Patent Application
20070110763
The present invention is related to SHB-GAS-102, SHB-GAS-103, and SHB-GAS-104 polypeptides of S. pyogenes (Group A Streptococcus) and corresponding DNA fragments, which may be used to prevent, diagnose and/or treat S. pyogenes infections.
Streptococci are gram (+) bacteria which are differentiated by group specific carbohydrate antigens A through O which are found at the cell surface. S. pyogenes isolates are further distinguished by type-specific M protein antigens. M proteins are important virulence factors which are highly variable both in molecular weights and in sequences. Indeed, more than 100-M protein types have been identified on the basis of antigenic differences.
S. pyogenes is responsible for many diverse infection types, including pharyngitis, erysipelas and impetigo, scarlet fever, and invasive diseases such as bacteremia and necrotizing fasciitis. A resurgence of invasive disease in recent years has been documented in many countries, including those in North America and Europe. Although the organism is sensitive to antibiotics, the high attack rate and rapid onset of sepsis results in high morbidity and mortality.
To develop a vaccine that will protect individuals from S. pyogenes infection, efforts have focused on virulence factors such as the type-specific M proteins. However, the carboxy-terminal portion of M proteins was found to induce cross-reactive antibodies which reacted with human myocardium, tropomyosin, myosin, and vimentin, which might be implicated in autoimmune diseases. Others have used recombinant techniques to produce complex hybrid proteins containing amino-terminal peptides of M proteins from different serotypes. However, a safe vaccine containing all S. pyogenes serotypes will be highly complex to produce and standardize.
In addition to the serotype-specific antigens, other S. pyogenes proteins have generated interest as potential vaccine candidates. The C5a peptidase, which is expressed by at least S. pyogenes 40 serotypes, was shown to be immunogenic in mice, but its capacity to reduce the level of nasopharyngeal colonization was limited. Other investigators have also focused on the streptococcal pyrogenic exotoxins which appear to play an important role in pathogenesis of infection. Immunization with these proteins prevented the deadly symptoms of toxic shock, but did not prevent colonization.
Therefore there remains an unmet need for S. pyogenes antigens that may be used as vaccine components for the prophylaxis and/or therapy of S. pyogenes infection.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising SEQ ID No : 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
According to one aspect, the present invention relates to polypeptides comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
In other aspects, there are provided polypeptides encoded by polynucleotides of the invention, pharmaceutical compositions, vectors comprising polynucleotides of the invention operably linked to an expression control region, as well as host cells transfected with said vectors and processes for producing polypeptides comprising culturing said host cells under conditions suitable for expression.
The present invention provides purified and isolated polynucleotides, which encode S. pyogenes polypeptides which may be used to prevent, diagnose and/or treat S. pyogenes infection.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 90% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 98% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 99% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 70% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 80% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 90% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 95% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 98% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44.
According to one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide having at least 99% identity to a second polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44.
According to one aspect, the present invention relates to polypeptides comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
According to one aspect, the present invention relates to polypeptides comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44.
According to one aspect, the present invention provides a polynucleotide encoding an epitope bearing portion of a polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
According to one aspect, the present invention provides a polynucleotide encoding an epitope bearing portion of a polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44
According to one aspect, the present invention relates to epitope bearing portions of a polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
According to one aspect, the present invention relates to epitope bearing portions of a polypeptide comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44.
According to one aspect, the present invention provides an isolated polynucleotide comprising a polynucleotide chosen from:
According to one aspect, the present invention provides an isolated polynucleotide comprising a polynucleotide chosen from:
According to one aspect, the present invention provides an isolated polynucleotide consisting essentially of a polynucleotide chosen from:
According to one aspect, the present invention provides an isolated polynucleotide consisting essentially of a polynucleotide chosen from:
According to one aspect, the present invention provides an isolated polypeptide comprising a polypeptide chosen from:
According to one aspect, the present invention provides an isolated polypeptide comprising a polypeptide chosen from:
According to one aspect, the present invention provides an isolated polypeptide consisting essentially of a polypeptide chosen from:
According to one aspect, the present invention provides an isolated polypeptide comprising a polypeptide chosen from:
Those skilled in the art will appreciate that the invention includes DNA molecules, i.e. polynucleotides and their complementary sequences that encode analogs such as mutants, variants, homologues and derivatives of such polypeptides, as described herein in the present patent application. The invention also includes RNA molecules corresponding to the DNA molecules of the invention. In addition to the DNA and RNA molecules, the invention includes the corresponding polypeptides and monospecific antibodies that specifically bind to such polypeptides.
In accordance with the present invention, all polynucleotides encoding polypeptides of the present invention are within the scope of the present invention.
In a further embodiment, the polypeptides in accordance with the present invention are antigenic, i.e. are able to bind specifically to components of the immune response, such as antibodies and lymphocytes.
In a further embodiment, the polypeptides in accordance with the present invention are immunogenic or can elicit an immune response in a host.
In a further embodiment, the present invention also relates to polypeptides which are able to raise antibodies having binding specificity to the polypeptides of the present invention as defined above.
An antibody that “has binding specificity” is an antibody that recognizes and binds the selected polypeptide but which does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, which naturally includes the selected peptide. Specific binding can be measured using an ELISA assay in which the selected polypeptide is used as an antigen.
In a further embodiment, the polypeptides in accordance with the present invention can elicit a B cell response in a host.
In a further embodiment, the polypeptides in accordance with the present invention can elicit a T cell response in a host.
In accordance with the present invention, the polypeptides of the invention can also be effective when administered to an host to protect against the bacteria. “Protection” in the biological studies is defined by a significant increase in the survival curve, rate or period. Statistical analysis using the Log rank test to compare survival curves, and Fisher exact test to compare survival rates and numbers of days to death, respectively, can be used to calculate P values and determine whether the difference between the two groups is statistically significant. P values greater than 0.05 are regarded as not significant.
In an additional aspect of the invention there are provided antigenic/immunogenic fragments of the polypeptides of the invention, or of analogs thereof.
The present invention also relates to fragments which are specific to, or for SEQ ID NOS. 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 or 44. A specific fragment contains a defined order of amino acids which occurs in a target polypeptide, and which is characteristic of that target polypeptide, but substantially no other non-target polypeptides. Such polypeptide fragments can be of any size which is necessary to confer specificity, e.g., comprising or consisting of at least 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 amino acids, etc.
Specific fragments can also be described as being specific for Streptococcus pyogenes, indicating that it occurs in that bacteria, but not in other organisms, especially not in GBS or other bacteria.
The fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain substantially their antigenic/immunogenic properties. Thus, for fragments according to the present invention the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a polypeptide or analog thereof as described herein.
The present invention further provides fragments having a smaller sequence than the ones described in the figures.
The present invention further provides fragments having at least 10 contiguous amino acid residues from the polypeptide sequences of the present invention.
In one embodiment, at least 15 contiguous amino acid residues.
In one embodiment, at least 20 contiguous amino acid residues.
In one embodiment, at least 25 contiguous amino acid residues.
In one embodiment, at least 30 contiguous amino acid residues.
In one embodiment, at least 35 contiguous amino acid residues.
In one embodiment, at least 40 contiguous amino acid residues.
In one embodiment, at least 45 contiguous amino acid residues.
In one embodiment, at least 50 contiguous amino acid residues.
In one embodiment, at least 55 contiguous amino acid residues.
In one embodiment, at least 60 contiguous amino acid residues.
In one embodiment, at least 65 contiguous amino acid residues.
In one embodiment, at least 70 contiguous amino acid residues.
In one embodiment, at least 75 contiguous amino acid residues.
In one embodiment, at least 80 contiguous amino acid residues.
In one embodiment, at least 85 contiguous amino acid residues.
In one embodiment, at least 90 contiguous amino acid residues.
In one embodiment, at least 95 contiguous amino acid residues.
In one embodiment, at least 100 contiguous amino acid residues.
In one embodiment, at least 105 contiguous amino acid residues.
In one embodiment, at least 110 contiguous amino acid residues.
In one embodiment, at least 115 contiguous amino acid residues.
In one embodiment, at least 120 contiguous amino acid residues.
In one embodiment, at least 125 contiguous amino acid residues.
In one embodiment, at least 130 contiguous amino acid residues.
In one embodiment, at least 135 contiguous amino acid residues.
In one embodiment, at least 140 contiguous amino acid residues.
In one embodiment, at least 145 contiguous amino acid residues.
In one embodiment, at least 150 contiguous amino acid residues.
In one embodiment, at least 155 contiguous amino acid residues.
In one embodiment, at least 160 contiguous amino acid residues.
In one embodiment, at least 165 contiguous amino acid residues.
In one embodiment, at least 170 contiguous amino acid residues.
In one embodiment, at least 175 contiguous amino acid residues.
In one embodiment, at least 180 contiguous amino acid residues.
In one embodiment, at least 185 contiguous amino acid residues.
In one embodiment, at least 190 contiguous amino acid residues.
In one embodiment, at least 195 contiguous amino acid residues.
In one embodiment, at least 200 contiguous amino acid residues.
In one embodiment, at least 205 contiguous amino acid residues.
In one embodiment, at least 210 contiguous amino acid residues.
In one embodiment, at least 215 contiguous amino acid residues.
In one embodiment, at least 220 contiguous amino acid residues.
In one embodiment, at least 225 contiguous amino acid residues.
In one embodiment, at least 230 contiguous amino acid residues.
In one embodiment, at least 235 contiguous amino acid residues.
In one embodiment, at least 240 contiguous amino acid residues.
In one embodiment, at least 245 contiguous amino acid residues.
In one embodiment, at least 250 contiguous amino acid residues.
In one embodiment, at least 255 contiguous amino acid residues.
In one embodiment, at least 260 contiguous amino acid residues.
In one embodiment, at least 265 contiguous amino acid residues.
In one embodiment, at least 270 contiguous amino acid residues.
In one embodiment, at least 275 contiguous amino acid residues.
In one embodiment, at least 280 contiguous amino acid residues.
In one embodiment, at least 285 contiguous amino acid residues.
In one embodiment, at least 290 contiguous amino acid residues.
In one embodiment, at least 295 contiguous amino acid residues.
In one embodiment, at least 300 contiguous amino acid residues.
In one embodiment, at least 305 contiguous amino acid residues.
In one embodiment, at least 310 contiguous amino acid residues.
In one embodiment, at least 315 contiguous amino acid residues.
In one embodiment, at least 320 contiguous amino acid residues.
In one embodiment, at least 325 contiguous amino acid residues.
In one embodiment, at least 330 contiguous amino acid residues.
In one embodiment, at least 335 contiguous amino acid residues.
In one embodiment, at least 340 contiguous amino acid residues.
In one embodiment, at least 345 contiguous amino acid residues.
In one embodiment, at least 350 contiguous amino acid residues.
In one embodiment, at least 355 contiguous amino acid residues.
In one embodiment, at least 360 contiguous amino acid residues.
In one embodiment, at least 365 contiguous amino acid residues.
In one embodiment, at least 370 contiguous amino acid residues.
In one embodiment, at least 375 contiguous amino acid residues.
In one embodiment, at least 380 contiguous amino acid residues.
In one embodiment, at least 385 contiguous amino acid residues.
In one embodiment, at least 390 contiguous amino acid residues.
In one embodiment, at least 395 contiguous amino acid residues.
In one embodiment, at least 400 contiguous amino acid residues.
In one embodiment, at least 405 contiguous amino acid residues.
In one embodiment, at least 410 contiguous amino acid residues.
In one embodiment, at least 415 contiguous amino acid residues.
In one embodiment, at least 420 contiguous amino acid residues.
Polypeptide fragments of the invention may be of any size that is compatible with the invention. They may range in size from the smallest specific epitope (e.g., about 6 amino acids) to a nearly full-length gene product (e.g., a single amino acid shorter than SEQ ID Nos: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 or 44).
The skilled person will appreciate that analogs of the polypeptides of the invention will also find use in the context of the present invention, i.e. as antigenic/immunogenic material. Thus, for instance proteins or polypeptides which include one or more additions, deletions, substitutions or the like are encompassed by the present invention.
As used herein, “fragments”, “analogs” or “derivatives” of the polypeptides of the invention include those polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably conserved) and which may be natural or unnatural.
These substitutions are those having a minimal influence on the secondary structure and hydropathic nature of the polypeptide. Preferred substitutions are those known in the art as conserved, i.e. the substituted residues share physical or chemical properties such as hydrophobicity, size, charge or functional groups. These include substitutions such as those described by Dayhoff, M. in Atlas of Protein Sequence and Structure 5, 1978 and by Argos, P. in EMBO J. 8, 779-785, 1989. For example, amino acids, either natural or unnatural, belonging to one of the following groups represent conservative changes:
The preferred substitutions also include substitutions of D-enantiomers for the corresponding L-amino acids.
In one embodiment, preferred base substitutions are located where there is less sequence homology on
In one embodiment, preferred amino acids substitutions are located where there is less sequence homology on
The additional amino acid residues may be from a heterologous source or may be endogenous to the natural gene.
In an alternative approach, the analogs could be fusion polypeptides, incorporating moieties which render purification easier, for example by effectively tagging the desired polypeptide. It may be necessary to remove the “tag” or it may be the case that the fusion polypeptide itself retains sufficient antigenicity to be useful.
The percentage of homology is defined as the sum of the percentage of identity plus the percentage of similarity or conservation of amino acid type.
In one embodiment, analogs of polypeptides of the invention will have about 70% identity with those sequences illustrated in the figures or fragments thereof. That is, 70% of the residues are the same. In a further embodiment, polypeptides will have greater than 80% identity. In a further embodiment, polypeptides will have greater than 85% identity. In a further embodiment, polypeptides will have greater than 90% identity. In a further embodiment, polypeptides will have greater than 95% identity. In a further embodiment, polypeptides will have greater than 98% identity. In a further embodiment, polypeptides will have greater than 99% identity. In a further embodiment, analogs of polypeptides of the invention will have fewer than about 20 amino acid residue substitutions, modifications or deletions and more preferably less than 10.
In one embodiment, analogs of polypeptides of the invention will have about 70% similarity with those sequences illustrated in the figures or fragments thereof. That is, 70% of the residues are the same. In a further embodiment, polypeptides will have greater than 80% similarity. In a further embodiment, polypeptides will have greater than 85% similarity. In a further embodiment, polypeptides will have greater than 90% similarity. In a further embodiment, polypeptides will have greater than 95% similarity. In a further embodiment, polypeptides will have greater than 98% similarity. In a further embodiment, polypeptides will have greater than 99% similarity. In a further embodiment, analogs of polypeptides of the invention will have fewer than about 20 amino acid residue substitutions, modifications or deletions and more preferably less than 10.
In one embodiment, analogs of polypeptides of the invention will have about 70% homology with those sequences illustrated in the figures or fragments thereof. In a further embodiment, polypeptides will have greater than 80% homology. In a further embodiment, polypeptides will have greater than 85% homology. In a further embodiment, polypeptides will have greater than 90% homology. In a further embodiment, polypeptides will have greater than 95% homology. In a further embodiment, polypeptides will have greater than 98% homology. In a further embodiment, polypeptides will have greater than 99% homology. In a further embodiment, analogs of polypeptides of the invention will have fewer than about 20 amino acid residue substitutions, modifications or deletions and more preferably less than 10.
One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or homology for an optimal alignment. A program like BTLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.
In an alternative approach, the analogs or derivatives could be fusion polypeptides, incorporating moieties which render purification easier, for example by effectively tagging the desired protein or polypeptide, it may be necessary to remove the “tag” or it may be the case that the fusion polypeptide itself retains sufficient antigenicity to be useful.
It is well known that it is possible to screen an antigenic polypeptide to identify epitopic regions, i.e. those regions which are responsible for the polypeptide's antigenicity or immunogenicity. Methods for carrying out such screening are well known in the art. Thus, the fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties.
The invention also encompasses polypeptides having a lower degree of sequence identity, but having sufficient similarity so as to perform one or more of the functions or activities exhibited by the native polypeptides.
Thus, what is important for analogs, derivatives and fragments is that they possess at least a degree of the antigenicity/immunogenicity of the protein or polypeptide from which they are derived.
Also included are polypeptides in which one or more of the amino acid residues includes a substituent group. These polypeptides include, e.g., modified polypeptides. Known polypeptide modifications include, but are not limited to, glycosylation, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formatin, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
Such modifications are well-known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in many basic texts, such as Proteins—Structure and Molecular Properties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslationail Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (1990) Meth. Enzymol. 182:626-646 and Rattan et al. (1992) Ann. N.Y. Acad. Sci. 663:48-62.
Also included are polypeptides which have fused thereto other compounds which alter the polypeptides biological or pharmacological properties i.e. polyethylene glycol (PEG) to increase half-life; leader or secretory amino acid sequences for ease of purification; prepro- and pro-sequences; and (poly)saccharides.
Furthermore, in those situations where amino acid regions are found to be polymorphic, it may be desirable to vary one or more particular amino acids to more effectively mimic the different epitopes of the different S. pyogenes strains.
Moreover, the polypeptides of the present invention can be modified by terminal —NH2 acylation (eg. by acetylation, or thioglycolic acid amidation, terminal carboxy amidation, e.g. with ammonia or methylamine) to provide stability, increased hydrophobicity for linking or binding to a support or other molecule.
Also contemplated are hetero and homo polypeptide multimers of the polypeptide fragments and analogues. These polymeric forms include, for example, one or more polypeptides that have been cross-linked with cross-linkers such as avidin/biotin, gluteraldehyde or dimethylsuperimidate. Such polymeric forms also include polypeptides containing two or more tandem or inverted contiguous sequences, produced from multicistronic mRNAs generated by recombinant DNA technology.
In a further embodiment, the present invention also relates to chimeric polypeptides which comprise one or more polypeptides or fragments or analogs thereof as defined in the figures of the present application.
In a further embodiment, the present invention also relates to chimeric polypeptides comprising two or more polypeptides comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof; provided that the polypeptides are linked as to form a chimeric polypeptide.
In a further embodiment, the present invention also relates to chimeric polypeptides comprising two or more polypeptides comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 provided that the polypeptides are linked as to form a chimeric polypeptide.
Preferably, a fragment, analog or derivative of a polypeptide of the invention will comprise at least one antigenic region i.e. at least one epitope.
In order to achieve the formation of antigenic polymers (i.e. synthetic multimers), polypeptides may be utilized having bishaloacetyl groups, nitroarylhalides, or the like, where the reagents being specific for thio groups. Therefore, the link between two mercapto groups of the different polypeptides may be a single bond or may be composed of a linking group of at least two, typically at least four, and not more than 16, but usually not more than about 14 carbon atoms.
In a particular embodiment, polypeptide fragments and analogs of the invention do not contain a starting residue, such as methionine (Met) or valine (Val). Preferably, polypeptides will not incorporate a leader or secretory sequence (signal sequence). The signal portion of a polypeptide of the invention may be determined according to established molecular biological techniques. In general, the polypeptide of interest may be isolated from a S. pyogenes culture and subsequently sequenced to determine the initial residue of the mature protein and therefore the sequence of the mature polypeptide.
It is understood that polypeptides can be produced and/or used without their start codon (methionine or valine) and/or without their leader peptide to favor production and purification of recombinant polypeptides. It is known that cloning genes without sequences encoding leader peptides will restrict the polypeptides to the cytoplasm of E. coli and will facilitate their recovery (Glick, B. R. and Pasternak, J. J. (1998) Manipulation of gene expression in prokaryotes. In “Molecular biotechnology: Principles and applications of recombinant DNA”, 2nd edition, ASM Press, Washington D.C., p.109-143).
In another embodiment, the polypeptides of the invention may be lacking an N-terminal leader peptide, and/or a transmembrane domain and/or a C-terminal anchor domain.
The present invention further provides a fragment of the polypeptide comprising substantially all of the extra cellular domain of a polypeptide which has at least 70% identify, preferably 80% identity, more preferably 95% identity, to a sequence chosen from SEQ ID NOs: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof, over the entire length of said sequence.
According to another aspect of the invention, there are also provided (i) a composition of matter containing a polypeptide of the invention, together with a carrier, diluent or adjuvant; (ii) a pharmaceutical composition comprising a polypeptide of the invention and a carrier, diluent or adjuvant; (iii) a vaccine comprising a polypeptide of the invention and a carrier, diluent or adjuvant; (iv) a method for inducing an immune response against S. pyogenes, in a host, by administering to the host, an immunogenically effective amount of a polypeptide of the invention to elicit an immune response, e.g., a protective immune response to S. pyogenes; and particularly, (v) a method for preventing and/or treating a S. pyogenes infection, by administering a prophylactic or therapeutic amount of a polypeptide of the invention to a host in need.
According to another aspect of the invention, there are also provided (i) a composition of matter containing a polynucleotide of the invention, together with a carrier, diluent or adjuvant; (ii) a pharmaceutical composition comprising a polynucleotide of the invention and a carrier, diluent or adjuvant; (iii) a method for inducing an immune response against S. pyogenes, in a host, by administering to the host, an immunogenically effective amount of a polynucleotide of the invention to elicit an immune response, e.g., a protective immune response to S. pyogenes; and particularly, (iv) a method for preventing and/or treating a S. pyogenes infection, by administering a prophylactic or therapeutic amount of a polynucleotide of the invention to a host in need.
Before immunization, the polypeptides of the invention can also be coupled or conjugated to carrier proteins such as tetanus toxin, diphtheria toxin, hepatitis B virus surface antigen, poliomyelitis virus VP1 antigen or any other viral or bacterial toxin or antigen or any suitable proteins to stimulate the development of a stronger immune response. This coupling or conjugation can be done chemically or genetically. A more detailed description of peptide-carrier conjugation is available in Van Regenmortel, M. H. V., Briand J. P., Muller S., Plaué S., <<Synthetic Polypeptides as antigens>> in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 19 (ed.) Burdou, R. H. & Van Knippenberg P. H. (1988), Elsevier N.Y.
According to another aspect, there are provided pharmaceutical compositions comprising one or more S. pyogenes polypeptides or chimeric polypeptides of the invention in a mixture with a pharmaceutically acceptable carrier or diluent.
According to another aspect, there are provided pharmaceutical compositions comprising one or more S. pyogenes polypeptides or chimeric polypeptides of the invention in a mixture with a pharmaceutically acceptable adjuvant. Suitable adjuvants include (1) oil-in-water emulsion formulations such as MF59™, SAF™, Ribi™; (2) Freund's complete or incomplete adjuvant; (3) salts i.e. AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4)2, Al(OH)3, AlPO4, silica, kaolin; (4) saponin derivatives such as Stimulon™ or particles generated therefrom such as ISCOMs (immunostimulating complexes); (5) cytokines such as interleukins, interferons, macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF); (6) other substances such as carbon polynucleotides i.e. poly IC and poly AU, detoxified cholera toxin (CTB)and E. coli heat labile toxin for induction of mucosal immunity; and (7) liposomes. A more detailed description of adjuvants is available in a review by M. Z. I Khan et al. in Pharmaceutical Research, vol. 11, No. 1 (1994) pp 2-11, and also in another review by Gupta et al., in Vaccine, Vol. 13, No. 14, pp 1263-1276 (1995) and in WO 99/24578. Preferred adjuvants include QuilA™, QS21™, Alhydrogel™ and Adjuphos™.
Pharmaceutical compositions of the invention may be administered parenterally by injection, rapid infusion, nasopharyngeal absorption, dermoabsorption, or buccal or oral.
The term pharmaceutical composition is also meant to include antibodies. In accordance with the present invention, there is also provided the use of one or more antibodies having binding specificity for the polypeptides of the present invention for the treatment or prophylaxis of streptococcus infection and/or diseases and symptoms mediated by streptococcus infection.
Pharmaceutical compositions of the invention are used for the prophylaxis of S. pyogenes infection and/or diseases and symptoms mediated by S. pyogenes infection as described in Manual of Clinical Microbiology, P. R. Murray (Ed, in chief), E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken. ASM Press, Washington, D.C. seventh edition, 1999, 1773 p.
In one embodiment, pharmaceutical compositions of the present invention are used for the prophylactic or therapeutic treatment or of many diverse infection types, including pharyngitis, erysipelas and impetigo, scarlet fever, and invasive diseases such as bacteremia and necrotizing fasciitis.
In one embodiment, pharmaceutical compositions of the invention are used for the prophylactic or therapeutic treatment of Streptococcus infection and/or diseases and symptoms mediated by Streptococcus infection, in particular group A Streptococcus (Streptococcus pyogenes), group B Streptococcus (GBS or S. agalactiae), S. pneumoniae, S. dysgalactiae, S. uberis, S. nocardia as well as Staphylococcus aureus.
In one embodiment, pharmaceutical compositions of the invention are used for the prophylactic or therapeutic treatment of S. pyogenes infection and/or diseases and symptoms mediated by S. pyogenes infection. In a further embodiment, the S. pyogenes infection is nontypeable S. pyogenes.
Pharmaceutical compositions can also be specific or selective for one or more of the mentioned bacteria. For example, a composition can be selective or specific for a group A Streptococcus (Streptococcus pyogenes) and not react with group B Streptococcus (GBS or S. agalactiae), S. pneumoniae, S. dysgalactiae, S. uberis, S. nocardia nor Staphylococcus aureus.
In a further embodiment, the invention provides a method for prophylactic or therapeutic treatment of streptococcus infection in a host susceptible to streptococcus infection comprising administering to said host a prophylactic or therapeutic amount of a composition of the invention.
In a further embodiment, the invention provides a method for prophylactic or therapeutic treatment of S. pyogenes infection in a host susceptible to S. pyogenes infection comprising administering to said host a prophylactic or therapeutic amount of a composition of the invention.
As used in the present application, the term “host” includes mammals. In a further embodiment, the mammal is human.
In a particular embodiment, pharmaceutical compositions are administered to those hosts at risk of S. pyogenes infection such as neonates, infants, children, elderly and immunocompromised hosts.
In a particular embodiment, pharmaceutical compositions are administered to those hosts at risk of S. pyogenes infection such as adults.
Pharmaceutical compositions are preferably in unit dosage form of about 0.001 to 100 μg/kg (antigen/body weight) and more preferably 0.01 to 10 μg/kg and most preferably 0.1 to 1 μg/kg 1 to 3 times with an interval of about 1 to 6 week intervals between immunizations.
Pharmaceutical compositions are preferably in unit dosage form of about 0.1 μg to 10 mg and more preferably 1 μg to 1 mg and most preferably 10 to 100 μg 1 to 3 times with an interval of about 1 to 6 week intervals between immunizations.
According to another aspect, there are provided polynucleotides encoding polypeptides characterized by the amino acid sequence comprising SEQ ID No: 2, 4, 6, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or fragments or analogs thereof.
In one embodiment, polynucleotides are those illustrated in SEQ ID No: 1, 3, 5, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 which may include the open reading frames (ORF), encoding the polypeptides of the invention.
Many types of variants of polynucleotides are encompassed by the invention including, e.g., (i) one in which one or more of the nucleotides is substituted with another nucleotide, or which is otherwise mutated; or (ii) one in which one or more of the nucleotides is modified, e.g., includes a subtituent group; or (iii) one in which the polynucleotide is fused with another compound, such as a compound to increase the half-life of the polynucleotide; or (iv) one in which additional nucleotides are covalently bound to the polynucleotide, such a sequences encoding a leader or secretory sequence or a sequence which is employed for purification of the polypeptide. The additional nucleotides may be from a heterologous source, or may be endogenous to the natural gene.
Polynucleotide variants belonging to type (i) above include, e.g., polymorphisms, including single nucleotide polymorphisms (SNPs), and mutants. Variant polynucleotides can comprise, e.g., one or more additions, insertions, deletions, substitutions, transitions, transversions, inversions, or the like, or any combinations thereof.
Polynucleotide variants belonging to type (ii) above include, e.g., modifications such as the attachment of detectable markers (avidin, biotin, radioactive elements, fluorescent tags and dyes, energy transter labels, energy-emitting labels, binding partners, etc.) or moieties which improve expression, uptake, cataloging, tagging, hybridization, detection, and/or stability. The polynucleotides can also be attached to solid supports, e.g., nitrocellulose, magnetic or paramagnetic microspheres (e.g., as described in U.S. Pat. No. 5,411,863; U.S. Pat. No. 5,543,289; for instance, comprising ferromagnetic, supermagnetic, paramagnetic, superparamagnetic, iron oxide and polysaccharide), nylon, agarose, diazotized cellulose, latex solid microspheres, polyacrylamides, etc., according to a desired method. See, e.g., U.S. Pat. Nos. 5,470,967; 5,476,925; 5,478,893.
Polynucleotide variants belonging to type (iii) above are well known in the art and include, e.g., various lengths of polyA+ tail, 5′ cap structures, and nucleotide analogs, e.g., inosine, thionucleotides, or the like.
Polynucleotide variants belonging to type (iv) above include, e.g., a variety of chimeric, hybrid or fusion polynucleotides. For example a polynucleotide of the invention can comprise a coding sequence and additional non-naturally occurring or heterologous coding sequence (e.g., sequences coding for leader, signal, secretory, targeting, enzymatic, fluorescent, antibiotic resistance, and other functional or diagnostic peptides); or a coding sequence and non-coding sequences, e.g., untranslated sequences at either a 5′ or 3′ end, or dispersed in the coding sequence.
It will be appreciated that the polynucleotide sequences illustrated in the figures may be altered with degenerate codons yet still encode the polypeptides of the invention. Accordingly the present invention further provides polynucleotides which hybridize to the polynucleotide sequences herein above described (or the complement sequences thereof) having 70% identity between sequences. In one embodiment, at least 80% identity between sequences. In one embodiment, at least 85% identity between sequences. In one embodiment, at least 90% identity between sequences. In a further embodiment, polynucleotides are hybridizable under stringent conditions i.e. having at least 95% identity. In a further embodiment, more than 97% identity. In a further embodiment, more than 98% identity. In a further embodiment, more than 99% identity. In a further embodiment, polynucleotides are hybridizable under stringent conditions.
Suitable stringent conditions for hybridization can be readily determined by one of skilled in the art (see for example Sambrook et al., (1989) Molecular cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology, (1999) Edited by Ausubel F. M. et al., John Wiley & Sons, Inc., New York).
“Suitable stringent conditions”, as used herein, means, for example, incubating a blot overnight (e.g., at least 12 hours) with a long polynucleotide probe in a hybridization solution containing, e.g., about 5×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 50% formamide, at 42° C. Blots can be washed at high stringency conditions that allow, e.g., for less than 5% bp mismatch (e.g., wash twice in 0.1×SSC and 0.1% SDS for 30 min at 65° C.), thereby selecting sequences having, e.g., 95% or greater sequence identity.
Other non-limiting examples of suitable stringent conditions include a final wash at 65° C. in aqueous buffer containing 30 mM NaCl and 0.5% SDS. Another example of suitable stringent conditions is hybridization in 7% SDS, 0.5 M NaPO4, pH 7, 1 mM EDTA at 50° C., e.g., overnight, followed by one or more washes with a 1% SDS solution at 42° C. Whereas high stringency washes can allow for less than 5% mismatch, reduced or low stringency conditions can permit up to 20% nucleotide mismatch. Hybridization at low stringency can be accomplished as above, but using lower formamide conditions, lower temperatures and/or lower salt concentrations, as well as longer periods of incubation time.
According to another aspect of the invention, there are also provided isolated polynucleotides comprising a sequence that hybridize under stringent conditions to either
In a further embodiment, the present invention provides polynucleotides that hybridize under stringent conditions to either
According to another aspect of the invention, there are also provided isolated polynucleotides comprising a sequence that hybridize under stringent conditions to either
In a further embodiment, the present invention provides polynucleotides that hybridize under stringent conditions to either
In a further embodiment, the present invention provides polynucleotides that hybridize under stringent conditions to either
In a further embodiment, the present invention provides polynucleotides that hybridize under stringent conditions to either
In a further embodiment, the present invention provides polynucleotides that hybridize under stringent conditions to either
In a further embodiment, the present invention provides polynucleotides that hybridize under stringent conditions to either
In a further embodiment, polynucleotides are those illustrated in SEQ ID NO: 1, 3, 5, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 or fragments or analogs thereof encoding polypeptides of the invention.
In a further embodiment, polynucleotides are those illustrated in SEQ ID NO: 1, 3, 5, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 encoding polypeptides of the invention.
As will be readily appreciated by one skilled in the art, polynucleotides include both DNA and RNA.
The present invention also includes polynucleotides complementary to the polynucleotides described in the present application.
In a further aspect, polynucleotides encoding polypeptides of the invention, or fragments, analogs or derivatives thereof, may be used in a DNA immunization method. That is, they can be incorporated into a vector which is replicable and expressible upon injection thereby producing the antigenic polypeptide in vivo. For example polynucleotides may be incorporated into a plasmid vector under the control of the CMV promoter which is functional in eukaryotic cells. Preferably the vector is injected intramuscularly.
According to another aspect, there is provided a process for producing polypeptides of the invention by recombinant techniques by expressing a polynucleotide encoding said polypeptide in a host cell and recovering the expressed polypeptide product.
Alternatively, the polypeptides can be produced according to established synthetic chemical techniques i.e. solution phase or solid phase synthesis of oligopeptides which are ligated to produce the full polypeptide (block ligation).
General methods for obtention and evaluation of polynucleotides and polypeptides are described in the following references: Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F. M. et al., John Wiley and Sons, Inc. New York; PCR Cloning Protocols, from Molecular Cloning to Genetic Engineering, Edited by White B. A., Humana Press, Totowa, N.J., 1997, 490 pages; Protein Purification, Principles and Practices, Scopes R. K., Springer-Verlag, New York, 3rd Edition, 1993, 380 pages; Current Protocols in Immunology, Edited by Coligan J. E. et al., John Wiley & Sons Inc., New York.
The present invention provides a vector comprising a polynucleotide of the invention, wherein said DNA is operably linked to an expression control region.
The present invention provides a host cell transfected with the vector of the invention.
The present invention provides a process for producing a polypeptide comprising culturing a host cell of the invention under conditions suitable for expression of said polypeptide.
For recombinant production, host cells are transfected with vectors which encode the polypeptides of the invention, and then cultured in a nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes. Suitable vectors are those that are viable and replicable in the chosen host and include chromosomal, non-chromosomal and synthetic DNA sequences e.g. bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA. The polypeptide sequence may be incorporated in the vector at the appropriate site using restriction enzymes such that it is operably linked to an expression control region comprising a promoter, ribosome binding site (consensus region or Shine-Dalgarno sequence), and optionally an operator (control element). One can select individual components of the expression control region that are appropriate for a given host and vector according to established molecular biology principles (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F. M. et al., John Wiley and Sons, Inc. New York). Suitable promoters include but are not limited to LTR or SV40 promoter, E. coli lac, tac or trp promoters and the phage lambda PL promoter. Vectors will preferably incorporate an origin of replication as well as selection markers i.e. ampicilin resistance gene. Suitable bacterial vectors include pET, pQE70, pQE60, pQE-9, pD10 phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 and eukaryotic vectors pBlueBacIII, pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG and pSVL. Host cells may be bacterial i.e. E. coli, Bacillus subtilis, Streptomyces; fungal i.e. Aspergillus niger, Aspergillus nidulins; yeast i.e. Saccharomyces or eukaryotic i.e. CHO, COS.
Upon expression of the polypeptide in culture, cells are typically harvested by centrifugation then disrupted by physical or chemical means (if the expressed polypeptide is not secreted into the media) and the resulting crude extract retained to isolate the polypeptide of interest. Purification of the polypeptide from culture media or lysate may be achieved by established techniques depending on the properties of the polypeptide i.e. using ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and lectin chromatography. Final purification may be achieved using HPLC.
The polypeptides may be expressed with or without a leader or secretion sequence. In the former case the leader may be removed using post-translational processing (see U.S. Pat. No. 4,431,739; U.S. Pat. No. 4,425,437; and U.S. Pat. No. 4,338,397) or be chemically removed subsequent to purifying the expressed polypeptide.
According to a further aspect, the S. pyogenes polypeptides of the invention may be used in a diagnostic test for Streptococcus infection, in particular S. pyogenes infection.
Several diagnostic methods are possible, for example detecting S. pyogenes organism in a biological sample, the following procedure may be followed:
Alternatively, a method for the detection of antibody specific to a S. pyogenes antigen in a biological sample containing or suspected of containing said antibody may be performed as follows:
One of skill in the art will recognize that this diagnostic test may take several forms, including an immunological test such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay or a latex agglutination assay, essentially to determine whether antibodies specific for the polypeptide are present in an organism.
The DNA sequences encoding polypeptides of the invention may also be used to design DNA probes f or use in detecting the presence of S. pyogenes in a biological sample suspected of containing such bacteria. The detection method of this invention comprises:
The DNA probes of this invention may also be used for detecting circulating S. pyogenes i.e. S. pyogenes nucleic acids in a sample, for example using a polymerase chain reaction, as a method of diagnosing S. pyogenes infections. The probe may be synthesized using conventional techniques and may be immobilized on a solid phase, or may be labelled with a detectable label. A preferred DNA probe for this application is an oligomer having a sequence complementary to at least about 6 contiguous nucleotides of the S. pyogenes polypeptides of the invention. In a further embodiment, the preferred DNA probe will be an oligomer having a sequence complementary to at least about 15 contiguous nucleotides of the S. pyogenes polypeptides of the invention. In a further embodiment, the preferred DNA probe will be an oligomer having a sequence complementary to at least about 30 contiguous nucleotides of the S. pyogenes polypeptides of the invention. In a further embodiment, the preferred DNA probe will be an oligomer having a sequence complementary to at least about 50 contiguous nucleotides of the S. pyogenes polypeptides of the invention.
Another diagnostic method for the detection of S. pyogenes in a host comprises:
A further aspect of the invention is the use of the S. pyogenes polypeptides of the invention as immunogens for the production of specific antibodies for the diagnosis and in particular the treatment of S. pyogenes infection. Suitable antibodies may be determined using appropriate screening methods, for example by measuring the ability of a particular antibody to passively protect against S. pyogenes infection in a test model. One example of an animal model is the mouse model described in the examples herein. The antibody may be a whole antibody or an antigen-binding fragment thereof and may belong to any immunoglobulin class. The antibody or fragment may be of animal origin, specifically of mammalian origin and more specifically of murine, rat or human origin. It may be a natural antibody or a fragment thereof, or if desired, a recombinant antibody or antibody fragment. The term recombinant antibody or antibody fragment means antibody or antibody fragment which was produced using molecular biology techniques. The antibody or antibody fragments may be polyclonal, or preferably monoclonal. It may be specific for a number of epitopes associated with the S. pyogenes polypeptides but is preferably specific for one.
According to one aspect, the present invention provides the use of an antibody for treatment and/or prophylaxis of S. pyogenes infections.
A further aspect of the invention is the use of the antibodies directed to the polypeptides of the invention for passive immunization. One could use the antibodies described in the present application.
A further aspect of the invention is a method for immunization, whereby an antibody raised by a polypeptide of the invention is administered to a host in an amount sufficient to provide a passive immunization.
The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method or system such as direct injection of plasmid DNA into muscles [Wolf et al. H M G (1992) 1: 363; Turnes et al., Vaccine (1999), 17: 2089; Le et al., Vaccine (2000) 18: 1893; Alves et al., Vaccine (2001) 19: 788], injection of plasmid DNA with or without adjuvants [Ulmer et al., Vaccine (1999) 18: 18; MacLaughlin et al., J. Control Release (1998) 56: 259; Hartikka et al., Gene Ther. (2000) 7: 1171-82; Benvenisty and Reshef, PNAS USA (1986) 83:9551; Singh et al., PNAS USA (2000) 97: 811], targeting cells by delivery of DNA complexed with specific carriers [Wa et al., J Biol Chem (1989) 264: 16985; Chaplin et al., Infect. Immun. (1999) 67: 6434], injection of plasmid completed or encapsulated in various forms of liposomes [Ishii et al., AIDS Research and Human Retroviruses (1997) 13: 142; Perrie et al., Vaccine (2001) 19: 3301], administration of DNA with different methods of bombardment [Tang et al., Nature (1992) 356: 152; Eisenbraun et al., DNA Cell Biol (1993) 12: 791; Chen et al., Vaccine (2001) 19: 2908], and administration of DNA with lived vectors [Tubulekas et al., Gene (1997) 190: 191; Pushko et al., Virology (1997) 239: 389; Spreng et al. FEMS (2000) 27: 299; Dietrich et al., Vaccine (2001) 19: 2506].
In a further embodiment, the invention provides the use of a pharmaceutical composition of the invention in the manufacture of a medicament for the prophylactic or therapeutic treatment of S. pyogenes infection.
In a further embodiment, the invention provides a kit comprising a polypeptide of the invention for detection or diagnosis of S. pyogenes infection.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
This example illustrates the cloning and molecular characteristics of SHB-GAS-102 gene and corresponding polypeptide
The coding region of S. pyogenes SHB-GAS-102 (SEQ ID NO: 1) gene was amplified by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene, La Jolla, Calif.) from genomic DNA of serotype M1 S. pyogenes strain ATCC700294 using the following oligonucleotide primers that contained base extensions for the addition of restriction sites NdeI (CATATG) and XhoI (CTCGAG): DMAR2174 and DMAR2175, which are presented in Table 1. PCR products were purified from agarose gel using a QIAquick gel extraction kit from QIAgen following the manufacturer's instructions (Chatsworth, Calif.), and digested with NdeI and XhoI (Amersham Biosciences Inc, Baie d'Urfé, Canada). The pET-19b(+) vector (Novagen, Madison, Wis.) was digested with NdeI and XhoI and purified from agarose gel using a QIAquick gel extraction kit from QIAgen (Chatsworth, Calif.). The NdeI-XhoI PCR products were ligated to the NdeI-XhoI pET-19b(+) expression vector. The ligated products were transformed into E. coli strain DH5α [φ80dlacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17 (rK−mK+) deoR thi-1 supE44 λ−gyrA96 relA1] (Gibco BRL, Gaithersburg, Md.) according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D. M. Glover (ed), pp. 109-135). Recombinant pET-19b(+) plasmid (rpET19b(+)) containing SHB-GAS-102 gene was purified using the GenElute plasmid kit (Sigma-Aldrich Company Ltd, Mo.) and DNA insert was sequenced (Taq Dye Deoxy Terminator Cycle Sequencing kit, ABI, Foster City, Calif.).
1The underlined amino acid residue represents the modification in the DNA sequence.
2PCR oligonucleotide primer sets used to remove the BamHI restriction site present in the SHB-GAS-102 gene.
It was determined that the 537-bp including a stop codon (TAA) open reading frame (ORF) of SHB-GAS-102 encodes a 178 amino-acid residues polypeptide with a predicted pI of 9.55 and a predicted molecular mass of 19,431.0 Da. Analysis of the predicted amino acid residues sequence (SEQ ID NO: 2) using the Spscan sofware (Wisconsin Sequence Analysis Package; Genetics Computer Group) did not reveal the existence of a signal peptide.
To confirm the presence by PCR amplification of SHB-GAS-102 (SEQ ID NO:1) gene, the following 4 serologically distinct S. pyogenes strains were used: the serotype M1 S. pyogenes strain ATCC700294, the serotype M3 S. pyogenes strain ATCC12384, and the serotype M18 S. pyogenes strain ATCC12357 were obtained from the American Type Culture Collection (Rockville, Md.), and the serotype M6 S. pyogenes SPY67 clinical isolate was provided by the Centre de recherche en infectiologie du Centre hospitalier de l'Université Laval, Sainte-Foy, Canada. Chromosomal DNA was isolated from each strain as previously described (Jayarao B M et al. 1991. J. Clin. Microbiol. 29:2774-2778). SHB-GAS-102 (SEQ ID NO:1) gene was amplified by PCR (Robocycler Gradient 96 Temperature cycler) from the genomic DNA purified from the 4 S. pyogenes strains using the oligonucleotide primers DMAR2174 and DMAR2175 (Table 1). PCR was performed with 35 cycles of 45 sec at 95° C., 45 sec at 45° C. and 75 sec at 72° C. and a final elongation period of 10 min at 72° C. The PCR products were size fractionated in 1% agarose gels and were visualized by ethidium bromide staining. The results of these PCR amplifications are presented in Table 2. The analysis of the amplification products revealed that SHB-GAS-102 (SEQ ID NO:1) gene was present in the genome of all of the 4 S. pyogenes strains tested. These PCR data presented in the previous paragraphs clearly demonstrated that the SHB-GAS-102 gene is highly conserved among streptococcal strains.
In order to evaluate the distribution of the SHB-GAS-102 polypeptide among S. pyogenes isolates, the reactivity of pooled mouse anti-SHB-GAS-102 sera to a collection of 13 strains of S. pyogenes representing 13 M serotypes ( Table 3) was tested by immunoblots. These sera were collected from mice immunized three times at two-week intervals with 20 μg of purified recombinant SHB-GAS-102 polypeptides mixed with Quil A adjuvant. The recombinant SHB-GAS-102 polypeptide was produced and purified as described in Example 6. The S. pyogenes cells used for this study were prepared following this protocol: bacteria were grown in Todd Hewitt (TH) broth with 0.5% Yeast extract and 1% peptone extract overnight at 37° C. in a 8% CO2 atmosphere. The bacterial suspension was adjusted to an OD600nm of 1.0 and the S. pyogenes cells were fixed with 0.3% formaldehyde in PBS buffer for 18 h at 4° C. After incubation, this solution was treated with mutanolysin (7.5 U/500 μl) for 30 min at 37° C., and boiled for 5 min in SDS-PAGE sample buffer. Antibodies present in the pooled mouse sera recognized the SHB-GAS-102 polypeptide band (≈22 kDa) in all the 13 S. pyogenes preparations tested (Table 3). In order to confirm the nature of the polypeptide band, anti-SHB-GAS-102 sera were adsorbed with 12.5 μg of purified recombinant SHB-GAS-102 polypeptides (overnight, 4° C.). As expected, this adsorbed sera did not recognize, in the S. pyogenes whole-cell extract applied onto the gel, the polypeptide band at ≈22 kDa confirming that this polypeptide band was the SHB-GAS-102 polypeptide.
These data as well as the data presented in the previous paragraphs clearly demonstrated that the SHB-GAS-102 gene and the SHB-GAS-102 polypeptide are highly conserved among streptococcal strains.
1Sera were collected from mice immunized three times at two-week intervals with 20 μg of purified recombinant polypeptides mixed with Quil A adjuvant. Sera were diluted 1/1000 to perform the immunoblots.
This example illustrates the cloning and molecular characteristics of SHB-GAS-103 gene and corresponding polypeptide
The coding region of S. pyogenes SHB-GAS-103 gene (SEQ ID NO: 3) was amplified by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene, Lajolla, Calif.) from genomic DNA of serotype M1 S. pyogenes strain ATCC700294 using the following oligos that contained base extensions for the addition of restriction sites NdeI (CATATG) and XhoI (CTCGAG): DMAR1878 and DMAR1879, which are presented in Table 1. The methods used for cloning SHB-GAS-103 into an expression vector and sequencing are similar to the methods described in Example 1.
It was determined that the 1269-bp including a stop codon (TAA) open reading frame (ORF) of SHB-GAS-103 encodes a 422 amino-acid-residues polypeptide with a predicted pI of 9.11 and a predicted molecular mass of 46,605.3 Da. Analysis of the predicted amino acid residues sequence (SEQ ID NO: 4) using the Spscan sofware (Wisconsin Sequence Analysis Package; Genetics Computer Group) suggested the existence of a 27 amino acid residues signal peptide (MFQLRKKMTRKQLALLSAGVLTCVVGG).
The SHB-GAS-103 gene was shown to be present after PCR amplification using the oligonucleotide primers DMAR1878 and DMAR1879 in the 4 serologically S. pyogenes strains tested (Table 2). In addition, the SHB-GAS-103 polypeptide was shown to be present in all the 13 S. pyogenes isolates tested by immunoblots as described in Example 1 (Table 3). Indeed, antibodies present in the pooled mouse sera recognized the SHB-GAS-103 polypeptide band (≈52 kDa) in all the 13 S. pyogenes preparations tested (Table 3). In order to confirm the nature of the polypeptide band, anti-SHB-GAS-103 sera were adsorbed with 12.5 μg of purified recombinant SHB-GAS-103 polypeptides (overnight, 4° C.). As expected, this adsorbed sera did not recognize, in the S. pyogenes whole-cell extract applied onto the gel, the polypeptide band at ≈52 kDa confirming that this polypeptide band was the SHB-GAS-103 polypeptide.
These data presented in the previous paragraphs clearly demonstrated that the SHB-GAS-103 gene and the SHB-GAS-103 polypeptide are highly conserved among streptococcal strains.
This example illustrates the cloning and molecular characteristics of SHB-GAS-104 gene and corresponding polypeptide
The coding region of S. pyogenes SHB-GAS-104 gene (SEQ ID NO: 5) was amplified by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene, LaJolla, Calif.) from genomic DNA of serotype M1 S. pyogenes strain ATCC700294 using the following oligos that contained base extensions for the addition of restriction sites NdeI (CATATG) and XhoI (CTCGAG): DMAR1976 and DMAR1977, which are presented in Table 1. The methods used for cloning SHB-GAS-104 into an expression vector and sequencing are similar to the methods described in Example 1.
It was determined that the 885-bp including a stop codon (TAA) open reading frame (ORF) of SHB-GAS-104 encodes a 294 amino-acid-residues polypeptide with a predicted pI of 5.83 and a predicted molecular mass of 33,381.9 Da. Analysis of the predicted amino acid residues sequence (SEQ ID NO: 6) using the Spscan sofware (Wisconsin Sequence Analysis Package; Genetics Computer Group) suggested the existence of a 19 amino acid residues signal peptide (MIKRCKGIGLALMAFFLVA).
The SHB-GAS-104 gene was shown to be present after PCR amplification using the oligonucleotide primers DMAR1976 and DMAR1977 in the 4 serologically S. pyogenes strains tested (Table 2). The methods used for PCR amplification of the SHB-GAS-104 gene were similar to the methods presented in Example 1.
In addition, the distribution of the SHB-GAS-104 polypeptide among S. pyogenes isolates have been evaluated as described in Example 1 using a S. pyogenes extract prepared by the following method: bacteria were grown in TH broth with 0.5% Yeast extract and 1% peptone extract overnight at 37° C. in a 8% CO2 atmosphere. The bacterial suspension was adjusted to an OD600 nm of 0.8. After centrifugation, the bacterial pellet was resuspended in 500 μl of extraction buffer (0.1 M Tris-HCl pH 7.6) and pelleted by centrifugation. The pellet was frozen for 10 min at −80° C. and thawed at 37° C. for 5 min. The freeze-thaw cycle was repeated three times. Bacterial pellet was resuspended in 500 μl of extraction buffer and sonicated 8×15 sec. Samples were centrifuged (14 000×g) at 4° C. for 15 min and supernatant was boiled for 5 min in SDS-PAGE sample buffer. Antibodies present in the pooled mouse anti-SHB-GAS-104 sera recognized the SHB-GAS-104 polypeptide band (≈37.5 kDa) in all of the 13 S. pyogenes preparations tested (Table 3). In order to confirm the nature of the polypeptide band, anti-SHB-GAS-104 sera were adsorbed with 12.5 μg of purified recombinant SHB-GAS-104 polypeptide (overnight, 4° C.). As expected, this adsorbed serum did not recognize, in the S. pyogenes extract applied onto the gel, the polypeptide band at ≈37.5 kDa confirming that this polypeptide band was the SHB-GAS-104 polypeptide.
These PCR data presented in the previous paragraphs clearly demonstrated that the SHB-GAS-104 gene is highly conserved among streptococcal strains.
This example illustrates the cloning of S. pyogenes genes in CMV plasmid pCMV-GH.
The DNA coding regions of S. pyogenes polypeptides were inserted in phase downstream of a human growth hormone (hGH) gene which was under the transcriptional control of the cytomegalovirus (CMV) promotor in the plasmid vector pCMV-GH (Tang et al., Nature, 1992, 356:152). The CMV promotor is a non functional plasmid in E. coli cells but active upon administration of the plasmid in eukaryotic cells. The vector also incorporated the ampicillin resistance gene.
In order to remove the BamHI site into the SHB-GAS-102 gene, mutagenesis experiments using the Quickchange Site-Directed Mutagenesis kit from Stratagene were performed according to the manufacturer's recommendations. The oligonucleotides DMAR2841 and DMAR2842 (Table 1) and the SHB-GAS-102 gene cloned into pET vector as DNA template were used to perform the mutagenesis experiments. The modification on the SHB-GAS-102 gene generated by site-directed mutagenesis is presented in the Table 1.
The coding regions of modified SHB-GAS-102, SHB-GAS-103 (SEQ ID NO: 3), and SHB-GAS-104 (SEQ ID NO: 5) genes were amplified by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene, LaJolla, Calif.) from pET vector containing the modified SHB-GAS-102 gene or the genomic DNA of serotype M1 S. pyogenes strain ATCC700294 (for SHB-GAS-103 and SHB-GAS-104 genes) using oligonucleotide primers that contained base extensions for the addition of restriction sites BamHI (GGATCC) and SalI (GTCGAC) which are described in Table 1. The PCR products were purified from agarose gel using a QIAquick gel extraction kit from QIAgen (Chatsworth, Calif.) and digested with restriction enzymes (Amersham Biosciences Inc, Baie d'Urfé, Canada). The pCMV-GH vector (Laboratory of Dr. Stephen A. Johnston, Department of Biochemistry, The University of Texas, Dallas, Tex.) was digested with BamHI and SalI and purified from agarose gel using the QIAquick gel extraction kit from QIAgen (Chatsworth, Calif.). The BamHI-SalI DNA fragments were ligated to the BamHI-SalI pCMV-GH vector to create the hGH-SHB-GAS-102, hGH-SHB-GAS-103, and hGH-SHB-GAS-104 fusion polypeptides under the control of the CMV promoter. The ligated products were transformed into E. coli strain DH5α [φ80dlacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(rK−mK+) deoR thi-1 supE44 λ−gyrA96 relA1] (Gibco BRL, Gaithersburg, Md.) according to the method of Simanis (Hanahan, D. DNA Cloning, 1985, D. M. Glover (ed), pp. 109-135). The recombinant pCMV plasmids were purified using the GenElute plasmid kit (Sigma-Aldrich company Ltd, Mo.) and the nucleotide sequences of the DNA inserts were verified by DNA sequencing.
This example illustrates the use of DNA to elicit an immune response to S. pyogenes polypeptide antigens.
Groups of 8 female BALB/c mice (Charles River, St-Constant, Québec, Canada) were immunized by intramuscular injection of 100 μl three times at two- or three-week intervals with 50 μg of recombinant pCMV-GH encoding modified SHB-GAS-102, SHB-GAS-103 (SEQ ID NO: 3), and SHB-GAS-104 (SEQ ID NO: 5) genes in presence of 50 μg of granulocyte-macrophage colony-stimulating factor (GM-CSF)—expressing plasmid pCMV-GH-GM-CSF (Laboratory of Dr. Stephen A. Johnston, Department of Biochemistry, The University of Texas, Dallas, Tex.). As control, groups of mice were injected with 50 μg of pCMV-GH in presence of 50 μg of pCMV-GH-GM-CSF. Blood samples were collected from the orbital sinus prior to each immunization and seven days following the third injection and serum antibody responses were determined by ELISA using the corresponding His-tagged labeled S. pyogenes recombinant polypeptides as coating antigens. The production and purification of these His-tagged labeled S. pyogenes recombinant polypeptides are presented in Example 6.
This example illustrates the production and purification of S. pyogenes recombinant polypeptides.
The recombinant pET-19b(+)plasmids with SHB-GAS-102 (SEQ ID NO: 1), SHB-GAS-103 (SEQ ID NO: 3), and SHB-GAS-104 (SEQ ID NO: 5) genes were used to transform by electroporation (Gene Pulser II apparatus, BIO-RAD Labs, Mississauga, Canada) E. coli strains BL21 (DE3) (F−ompT hsdSB (r−Bm−B) gal dcm (DE3)) or BL21 star (DE3) (F−ompT hsdSB (r−Bm−B) gal dcm rne131 (DE3)) (Novagen, Madison, Wis.). In these strains of E. coli , the T7 promotor controlling expression of the recombinant polypeptide is specifically recognized by the T7 RNA polymerase (present on the λDE3 prophage) whose gene is under the control of the lac promotor which is inducible by isopropyl-β-d-thio-galactopyranoside (IPTG). The transformants BL21(DE3)/rpET or BL21 star (Des)/rpET were grown at 37° C. with agitation at 250 rpm in LB broth (peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) containing 100 μg of carbenicillin (Sigma-Aldrich Canada Ltd., Oakville, Canada) per ml until the A600 reached a value of 0.6. In order to induce the production of His-tagged S. pyogenes recombinant polypeptides, the cells were incubated for 2 additional hours in the presence of IPTG at a final concentration of 1 mM. Induced cells from a 500 ml culture were pelleted by centrifugation and frozen at −70° C.
The purification of the recombinant polypeptides from the soluble fraction of IPTG-induced BL21(DE3)/rpET19b(+) or BL21 star(DE3)/rpET19b(+) was done by affinity chromatography based on the properties of the His●Tag sequence (10 consecutive histidine residues) to bind to divalent cations (Ni2+) immobilized on the His●Bind metal chelation resin. Briefly, the pelleted cells obtained from a 400 mL culture induced with IPTG was resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 7.9), sonicated and centrifuged at 12,000×g for 20 min to remove debris. The supernatant was incubated with Ni—NTA agarose resin (QIAgen) for 45 min at 4° C. The S. pyogenes recombinant polypeptides were eluted from the resin with a solution of 250 mM imidazole-500 mM NaCl-20 mM Tris, pH 7.9. The removal of the salt and imidazole from the samples was done by dialysis against PBS buffer overnight at 4° C. The amount of recombinant polypeptide was estimated by MicroBCA (Pierce, Rockford, Ill.).
This example illustrates the accessibility to antibodies of the S. pyogenes recombinant polypeptides at the surface of intact streptococcal cells.
Bacteria were grown in Todd Hewitt (TH) broth (Difco Laboratories, Detroit, Mich.) with 0.5% Yeast extract (Difco Laboratories) and 1% peptone extract (Merck, Darmstadt, Germany) at 37° C. in a 8% CO2 atmosphere to give an OD600 nm of 0.600 (˜109 CFU/ml). Dilutions of anti-SHB-GAS-102, anti-SHB-GAS-103, anti-SHB-GAS-104, or control sera were then added and allowed to bind to the cells, which were incubated for 2 h at 4° C. Samples were washed 2 times in blocking buffer [phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA)], and then 0.5 ml of goat fluorescein (FITC)-conjugated anti-mouse IgG+IgM diluted in blocking buffer was added. After an additional incubation of 60 min at room temperature, samples were washed 2 times in blocking buffer and fixed with 0.3% formaldehyde in PBS buffer for 18-24 h at 4° C. Cells were kept in the dark at 4° C. until analyzed by flow cytometry (Epics® XL; Beckman Coulter, Inc.). Ten thousands intact S. pyogenes cells were analyzed per sample.
This example illustrates the protection against fatal S. pyogenes infection induced by passive immunization of mice with rabbit hyper-immune sera.
New Zealand rabbit (Charles River) was immunized subcutaneously three times at 3-week intervals at multiple sites with 100 μg of His-tagged recombinant SHB-GAS-102 protein in the presence of Freund's incomplete adjuvant (Gibco-BRL). Blood samples were collected three weeks after the third injection. The antibodies present in the serum were partially purified by precipitation using 40% saturated ammonium sulfate solution. Groups of 6 female Balb/c mice (Charles River) were injected intravenously with 500 μl of partially purified serum collected from a rabbit immunized with the recombinant SHB-GAS-102 protein, or a rabbit immunized with an unrelated control recombinant protein. Eighteen hours later the mice were challenged with approximately 2×107 CFU of the type 3 S. pyogenes strain ATCC12384. Samples of the S. pyogenes challenge inoculum were plated on blood agar plates to determine the CFU and to verify the challenge dose. Deaths were recorded for a period of 8 days.
As presented in Table 4, 67% (4/6) mice immunized with partially purified serum collected from rabbit immunized with the recombinant SHB-GAS-102 protein were protected against a lethal challenge. On the contrary, immunization of mice with serum collected from rabbit immunized with an irrelevant protein did not confer such protection (Table 4).
This example illustrates the protection of mice against fatal S. pyogenes infection induced by immunization with recombinant polypeptides.
Groups of female Balb/c mice (Charles River) were immunized subcutaneously three times at two-week intervals with 20 μg of affinity purified SHB-GAS-102, SHB-GAS-103, or SHB-GAS-104 His-tagged recombinant polypeptides in presence of 10 μg of QuilA adjuvant (Cedarlane Laboratories Ltd, Hornby, Canada) or, as control, with QuilA adjuvant alone in PBS or with 20 μg of irrelevant polypeptide in presence of QuilA. Blood samples were collected from the orbital sinus on day 1, 14 and 28 prior to each immunization and 14 days (day 42) following the third injection. One week later the mice were challenged with approximately 4-8×106 CFU of the type 3 S. pyogenes strain ATCC12384. Samples of the S. pyogenes challenge inoculum were plated on blood agar plates to determine the CFU and to verify the challenge dose. Deaths were recorded for a period of 7 days.
As presented in Table 5, more than 83% (10/12) mice immunized with SHB-GAS-102 polypeptide were protected against a lethal challenge. Mice immunized with SHB-GAS-103 and SHB-GAS-104 were also protected against a lethal challenge (7/12). On the contrary, immunization of mice with adjuvant only or with an irrelevant protein did not confer such protection (Table 5).
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA04/01510 | 8/16/2004 | WO | 1/3/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60495094 | Aug 2003 | US |
