaxic injection). In addition, pharmaceutical compositions of the present invention may be placed within containers, along with packaging material which provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents {e.g., water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition.
As previously mentioned, protein SIC is involved in S. pyogenes pathogenicity and moreover, it is an extracellular protein. Hence, by carrying out an analysis of protein SIC, it is possible determine the presence of virulent Streptococcus pyogenes bacteria in a sample. Consequently, other objects of the present invention relates to methods and kits for analysis of protein SIC and/or corresponding antibodies based on for example enzyme-linked immonosorbent assay (ELISA), radioimmunological assay (RIA) and the polymerase chain reaction (PCR).
As previously mentioned, the present patent application relates to protein SIC, a novel extracellular protein of group A streptococci. Analogous to other proteins encoded by genes under control of mga, protein SIC contains repeated sequences. However, the sequence of protein SIC, including the repeats, shows no homology to previously sequenced genes. It is also noteworthy that in contrast to all other described products of the mga regulon, protein SIC does not have the typical structural features of cell wall proteins in Gram-positive bacteria; i.e. a COOH-terminal region anchored to the cell wall through an LPXTG motif (Fischetti et al., 1990: Mol. Microbiol. 4, 1603-1605; Schneewind et al., 1995: Science 268, 103-106; Schneewind et al., 1992: Cell 70, 267-281), followed further towards the COOH-terminus by a hydrophobic membrane-spanning domain and a tail of mostly positively charged amino acid residues. The missing cell wall anchor, the occurrence of a typical signal sequence, and the fact that considerable amounts of protein SIC is found in the growth medium, suggest that the molecule is secreted and has extracellular function(s).
Previous work has demonstrated several interactions between components of the complement system and proteins encoded by genes of the mga regulon. Members of the M protein family (M protein, protein Arp, protein Sir, and protein H) have been reported to bind complement factor H, CD46, and/or the C4b-binding protein (Horstmann et al., 1988: Proc. Natl. Acad. Sci. U.S.A. 85, 1657-61; Okada et al., 1995: Proc. Natl. Acad. Sci. U.S.A. 92, 2489-2493; Thern et al., 1995: J. Immunol. 154, 375-386; WO 91/19740; EP 0 371 199; EP 0 367 890), three proteins with regulatory functions in the complement system. Furthermore, the C5a peptidase (Wexler et al., 1985: Infect. Immun. 39, 239-246) which can be released from the streptococcal cell wall by a cysteine proteinase produced by the bacteria (Berge et al., 1995: J. Biol. Chem. 270, 9862-9867), cleaves the C5-derived fragment C5a and destroys its chemoattractant activity for polymorphonuclear leukocytes (Wexler et al., 1985: Proc. Natl. Acad. Sci. U.S.A. 82, 8144-8148).
The specific interactions between protein SIC and the plasma proteins clusterin and HRG resulted in a study of the final cytolytic step in the complement cascade. Clusterin is known to inhibit the hemolytic activity of complement by binding to MAC (Membrane Attack Complex) (Tschopp et al., 1993: J. Immunol. 151, 2159-2165; Tschopp et al., 1994: Clin. Exp. Immunol. 97 suppl 2, 11-14), whereas the influence of HRG on MAC is biphasic, inhibitory or stimulatory, depending on the experimental conditions (Chang et al., 1992: Blood 79, 2973-2980). As demonstrated here, protein SIC was inhibitory to hemolysis in classical pathway as well as alternative pathway systems. Furthermore, protein SIC was shown to be incorporated into C5b-C9 complexes formed in serum. Although other mechanisms may also be considered, the findings suggest that the anticomplementary action of protein SIC is focused on the terminal cytotoxic functions of complement.
The metabolically inert erythrocyte is a very sensitive target for MAC whereas most pathogenic bacteria including streptococci are resistant to complement-mediated cytolysis. However, apart from its cytolytic activity, MAC also has pro-inflammatory effects by stimulating the production and release of inflammatory mediators such as reactive oxygen metabolites, metabolites of arachidonic acid, and cytokines (Morgan 1989: Biochem. J.:264, 1-14). Bacterial products affecting the various functions of MAC, directly or indirectly, could therefore influence the host-parasite relationship. The molecular complexity necessary to establish and maintain this relationship, makes it difficult to predict the consequences of any isolated interaction. However, in the case of pathogenic and virulent bacteria like S. pyogenes, the balance is disturbed, and there is circumstantial evidence that protein SIC may contribute to the imbalance of the host-parasite relationship in S. pyogenes infections.
To our knowledge protein SIC is the first bacterial protein reported to interact with clusterin or HRG. As mentioned, glomerulonephritis represents a medically significant sequelae following infections with S. pyogenes. In these cases certain M. serotypes are more common, including the two protein SIC-producing serotypes M1 and M57 (Holm, 1988: Acta Pathol. Microbial. Scand. 96, 189-193). In post-streptococcal glomerulonephritis immunoglobulin deposits are found in the glomeruli. Interestingly, MAC is regarded as a mediator of glomerular injury in immune complex-related disease (Couser et al., 1985: Kidney Int. 28, 879-890), and clusterin was found to be co-localized with MAC in biopsies from glomerulonephritic kidneys (French et al., 1992: Clin. Exp. Immunol. 88, 389-393).
Also, plasma depleted of clusterin (to <30%) enhanced proteinuria and deposition of MAC components in perfused kidneys (Saunders et al., 1994: Kidney Int. 45, 817-827). The association of protein SIC to nephritogenic M serotypes, and its binding of clusterin in human plasma, makes it interesting to test if protein SIC can induce kidney damage in an animal model.
Since the late 1980's a world-wide increase of hyperacute, toxic, and often lethal S. pyogenes infections, has attracted also public attention (Nowak, 1994: Science 264, 1665). These systemic infections have particularly been associated with streptococci of the M1 serotype, and the observation that all M1 strains tested, including isolates from Swedish patients with toxic and severe infections (Holm et al., 1992: J. Infect. Dis. 166, 31-37), carry and express the sic gene, supports the notion that protein SIC plays a role in pathogenicity and virulence. The present invention may therefor be used for preventing and/or treating S. pyogenes infections.
As previously mentioned, effective amounts of the protein or fragments or variants thereof can be used as active ingredients in pharmaceutical compositions, especially in vaccine compositions against streptococcal infections, possibly together with pharmaceutically acceptable adjuvants and excipients. Suitable pharmaceutically acceptable adjuvants are those conventionally used in this field. Examples of suitable excipients are mannitol, lactose, starch, cellulose, glucose, etc., only to mention a few. The examples given of the adjuvant and the excipients are not to be regarded as limiting the invention.
The invention will now be described in more detail with reference to the accompanying drawings, in which
SEQ ID NO:1 and SEQ ID NO:2, respectively, relate to the DNA sequence and the deduced amino acid sequence of a gene encoding protein SIC that has been isolated from Streptococcus pyogenes, strain AP1 (40/58). SEQ ID NO: 3 and SEQ ID NO: 5 relate to a partial DNA sequence and a deduced amino acid sequence of a gene encoding protein SIC that has been isolated from Streptococcus pyogenes, strain U1034. SEQ ID NO: 4 and SEQ ID NO: 6 relate to a partial DNA sequence and a deduced amino acid sequence of a gene encoding protein SIC that has been isolated from Streptococcus pyogenes, strain U1004.
The invention vill now be further described with reference to the following examples. These examples are only given for the purpose of illustration and are not intended to limit the scope of the invention claimed herein.
Bacterial Strains, Bacteriophage, and Plasmids
Protein SIC and the sic gene were isolated from S. pyogenes strain AP1 (40/58) of the M1 serotype from the Institute of Hygiene and Epidemiology, Prague, Czech Republic. Forty eight additional strains of different M serotypes were also obtained from this Institute. These strains have been described (Ben Nasr et al., 1995: Biochem. J. 305, 173-180). S. pyogenes strains of serotypes M3, M33, M42, M52, M61, and M67 were kindly provided by Dr. U. Sjöbring, Lund University, and a collection of 35 M1 strains isolated in Sweden 1980-89 were kindly provided by Dr. Stig Holm, Ume{dot over (A)} University. One S. pyogenes strain of serotype M1 and two of type M57 were from the late Dr. L. W. Wannamaker. The sic gene was cloned from a λEMBL3 clone previously described ({dot over (A)}kesson et al., 1994: Biochem. J. 300, 877-886) Plasmid vectors pUC18/19 (Yannisch-Perron et al., 1985: Gene 33, 103-119) and pK18/19 (Pridmore, 1987: Gene 56, 309-312) were used in subcloning experiments. PCR products were cloned into the inducible secretion vector pHD389 (Dalboge et al., 1989: Gene 79, 325-332). The E. coli strain JM109 was used as host for recombinant plasmids. Streptococci were grown in Todd-Hewitt broth supplement with 0.2% yeast extract and Escherichia coli in LB broth.
Protein and Antisera
Recombinant M1 protein ({dot over (A)}kesson et al., 1994: Biochem. J. 300, 877-886) and protein PAB (de Château and Björck, 1994: J. Biol. Chem. 269, 12147-12151) were purified as previously described. Human serum albumin (HSA) was from Sigma (St. Louis, Mo.). The monoclonal antibody MCaE11 directed against a neoantigen of polymerized C9 within the C5b-C9 membrane attack complex (MAC) of complement (Mollnes et al., 1985: Scand. J. Immunol. 22, 197-202) was kindly provided by Dr. T. E. Mollnes, the National Hospital, Norway. Antisera against purified streptococcal protein SIC were raised in rabbits. Sheep anti-rat clusterin (polyclonal IgG fraction) crossreacting with human clusterin was purchased from Quidel (San Diego, Calif.). Goat antisera against human histidine-rich glycoprotein was a kind gift from Dr. William T. Morgan (University of Missouri, Kansas City, Mo.). Peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad, Richmond, Calif.) and donkey antisheep IgG (ICN, Aurora, Ohio) were used as secondary antibodies.
Purification of Clusterin and HRG
For the purification of clusterin and histidine-rich glycoprotein (HRG) fresh human plasma supplemented with 1 mM benzamidine and 0.4 mM phenylmethylsulfonyl fluoride was used. Clusterin was purified by a method modified from a previous study (Wilson and Easterbrook-Smith, 1992: Biochim. Biophys. Acta 1159, 319-326). Thus, Human plasma was differentially precipitated with polyethyleneglycol (12-23% w/v). The precipitate was then passed over an IgG-Sepharose column (Pharmacia). The column was washed with PBSA and bound proteins were eluted with 0.1 M glycine-HCl, pH 2.0. The material was dialyzed against 20 mM Tris, pH 7.5, and subjected to a Mono Q column. Using a linear NaCl gradient a protein peak eluted at 0.3 M NaCl was found to contain a single band of 80 kDa when analyzed by SDS-PAGE under non-reducing conditions. The material gave rise to a single band of 35 kDa when run under reducing conditions in the presence of 5% β-mercapto-ethanol. The bands were identified as clusterin in Western blot experiments using anti-clusterin antibodies. HRG was purified using the protocol of Saigo et al (J. Biol. Chem. 264, 8249-8253 (1989)). Briefly, plasma was absorbed with CM-Sephadex (Pharmacia). The matrix was washed extensively with distilled water and adsorbed proteins were eluted with 0.5 M NH4HCO3. After dialysis against PBS the material was applied to a HiTrap Heparin column (Pharmacia). The column was washed with PBS and proteins were eluted with 1.0 M NaCl. The eluate was dialyzed against 20 mM Tris, pH 7.5, and subjected to ion-exchange chromatography on a Mono Q column. A linear NaCl gradient was used and the peak at 0.25 M NaCl was collected. The resulting preparation consisted of two major bands of 70 and 63 kDa when examined by SDS-PAGE under non-reducing conditions. The identity of the bands with HRG was confirmed by Western blotting using anti-HRG antisera.
Electrophoresis and Electroblotting
SDS-PAGE was performed as described (Neville, 1971: J. Immunol. Meth. 72, 49-59) using gels of 8% or 12% acrylamide content and 3.3% crosslinking. Before loading, samples were boiled 3.5 min in an equal volume of buffer containing 2% SDS. Samples run under reducing conditions were boiled in the presence of 5% 2-mercaptoethanol. Western blotting was performed by transferring proteins to polyvinylidene difluoride (PVDF) membranes (Immobilon, Millipore, Bedford, Mass.) as described by Towbin (Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354 (1979)). After blocking, membranes were incubated 1 h with antisera against protein SIC (1:200 v/v), HRG (1:500 v/v), or antibodies towards clusterin (5 μg/ml), followed by a peroxidase-labeled secondary antibody (1:1000 v/v). For visualization of bound antibody, membranes were incubated in 0.02% (w/v) 3-amino-9-ethylcarbazol, 0.06% (v/v) H2O2 in 50 mM sodium acetate buffer, pH 5.0.
Cloning Techniques and Sequence Determination
The PCR product amplified for the cloning of the sic gene was generated with primers containing recognition sites for NarI and XbaI, and cloned into pHD389 digested with the same enzymes. Standard cloning procedures were used (Sambrook et al., 1989: Molecular cloning: A laboratory-manual, 2nd ed. Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y., USA). Restriction enzymes and T4 DNA ligase were obtained from Promega. DNA sequencing was performed with Sequenase (Pharmacia) according to the manufacturer's instructions. Ordered sets of deletions in template plasmid DNA were prepared using exonuclease BAL-31 (Promega). Computer search for sequence homology was made in the GenBank and EMBL databases. Analysis of sequence data was performed using the GeneWorks program (Intelligenetics, Mountain View, Calif., USA)
Other Methods
NH2-terminal amino acid sequence analysis of proteins electrotransferred to PVDF membranes were performed on an Applied Biosystems 470A gas-liquid solid phase sequenator. Clusterin, HRG, and albumin were labeled with 125I using the Bolton and Hunter reagent (Amersham Corp. Buckinghamshire, Great Britain). The specific activities were between 3 and 10 μCi per μg of protein.
Streptococcal protein SIC was purified by growing AP1 bacteria to mid-log phase (A600=0.5) and precipitating the culture supernatant with 30% ammonium sulfate. The resulting precipitate was resuspended to 1/100 of the starting volume with 20 mM Tris, pH 7.5, and dialyzed against the same buffer.
This material was applied to a Mono Q column on an FPLC system (Pharmacia, Uppsala, Sweden). Using a linear NaCl gradient, a single sharp peak was eluted at 0.3 NaCl. Corresponding fractions were pooled, concentrated, and subjected to gel filtration chromatography on a Superose 12 column (Pharmacia) in PBSA (0.03 M phosphate, 0.12 M NaCl, 0.02% NaN3, pH 7.2). Fractions containing the suspected protein SIC were pooled and the identity of the protein was confirmed by NH2-terminal amino acid sequencing and later by Western blotting using antisera against protein SIC. The yield was 0.5-2 mg purified protein SIC per litre of culture.
Recombinant protein SIC was purified from an E. coli strain JM109 harbouring the sic gene in pHD389. The strain was grown to mid-log phase at 30° C. before inducing protein production by raising the temperature to 40° C. for 4 hours. The culture supernatant was collected and purification of protein SIC was performed as described above. In order to examine the presence of protein SIC in other group A streptococcal strains, cultures grown to mid-logarithmic phase were precipitated with 30% ammonium sulfate, the precipitates were washed with ice cold ethanol, dissolved in 100 μl SDS-PAGE sample buffer, and subjected to Western blot analysis using rabbit antiserum against protein SIC.
The previously reported sequence of the protein H gene (sph) revealed the first 138 bp of an open reading frame 188 bp downstream of the stop codon for sph (Gomi et al., 1990: J. Immunol. 144, 4046-4052; EP 371 199). To determine the structure of the possible downstream gene, a phage clone, λ1:4 ({dot over (A)}kesson et al., 1994: Biochem. J. 300, 877-886) harbouring both the M1 gene (emm1) and sph was employed for subcloning. A 2.2 kbp SspI/EcoRI fragment of λ1:4 that contained the open reading frame except for the first six nucleotides was cloned into the vector pUC19. Sequencing of this subclone revealed that the entire open reading frame was 915 bp encoding a putative amino acid sequence of 305 residues (
The first 32 amino acid residues represents a typical bacterial signal sequence including a positively charged NH2-terminal region, a hydrophobic core, and a polar cleavage region. The predicted mature protein has 273 amino acid residues and a calculated molecular mass of 30.677 kDa. The most characteristic feature of the sequence is a central repeat region consisting of three tandem repeats of 29, 29, and 21 amino acid residues each (R1-R3), showing 90-95% internal homology.
The sequence preceding the R repeats also contains repeats although these are only five or nine amino acids long. In
Computer-assisted analysis of the sequence predicted the protein to be highly hydrophilic with a pI of 4.2. A thorough search of the data bases with the entire sequence, or fragments of the sequence including all kinds of repeats, did not reveal any obvious homology either at the nucleotide or the amino acid level.
The presence of sic in the mga locus of the AP1 strain was further demonstrated by PCR of chromosomal DNA with two sets of primers (
The PCR performed with the S3 primer from the 3′ end of sic and the C1 primer corresponding to a part of the signal sequence of the C5a peptidase gene (scpA) generated a 2.2 kbp product. These experiments demonstrate that sic is located 188 bp downstream of sph and approximately 2.1 kbp upstream of scpA (
Oligonucleotides were constructed from the SIC sequence to generate a PCR fragment corresponding to the mature protein. This fragment was amplified by PCR using Taq DNA polymerase (Promega, Madison, Wis.). For amplification of the sic gene for cloning, primers S1 and S2 containing recognition sites NarI and XbaI respectively (
To investigate the occurrence of sic in other group A streptococcal isolates, PCR was performed with the S1 and S2 primer with an annealing temperature at 20° C. below the Tm of the shortest primer (46° C.). Templates for the reactions were prepared by resuspending a single bacterial colony in 100 μl water, vortexing the sample vigorously, and the boiling it for 5 min. Cell debris was removed by centrifugation and 5 μl of the resulting supernatant were used in a 50 μl reaction. In order to determine the relationship of sic to other genes of the streptococcal mga locus, PCR's with two different primer pairs outlined in
The amplified fragment was cloned into the inducible expression vector pHD389. After growing the clone and inducing expression, a periplasmic lysate was examined by SDS-PAGE which showed the overexpression of a 34 kDa protein as compared to the host E. coli strain. Even higher amounts of the 34 kDa product was detected when analyzing the culture media after precipitation with 80% ammonium sulfate. The protein was purified using precipitation of the culture media with 30% ammonium sulfate, ion-exchange chromatography and gel filtration (
Both the presence of a signal sequence and the absence of cell-wall anchoring or membrane-spanning sequences in the gene, suggested that the protein is secreted by streptococci of the AP1 strain. Consequently, a similar purification scheme as used for the recombinant protein was applied to the culture medium of streptococcal AP1 bacteria, and a protein with identical migration on SDS-PAGE was obtained (
Being a secreted product protein SIC is most likely active outside the bacterial cell. On the streptococcal chromosome protein SIC is found in a locus encoding proteins which interact with host molecules, particularly plasma proteins. In order to examine if protein SIC also has affinity for plasma proteins, fresh human plasma was mixed with the protease inhibitors benzamidine iodocetic acid to final concentrations of 5 mM each. 5 ml of undiluted plasma was immediately added to 1 mg protein SIC coupled to Sepharose. After end to end rotation for 4 hours at room temperature the protein SIC-Sepharose was extensively washed with PBS (200 times the column volume) and the proteins bound to the column were eluted with 0.1 M glycine-HCl, pH 2.0. As a control 5 ml plasma was simultaneously absorbed with glycine-Sepharose. pH was adjusted to 7.5 by adding 1 M Tris, and the eluates were concentrated about 100 fold.
The eluted material was lyophilized, resuspended in PBS at fifty times concentration and examined on SDS-PAGE. As seen in
The band of 80 kDa (labelled I in
The band of 70 kDa (labelled II in
The binding of protein SIC to clusterin and HRG was now further analyzed. Firstly, clusterin and HRG were purified from human plasma. The observations made with human plasma were then re-examined by affinity chromatography of radiolabeled clusterin and HRG on protein SIC-Sepharose.
Purified radiolabeled clusterin, HRG, or HSA (4×105 cpm in 1.0 ml PBS) were run on columns of Sepharose coupled with protein SIC, M1 protein, HSA. After washing with PBS bound proteins were eluted with 0.1 M glycine-HCl, pH 2.0 and the radioactivity in the fractions measured in a gammacounter.
About 30% of the 125I-clusterin and about 60% of the 125I-HRG were retained after chromatography on a protein SIC-Sepharose column. In contrast, less than 5% of iodine labeled albumin was eluted from the column (
The interactions were also tested by indirect ELISA (Engvall and Perlmann, 1971: Immunochemistry 8, 871-874), which analysis was performed by coating microtiter plates (Maxisorb, NUNC, Denmark) over night at 4° C. with serial dilutions of protein SIC (starting concentration 2 μg/ml). The plates were washed in PBST and incubated with clusterin (5 μg/ml in PBST containing 2% bovine serum albumin) or HRG (1.25 μg/ml) diluted in PBST containing 1% gelatin (PBSTG). Bound proteins were detected by purified antibodies to clusterin (10 μg/ml) or antisera against HRG (1:2000 v/v), and binding was visualized by a horseradish peroxidase-conjugated secondary antibody against rabbit or sheep IgG (1:5000 v/v). All incubations were done at 37° C. for 1 h and followed by a washing step. Substrate solution, 0.1% (w/v) diammonium-2,2-azino-bis-(3-ethyl-2,3-dihydrobenzthiazoline)-6-sulfonate (ABTS), 0.012% (v/v) H2O2 in 100 mM citric acid, 100 mM NaH2PO4, pH 4.5, was added and the change in absorbance at 405 nm was determined after 30 min.
A competitive ELISA was performed using the same procedure specified above except for the following: microtiter plates coated with either clusterin (1.5 μg/ml) or HRG (0.5 μg/ml) were incubated with a mixture of protein SIC (2 μg/ml for binding to clusterin; 1 μg/ml for binding to HRG) and serial dilutions of the competitor protein (starting concentration 20 μg/ml). Bound protein SIC was detected by a specific rabbit antisera (1:1000 v/v) followed by a secondary antibody towards rabbit IgG (1:3000 v/v) and ABTS/H2O2.
The results of the indirect ELISAs show that the binding of clusterin or HRG to protein SIC is concentration-dependent (
Together with vitronectin, clusterin and HRG bind to the C5b-C9 complex in serum and thereby regulate the cytotoxic action of MAC (Tschopp et al., 1993: supra; Chang et al., 1992: supra). Clusterin partly inhibits hemolysis, while HRG modulates complement function in a biphasic fashion. A possible role for protein SIC in classical pathway-mediated hemolysis was assessed by the results of complement-mediated hemolytic assays and binding experiments.
Complement-mediated hemolytic assays in free solution were performed essentially as reported (Nilsson and Nilsson, 1984: J. Immunol. Meth. 72, 49-59) with target cells added in excess. Sheep erythrocytes, treated with rabbit antibody (NBL, Stockholm, Sweden) to yield optimally sensitized cells (EA cells), were used to measure the function of the classical pathway function. Assays were performed with veronal buffered saline (VBS) containing 0.15 mM Ca2+ and 0.5 Mg2+.
Alternative pathway-mediated hemolysis of guinea-pig erythrocytes (GpE) was performed in VBS containing EGTA at a final concentration of 16 mM and Mg2+ at 4 mM. Various quantities of protein SIC were added to human serum diluted to a concentration causing 10% lysis of erythrocytes. After incubation for 30 min at room temperature EA or GpE were added at a concentration of 5×109 cells per ml and the mixture was incubated at 37.degree. C. for 20 min. The reaction was stopped by adding a fifteen-fold volume of cold VBS containing 10 mM EDTA.
After centrifugation hemolysis was measured as the absorbance of the supernatant at 541 nm. The final human serum dilutions were 1:40 for assays of the classical pathway and 1:20 for the alternative pathway. The serum used was free from antibodies towards protein SIC. Complement-mediated hemolysis in gel was studied according to the procedure previously described (Truedsson et al., 1981: Acta Pathol. Microbial. Scand., C. Immunol. 89, 161-166) using EA or GpE in agarose as target cells. 5 μl undiluted human serum and 1.5 μg (in 5 μl) protein SIC or protein PAB were applied to adjacent wells in the gels with a distance of 2-4 mm. Additionally, 1.5 μg bacterial proteins were added after 2, 4, 6, and 8 h incubation. After incubation of the gels at 4° C. for 16 h lysis was induced at 37° C. for 3 h.
A dose dependent inhibition of complement hemolysis was obtained (
As shown in
The interaction between protein SIC and the terminal complement proteins in serum was examined with a capture ELISA modified from Mollnes et al., 1985: Scand. J. Immunol. 22, 183-202. Microtiter plates were coated with 1.0 μg/ml of MCaE11, a monoclonal antibody against a neoantigen of polymerized C9 that is formed during the assembly of the C5b-C9 complex. Incubation of serum gives a high level of such complexes binding to the antibody due to spontaneous in vivo. activation of complement (Mollnes et al., 1985: supra). Serum diluted 1/50 in PBSTG was added to the wells followed by serial dilutions of protein SIC or SCP (starting concentrations 10 μg/ml), and the plates were incubated for 3 h at room temperature. Diluted serum was also preincubated with protein SIC or SCP at various concentrations for 3 h, after which generation of C5b-C9 complexes were stopped by addition of EDTA at 10 mM. The incubation mixtures were then transferred to antibody-coated microtiter plates for analysis. Detection of protein SIC and SCP were performed as described in Example 5 using specific rabbit antisera diluted 1/1000.
In
A collection of 55 group A streptococcal strains of different, M types were tested for the presence of the sic gene and the expression of protein SIC. At the DNA level sic was identified by PCR using primers from the start of the coding sequence and the repeat regions and by performing the reaction under low stringency conditions. The expression of protein SIC was tested by growing the strains to mid-logarithmic phase, precipitating the culture media with 30% ammonium sulfate, and examining the precipitate in a Western blot using polyclonal antisera against protein SIC. The strains of M type 1 and 57 were positive both in the PCR and in Western blot experiments. The rest of the strains gave no PCR products and were negative in the Western blot analyses. After the initial screening, 35 additional M type 1 isolates and two M 57 strains were tested. All of these strains contained sic and expressed protein SIC. The results suggest that the sic gene is highly restricted among various M serotypes, whereas within these serotypes all isolates have and express the gene.
An indirect ELISA was performed by coating microtiter plates (Maxisorb, NUNC) over night at +4° C. with 5 μg/ml protein SIC or 5 μg/ml fibrinogen (negative control). The plates were washed with phosphate buffered saline containing 0.25% Tween, and incubated for 1 h at 37° C. with two-fold dilutions of serum from two patients (starting dilution of serum was 1:10).
Patient A is infected by a virulent Streptococcus pyogenes strain and patient B is infected by a lesser virulent strain. Bound antibodies were detected by peroxidase-labelled anti-bodies towards human κ light chain (DAKO). Substrate solution, 0.1% (W/v) diammonium-2,2-azino-bis-(3-e-thyl-2,3-dihydro-benzthiazoline)-6-sulfonate (ABTS), 0.012% (v/v) H2O2 in 100 mM citric acid, 100 mM NaH2PO4, pH 4.5, was added and the change in absorbance at 405 nm was determined after 30 min.
The results are shown in
PCR experiments were performed using genomic DNA isolated from 35 different M type 1 strains and 2 M type 57 strains, and oligonucleotides from the 5′ and 3′ end of the coding sequence of sic. The size of the products from most strains were 830 bp, the same size as of the sic gene originally isolated (strain AP1, SEQ ID NO: 1). However, in 6 of the strains, sic was approximately 100 bp bigger, and in one strain 100 bp smaller. Four of the PCR-amplified products were subjected to DNA sequencing, including one bigger and one smaller sic gene. These results, which partially are disclosed in
Protein SIC variants encoded by the tour sequenced genes were isolated from the corresponding S. pyogenes strains. These were shown to have similar protein binding properties as protein SIC isolated from AP1.
| Number | Date | Country | Kind |
|---|---|---|---|
| 9503495 | Oct 1995 | SE | national |
This application is a divisional of U.S. patent application Ser. No. 10/374,535, filed on Feb. 26, 2003, now U.S. Pat. No. 7,361,750, which is a divisional of U.S. patent application Ser. No. 09/051,380, filed Sep. 8, 1998, now U.S. Pat. No. 6,576,741, which is a national stage application of PCT/SE96/01238, filed on Oct. 2, 1996. The present application claims the benefits of the filing dates of each of the aforementioned applications and incorporates them by reference as if they were each set forth fully herein.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5180810 | Gomi et al. | Jan 1993 | A |
| Number | Date | Country |
|---|---|---|
| 367 890 | May 1990 | EP |
| 371 199 | Oct 1994 | EP |
| WO 9119740 | Dec 1991 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20060205925 A1 | Sep 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10374535 | Feb 2003 | US |
| Child | 11418770 | US | |
| Parent | 09051380 | US | |
| Child | 10374535 | US |
