Compositions and methods for making and using therapeutic formulations of multivalent hybrid polypeptides comprising immunogenic peptides of M proteins from various different serotypes of group A streptococci and antibodies thereto are provided. Also provided are nucleic acids encoding such hybrid polypeptides. The hybrid polypeptide formulations may be used, for example, in methods for treating or preventing a microbial infection and eliciting a protective immune response having broadly protective opsonic antibodies in the absence of tissue cross-reactive antibodies.

SUMMARY OF THE INVENTION

The present invention provides the discovery of therapeutic formulations of multivalent hybrid polypeptides, particularly a cocktail of hybrid polypeptides useful for eliciting a protective immune response having broadly protective opsonic antibodies in the absence of tissue cross-reactive antibodies. Hybrid polypeptides of the invention comprise at least six different linked immunogenic peptides, wherein each peptide comprises an amino-terminal portion of a streptococcal M protein of at least 30 amino acids, and wherein the polypeptide has an amino-terminal peptide that is reiterated as a carboxy-terminal peptide and the polypeptide is capable of eliciting an immune response against more than one serotype of group A streptococci.

In one aspect, the invention provides a hybrid polypeptide, comprising at least six different linked immunogenic peptides, wherein each peptide comprises an amino-terminal portion of a streptococcal M protein of at least 30 amino acids, and wherein the polypeptide has an amino-terminal peptide that is reiterated as a carboxy-terminal peptide and the polypeptide is capable of eliciting an immune response against more than one antigen of group A streptococci comprising at least M5, M6, M14, M19, M24, and M29. In other embodiments, the polypeptide elicits an immune response against more than one antigen of group A streptococci comprising at least M2, M11, M22, M33, M43, M59, and M94, or at least M75, M76, M77, M89, M92, M101, and M114, or at least Spa, M1.0, M1.2, M3, M12, M18, and M28, or against 27 or more antigens of group A streptococci. In more embodiments, the amino-terminal immunogenic peptide of the hybrid polypeptide is M24, M1, M2, or M89. In still other embodiments, the polypeptides are recombinant and the immunogenic peptides are linked in tandem, the polypeptides having a structure of M24-M5-M6-M19-M29-M14-M24, M2-M43-M94-M22-M11-M59-M33-M2, M89-M101-M77-M114-M75-M76-M92-M89, or M1.0-M12-Spa-M28-M3-M1.2-M18-M1.0. In yet other embodiments, the hybrid polypeptides comprise the amino acid sequence of SEQ ID NOS:2, 4, 6, or 8. In other embodiments, the immunogenic peptides are linked by at least two amino acids encoded by a nucleic acid sequence that is a restriction enzyme recognition site, wherein the recognition site includes at least one of BamHI, ClaI, EcoRI, HindIII, KpnI, NcoI, NheI, PmlI, PstI, SalI, and XhoI. In further embodiments, any of the aforementioned hybrid polypeptides capable of eliciting at least one opsonic antibody that is not a tissue cross-reactive antibody in a subject, wherein the subject is a human or an animal. In more embodiments, any of the aforementioned hybrid polypeptides further comprising a carboxy-terminal tag, wherein the carboxy-terminal tag is selected from the group consisting of alkaline phosphatase, .beta.-galactosidase, hexahistidine, FLAG.RTM., XPRESS.RTM., and GST. In still more embodiments, any of the aforementioned hybrid polypeptides or fusion proteins further comprise at least one additional carboxy-terminal amino acid, wherein the additional carboxy-terminal amino acid is a D-amino acid or cysteine.

In another aspect, the invention provides any of the aforementioned hybrid polypeptides or fusion proteins wherein the polypeptides are synthetic. In certain embodiments, the synthetic hybrid polypeptides have one or more amino acids altered to a corresponding D-amino acid or are linked to an alkane such as acrylamide or an analog or derivative thereof. In a further embodiment, the synthetic hybrid polypeptides have immunogenic peptides linked to form a lysine core-branched peptide.

In still another aspect, the invention includes a nucleic acid molecule comprising a sequence encoding a hybrid polypeptide of SEQ ID NOS:2, 4, 6, or 8. In a related embodiment, there is provided a nucleic acid expression construct comprising an expression control sequence operably linked to a polynucleotide encoding a hybrid polypeptide of SEQ ID NOS:2, 4, 6, or 8. In other embodiments, the expression construct comprises a nucleic acid expression vector selected from the group comprising a plasmid, phagemid, shuttle vector, cosmid, or virus. In one embodiment, the vector is plasmid pT5 (SEQ ID NO:17). In still another embodiment, there is provided a host cell containing any of the aforementioned nucleic acid constructs. In yet other embodiments, the host cell is selected from a bacterium, a yeast cell, a nematode cell, an insect cell, or a mammalian cell. In one embodiment, the host cell is the bacterium Escherichia coli.

In a further aspect, the present invention provides a plurality of antibodies, comprising two or more different antibodies wherein each antibody is specific for a different immunogenic peptide of a hybrid polypeptide, the polypeptide comprises at least six different immunogenic peptides linked in tandem, each peptide comprises at least 30 amino acids and the amino-terminal peptide is reiterated as a carboxy-terminal peptide, wherein the polypeptide is capable of eliciting an immune response against more than one antigen of group A streptococci comprising at least M5, M6, M14, M19, M24, and M29. In other embodiments, the plurality of antibodies are specific for more than one antigen of group A streptococci comprising at least M2, M11, M22, M33, M43, M59, and M94, or at least M75, M76, M77, M89, M92, M101, and M114, or at least Spa, M1.0, M1.2, M3, M12, M18, and M28, or specific for 27 or more antigens of group A streptococci. In yet other embodiments, any of the aforementioned antibodies wherein the hybrid polypeptides comprise the amino acid sequence of SEQ ID NOS:2, 4, 6, or 8. In other embodiments, any of the aforementioned antibodies are opsonic and not tissue cross-reactive in a subject. In one embodiment, the antibodies are polyclonal.

In yet another aspect, there is provided a method of producing a hybrid polypeptide, comprising culturing a host cell containing any of the aforementioned nucleic acid expression vectors comprising at least one expression control sequence operably linked to a nucleic acid molecule encoding a hybrid polypeptide of SEQ ID NOS:2, 4, 6, or 8, under conditions and for a time sufficient for expression of the polypeptide. In a related aspect, the invention provides any of the aforementioned hybrid polypeptides produced by the aforementioned method.

In another aspect, there is provided a composition comprising a pharmaceutically acceptable carrier and any of the aforementioned hybrid polypeptides. In other aspects, the invention is a cocktail composition comprising a pharmaceutically acceptable carrier and a mixture of at least two or three of any of the aforementioned hybrid polypeptides. In one embodiment, the cocktail compositions include at least one of the hybrid polypeptides having a Spa immunogenic peptide. In other embodiments, provided are any of the aforementioned compositions wherein the hybrid polypeptides are mixed in equimolar amounts. In further embodiments, any of the aforementioned compositions further comprise an adjuvant such as alum or Freund's.

In still another aspect, the invention provides a method for preventing a microbial infection, comprising administering to a subject any of the aforementioned compositions at a dose sufficient to elicit antibodies specific for one or more hybrid polypeptide, wherein the antibodies are opsonic and are not tissue cross-reactive. In certain embodiments, the microbial infection being prevented is a streptococcal infection and in a related embodiment is a group A streptococcal infection. In some embodiments, any of the aforementioned compositions are administered to a subject by a route selected from enteral, parenteral, transdermal, transmucosal, or inhalation. In further embodiments, the compositions are administered to a human or animal subject and the compositions further comprise an adjuvant such as alum or Freund's. In another related aspect, there is provided isolated antibodies produced by the aforementioned methods for preventing a microbial infection. In certain embodiments, the antibodies produced by these methods will comprise at least one antibody specific for a M serotype not represented in a hybrid polypeptide, such as M4. In still another embodiment, there is provided a method for treating or preventing a microbial infection comprising administering to a subject a composition comprising a pharmaceutically acceptable carrier and any of the aforementioned plurality of antibodies. In one embodiment, the subject is an animal or human.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed to hybrid polypeptides of streptococcal antigens and compositions thereof, which are capable of eliciting protective antibodies against streptococci. Furthermore, the compositions may include a single hybrid polypeptide or a combination of several different hybrid polypeptides, which may be useful to elicit an immune response against group A streptococci. In one aspect, one or more hybrid polypeptide may be formulated as a composition for simultaneously treating or preventing an infection by several different group A streptococcal serotypes, as well as treating or preventing infection by serotypes not represented in the hybrid polypeptide(s). The present invention also provides isolated nucleic acids that encode such hybrid polypeptides, as well as methods of expressing and purifying such hybrid polypeptides. A hybrid polypeptide, as described herein, comprises several different linked immunogenic peptides wherein each peptide comprises, for example, an amino-terminal portion of a streptococcal M protein and can elicit opsonic antibodies specific for a particular group A streptococcal serotype without eliciting antibodies that are cross-reactive with host tissue.

The present invention also provides antibodies specific for each immunogenic peptide serotype included in a hybrid polypeptide or in a combination of hybrid polypeptides, as well as antibodies specific for serotypes not included in the hybrid polypeptide(s) (i.e., antibodies that confer cross-protection between serotypes). The invention, therefore, relates generally to the surprising discovery that highly complex, multivalent hybrid polypeptide-based vaccines are feasible to simultaneously elicit broadly protective antibodies against several different streptococcal serotypes. Moreover, a composition comprising a cocktail of more than one multivalent hybrid polypeptide used as a vaccine unexpectedly elicits a much more robust antibody response in humans than does a composition comprising a single multivalent hybrid polypeptide, wherein the increase in antibody response also results in an increase in antibody function (i.e., increased ability to opsonize and/or to kill microorganisms). As used herein, a "cocktail" of hybrid polypeptides refers to a composition comprising at least two different hybrid polypeptides of this invention. Hence, the invention also relates generally to the surprising discovery that a cocktail of multivalent hybrid polypeptides can function synergistically to elicit a greater antibody response that is a protective immune response. Accordingly, the compositions and methods of the present invention may be readily used to treat or prevent streptococcal infections. Discussed in more detail below are hybrid polypeptides and assorted compositions thereof (including admixtures or cocktails) suitable for use within the present invention, as well as exemplary methods of making such hybrid polypeptides and compositions, and therapeutic uses thereof.

Any numerical ranges recited herein are to be understood to include any integer within the range and, where applicable (e.g., concentrations), fractions thereof, such as one tenth and one hundredth of an integer (unless otherwise indicated).

Hybrid Polypeptides

The present invention is directed generally to multivalent immunogenic hybrid polypeptides of M proteins and M-like proteins, including fusions to other proteins. The immunogenic M and M-like peptides may comprise any portion of an M protein that is immunogenic, which may or may not confer serotype specificity. A plurality of different multivalent immunogenic hybrid polypeptides can be mixed or combined into a cocktail composition for use in eliciting a protective immune response. The present invention further provides methods for producing synthetic or recombinant multivalent immunogenic hybrid M polypeptides, including fusion proteins. For example, host cells containing hybrid polypeptide-encoding nucleic acid expression constructs may be cultured to produce recombinant hybrid polypeptides. Also contemplated are methods for treating or preventing a microbial infection or eliciting an immune response using a hybrid polypeptide or a combination of hybrid polypeptides (including fusion proteins).

By way of background and not wishing to be bound by theory, streptococcal species are Gram-positive bacteria that are grouped based on immunological differences in their cell wall polysaccharides and are designated, for example, group A, B, C, F, and G. Specifically, group A streptococci (GrAS) are clinically important microorganisms that colonize the throat and skin. GrAS are responsible for a variety of suppurative infections (e.g., strep throat, necrotizing fasciitis) and non-suppurative sequelae (e.g., acute rheumatic fever, reactive arthritis) (see generally Cunningham, Clin. Microbiol. Rev. 13:470, 2000). GrAS have two major, surface-exposed anti-phagocytic factors, capsule and M protein, which allow these organisms to colonize and survive in a host. The M protein, which is encoded by the emm gene, extends from the cell surface as an .alpha.-helical coiled-coil dimer that appears as a fibril on the surface of GrAS. The M proteins are a diverse group, which have been serologically separated into M protein serotypes.

Currently, more than 120 M protein serotypes have been identified, and within some serotypes there have been identified several subtypes. For example, without limitation, type 1 M protein has related subtypes 1.1-1.15, and type 12 has subtypes 12.1-12.9. In addition, as is known in the art, unclassified serotypes may have an initial designation (such as st2967) and ultimately receive a "final" classification (such as st2967 now being classified as M type 114), or previously classified serotypes may be reclassified with a different number (such as M13W is now reclassified as M94). In certain embodiments, amino-terminal portions of any one or more of known M protein from serotypes 1-120+, or from unknown serotypes, can be used to generate immunogenic peptides for inclusion in multivalent immunogenic hybrid polypeptides of the instant invention.

Furthermore, the M protein is part of a superfamily of proteins, which includes without limitation, immunoglobulin binding proteins (e.g., FrcA), M-like proteins (e.g., Mrp and Spa), and M proteins. Accordingly, as used herein, "M protein" refers to the superfamily of proteins, which includes any known or unknown M protein (with or without a designated serotype), as well as M-like proteins, such as Spa (see, e.g., Dale et al., J. Clin. Invest. 103:1261, 1999 and McLellan et al., Infect. Immun. 69:2943, 2001), Mrp (see, e.g., Boyle et al., J. Infect. Dis. 177:991, 1998), immunoglobulin binding proteins (see, e.g., Podbielski et al., Mol. Microbiol. 12:725, 1994; Whatmore and Kehoe, Mol. Microbiol. 11:363, 1994), and the like. As described herein and is understood in the art, the serotype of an M protein may be reclassified and result in a change to a different type, a new type, or even a subtype. Also, as used herein, "M protein serotype" or "M type" refers to all M proteins within that serotype, including all subtypes, related proteins, and the like. Furthermore, reference to a particular M type may be applied interchangeably as follows, by way of illustration and not limitation, serotype 1 M protein, type 1, M1, and the like, which as set forth above, includes all subtypes as well.

As described above, group A streptococci have developed a system for avoiding some of the antimicrobial defenses of a human host. Strains of streptococci that express capsule and M protein can evade phagocytosis by, for example, polymorphonuclear leukocytes or neutrophils and multiply in a host that has not been exposed to streptococci (i.e., have non-immune blood). Yet, a subject may develop resistance to a streptococcal infection if the host can produce protective antibodies directed against streptococci. Protection against GrAS generally correlates with the presence of opsonizing antibody against type-specific M protein (see, e.g., Lancefield, J. Immunol. 89:307, 1962), and the development of secretory or mucosal antibodies is suspected of playing an important role in preventing initial colonization by streptococci. However, the pathogenesis of some of the non-suppurative sequelae may be due to host tissue cross-reaction with streptococcal antibodies. As used herein, "tissue cross-reactivity" means a host antigen shares at least one epitope with a foreign antigen, such as a streptococcal antigen. For example, the different antigens may share an identical amino acid sequence, may be homologous but non-identical, or may be dissimilar molecules with a shared epitope (e.g., protein and carbohydrate or protein and nucleic acid molecule). Thus, an effective vaccine against streptococcal infections would preferably elicit an immune response that includes antibodies that are not tissue cross-reactive (i.e., do not cause autoimmune disease) and are protective (i.e., opsonic) against many of the clinically important serotypes.

The present invention pertains to hybrid polypeptides or variants thereof having a plurality of immunogenic peptides from M or M-like proteins, or nucleic acid molecules encoding such polypeptides or variants thereof. As used herein, "immunogenic peptide" refers to any streptococcal peptide or polypeptide having at least one epitope capable of eliciting an immune response, which are the component units of the hybrid polypeptides. Preferably, the epitope is within an amino-terminal portion of a streptococcal M protein or Spa protein and more preferably is an opsonic epitope that does not elicit tissue cross-reactive antibodies.

The present invention also provides a rational vaccine design approach for selection of the streptococcal M protein serotypes to be included in the multivalent hybrid polypeptide vaccine. Some criteria that may be used, without limitation, include identifying the M protein serotypes that are frequent causes of uncomplicated pharyngitis, identifying the serotypes that are commonly recovered from normally sterile sites (i.e., invasive strains) (e.g., useful data may obtained from the Active Bacterial Core Surveillance of the Emerging Infections Program Network, supported by the Centers for Disease Control and Prevention; see also Beall et al., J. Clin. Microbiol. 35:1231, 1997; Schuchat et al., Emerg. Infect. Dis. 7:92, 2001), and identifying serotypes that are considered currently or historically "rheumatogenic" (Bisno A L. The concept of rheumatogenic and nonrheumatogenic group A streptococci. In: Reed S E, and J. B. Zabriskie, ed. Streptococcal Diseases and the Immune Response. New York: Academic Press, 1980:789-803). Also useful is the amino-terminal peptide fragment of Spa, a new protective antigen that is expressed by several serotypes of group A streptococci (Dale et al., J. Clin. Invest. 103:1261, 1999), or any other streptococcal antigen capable of eliciting protective antibodies may be used.

As described herein and known in the art, M protein amino acid sequences selected for inclusion in a hybrid polypeptide are available at the CDC emm typing center website (see Internet at cdc.gov/ncidod/biotech/strep/emmtypes). In addition, to eliminate the possibility of eliciting tissue cross-reactive antibodies, the amino-terminal regions of a mature M protein and Spa may be compared to known human proteins to detect any homology (e.g., using BLASTP). Preferably, amino-terminal M protein portions having five or more contiguous amino acid matches with a human protein are excluded. In addition, the selected regions of the M peptides and Spa are preferably analyzed by the method of Hopp and Woods (Mol. Immunol. 20:483, 1983) to ensure the integrity of hydrophilic peaks. For example the Hopp and Woods method may be helpful in predicting that a particular immunogenic peptide from an M protein amino-terminal may best be fused with a certain subset of other immunogenic peptides and not with others.

In preferred embodiments, the hybrid polypeptides or variants, and combinations thereof, may be designed to be vaccines specific for streptococci associated with, for example, pharyngitis, scarlet fever, acute rheumatic fever, necrotizing faciitis, cellulitis, meningitis, pneumonia, toxic shock syndrome, bacteremia, septicemia, septic arthritis, pyoderma, skin infections, impetigo, erysipelas, soft-tissue infection, nephritis, pyrogenic reactions, and the like. Additionally, the vaccines may be designed to treat or prevent streptococcal infections in particular populations (e.g., immunocompromised patients, children, and elderly) particular geographic populations (e.g., Australian aborigines), and particular geographic locations (e.g., temperate regions or Scandinavian countries). Preferably, the amino-terminal portions of the different M proteins that comprise an immunogenic peptide can elicit opsonic antibodies that do not cross-react with host tissue.

In another preferred aspect, the M protein serotype is selected based on its prevalence on the most common streptococci known to currently be associated with a particular disease (e.g., serotypes associated with pharyngitis or skin infections) or sequelae. In a further preferred aspect, the instant invention may be used to design and generate a variety of different multivalent immunogenic hybrid polypeptides that may be directed to, or be admixed in particular groupings to address, shifts in prevalent streptococci. For example, as is known in the art, the most prevalent streptococci serotype associated with a disease today, such as ARF, may not be as prevalent or important 5, 10, or 15 years. In another example, a particular streptococcal serotype may be prevalent in Europe and suddenly become important in South America. Hence the immunogenic peptides that comprise a multivalent hybrid polypeptide may be changed, or different multivalent hybrid polypeptides may be mixed and matched to create a desired cocktail, designed for use in a particular population or location to attack the most clinically relevant streptococci as needed.

In certain preferred embodiments, an immunogenic peptide comprises an amino-terminal portion of a M protein or Spa protein having 10-70 amino acids, or any integer in that range; more preferably having 20-65 amino acids, or any integer in that range; and most preferably 30-60 amino acids, or any integer in that range. In particularly preferred embodiments, a hybrid polypeptide comprises at least six or seven different linked immunogenic peptides, wherein each peptide comprises an amino-terminal portion of a streptococcal M protein of at least 30 amino acids, and wherein the polypeptide has an amino-terminal peptide that is reiterated as a carboxy-terminal peptide and the polypeptide is capable of eliciting an immune response against more than one serotype of group A streptococci. Preferably, a hybrid polypeptide is capable of eliciting an immune response against at least serotypes 5, 6, 14, 19, 24, and 29; or at least against serotypes 2, 11, 22, 33, 43, 59, and 94; or at least against serotypes 75, 76, 77, 89, 92, 101, and 114; or at least against serotypes 1.0, 1.2, 3, 12, 18, and 28. In another preferred embodiment, a hybrid polypeptide is capable of eliciting an immune response against a M serotype not represented in the hybrid polypeptide, such as serotype 4.

In certain embodiments, the hybrid polypeptides have at least 50% to 100% amino acid identity, or any integer in that range, to the amino acid sequence as set forth in SEQ ID NOS:2, 4, 6, and 8; preferably 60%-99% identity or any integer in that range, more preferably 70%-97% identity or any integer in that range, and most preferably 80%-95% identity or any integer in that range. As used herein, "percent identity" or "% identity" is the percentage value returned by comparing the whole of the subject polypeptide, peptide, or variant thereof sequence to a test sequence using a computer implemented algorithm, typically with default parameters. Sequence comparisons can be performed using any standard software program, such as BLAST, tBLAST, pBLAST, or MegAlign. Still others include those provided in the Lasergene bioinformatics computing suite, which is produced by DNASTAR.RTM. (Madison, Wis.). References for algorithms such as ALIGN or BLAST may be found in, for example, Altschul, J. Mol. Biol. 219:555-565, 1991; or Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992. BLAST is available at the NCBI website (see Internet at ncbi.nlm.nih.gov/BLAST). Other methods for comparing multiple nucleotide or amino acid sequences by determining optimal alignment are well known to those of skill in the art (see, e.g., Peruski and Peruski, The Internet and the New Biology: Tools for Genomic and Molecular Research (ASM Press, Inc. 1997); Wu et al. (eds.), "Information Superhighway and Computer Databases of Nucleic Acids and Proteins," in Methods in Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997); and Bishop (ed.), Guide to Human Genome Computing, 2nd Edition, Academic Press, Inc., 1998). As used herein, "similarity" between two peptides or polypeptides is generally determined by comparing the amino acid sequence of one peptide or polypeptide to the amino acid sequence and conserved amino acid substitutes thereto of a second peptide or polypeptide. Fragments or portions of the hybrid polypeptides of the present invention may be employed for producing the corresponding full-length hybrid polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length hybrid polypeptides. Similarly, fragments or portions of the nucleic acids of the present invention may be used to synthesize full-length nucleic acids of the present invention.

The hybrid polypeptides and corresponding nucleic acids of the present invention are preferably provided in an isolated form, and in certain preferred embodiments, are purified to homogeneity. As used herein, the term "isolated" means that the material is removed from its original or natural environment. For example, a naturally occurring nucleic acid molecule or polypeptide present in a living animal or cell is not isolated, but the same nucleic acid molecule or polypeptide is isolated when separated from some or all of the co-existing materials in the natural system. The nucleic acid molecules, for example, could be part of a vector and/or such nucleic acids or polypeptides could be part of a composition and still be isolated in that such vector or composition is not part of its natural environment.

The present invention also pertains to hybrid polypeptides and variants thereof produced synthetically or recombinantly, and preferably recombinantly. The immunogenic peptide components of the hybrid polypeptides may be synthesized by standard chemical methods, including synthesis by automated procedure. In general, immunogenic peptides are synthesized based on the standard solid-phase Fmoc protection strategy with HATU as the coupling agent. The immunogenic peptide is cleaved from the solid-phase resin with trifluoroacetic acid containing appropriate scavengers, which also deprotects side chain functional groups. Crude immunogenic peptide is further purified using preparative reversed-phase chromatography. Other purification methods, such as partition chromatography, gel filtration, gel electrophoresis, or ion-exchange chromatography may be used. Other synthesis techniques known in the art may be employed to produce similar immunogenic peptides, such as the tBoc protection strategy, use of different coupling reagents, and the like. In addition, any naturally occurring amino acid or derivative thereof may be used, including D- or L-amino acids and combinations thereof. In particularly preferred embodiments, a synthetic hybrid polypeptide of the invention will have a M2, M12, M24, or M89 immunogenic peptide at the amino-terminus.

As described herein, the hybrid polypeptides of the invention may be recombinant, wherein the hybrid polypeptide is expressed from a polynucleotide encoding a desired hybrid polypeptide that is operably linked to an expression control sequence (e.g., promoter) in a nucleic acid expression construct. In particularly preferred embodiments, a recombinant hybrid polypeptide of the invention will have a M2, M12, M24, or M89 immunogenic peptide at the amino-terminus. Some preferable recombinant hybrid polypeptides comprise immunogenic peptides linked in tandem having the structure of M24-M5-M6-M19-M29-M14-M24, M2-M43-M94-M22-M 11-M59-M33-M 2, M89-M101-M77-M114-M75-M76-M92-M89, or M1.0-M12-Spa-M28-M3-M1.2-M18-M1.0, and any combination thereof. Most preferably, a hybrid polypeptide comprises the amino acid sequence of SEQ ID NOS:2, 4, 6, or 8, and variants thereof. As set forth above and herein, any M serotype may be included in the present invention, preferably serotypes 1.0, 1.2, 2, 3, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 33, 43, 59, 75, 76, 77, 89, 92, 94, 101, and 114 are included in the hybrid polypeptides. In certain preferred embodiments as described herein, hybrid polypeptides of the subject invention capable of eliciting at least one opsonic antibody that is not a tissue cross-reactive antibody in a subject, wherein the subject is an animal or a human.

In another preferred embodiment, the hybrid polypeptides are linked by at least two amino acids encoded by a nucleic acid sequence that is a restriction enzyme recognition site, wherein the restriction sites may be any one or more of BamHI, ClaI, EcoRI, HindIII, KpnI, NcoI, NheI, PmlI, PstI, SalI, XhoI, and the like. Additional amino acid linkers may also be added synthetically as described herein. Preferably, the additional amino acids do not create any identity in sequence within a five amino acid stretch of a human protein. In addition, the hybrid polypeptides of the subject invention may further comprise at least one additional carboxy-terminal amino acid, wherein the additional amino acid is a D- or an L-amino acid. Any of the twenty naturally occurring amino acids or derivatives thereof may be added, such as cysteine, histidine, leucine, and glutamic acid. For example, the addition of cysteine may be useful to attach other constituents, such as a lipid, a carrier protein, and the like.

As described herein, the invention also provides hybrid polypeptide fusion proteins comprising hybrid polypeptides fused to an additional functional or non-functional polypeptide sequence that permits, for example by way of illustration and not limitation, detection, isolation and/or purification of the hybrid polypeptide fusion proteins. For instance, an additional functional polypeptide sequence may be a tag sequence, which includes fusion proteins that may in certain embodiments be detected, isolated and/or purified by protein-protein affinity (e.g., receptor-ligand), metal affinity or charge affinity methods. In certain other embodiments the hybrid polypeptide fusion proteins may be detected by specific protease cleavage of a fusion protein having a sequence that comprises a protease recognition sequence, such that the hybrid polypeptides may be separable from the additional polypeptide sequence. In addition, the hybrid polypeptides may be made synthetically including additional amino acids, a carrier protein, or a tag sequence, which may be located at either the amino- or carboxy-terminal end. In particularly preferred embodiments, for example, recombinant hybrid polypeptides are fused in-frame to a carboxy-terminal tag, which tag may be any one of alkaline phosphatase, .beta.-galactosidase, hexahistidine (6.times.His), FLAG.RTM. epitope tag (DYKDDDDK, SEQ ID NO:18), or GST, and the like. Most preferred are hybrid polypeptide fusion proteins that facilitate affinity detection and isolation of the hybrid polypeptides and may include, for example, poly-His or the defined antigenic peptide epitopes described in U.S. Pat. No. 5,011,912 and in Hopp et al., (1988 Bio/Technology 6:1204), or the XPRESS.TM. epitope tag (DLYDDDDK, SEQ ID NO:19; Invitrogen, Carlsbad, Calif.). The affinity sequence may be a hexa-histidine tag as supplied by a vector. For example, a pBAD/His (Invitrogen) or a pQE-30 (Qiagen, Valencia, Calif.) vector can provide a polyhistidine tag for purification of the mature protein fusion from a particular host, such as a bacterium. Alternatively, the affinity sequence may be added either synthetically or engineered into the primers used to recombinantly generate the nucleic acid sequence (e.g., using the polymerase chain reaction) encoding an immunogenic peptide of the multivalent hybrid polypeptide. Preferably, a multivalent hybrid polypeptide is fused to a polyhistidine and is encoded by a recombinant nucleic acid sequence encoding such a fusion protein.

The immunogenic peptides may also be chemically linked to form the desired hybrid polypeptides, including additional amino acid sequences, by a variety of methods, as provided herein and known in the art (see, generally, Jackson et al., Vaccine 18:355, 2000). Recombinant or synthetic peptides may be linked to form linear (see, e.g., Leclerc, et al., Eur. J. Immunol. 17:26, 1987 and Francis, et al., Nature 330:168, 1987) or branched (see, e.g., Fitzmaurice, et al., Vaccine 14:553, 1996) constructs, or may be linked using chemical ligation of epitopes (see, e.g., Tam and Spetzler, Biomed. Pept. Proteins Nucleic Acids 1:123, 1995; Rose, J. Am. Chem. Soc. 116:30, 1994; and Dawson, et al., Science 266:776, 1994). Peptides may also be linked via the multiple antigen peptide system to form branched hybrid polypeptides (see, e.g., Tam, Proc. Natl. Acad. Sci. USA 85:5409, 1988; U.S. Pat. No. 5,229,490). The multiple antigen peptide system makes use of multifunctional core molecules where each functional group on the core molecule forms at least two branches, the principal units of which are also multifunctional. For example, lysine may be used as the core peptide because it has one carboxyl functional group and two (.alpha. and .epsilon.) amine functional groups. Each multifunctional unit in a branch provides a base for added growth, resulting in exponential growth of the dendritic polymer. Peptides may then be joined to the dendritic core using a linking molecule (e.g., glycine). The multiple antigen peptide system links a large number of synthetic peptides to the functional group of a dendritic core molecule providing a high concentration of synthetic peptides in a low molecular volume. Preferably, either two or three levels of geometrically branched lysines are used, wherein these lysine cores form a tetrameric and octameric core structure, respectively. The multiple antigen peptide system may also include a lipophilic anchoring moiety attached to the core molecule, thereby eliminating the need for an adjuvant formulated in a peptide vaccine otherwise requiring one for immunostimulation (see, e.g., U.S. Pat. No. 5,580,563). In one preferred embodiment, a hybrid polypeptide of the invention comprises immunogenic peptides linked to form a lysine core-branched polypeptide.

Additionally, similar or different synthetic peptides may be linked by controlled polymerization through derivatization of the amino-terminus of a peptide with the acryloyl (CH.sub.2.dbd.CH--) group using acryloyl chloride (see, e.g., O'Brien-Simpson, et al., J. Am. Chem. Soc. 119:1183, 1997; Jackson, et al., Vaccine 15:1697, 1997). The derivatized peptides are then polymerized singly, or in admixture with similarly derivatized peptides, by free radical initiation of chain elongation. As a result, peptides are assembled into polymers in which the peptide determinants form side chains pendant from an alkane backbone. The hybrid polypeptides and fusion proteins as described herein may be constructed as set forth above. In one preferred embodiment, a hybrid polypeptide of the invention comprises immunogenic peptides linked by an alkane backbone. In certain embodiments, the alkane backbone is acrylamide or an analog or derivative thereof.

Therapeutic Formulations and Methods of Use

The invention also relates to pharmaceutical compositions that contain one or more hybrid multivalent polypeptides, which may be used to elicit an immune response. The invention further relates to methods for treating and preventing microbial infections by administering to a subject a hybrid polypeptide or a mixture of hybrid polypeptides at a dose sufficient to elicit antibodies specific for one or more hybrid polypeptide, as described herein. A hybrid polypeptide or a cocktail of hybrid polypeptides is preferably part of a pharmaceutical composition when used in the methods of the present invention.

In certain aspects, the invention provides a composition comprising a pharmaceutically acceptable carrier, diluent, or excipient, and any of the multivalent hybrid polypeptides of the subject invention and any combination thereof. A preferred embodiment is a pharmaceutically acceptable carrier and a mixture of at least two or three hybrid polypeptides of the subject invention. In yet another preferred embodiment, a hybrid polypeptide that comprises at least seven different immunogenic peptides linked in tandem, wherein each peptide comprises an amino-terminal portion of a streptococcal M protein of at least 50 amino acids, and wherein the polypeptide has an amino-terminal peptide that is reiterated as a carboxy-terminal peptide and the polypeptide is capable of eliciting an immune response against more than one serotype of group A streptococci comprising at least serotypes 1.0, 1.2, 3, 12, 18, and 28, is combined with at least one other hybrid polypeptide of the subject invention and a pharmaceutically acceptable carrier. In a more preferred embodiment, a composition comprises a pharmaceutically acceptable carrier and a mixture of the hybrid polypeptides of SEQ ID NOS:2, 4, 6, and 8 or variants thereof, and in other embodiment these four polypeptides or variants thereof are mixed in equimolar amounts and at least one of the hybrid polypeptides has a Spa peptide. In other embodiments, SEQ ID NOS:2, 4, 6, and 8 or variants thereof are provided with a polyhistidine tag or further comprise at least one additional amino acid, such as cysteine. Each of these formulations may further comprise an adjuvant, such as alum or Freund's, and a diluent such as water or PBS. Further, therapeutic compositions of the present invention should preferably be stable for several months and capable of being produced and maintained under sterile conditions.

The pharmaceutical composition will include at least one of a pharmaceutically acceptable vehicle, carrier, diluent, or excipient, in addition to one or more hybrid multivalent polypeptide or fusion protein thereof and, optionally, other components. For example, pharmaceutically acceptable carriers suitable for use with a composition of a hybrid polypeptide or fusion protein thereof, or cocktail of two or more hybrid polypeptide or fusion protein thereof, may include, for example, a thickening agent, a buffering agent, a solvent, a humectant, a preservative, a chelating agent, an adjuvant, and the like, and combinations thereof. Exemplary adjuvants are alum (aluminum hydroxide, REHYDPAGEL.RTM.), aluminum phosphate, proteosome adjuvant (see, e.g., U.S. Pat. Nos. 5,726,292 and 5,985,284), virosomes, liposomes with and without lipid A, DETOX.RTM. (Ribi/Corixa), MF59, or other oil and water emulsions type adjuvants, such as nanoemulsions (see, e.g, U.S. Pat. No. 5,716,637) and submicron emulsions (see, e.g., U.S. Pat. No. 5,961,970), and Freund's complete and incomplete. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and as described herein and, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, ed., 18.sup.th Edition, 1990) and in CRC Handbook of Food, Drug, and Cosmetic Excipients, CRC Press LLC (S. C. Smolinski, ed., 1992).

"Pharmaceutically acceptable salt" refers to salts of the compounds of the present invention derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts). The compounds of the present invention may be used in either the free base or salt forms, with both forms being considered as being within the scope of the present invention.

In addition, the pharmaceutical composition may further include a diluent such as water or phosphate buffered saline (PBS). Preferably, diluent is PBS with a final phosphate concentration ranges from about 0.1 mM to about 1 M, more preferably from about 0.5 mM to about 500 mM, even more preferably from about 1 mM to about 50 mM, and most preferably from about 2.5 mM to about 10 mM; and the final salt concentration ranges from about 100 mM to about 200 mM and most preferably from about 125 mM to about 175 mM. Preferably, the final PBS concentration is about 5 mM phosphate and about 150 mM salt (such as NaCl). In certain embodiments, any of the aforementioned pharmaceutical compositions comprising a cocktail of multivalent hybrid polypeptides of the instant invention are preferably sterile.

The compositions can be sterile either by preparing them under an aseptic environment and/or they can be terminally sterilized using methods available in the art. Many pharmaceuticals are manufactured to be sterile and this criterion is defined by the USP XXII <1211>. Sterilization in this embodiment may be accomplished by a number of means accepted in the industry and listed in the USP XXII <1211>, including gas sterilization, ionizing radiation or filtration. Sterilization may be maintained by what is termed aseptic processing, defined also in USP XXII <1211>. Acceptable gases used for gas sterilization include ethylene oxide. Acceptable radiation types used for ionizing radiation methods include gamma, for instance from a cobalt 60 source and electron beam. A typical dose of gamma radiation is 2.5 MRad. When appropriate, filtration may be accomplished using a filter with suitable pore size, for example 0.22 .mu.m and of a suitable material, for instance TEFLON.RTM.. The term "USP" refers to U.S. Pharmacopeia (see the USP website: usp.org; Rockville, Md.).

The present invention also pertains to methods for preventing a microbial infection, comprising administering to a subject a composition of the subject invention at a dose sufficient to elicit antibodies specific for one or more hybrid polypeptide, wherein the antibodies are preferably opsonic and are not tissue cross-reactive. In certain embodiments an infection is a streptococcal infection, such as a group A streptococcal infection. A subject suitable for treatment with a hybrid polypeptide formulation may be identified by well-established indicators of risk for developing a disease or well-established hallmarks of an existing disease. For example, indicators of an infection include fever, pus, microorganism positive cultures, inflammation, and the like. Infections that may be treated with a hybrid polypeptide of the subject invention include, without limitation, those caused by or due to microorganisms, whether the infection is primary, secondary, opportunistic, or the like. Examples of microorganisms include Gram-positive bacteria, such as streptococci.

The pharmaceutical compositions that contain one or more hybrid polypeptides may be in any form that allows for the composition to be administered to a subject, such as a human or animal. For example, multivalent hybrid polypeptide compositions of the present invention may be prepared and administered as a liquid solution or prepared as a solid form (e.g., lyophilized), which may be administered in solid form, or resuspended in a solution in conjunction with administration. The hybrid polypeptide composition is formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a subject or patient or bioavailable via slow release. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units. In certain preferred embodiments, any of the aforementioned pharmaceutical compositions comprising a hybrid polypeptide or cocktail of hybrid polypeptides of the invention are in a container, preferably in a sterile container.

In one embodiment, the therapeutic composition is administered orally, and a hybrid polypeptide or cocktail composition of the invention is taken up by cells, such as cells located in the lumen of the gut. Other typical routes of administration include, without limitation, enteral, parenteral, transdermal/transmucosal, and inhalation. The term enteral, as used herein, is a route of administration in which the agent is absorbed through the gastrointestinal tract or oral mucosa, including oral, rectal, and sublingual. The term parenteral, as used herein, describes administration routes that bypass the gastrointestinal tract, including intraarterial, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intravenous, subcutaneous, submucosal, and intravaginal injection or infusion techniques. The term transdermal/transmucosal, as used herein, is a route of administration in which the agent is administered through or by way of the skin, including topical. The term inhalation encompasses techniques of administration in which an agent is introduced into the pulmonary tree, including intrapulmonary or transpulmonary. Preferably, the compositions of the present invention are administered intramuscularly.

Depending upon the application, the dose of hybrid polypeptide in the compositions will vary, generally, from about 10 .mu.g to about 10 mg, preferably from about 100 .mu.g to 1 mg, more preferably from about 150 .mu.g to 500 .mu.g, and most preferably from about 200 .mu.g to about 400 .mu.g, in combination with the biologically acceptable excipient, adjuvant, binder, and/or diluent, including any integer with the dosing range. As used herein, the term "about" or "consists essentially of" refers to .+-.10% of any indicated structure, value, or range. Booster immunizations may be given at multiple times, at desired intervals ranging from about 2 weeks to about 24 weeks, preferably 2, 4 and 8 week intervals, and more preferably 2, 4, and 16 week intervals, and even more preferably 0, 4, and 24 week intervals to maximize the immune response.

The invention further provides a plurality of antibodies produced by the method for preventing a microbial infection that comprises administering to a subject a composition of the subject invention at a dose sufficient to elicit antibodies specific for one or more hybrid polypeptide, wherein the antibodies are opsonic and are not tissue cross-reactive. In one embodiment, the antibodies comprise at least one antibody specific for a M serotype not represented in a hybrid polypeptide, such as type 4 M protein. In another embodiment, a method for treating or preventing a microbial infection comprises administering to a subject a composition comprising a pharmaceutically acceptable carrier and a plurality of antibodies of the subject invention.

Claim 1 of 15 Claims

1. A composition comprising a pharmaceutically acceptable carrier and a mixture of at least two hybrid polypeptides selected from (a) a hybrid polypeptide consisting of six different immunogenic amino terminal peptides from six different group A streptococcal M proteins M24, M5, M6, M19, M29 and M14, wherein the six different immunogenic amino terminal peptides are linked in tandem by at least two amino acids, wherein each of the immunogenic amino terminal peptides comprises at least 30 contiguous amino acids of each of said group A streptococcal M proteins, wherein the immunogenic amino terminal peptide at the amino terminus of the hybrid polypeptide is reiterated at the carboxy terminus of the hybrid polypeptide and wherein the hybrid polypeptide is capable of eliciting an immune response against more than one of said group A streptococcal M proteins M5, M6, M14, M19, M24 and M29; (b) a hybrid polypeptide consisting of seven different immunogenic amino terminal peptides from seven different group A streptococcal M proteins M2, M43, M94, M22, M11, M59 and M33, wherein the seven different immunogenic amino terminal peptides are linked in tandem by at least two amino acids, wherein each of the immunogenic amino terminal peptides comprises at least 35 contiguous amino acids of each of said group A streptococcal M proteins, wherein the immunogenic amino terminal peptide at the amino terminus of the hybrid polypeptide is reiterated at the carboxy terminus of the hybrid polypeptide and wherein the hybrid polypeptide is capable of eliciting an immune response against more than one of said group A streptococcal M proteins M2, M11, M22, M33, M43, M59 and M94; (c) a hybrid polypeptide consisting of seven different immunogenic amino terminal peptides from seven different group A streptococcal M proteins M89, M101, M77, M114, M75, M76 and M92, wherein the seven different immunogenic amino terminal peptides are linked in tandem by at least two amino acids, wherein each of the immunogenic amino terminal peptides comprises at least 40 contiguous amino acids of each of said group A streptococcal M proteins, wherein the immunogenic amino terminal peptide at the amino terminus of the hybrid polypeptide is reiterated at the carboxy terminus of the hybrid polypeptide and wherein the hybrid polypeptide is capable of eliciting an immune response against more than one of said group A streptococcal M proteins M75, M76, M77, M89, M92, M101 and M114; and (d) a hybrid polypeptide consisting of seven different immunogenic amino terminal peptides from seven different group A streptococcal proteins M1.0, M12, Spa, M28, M3, M1.2, M18 and M1.0, wherein the seven different immunogenic amino terminal peptides are linked in tandem by at least two amino acids, wherein each of the immunogenic amino terminal peptides comprises at least 50 contiguous amino acids of each of said group A streptococcal proteins, wherein the immunogenic amino terminal peptide at the amino terminus of the hybrid polypeptide is reiterated at the carboxy terminus of the hybrid polypeptide and wherein the hybrid polypeptide is capable of eliciting an immune response against more than one of said group A streptococcal proteins Spa, M1.0, M1.2, M3, M12, M18 and M28.

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