HK1215452B - Antibodies to s. aureus surface determinants - Google Patents

Description

Antibodies to surface determinants of Staphylococcus aureus

The present disclosure generally relates to antibodies that bind to surface determinants of Staphylococcus aureus (S. aureus) and antibodies that bind to toxins secreted by S. aureus. The present disclosure also relates to combinations of antibodies that bind to surface determinants of S. aureus together with antibodies that bind to toxins secreted by S. aureus, compositions comprising such combinations of antibodies, and methods for preventing S. aureus-associated diseases comprising administering such combinations of antibodies.

Staphylococcus aureus is a gram-positive, aerobic, cluster-forming coccus bacterium that typically colonizes the nose and skin of healthy people. At any given time, approximately 20% to 30% of the human population is colonized with S. aureus. Staphylococcus aureus bacteria (sometimes also referred to as "staph," "Staph. aureus," or "S. aureus") are considered opportunistic pathogens that cause minor infections (e.g., pimples, boils, and other soft tissue infections) and systemic infections (e.g., pneumonia, sepsis, osteomyelitis, and endocarditis).

In some embodiments, the present invention relates to the staphylococcus aureus infection of the present invention. Generally, mucous membrane and epidermal barrier (skin) protect human body from staphylococcus aureus infection. Due to the destruction of these natural barriers caused by damage (for example, burns, trauma and surgical operation) significantly increased infection risk. The disease that damages immune system (for example, diabetes, end-stage renal disease and cancer) also increases infection risk. Opportunistic staphylococcus aureus bacterial infection can become serious, causing various diseases or illness, and its non-limiting examples include bacteremia, cellulitis, eyelid infection, food poisoning, joint infection, skin infection, scalded skin syndrome, toxic shock syndrome, pneumonia, osteomyelitis, endocarditis, meningitis and abscess formation.

Staphylococcus aureus expresses a variety of surface determinant antigens, including the serine-aspartate repeat proteins SdrC, SdrD, and SdrE, the aggregation factor proteins ClfA and ClfB, the iron-regulated surface determinant proteins IsdA, IsdB, IsdC, IsdE, and IsdH, Staphylococcus aureus protein A (SpA), and the polysaccharide poly-N-acetylglucosamine (PNAG). These surface antigens play a role in the colonization of host tissues, circumvention of host immune responses, and bacterial fitness. Mutations in ClfA, SpA, IsdA, IsdB, and IsdH have been shown to reduce the virulence of Staphylococcus aureus.

Protein (such as IsdH) plays a role in the ability of Staphylococcus aureus to circumvent the host immune response, such as neutrophil-mediated phagocytosis, a key process for Staphylococcus aureus to cause infection. IsdH is a part of the following complex, which is activated under iron-limited conditions for binding hemoglobin and haptoglobin-hemoglobin complex, and then extracts and transports heme into the cytoplasm. Three N-terminal NEAr transporter (NEAT) motifs are present in IsdH, and the determined structure of NEAT1 indicates that certain residues in this motif are involved in ligand binding. It has been shown that compared with the wild type, the IsdH-deficient mutant of Staphylococcus aureus has reduced toxicity and is more rapidly engulfed by human neutrophils in the presence of serum opsonins. The protective mechanism of IsdH seems to be derived from the accelerated degradation of serum opsonin C3b. Therefore, IsdH plays a role in the anti-phagocytic properties of Staphylococcus aureus organisms.

ClfA is a virulence factor that binds fibrinogen. This function of ClfA appears to further contribute to the anti-phagocytic properties of S. aureus. In addition, ClfA also promotes S. aureus aggregation in blood and biofilm formation on biomaterial surfaces.

Staphylococcus aureus also expresses several other virulence factors, including capsular polysaccharides and protein toxins. A virulence factor often associated with Staphylococcus aureus infection is alpha toxin (also known as α-hemolysin or Hla), a pore-forming and hemolytic extracellular protein produced by the most virulent strains of Staphylococcus aureus. The toxin forms heptamer pores in the membranes of susceptible cells (e.g., leukocytes, platelets, erythrocytes, peripheral blood mononuclear cells, macrophages, keratinocytes, fibroblasts, and endothelial cells). Alpha toxin pore formation often leads to cell dysfunction or dissolution. It can also cause destruction of epithelial and endothelial tight junctions and immune regulation abnormalities.

Currently, S. aureus is the leading cause of infection-related mortality in the United States and is the leading cause of hospital-acquired infections. Furthermore, increasing resistance of S. aureus to antibiotics has compounded this problem. Therefore, it would be desirable to develop effective alternative methods for diagnosing and treating S. aureus infections, including combined antibody therapies.

As previously disclosed in U.S. Provisional Application No. 61/440,581 and in International Application No. PCT/US2012/024201 (published as WO 2012/109205), the contents of each of which are incorporated herein by reference, antibodies that bind to S. aureus α-toxin have been shown to reduce CA-MRSA disease severity in a murine skin necrosis model and promote bacterial clearance in a mouse staphylococcal pneumonia model. Therefore, such antibodies can be utilized to treat S. aureus-associated diseases.

In addition to antibodies that bind to Staphylococcus aureus alpha-toxin, the present disclosure also provides antibodies and combinations thereof directed against Staphylococcus aureus surface determinant antigens. The present invention provides compositions comprising such antibodies or combinations of such antibodies, and methods for preventing and/or treating Staphylococcus aureus-associated diseases using such antibodies or combinations of such antibodies. Methods for preventing and/or treating Staphylococcus aureus-associated diseases using antibodies that bind to Staphylococcus aureus alpha-toxin are described in U.S. Provisional Application No. 61/440,581 and in International Application No. PCT/US2012/024201 (published as WO 2012/109205) (the contents of which are incorporated herein by reference), as well as in the U.S. Provisional Application (the contents of which are incorporated herein by reference) filed together with the present application of Salman et al., entitled "Methods of Treating S.Aureus Associated Diseases."

The present invention also provides certain combinations of antibodies, such as an antibody that binds to a surface determinant of S. aureus combined with an antibody that binds to S. aureus alpha toxin, wherein such combinations work together synergistically. The present invention also provides for combining antibodies that target surface determinant antigens of S. aureus involved in circumventing the host's opsonophagocytic function with antibodies that target a toxin secreted by S. aureus involved in directly damaging host cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the percentage of opsonophagocytic killing (OPK) induced by anti-IsdH antibodies B11, 2F4, and A7 compared to the percentage OPK induced by control antibody R347 when tested with S. aureus strains Newman and USA300.

Figure 2 shows the binding of anti-IsdH antibodies B11, 2F4, A7 and 1C1 to S. aureus strains ARC2081 and USA300 compared to control antibody R347.

Figure 3A is a graph showing the competitive binding between antibody 1C1 and haptoglobin (Hp) for the subunit Neat-1 bound to IsdH, compared to the competitive binding between control antibody R347 and Hp. Figure 3B is a graph showing the competitive binding between antibody 2F4 and Hp for the subunit Neat-2 bound to IsdH, compared to the competitive binding between control antibody R347 and Hp.

Figure 4A shows the concentration of S. aureus colony forming units (CFU) measured in a mouse bacteremia model in the presence of antibody 1C1. The CFU concentration is reported as log ₁₀ [CFU/ml]. Figure 4B shows the concentration of S. aureus CFU measured in a mouse bacteremia model in the presence of antibody A7. The CFU concentration is reported as log ₁₀ [CFU/ml]. Figure 4C shows the concentration of S. aureus CFU measured in a mouse bacteremia model in the presence of antibody IsdH0016. The CFU concentration is reported as log ₁₀ [CFU/ml]. Figure 4D shows the concentration of S. aureus CFU measured in a mouse bacteremia model in the presence of antibody IsdH003. The CFU concentration is reported as log ₁₀ [CFU/ml].

Figure 5 shows the concentration of Staphylococcus aureus colony forming units (CFU) measured in a mouse bacteremia model in the presence of antibody 2F4. CFU concentrations are reported as log ₁₀ [CFU/ml].

Figure 6 graphically depicts 2F4 binding measured as a percentage of maximal binding in S. aureus strains ARC2379 (USA100), ARC2081 (USA200), and BAA-1556 (USA300) at times _To , _Tihr , and _T4hr compared to control antibody R347.

Figure 7A shows the percent OPK induced by antibody 2F4 when tested with S. aureus strain Newman, compared to the percent OPK induced by control antibody R347. Figure 7B shows the percent OPK induced by antibody 2F4 when tested with S. aureus strain ARC634, compared to the percent OPK induced by control antibody R347. Figure 7C shows the percent OPK induced by antibody 2F4 when tested with S. aureus strain ARC2081 (USA200), compared to the percent OPK induced by control antibody R347. Figure 7D shows the percent OPK induced by antibody 2F4 when tested with S. aureus strain BAA-1556 (USA300), compared to the percent OPK induced by control antibody R347.

Figure 8 shows the evaluation of the affinity of 2F4 for IsdH and for Neat-2 subunits in a hu1gGFc capture assay.

Figure 9A shows the kidney CFU concentration of S. aureus strain USA300 measured in the organ burden model after treatment with 2F4 alone, 2A3 alone, or a combination of 2F4 and 2A3 (given at an initial concentration of 2.05e8). CFU concentrations are reported as log ₁₀ [CFU/organ]. Figure 9B shows the kidney CFU concentration of S. aureus strain USA300 measured in the organ burden model after treatment with 2F4 alone, 2A3 alone, or a combination of 2F4 and 2A3 (given at an initial concentration of 2.05e8). CFU concentrations are reported as log ₁₀ [CFU/organ].

Figure 10 shows that anti-CIfA antibodies inhibit CIfA binding to immobilized fibrinogen. Anti-CIfA antibodies 23D6, 27H4, 11H10 showed increased inhibition of binding to fibrinogen compared to the control R347.

Figure 11 shows that anti-ClfA antibodies inhibit the aggregation of Staphylococcus aureus in human plasma. The ability of anti-ClfA antibodies 23D6, 27H4, and 11H10 to inhibit the aggregation of three different Staphylococcus aureus strains (NR S112, BAA 1556, and UAMS-1) compared to the control R347. mAbs and ClfA proteins were not tested. Anti-ClfA antibody 11H10 demonstrated the greatest strain coverage in this assay.

Figure 12 shows that anti-CIfA antibody 11H10 binds to a different epitope on CIfA compared to anti-CIfA antibodies 23D6 and 27H4.

Figure 13 demonstrates that anti-CIfA antibody 11H10 and anti-AT antibody LC10 reduce bacterial burden in the heart.

Figure 14 shows the effect of anti-ClfA antibody 11H10 in a murine sepsis model.

FIG15 shows the effects of anti-ClfA antibody 11H10, anti-AT antibody LC10, and the combination of anti-ClfA antibody 11H10 and anti-AT antibody LC10 in a murine sepsis model challenged with CA-MRSA USA300 (IV lethal challenge).

FIG16 shows the effect of the combination of anti-ClfA antibody 11H10 and anti-At antibody LC10 in a murine sepsis model challenged with HA-MRSA USA100 (IV lethal challenge).

FIG17 shows the effect of the combination of anti-ClfA antibody 11H10 and anti-AT antibody LC10 in a murine sepsis model challenged with HA-MRSA USA200 (IV lethal challenge).

FIG18 shows the effect of the combination of anti-IsdH antibody 2F4 and anti-AT antibody LC10 in a murine sepsis model challenged with HA-MRSA USA100 (IV lethal challenge).

Description of Illustrative Embodiments

Reference will now be made in detail to certain exemplary embodiments according to the present disclosure, some examples of which are illustrated in the accompanying drawings.

Disclosed herein are antibodies that bind to surface determinant antigens of Staphylococcus aureus, including human, humanized and/or chimeric forms and fragments, derivatives/conjugates and compositions thereof, as well as antibodies that bind to toxins secreted by Staphylococcus aureus. Such antibodies can be useful for detecting and/or visualizing Staphylococcus aureus and can therefore be used in diagnostic methods and assays. The antibodies described herein also interfere with surface determinants of Staphylococcus aureus, thereby interfering with colonization and immune evasion, making these antibodies useful for therapeutic and preventive methods. Similarly, the antibodies described herein can bind to toxins secreted by Staphylococcus aureus, thereby reducing the virulence of Staphylococcus aureus infection. Combining antibodies that target both surface determinants and secreted toxins of Staphylococcus aureus can increase the therapeutic or preventive effect achieved by either antibody when administered alone.

Staphylococcus aureus expresses a variety of surface determinant antigens that are important for colonization, immune evasion, and fitness. Such surface determinants include SdrC, SdrD, SdrE, ClfA, ClfB, IsdA, IsdB, IsdC, IsdE, IsdH, SpA, FnbA, and PNAG. Antibodies disclosed herein can target surface determinant antigens.

S. aureus also produces a large number of secretory and cell-associated proteins, many of which are involved in pathogenesis, such as α-toxin (AT), β-toxin, γ-toxin, δ-toxin, leukocidin, toxic shock syndrome toxin (TSST), enterotoxin, coagulase, protein A, fibrinogen, etc. Alpha toxin is one of the virulence factors of S. aureus and is produced by most pathogenic S. aureus strains.

A. Antibodies against surface determinants and secreted toxins of Staphylococcus aureus

As used herein, the terms "an antibody," "antibodies" (also referred to as "immunoglobulins"), and "antigen-binding fragment" include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two different epitope-binding fragments, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments (e.g., antigen-binding portion) that exhibit a desired biological activity, disulfide-linked Fv (dsFv), and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies provided herein), intrabodies, and epitope-binding fragments of any of the above. Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain at least one antigen binding site. Immunoglobulin molecules can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), subisotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or allotype (e.g., Gm, such as G1m (f, z, a, or x), G2m (n), G3m (g, b, or c), Am, Em, and Km (1, 2, or 3)). Antibodies can be derived from any mammal, including but not limited to humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, and the like, or other animals, such as birds (e.g., chickens).

In certain embodiments, the antibodies of the present invention or their immunospecific fragments include antigen binding domains. Antigen binding domains are formed by antibody variable regions that vary from one antibody to another. Naturally occurring antibodies include at least two antigen binding domains, i.e., they are at least bivalent. As used herein, the term "antigen binding domain" includes sites that specifically bind to epitopes on antigens (e.g., cell surfaces of soluble antigens). The antigen binding domains of an antibody typically include at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region. The binding site formed by these variable regions determines the specificity of the antibody.

As used herein, unless otherwise specifically indicated, "mutation" encompasses the addition, deletion, substitution (including conservative substitutions) or other changes of at least one amino acid or nucleic acid. Unless otherwise specifically indicated, "conservative substitutions" refer to the replacement of a first amino acid with a second amino acid that does not significantly change the chemical, physical and/or functional properties of the antibody or its antigen-binding fragment (e.g., the antibody or its antigen-binding fragment retains the same charge, structure, polarity, hydrophobicity/hydrophilicity, and/or maintains function, such as the ability to bind to alpha toxin and thereby reduce the virulence of Staphylococcus aureus). Conservative substitutions also refer to the replacement of a first nucleic acid with a second nucleic acid encoding a previously described conservative amino acid substitution.

The antibodies provided herein include full-length or intact antibodies, antibody fragments, native sequence antibodies or amino acid variants of native antibodies, human, humanized, post-translationally modified, chimeric or fusion antibodies, immunoconjugates and functional fragments thereof. Antibodies can be modified in the Fc region, and certain modifications can provide desired effector functions or altered serum half-life.

Unless otherwise indicated, the numbering of amino acids in the variable domains, complementary determining regions (CDRs) and framework regions (FRs) of antibodies follows the Kabat definition as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, Public Health Service, 5th Ed., National Institutes of Health, Bethesda, Maryland (1991). Using this numbering system, the actual linear amino acid sequence may contain a shortening of the FR or CDR corresponding to the variable domain, or fewer or additional amino acids inserted. The Kabat numbering of residues can be determined for a given antibody by comparing the antibody sequence with a "standard" Kabat numbering sequence in a homologous region. The maximum alignment of framework residues often requires insertion of "interval" residues in the numbering system to be used for the Fv region. In addition, due to interspecies or allelic differences, the identity of certain individual residues at any given Kabat site numbering may vary from antibody chain to antibody chain.

In certain embodiments, an isolated antibody is provided. As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies and the molecules that are typically present in the natural cell environment. Therefore, in some embodiments, the antibody provided is an isolated antibody, wherein they are separated from antibodies with different antigenic specificities. The isolated antibody can be a monoclonal antibody or a polyclonal antibody. However, the isolated antibody that specifically binds to an epitope, isotype or variant of a staphylococcus aureus surface antigen or secreted toxin can have cross-reactivity with, for example, other related antigens from other species (for example, staphylococcal species homologues). The isolated antibody provided can be substantially free of one or more other cellular materials. In certain embodiments, the combination of "isolated" monoclonal antibodies is provided, and relates to antibodies with different specificities and combined in the compositions of definition.

Also disclosed are isolated nucleic acid sequences encoding the amino acid sequences of the disclosed antibodies and antigen-binding fragments thereof. Due to the degeneracy of the nucleotide code, more than one nucleotide may be present at any nucleic acid position, but still encode the same amino acid. In some embodiments, nucleic acid sequences encoding the following amino acid sequences are provided, which are 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% (or any percentage in between) identical to the amino acid sequences of the disclosed antibodies or antigen-binding fragments thereof that bind to Staphylococcus aureus surface antigens or secreted toxins. In further embodiments, these nucleic acid sequences encode the following amino acid sequences, which retain the functional ability of the disclosed antibodies and antigen-binding fragments thereof, such as binding to Staphylococcus aureus surface antigens or secreted toxins and thereby reducing Staphylococcus aureus colony growth, escape from opsonophagocytosis, or the toxicity of secreted toxins.

In various embodiments, the antibodies or fragments disclosed herein can specifically bind to a Staphylococcus aureus surface antigen or secreted toxin polypeptide, or an antigenic fragment thereof. In certain embodiments, the surface antigen is SdrC, SdrD, SdrE, ClfA, ClfB, IsdA, IsdB, IsdC, IsdE, IsdH, SpA, FnbA, or PNAG. In further embodiments, the surface antigen is IsdH. In other embodiments, the surface antigen is ClfA. In some embodiments, the secreted toxin is an alpha toxin or a phenol-soluble regulatory protein. In further embodiments, the secreted toxin is an alpha toxin. Certain amino acid and nucleic acid sequences useful for alpha toxin antibodies disclosed herein are disclosed in U.S. Provisional Application No. 61/440,581 and in International Application No. PCT/US2012/024201 (published as WO 2012/109205), the contents of each of which are hereby incorporated in their entirety.

The antibodies provided herein can specifically bind to one or more epitopes specific for a surface determinant antigen or secreted toxin protein of Staphylococcus aureus and generally do not specifically bind to other polypeptides. As used herein, the term "epitope" refers to a peptide, subunit, fragment, portion, oligomer, or any combination thereof that can be bound by an antibody.

In certain embodiments, antibodies or antigen-binding fragments thereof to surface determinant antigens or secreted toxins of Staphylococcus aureus can bind to epitopes that are conserved across species. In some embodiments, the antibodies or antigen-binding fragments thereof bind to surface determinant antigens or secreted toxins of Staphylococcus aureus or homologs or orthologs thereof from another bacterial species, and antigenic fragments thereof. In some embodiments, the antibodies or antigen-binding fragments thereof can bind to one or more isoforms of surface determinant antigens or secreted toxins.

In various embodiments, the epitope is composed of at least a portion of a surface determinant antigen from Staphylococcus aureus. These surface determinant antigens may include SdrC, SdrD, SdrE, ClfA, ClfB, IsdA, IsdB, IsdC, IsdE, IsdH, SpA, FnbA, or PNAG. In some embodiments, the antigen is IsdH. In other embodiments, the antigen is ClfA. In other embodiments, the epitope is composed of at least a portion of a toxin secreted by Staphylococcus aureus. In some embodiments, the secreted toxin is an alpha toxin, which is involved in the formation of an alpha toxin heptamer complex.

The epi-position can include any amino acid sequence, and this amino acid sequence includes at least 3 continuous amino acid residues from the amino acid sequence of the target antigen.This epi-position can include longer amino acid sequence, up to and include the whole amino acid sequence of the target antigen.In certain embodiments, this epi-position includes at least 4 amino acid residues, at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues or at least 15 amino acid residues from the amino acid sequence of the target antigen.In certain other embodiments, this epi-position includes 2,3,4,5,6,7,8,9,10,11,12,13,14 or 15 continuous or non-continuous amino acid residues from the amino acid sequence of the target antigen.

In certain embodiments, the following combination is provided, comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to a toxin secreted by Staphylococcus aureus and an isolated antibody that specifically binds to a surface determinant antigen of Staphylococcus aureus. In further embodiments, the antibody that binds to a surface determinant antigen of Staphylococcus aureus binds to an antigen selected from the group consisting of SdrC, SdrD, SdrE, ClfA, ClfB, IsdA, IsdB, IsdC, IsdE, IsdH, SpA, FnbA, or PNAG. In still further embodiments, the surface determinant antigen is IsdH. In certain embodiments, the antibody that binds to a toxin secreted by Staphylococcus aureus binds to a toxin selected from alpha toxin and phenol-soluble regulatory proteins. In further embodiments, the secreted toxin is alpha toxin.

In certain embodiments, the antibody or antibody combination is present in an aqueous solution. In other embodiments, the antibody or antibody combination is present in a powdered or lyophilized form. In certain embodiments, the antibody or antibody combination is at a concentration sufficient for therapeutic or diagnostic use. In some embodiments, the antibody or antibody combination is present in a sterile vessel or container.

In certain embodiments, antibodies or antigen-binding fragments thereof capable of binding to Staphylococcus aureus surface antigens or secreted toxins are prepared from parent antibodies. As used herein, the term "parent antibody" refers to an antibody encoded by an amino acid sequence for preparing a variant or derivative antibody as defined herein. A parent polypeptide may include a natural antibody sequence (i.e., a naturally occurring antibody polypeptide, including naturally occurring allelic variants) or a naturally occurring sequence with a pre-existing amino acid sequence modification (such as insertion, deletion, and/or substitution). The parent antibody may be a humanized antibody or a human antibody. In a specific embodiment, these Staphylococcus aureus surface antigens or secreted toxin antibodies and antigen-binding fragments thereof are variants of the parent antibody. As used herein, the term "variant" refers to an antibody or antigen-binding fragment thereof that is different from the "parent" antibody or its antigen-binding fragment amino acid sequence in its amino acid sequence by adding, deleting, and/or replacing one or more amino acid residues in the parent antibody sequence.

Antibodies against Staphylococcus aureus surface antigens or secreted toxins and antigen-binding fragments thereof of the present invention comprise at least one antigen-binding domain. The antigen-binding portion of the antibody comprises one or more fragments of the antibody that retain the ability to specifically bind to the antigen. These retained portions may include heavy chain variable regions and/or light chain variable regions from a parent antibody or a variant of the parent antibody.

B. Anti-IsdH Antibodies

As used herein, the term "percentage (%) of sequence identity" or "homology" is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in a reference sequence, after aligning the sequences and, if necessary, introducing gaps (so as to obtain maximum percentage sequence identity) and excluding conservative substitutions. In addition to manual alignment, optimal alignment of sequences for comparison can be generated by means of local homology algorithms known in the art or by means of computer programs that use these algorithms (e.g., GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA).

In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to the surface antigen IsdH comprises a heavy chain variable region (VH) that is 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 80, 82, 84, 86, or 88. In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds to IsdH comprises a light chain variable region (VL) that is 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 81, 83, 85, 87, or 89. In specific embodiments, an antibody or antigen-binding fragment thereof that specifically binds to IsdH comprises a VH having 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 80, 82, 84, 86, or 88, and a VL comprising the amino acid sequence of SEQ ID NO: 81, 83, 85, 87, or 89.

In specific embodiments, antibodies or antigen-binding fragments thereof that specifically bind to IsdH comprise the following VH and the following VL, wherein the VH and VL are selected from the group consisting of SEQ ID NOs: 80 and 81; SEQ ID NOs: 82 and 83; SEQ ID NOs: 84 and 85; SEQ ID NOs: 86 and 87; and SEQ ID NOs: 88 and 89. In certain embodiments, antibodies or antigen-binding fragments thereof that specifically bind to IsdH comprise the following VH and the following VL, wherein the VH and VL correspond to SEQ ID NOs: 80 and 81. Example 7, Table 12 provides representative VH and VL sequences as presented herein, which can be present in any combination to form anti-surface antigen antibodies or antigen-binding fragments thereof.

In further embodiments, an antibody or antigen-binding fragment thereof that specifically binds to IsdH comprises a VH amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (including additions, deletions, and substitutions, such as conservative substitutions) in the amino acid sequence of SEQ ID NO: 80, 82, 84, 86, or 88. In various embodiments, an antibody or antigen-binding fragment thereof that specifically binds to IsdH comprises a VL amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (including additions, deletions, and substitutions, such as conservative substitutions) in the amino acid sequence of SEQ ID NO: 81, 83, 85, 87, or 89.

In certain embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the surface antigen IsdH has one or more of the following characteristics:

(a) a dissociation constant ( _KD ) of about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 20 nm or less, about 10 nm or less, about 9, 8, 7, 6, 5, 4, 3, 2, or 1 nm (or any value therebetween) for a surface antigen of Staphylococcus aureus;

(b) reducing the ability of S. aureus to evade opsonophagocytosis by immune cells by at least 50%, 60%, 70%, 80%, 90% or 95% (or any percentage in between) as measured by an opsonophagocytic killing assay;

(c) reducing the concentration of S. aureus colony forming units (CFU) by at least 50%, 60%, 70%, 80%, 90% or 95% (or any percentage therebetween) as measured by a bacteremia model; or

(d) Reduced immune cell infiltration, bacterial load, and pro-inflammatory cytokine release.

The antibodies and antigen-binding fragments thereof of the present invention that specifically bind to surface antigens or secreted toxins of Staphylococcus aureus include at least one antigen-binding domain comprising at least one complementarity determining region (e.g., at least one of CDR1, CDR2, or CDR3). In some embodiments, the antibodies or antigen-binding fragments thereof include a VH comprising at least one VH CDR (e.g., VH CDR1, VH CDR2, or VH CDR3). In certain embodiments, the antibodies or antigen-binding fragments thereof include a VL comprising at least one VL CDR (e.g., VL CDR1, VL CDR2, or VL CDR3). In some embodiments, the antibodies or antigen-binding fragments thereof include a VH comprising at least one VH CDR and at least one VL CDR.

The CDR regions disclosed herein can be combined in a variety of combinations, as each CDR region can be independently selected and combined with any other CDR region for a given antibody. In certain embodiments, the VH and/or VL CDR sequences can be present in any combination to form an antibody or antigen-binding fragment thereof directed against a surface antigen or secreted toxin of Staphylococcus aureus.

In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to IsdH comprises (a) a VH CDR1 comprising an amino acid sequence identical to SEQ ID NO: 90, 96, 102, 108, or 114, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; (b) a VH CDR2 comprising an amino acid sequence identical to SEQ ID NO: 91, 97, 103, 109, or 115, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; and/or (c) a VH CDR3 comprising an amino acid sequence identical to SEQ ID NO: 92, 98, 104, 110, or 116, or comprising 1, 2, or 3 amino acid residue mutations relative thereto.

In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to IsdH comprises (a) a VL CDR1 comprising an amino acid sequence identical to SEQ ID NO: 93, 99, 105, 111, or 117, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; (b) a VL CDR2 comprising an amino acid sequence identical to SEQ ID NO: 94, 100, 106, 112, or 118, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; and/or (c) a VL CDR3 comprising an amino acid sequence identical to SEQ ID NO: 95, 101, 107, 113, or 119, or comprising 1, 2, or 3 amino acid residue mutations relative thereto.

In some embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to IsdH comprises a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3 comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue mutations in each CDR relative to, (a) a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 90, 96, 102, 108, or 114; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 91, 97, 103, 109, or 115; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 92, 98, 104, 110, or 116; (d) a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 91, 97, 103, 109, or 115; NO: 93, 99, 105, 111, or 117; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 94, 100, 106, 112, or 118; and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 95, 101, 107, 113, or 119.

In specific embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to IsdH comprises a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are selected from the group consisting of SEQ ID NOs: 90, 91, 92, 93, 94, and 95; SEQ ID NOs: 96, 97, 98, 99, 100, and 101; SEQ ID NOs: 102, 103, 104, 105, 106, and 107; SEQ ID NOs: 108, 109, 110, 111, 112, and 113; SEQ ID NOs: 114, 115, 116, 117, 118, and 119. In further embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to IsdH comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 correspond to SEQ ID NOs: 90, 91, 92, 93, 94 and 95.

In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to IsdH corresponds to any one of the isolated antibodies or antigen-binding fragments described above and has one or more of the following characteristics:

(a) a dissociation constant ( _KD ) for a Staphylococcus aureus surface antigen of about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, or about 40 nM or less, about 20 nm or less, about 10 nm or less, about 9, 8, 7, 6, 5, 4, 3, 2, or 1 nm (or any value in between);

(b) reducing the ability of S. aureus to evade opsonophagocytosis by immune cells by at least 50%, 60%, 70%, 80%, 90% or 95% (or any percentage in between) as measured by an opsonophagocytic killing assay;

(c) reducing the number of S. aureus colony forming units (CFU) by at least 50%, 60%, 70%, 80%, 90% or 95% (or any percentage therebetween) as measured by a bacteremia model; or

(d) Reduced immune cell infiltration, bacterial load, and pro-inflammatory cytokine release.

In further embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to the same IsdH epitope as any of the anti-CIfA antibodies or antigen-binding fragments described above.

C. Anti-ClfA Antibody

In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to the surface antigen CIfA comprises a heavy chain variable region (VH) that is 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 132 or 140. In certain embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to CIfA comprises a light chain variable region (VL) that is 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 136 or 144. In specific embodiments, an antibody or antigen-binding fragment thereof that specifically binds to CIfA comprises a VH having 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 132 or 136 and a VL comprising the amino acid sequence of SEQ ID NO: 140 or 144. In specific embodiments, an antibody or antigen-binding fragment thereof that specifically binds to CIfA comprises a VH and a VL, wherein the VH and VL are selected from the group consisting of SEQ ID NO: 132 and 140; and SEQ ID NO: 136 and 144. Example 7, Table 14 provides representative VH and VL sequences as presented herein, which sequences can be present in any combination to form an anti-surface antigen antibody or antigen-binding fragment thereof.

In further embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to CIfA comprises a VH amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (including additions, deletions, and substitutions, e.g., conservative substitutions) in the amino acid sequence of SEQ ID NO: 132 or 140. In various embodiments, an antibody or antigen-binding fragment thereof that specifically binds to CIfA comprises a VL amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (including additions, deletions, and substitutions, e.g., conservative substitutions) in the amino acid sequence of SEQ ID NO: 136 or 144.

In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to CIfA comprises (a) a VH CDR1 comprising an amino acid sequence identical to SEQ ID NO: 133 or 141, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; (b) a VH CDR2 comprising an amino acid sequence identical to SEQ ID NO: 134 or 142, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; and/or (c) a VH CDR3 comprising an amino acid sequence identical to SEQ ID NO: 135 or 143, or comprising 1, 2, or 3 amino acid residue mutations relative thereto.

In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to CIfA comprises (a) a VL CDR1 comprising an amino acid sequence identical to SEQ ID NO: 137 or 145, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; (b) a VL CDR2 comprising an amino acid sequence identical to SEQ ID NO: 138 or 146, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; and/or (c) a VL CDR3 comprising an amino acid sequence identical to SEQ ID NO: 139 or 147, or comprising 1, 2, or 3 amino acid residue mutations relative thereto.

In some embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to CIfA comprises a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3 comprising an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue mutations in each CDR relative to: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 133 or 141; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 134 or 142; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 135 or 143; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 137 or 145; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 138 or 146; and (f) a VL CDR3, the VL CDR3 comprising the amino acid sequence of SEQ ID NO: 139 or 147.

In specific embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to IsdH comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are selected from the group consisting of SEQ ID NOs: 133, 134, 135, 137, 138, and 139; and SEQ ID NOs: 141, 142, 143, 144, 145, 146, and 147.

In further embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to the same epitope of CIfA as any of the anti-CIfA antibodies or antigen-binding fragments described above.

D. Anti-α-toxin (AT) antibodies

In some embodiments, the antibodies or antigen-binding fragments thereof directed against a secreted toxin comprise a VH comprising the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. In certain embodiments, the antibodies or antigen-binding fragments thereof directed against a secreted toxin comprise a VL comprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 63. In yet another embodiment, the anti-alpha toxin antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and a VL comprising the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 63. See Example 7, Table 7 for a representative list of VH and VL sequences as presented herein, which sequences can be present in any combination to form an anti-alpha toxin antibody or antigen-binding fragment thereof or in combination to form a mAb of the invention. In some embodiments, the VH is SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. In various embodiments, the VL is SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 63.

Certain VH and VL nucleotide sequences encoding the VH and VL amino acid sequences discussed herein are presented in Example 7, Table 8.

In some embodiments, the isolated antibodies or antigen-binding fragments thereof disclosed herein comprise a VH and a VL, wherein the VH and VL have amino acid sequences represented by SEQ ID NOs: 20 and 19; SEQ ID NOs: 22 and 21; SEQ ID NOs: 24 and 23; SEQ ID NOs: 26 and 25; SEQ ID NOs: 28 and 27; SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46; SEQ ID NOs: 47 and 48; SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 50; SEQ ID NOs: 51 and 52; SEQ ID NOs: 51 and 52; SEQ ID NOs: 53 and 54; SEQ ID NOs: 55 and 56; SEQ ID NOs: 57 and 58; SEQ ID NOs: 59 and 60; SEQ ID NOs: 61 and 62; SEQ ID NOs: 62 and 63; SEQ ID NOs: 64 and 65; SEQ ID NOs: 66 and 67; SEQ ID NOs: 68 and 69; SEQ ID NOs: 70 and 71; SEQ ID NOs: 72 and 73; SEQ ID NOs: 74 and 75; SEQ ID NOs: 76 and 77; SEQ ID NOs: 78 and 79; SEQ ID NOs: 80 and 81; SEQ ID NOs: 82 and 83; SEQ ID NOs: 84 and 85 NO: 79 and 63.

In certain embodiments, antibodies or fragments directed against a surface antigen or secreted toxin of Staphylococcus aureus comprise a VH and/or VL having a given percentage identity to any one of the VH and/or VL sequences disclosed in Table 7.

In some embodiments, the anti-secretory toxin antibody or antigen-binding fragment thereof comprises a VH amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical (or any percentage in between) to the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (including additions, deletions, and substitutions, e.g., conservative substitutions) in the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. As used herein, a "conservative substitution" refers to the replacement of a first amino acid with a second amino acid that does not significantly alter the chemical, physical, and/or functional properties of the antibody or antigen-binding fragment thereof (e.g., the antibody or antigen-binding fragment thereof retains the same charge, structure, polarity, hydrophobicity/hydrophilicity, and/or maintains function, such as the ability to bind alpha toxin and thereby reduce the virulence of S. aureus). In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having a given percent identity to SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and has one or more of the following characteristics:

(a) a dissociation constant (K _D ) of about 13 nM or less for Staphylococcus aureus alpha toxin;

(b) inhibiting the formation of alpha toxin oligomers by at least 50%, 60%, 70%, 80%, 90%, or 95% (or any percentage therebetween);

(c) reducing alpha toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (or any percentage therebetween) (as determined by cytolysis and hemolysis assays); or

(d) Reduced immune cell infiltration, bacterial load, and pro-inflammatory cytokine release (e.g., in animal pneumonia models).

In certain embodiments, the anti-secretory toxin antibody or antigen-binding fragment thereof comprises a VL amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical (or any percentage in between) to the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 63. In various embodiments, the antibody or antigen-binding fragment thereof comprises a VL amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (including additions, deletions, and substitutions, e.g., conservative substitutions) in the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 63. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having a given percent identity to SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 63 and has one or more of the following characteristics:

(a) a dissociation constant (KD) for Staphylococcus aureus alpha toxin of about 13 nM or less;

(b) inhibiting the binding of alpha toxin to the cell surface by at least 50%, 60%, 70%, 80%, 90% or 95% (or any percentage therebetween), thereby disrupting the formation of alpha toxin oligomers;

(c) reducing alpha toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (or any percentage therebetween) (as determined by cytolysis and hemolysis assays); or

(d) Reduced immune cell infiltration, bacterial load, and pro-inflammatory cytokine release (e.g., in animal pneumonia models).

In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises (a) a VH CDR1 comprising an amino acid sequence identical to SEQ ID NO: 7, 10, 13, or 69, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; (b) a VH CDR2 comprising an amino acid sequence identical to SEQ ID NO: 8, 11, 14, 17, 70, or 75, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; and/or (c) a VH CDR3 comprising an amino acid sequence identical to SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76, or 78, or comprising 1, 2, or 3 amino acid residue mutations relative thereto.

In specific embodiments, an isolated antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue mutations in each CDR relative to, SEQ ID NOs: 7, 8, and 9; SEQ ID NOs: 10, 11, and 12; SEQ ID NOs: 13, 14, and 15; SEQ ID NOs: 7, 17, and 18; SEQ ID NOs: 7, 8, and 16; SEQ ID NOs: 7, 8, and 65; SEQ ID NOs: 7, 8, and 66; SEQ ID NOs 7, 8, and 67; SEQ ID NOs: 7, 8, and 78; SEQ ID NOs: 69, 70, and 71; SEQ ID NOs: 7, 8, and 72; SEQ ID NOs: 69, 75, and 71; SEQ ID NOs: 69, 75, and 76; or SEQ ID NOs: 69, 70, and 71.

In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises (a) a VL CDR1 comprising an amino acid sequence identical to SEQ ID NO: 1 or 4, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; (b) a VL CDR2 comprising an amino acid sequence identical to SEQ ID NO: 2, 5, 73 or 77, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; and/or (c) a VL CDR3 comprising an amino acid sequence identical to SEQ ID NO: 3, 6, 64, 68 or 74, or comprising 1, 2, or 3 amino acid residue mutations relative thereto.

In specific embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue mutations in each CDR relative to, SEQ ID NOs: 1, 2, and 3; SEQ ID NOs: 4, 5, and 6; SEQ ID NOs: 1, 2, and 64; SEQ ID NOs: 1, 2, and 68; SEQ ID NOs: 1, 73, and 74; or SEQ ID NOs: 1, 77, and 74.

In some embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3 comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue mutations in each CDR relative to, (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13, or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 70, or 75; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76, or 78; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: NO: 1 or 4; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73, or 77; and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68, or 74.

In specific embodiments, an isolated antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprising an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue mutations in each CDR relative to SEQ ID NOs: 7, 8, 9, 1, 2, and 3; SEQ ID NOs: 10, 11, 12, 1, 2, and 3; SEQ ID NOs: 13, 14, 15, 4, 5, and 6; SEQ ID NOs: 7, 17, 18, 1, 2, and 3; SEQ ID NOs: 7, 8, 16, 1, 2, and 64; SEQ ID NOs: 7, 8, 17, 18, 1, 2, and 65; SEQ ID NOs: 7, 8, 18, 1, 2, and 66; NO: 7, 8, 67, 1, 2, and 64; SEQ ID NO: 7, 8, 78, 1, 2, and 64; SEQ ID NO: 7, 8, 65, 1, 2, and 68; SEQ ID NO: 69, 70, 71, 1, 2, and 68; SEQ ID NO: 7, 8, 72, 1, 73, and 74; SEQ ID NO: 69, 75, 71, 1, 2, and 68; SEQ ID NO: 69, 75, 76, 1, 2, and 68; SEQ ID NO: 69, 75, 76, 1, 77, and 74; or SEQ ID NO: 69, 70, 71, 1, 77, and 74.

In some embodiments, a composition is provided, comprising an isolated antibody or antigen-binding fragment thereof, which (i) comprises a VH chain domain and a VL chain domain, the VH chain domain comprising three CDRs, the VL chain domain comprising three CDRs; and (ii) specifically binds to a Staphylococcus aureus alpha toxin polypeptide, wherein the three CDRs of the VH chain domain comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13, or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 70, or 75; and (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76, or 78. In specific embodiments, the VH CDR1, VH CDR2, and VH CDR3 correspond to SEQ ID NOs: 7, 8, and 9; SEQ ID NOs: 10, 11, and 12; SEQ ID NOs: 13, 14, and 15; SEQ ID NOs: 7, 17, and 18; SEQ ID NOs: 7, 8, and 16; SEQ ID NOs: 7, 8, and 65; SEQ ID NOs: 7, 8, and 66; SEQ ID NOs: 7, 8, and 67; SEQ ID NOs: 7, 8, and 78; SEQ ID NOs: 69, 70, and 71; SEQ ID NOs: 7, 8, and 72; SEQ ID NOs: 69, 75, and 71; SEQ ID NOs: 69, 75, and 76; or SEQ ID NOs: 69, 70, and 71.

In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to a toxin secreted by Staphylococcus aureus comprises (a) a VH CDR1 comprising an amino acid sequence consistent with, or comprising 1, 2, or 3 amino acid residue mutations relative to, SEQ ID NO: 7, 10, 13, or 69; (b) a VH CDR2 comprising an amino acid sequence consistent with, or comprising 1, 2, or 3 amino acid residue mutations relative to, SEQ ID NO: 8, 11, 14, 17, 70, or 75; and (c) a VH CDR3 comprising an amino acid sequence consistent with, or comprising 1, 2, or 3 amino acid residue mutations relative to, SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76, or 78; (d) a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 1 or 4; (e) a VL (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68, or 74 and having one or more of the following characteristics:

(a) a dissociation constant (KD) for alpha toxin of about 13 nM or less;

(b) binding to alpha toxin monomers;

(c) inhibiting the formation of alpha toxin oligomers by at least 50%, 60%, 70%, 80%, 90%, or 95% (or any percentage therebetween);

(d) reducing alpha toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (or any percentage therebetween) (as determined by cytolysis and hemolysis assays); or

(e) Reduced immune cell infiltration, bacterial load, and pro-inflammatory cytokine release (e.g., in animal pneumonia models).

In certain embodiments, an antibody or antibody fragment that specifically binds to a surface antigen or secreted toxin of S. aureus comprises a heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, 62, 80, 82, 84, 86, or 88, and a light chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 63, 81, 83, 85, 87, or 89. In further embodiments, the antibody or antigen-binding fragment thereof reduces the ability of S. aureus to evade opsonophagocytosis by at least 50%. In further embodiments, the antibody or antigen-binding fragment thereof reduces the concentration of S. aureus CFU by at least 50%. In other embodiments, the antibody or antigen-binding fragment thereof inhibits one or more alpha toxin monomers from binding to each other (e.g., inhibits oligomerization) and/or reduces the virulence of S. aureus.

In some embodiments, the isolated antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises (a) a VH CDR1 comprising an amino acid sequence identical to SEQ ID NO: 7, 10, 13, or 69, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; (b) a VH CDR2 comprising an amino acid sequence identical to SEQ ID NO: 8, 11, 14, 17, 70, or 75, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; and/or (c) a VH CDR3 comprising an amino acid sequence identical to SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76, or 78, or comprising 1, 2, or 3 amino acid residue mutations relative thereto.

In specific embodiments, an isolated antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue mutations in each CDR relative to, SEQ ID NOs: 7, 8, and 9; SEQ ID NOs: 10, 11, and 12; SEQ ID NOs: 13, 14, and 15; SEQ ID NOs: 7, 17, and 18; SEQ ID NOs: 7, 8, and 16; SEQ ID NOs: 7, 8, and 65; SEQ ID NOs: 7, 8, and 66; SEQ ID NOs 7, 8, and 67; SEQ ID NOs: 7, 8, and 78; SEQ ID NOs: 69, 70, and 71; SEQ ID NOs: 7, 8, and 72; SEQ ID NOs: 69, 75, and 71; SEQ ID NOs: 69, 75, and 76; or SEQ ID NOs: 69, 70, and 71.

In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises (a) a VL CDR1 comprising an amino acid sequence identical to SEQ ID NO: 1 or 4, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; (b) a VL CDR2 comprising an amino acid sequence identical to SEQ ID NO: 2, 5, 73 or 77, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; and/or (c) a VL CDR3 comprising an amino acid sequence identical to SEQ ID NO: 3, 6, 64, 68 or 74, or comprising 1, 2, or 3 amino acid residue mutations relative thereto.

In specific embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises a VL CDR1, a VL CDR2, and a VL CDR3 comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue mutations in each CDR relative to, SEQ ID NOs: 1, 2, and 3; SEQ ID NOs: 4, 5, and 6; SEQ ID NOs: 1, 2, and 64; SEQ ID NOs: 1, 2, and 68; SEQ ID NOs: 1, 73, and 74; or SEQ ID NOs: 1, 77, and 74.

In some embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3 comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid residue mutations in each CDR relative to, (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13, or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 70, or 75; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76, or 78; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: NO: 1 or 4; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73, or 77; and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68, or 74.

In specific embodiments, an isolated antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus alpha toxin polypeptide comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprising an amino acid sequence identical to or comprising 1, 2, or 3 amino acid residue mutations in each CDR relative to SEQ ID NOs: 7, 8, 9, 1, 2, and 3; SEQ ID NOs: 10, 11, 12, 1, 2, and 3; SEQ ID NOs: 13, 14, 15, 4, 5, and 6; SEQ ID NOs: 7, 17, 18, 1, 2, and 3; SEQ ID NOs: 7, 8, 16, 1, 2, and 64; SEQ ID NOs: 7, 8, 17, 18, 1, 2, and 65; SEQ ID NOs: 7, 8, 18, 1, 2, and 66; NO: 7, 8, 67, 1, 2, and 64; SEQ ID NO: 7, 8, 78, 1, 2, and 64; SEQ ID NO: 7, 8, 65, 1, 2, and 68; SEQ ID NO: 69, 70, 71, 1, 2, and 68; SEQ ID NO: 7, 8, 72, 1, 73, and 74; SEQ ID NO: 69, 75, 71, 1, 2, and 68; SEQ ID NO: 69, 75, 76, 1, 2, and 68; SEQ ID NO: 69, 75, 76, 1, 77, and 74; or SEQ ID NO: 69, 70, 71, 1, 77, and 74.

In some embodiments, a composition is provided, comprising an isolated antibody or antigen-binding fragment thereof, which (i) comprises a VH chain domain and a VL chain domain, the VH chain domain comprising three CDRs, the VL chain domain comprising three CDRs; and (ii) specifically binds to a Staphylococcus aureus alpha toxin polypeptide, wherein the three CDRs of the VH chain domain comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13, or 69; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 70, or 75; and (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76, or 78. In specific embodiments, the VH CDR1, VH CDR2, and VH CDR3 correspond to SEQ ID NOs: 7, 8, and 9; SEQ ID NOs: 10, 11, and 12; SEQ ID NOs: 13, 14, and 15; SEQ ID NOs: 7, 17, and 18; SEQ ID NOs: 7, 8, and 16; SEQ ID NOs: 7, 8, and 65; SEQ ID NOs: 7, 8, and 66; SEQ ID NOs: 7, 8, and 67; SEQ ID NOs: 7, 8, and 78; SEQ ID NOs: 69, 70, and 71; SEQ ID NOs: 7, 8, and 72; SEQ ID NOs: 69, 75, and 71; SEQ ID NOs: 69, 75, and 76; or SEQ ID NOs: 69, 70, and 71.

In certain embodiments, the combination of CDR sequences used to form an anti-secretory toxin antibody comprises a VH CDR1 comprising SEQ ID NO: 7, 10, 13, or 69, a VH CDR2 comprising SEQ ID NO: 8, 11, 14, 17, 70, or 75, and a VH CDR3 comprising SEQ ID NO: SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76, or 78, as depicted in Table 9. In some embodiments, the VL CDR1 comprises SEQ ID NO: 1 or 4, the VL CDR2 comprises SEQ ID NO: 2, 5, 73, or 77, and the VL CDR3 comprises SEQ ID NO: 3, 6, 64, 68, or 74, as depicted in Table 9.

Antibodies and antigen-binding fragments thereof disclosed herein may include one or more amino acid sequences substantially identical to the amino acid sequences described herein. Substantially identical amino acid sequences include sequences containing conservative amino acid substitutions and amino acid deletions and/or insertions.

E. Framework region

The variable domains of the heavy and light chains each include at least one framework region (FR1, FR2, FR3, FR4, or alternatively FW1, FW2, FW3, FW4). The framework regions of the heavy chain are designated herein as VH FRs, while the framework regions of the light chain are designated herein as VL FRs. In certain embodiments, these framework regions may comprise substitutions, insertions, or other alterations. In certain embodiments, these alterations result in improved or optimized binding affinity of the antibody. Non-limiting examples of framework region residues that may be modified include those that directly bind to the antigen non-covalently, interact with/affect the conformation of the CDRs, and/or participate in the VL-VH interface.

In certain embodiments, the framework region can include one or more amino acid changes for the purpose of "germlining". For example, the amino acid sequences of the selected antibody heavy and light chains can be compared with the germline heavy and light chain amino acid sequences, and wherein some framework residues of the selected VL and/or VH chains are different from the germline configuration (for example, due to somatic mutations of the immunoglobulin genes used to prepare the phage library), it may be desirable to "revert" the changed framework residues of the selected antibody to the germline configuration (that is, to change the framework amino acid sequence of the selected antibody so that they are identical to the germline framework amino acid sequence). This "revert" (or "germlining") of framework residues can be completed by standard molecular biology methods for introducing specific mutations (for example, site-directed mutagenesis or PCR-mediated mutagenesis). In certain embodiments, variable light and/or heavy chain framework residues are reversed. In certain embodiments, the variable heavy chain of the currently disclosed separated antibody or antigen-binding fragment is reversed. In certain embodiments, the variable heavy chain of the isolated antibody or antigen-binding fragment comprises at least one, at least two, at least three, at least four, or more back mutations.

In certain embodiments, the VH of the anti-alpha toxin antibody or antigen-binding fragment thereof can include FR1, FR2, FR3 and/or FR4 having an amino acid sequence that is about 65% to about 100% identical to the corresponding VH framework region within SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. In some embodiments, the anti-alpha toxin antibody or antigen-binding fragment thereof comprises a VH FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical (or any percentage in between) to the corresponding FR region of VH SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. In certain embodiments, the anti-alpha toxin antibody or antigen-binding fragment thereof comprises a VH FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to the corresponding FRs of VH SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62.

In certain embodiments, the anti-alpha toxin antibody or antigen-binding fragment thereof can include a VH FR (FR1, FR2, FR3, and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FR of VH SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62. Specifically, the FR1, FR2, FR3, or FR4 of VH can each have an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FR1, FR2, FR3, or FR4 of VH SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62.

In certain embodiments, the VL of the anti-alpha toxin antibodies or antigen-binding fragments thereof provided herein can include FR1, FR2, FR3 and/or FR4 having an amino acid sequence that is about 65% to about 100% identical to the corresponding VH framework region within the FRs of VL SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. In some embodiments, the anti-alpha toxin antibody or antigen-binding fragment thereof comprises a VL FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical (or any percentage in between) to the corresponding FR of VL SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63. In certain embodiments, the anti-alpha toxin antibody or antigen-binding fragment thereof comprises a VL FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to the corresponding FR of VL SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 63.

In certain embodiments, the anti-alpha toxin antibody or antigen-binding fragment thereof comprises a VL FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FR of VL SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 63. Specifically, the FR1, FR2, FR3, or FR4 of VL can each have an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FR1, FR2, FR3, or FR4 of VH SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 63.

In certain embodiments, the isolated antibody or antigen-binding fragment that specifically binds to a toxin secreted by Staphylococcus aureus comprises a VH FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FRs of VH SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62, and/or a VL FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FRs of VL SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 63.

In certain embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to a toxin secreted by Staphylococcus aureus comprises a VH FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to or comprising 1, 2, or 3 amino acid mutations relative to the corresponding FR of VH SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62, and/or a VL ...L SEQ ID NO: The antibody comprises one or more of the following amino acid mutations:

(a) an affinity constant (K _D ) for alpha toxin of about 13 nM or less;

(b) binding to alpha toxin monomers;

(c) inhibiting the formation of alpha toxin oligomers by at least 50%, 60%, 70%, 80%, 90%, or 95% (or any percentage therebetween);

(d) reducing alpha toxin cytolytic activity by at least 50%, 60%, 70%, 80%, 90% or 95% (or any percentage therebetween) (as determined by cytolysis and hemolysis assays); or

(e) Reduced immune cell infiltration, bacterial load, and pro-inflammatory cytokine release (e.g., in animal pneumonia models).

In certain embodiments, an isolated antibody or antigen-binding fragment that specifically binds to the IsdH surface antigen of Staphylococcus aureus is provided, the isolated antibody or antigen-binding fragment comprising a VH FR1, FR2, FR3, and/or FR4 region having an amino acid sequence about 65% to about 100% identical to the corresponding amino acid sequences of the four VH framework regions within SEQ ID NO: 80, 82, 84, 86, or 88. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH FR amino acid sequence (FR1, FR2, FR3, and/or FR4) that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical (or any percentage in between) to the corresponding amino acid sequences of the four FR regions of VH SEQ ID NO: 80, 82, 84, 86, or 88. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to the corresponding FR region of VH SEQ ID NO: 80, 82, 84, 86, or 88.

In certain embodiments, an isolated antibody or antigen-binding fragment that specifically binds IsdH is provided, comprising VL FR1, FR2, FR3, and/or FR4 regions having amino acid sequences from about 65% to about 100% identical to the corresponding amino acid sequences of the four VL framework regions within SEQ ID NO: 81, 83, 85, 87, or 89. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL FR amino acid sequence (FR1, FR2, FR3, and/or FR4) that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical (or any percentage in between) to the corresponding FR regions of VL SEQ ID NO: 81, 83, 85, 87, or 89. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VL FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to the corresponding FR region of VL SEQ ID NO: 81, 83, 85, 87, or 89.

In certain embodiments, the isolated antibody or antigen-binding fragment that specifically binds IsdH comprises a VH FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FRs of VH SEQ ID NO: 80, 82, 84, 86, or 88, and/or a VL FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FRs of VL SEQ ID NO: 81, 83, 85, 87, or 89.

In certain embodiments, an isolated antibody or antigen-binding fragment that specifically binds IsdH is provided, the isolated antibody or antigen-binding fragment comprising VH FR1, FR2, FR3, and/or FR4 regions having amino acid sequences from about 65% to about 100% identical to the corresponding amino acid sequences of the four VH framework regions within SEQ ID NO: 80, 82, 84, 86, or 88. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH FR amino acid sequence (FR1, FR2, FR3, and/or FR4) that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical (or any percentage in between) to the corresponding amino acid sequences of the four FR regions of VH SEQ ID NO: 80, 82, 84, 86, or 88. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to the corresponding FR region of VH SEQ ID NO: 80, 82, 84, 86, or 88.

In certain embodiments, an isolated antibody or antigen-binding fragment that specifically binds IsdH is provided, comprising VL FR1, FR2, FR3, and/or FR4 regions having amino acid sequences from about 65% to about 100% identical to the corresponding amino acid sequences of the four VL framework regions within SEQ ID NO: 81, 83, 85, 87, or 89. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL FR amino acid sequence (FR1, FR2, FR3, and/or FR4) that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical (or any percentage in between) to the corresponding FR regions of VL SEQ ID NO: 81, 83, 85, 87, or 89. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VL FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to the corresponding FR region of VL SEQ ID NO: 81, 83, 85, 87, or 89.

In certain embodiments, the isolated antibody or antigen-binding fragment that specifically binds IsdH comprises a VH FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FRs of VH SEQ ID NO: 80, 82, 84, 86, or 88, and/or a VL FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FRs of VL SEQ ID NO: 81, 83, 85, 87, or 89.

In certain embodiments, an isolated antibody or antigen-binding fragment that specifically binds to a S. aureus ClfA surface antigen is provided, the isolated antibody or antigen-binding fragment comprising a VH FR1, FR2, FR3, and/or FR4 region having an amino acid sequence that is about 65% to about 100% identical to the corresponding amino acid sequences of the four VH framework regions within SEQ ID NO: 132 or 140. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH FR amino acid sequence (FR1, FR2, FR3, and/or FR4) that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical (or any percentage therebetween) to the corresponding amino acid sequences of the four FR regions of VH SEQ ID NO: 132 or 140. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to the corresponding FR region of VH SEQ ID NO: 132 or 140.

In certain embodiments, an isolated antibody or antigen-binding fragment that specifically binds CIfA is provided, comprising VL FR1, FR2, FR3, and/or FR4 regions having amino acid sequences from about 65% to about 100% identical to the corresponding amino acid sequences of the four VL framework regions within SEQ ID NO: 136 or 144. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL FR amino acid sequence (FR1, FR2, FR3, and/or FR4) that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical (or any percentage in between) to the corresponding FR regions of VL SEQ ID NO: 136 or 144. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VL FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to the corresponding FR region of VL SEQ ID NO: 136 or 144.

In certain embodiments, the isolated antibody or antigen-binding fragment that specifically binds CIfA comprises a VH FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FRs of VH SEQ ID NO: 132 or 136, and/or a VL FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FRs of VL SEQ ID NO: 136 or 144.

In certain embodiments, an isolated antibody or antigen-binding fragment that specifically binds CIfA is provided, comprising VH FR1, FR2, FR3, and/or FR4 regions having amino acid sequences from about 65% to about 100% identical to the corresponding amino acid sequences of the four VH framework regions within SEQ ID NO: 132 or 140. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH FR amino acid sequence (FR1, FR2, FR3, and/or FR4) that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical (or any percentage in between) to the corresponding amino acid sequences of the four FR regions of VH SEQ ID NO: 132 or 140. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to the corresponding FR region of VH SEQ ID NO: 132 or 140.

In certain embodiments, an isolated antibody or antigen-binding fragment that specifically binds IsdH is provided, comprising VL FR1, FR2, FR3, and/or FR4 regions having amino acid sequences from about 65% to about 100% identical to the corresponding amino acid sequences of the four VL framework regions within SEQ ID NO: 136 or 144. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL FR amino acid sequence (FR1, FR2, FR3, and/or FR4) that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical (or any percentage in between) to the corresponding FR regions of VL SEQ ID NO: 136 or 144. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VL FR amino acid sequence (FR1, FR2, FR3 and/or FR4) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to the corresponding FR region of VL SEQ ID NO: 136 or 144.

In certain embodiments, the isolated antibody or antigen-binding fragment that specifically binds CIfA comprises a VH FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FRs of VH SEQ ID NO: 132 or 140, and/or a VL FR (FR1, FR2, FR3 and/or FR4) comprising an amino acid sequence identical to, or comprising 1, 2, or 3 amino acid mutations relative to, the corresponding FRs of VL SEQ ID NO: 136 or 144.

F. Nucleotide sequences encoding anti-alpha toxin antibodies and antigen-binding fragments thereof

In addition to the amino acid sequences described above, nucleotide sequences corresponding to the amino acid sequences disclosed herein are further provided. In certain embodiments, the nucleotide sequence encodes an antibody or antigen-binding fragment thereof directed against a surface antigen of Staphylococcus aureus or a secreted toxin. The nucleotide sequences are provided in Example 7 and Table 8. Therefore, polynucleotide sequences encoding the VH and VL regions (including FR regions and CDRs) of the antibodies or fragments described herein and expression vectors for their effective expression in cells (e.g., mammalian cells) are also provided.

Also disclosed herein are polynucleotides that are substantially identical to those encoding the amino acid sequences disclosed herein. The substantially identical sequence can be a polymorphic sequence, i.e., an alternative sequence or allele in a population. The substantially identical sequence can also comprise a mutagenic sequence, comprising a sequence containing a silent mutation. The sudden change can comprise the insertion of one or more residue changes, one or more residue deletions, or one or more other residues. The substantially identical sequence can also comprise different nucleotide sequences encoding the same amino acid at any given amino acid position in the amino acid sequence disclosed herein, owing to the degeneracy of the nucleic acid code.

Also disclosed herein are polynucleotides that hybridize to the following polynucleotides under high stringency or lower stringency hybridization conditions, which encode antibodies or antigen-binding fragments thereof against Staphylococcus aureus surface antigens or secreted toxins. The term "stringency" as used herein refers to the experimental conditions (e.g., temperature and salt concentration) of a hybridization experiment, indicating the degree of homology between two nucleic acids; the higher the stringency, the higher the percentage of homology between the two nucleic acids. As used herein, the phrase "hybridization" or its grammatical variants refers to the binding of a first nucleic acid molecule to a second nucleic acid molecule under low, medium, or high stringency conditions, or under nucleic acid synthesis conditions. Hybridization can include the binding of a first nucleic acid molecule to a second nucleic acid molecule, wherein the first and second nucleic acid molecules are complementary.

Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2X SSC/0.1% SDS at about 50° C.-65° C. Other stringent conditions include hybridization to filter-bound DNA in 6X SSC at about 45° C., followed by one or more washes in 0.1X SSC/0.2% SDS at about 65° C. Other hybridization conditions of known stringency are familiar to those of ordinary skill in the art and are encompassed herein.

In certain embodiments, the nucleic acids disclosed herein can encode the amino acid sequence of an antibody or antigen-binding fragment thereof against a surface antigen or secreted toxin of Staphylococcus aureus, or the nucleic acid can hybridize under stringent conditions to a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of the antibody or antigen-binding fragment thereof.

In certain embodiments, the polynucleotide sequence may include a nucleotide sequence encoding an amino acid sequence that is an amino acid sequence of an antibody or antigen-binding fragment thereof that can bind to a surface antigen or secreted toxin of Staphylococcus aureus and is at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the VH amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, 62, 80, 82, 84, 86, or 88 (or any percentage therebetween). In certain embodiments, the polynucleotide sequence may include a nucleotide sequence encoding an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (including additions, deletions, and substitutions, such as conservative substitutions) in the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, 62, 80, 82, 84, 86, or 88. In some embodiments, the polynucleotide sequence may include a nucleotide sequence that encodes an amino acid sequence of an antibody or antigen-binding fragment thereof that is capable of binding to a surface antigen or secreted toxin of Staphylococcus aureus and is at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the VH nucleotide sequence of SEQ ID NO: 30, 32, 34, 36, 38, 120, 122, 124, 126, or 128 (or any percentage in between).

In certain embodiments, the polynucleotide sequence may include a nucleotide sequence encoding an amino acid sequence that is an amino acid sequence of an antibody or antigen-binding fragment thereof that can bind to a surface antigen or secreted toxin of Staphylococcus aureus and is at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the VL amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 63, 81, 83, 85, 87, or 89 (or any percentage in between). In certain embodiments, the polynucleotide sequence may include a nucleotide sequence encoding an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (including additions, deletions, and substitutions, such as conservative substitutions) in the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 63, 81, 83, 85, 87, or 89. In some embodiments, the polynucleotide sequence may include a nucleotide sequence that encodes an amino acid sequence of an antibody or antigen-binding fragment thereof that is capable of binding to a surface antigen or secreted toxin of Staphylococcus aureus and is at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical (or any percentage in between) to the VL nucleotide sequence of SEQ ID NO: 29, 31, 33, 35, 37, 121, 123, 125, 127, or 129.

In a specific embodiment, the polynucleotide sequence may include a nucleotide sequence encoding an amino acid sequence that is an amino acid sequence of an antibody or antigen-binding fragment thereof that is capable of binding to a surface antigen or secreted toxin of Staphylococcus aureus and is at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the following VH amino acid sequence (or any percentage therebetween) and at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the following VL amino acid sequence (or any percentage therebetween), wherein the VH and VL sequences are represented by SEQ ID NOs: 20 and 19; SEQ ID NOs: 22 and 21; SEQ ID NOs: 24 and 23; SEQ ID NOs: 26 and 25; SEQ ID NOs: 28 and 27; SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46; SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 50; SEQ ID NOs: 51 and 52; SEQ ID NOs: 53 and 54; SEQ ID NOs: 55 and 56; SEQ ID NOs: 57 and 58; SEQ ID NOs: 59 and 60; SEQ ID NOs: 61 and 62; SEQ ID NOs: 63 and 64; SEQ ID NOs: 65 and 66; SEQ ID NOs: 67 and 68; SEQ ID NOs: 69 and 70; SEQ ID NOs: 71 and 72; SEQ ID NOs: 73 and 74; SEQ ID NOs: 75 and 76; SEQ ID NOs: NO: 47 and 48; SEQ ID NO: 47 and 48; SEQ ID NO: 49 and 50; SEQ ID NO: 51 and 52; SEQ ID NO: 51 and 52; SEQ ID NO: 53 and 54; SEQ ID NO: 55 and 56; SEQ ID NO: 57 and 58; SEQ ID NO: 59 and 60; SEQ ID NO: 61 and 58; SEQ ID NO: 62 and 58; SEQ ID NO: 62 and 63; SEQ ID NO: 79 and 63; SEQ ID NO: 80 and 81; SEQ ID NO: 82 and 83; SEQ ID NO: 84 and 85; SEQ ID NO: 86 and 87; SEQ ID NO: 88 and 89.

The nucleotide sequence of the polynucleotide of these disclosures and the polynucleotide of determination can be obtained by any method known in the art.For example, if the nucleotide sequence of the antibody is known, the polynucleotide encoding the antibody can be assembled by the oligonucleotide synthesized by chemical method.This will relate to the synthesis of the overlapping oligonucleotides of the part containing the sequence encoding the antibody, the annealing and connection of those oligonucleotides and then the oligonucleotides connected by pcr amplification.The polynucleotide of these disclosures can also be produced from any suitable nucleic acid source, for example, an antibody cDNA library or a cDNA library separated from any tissue or cell (for example, separated from the hybridoma cell selected for expressing the antibody) that expresses the antibody.

G. Functional Characterization of Antibodies or Fragments Targeting Staphylococcus aureus Surface Antigens or Secreted Toxins

In certain embodiments, antibodies or antigen-binding fragments thereof directed against a surface antigen of Staphylococcus aureus change the biological properties of Staphylococcus aureus cells expressing the surface antigen. In various embodiments, the antibody binds to a surface antigen of Staphylococcus aureus, thereby enhancing opsonophagocytosis by host cells. In further embodiments, opsonophagocytosis is increased by 50%, 60%, 70%, 80%, 90%, or 95% (or any percentage therebetween), as measured by an opsonophagocytic killing assay. In some embodiments, the antibody binds to the surface determinant antigen, preventing the interaction between the surface antigen and the surface adhesin, thereby reducing the concentration of colony-forming units (CFU) present in host tissue, as measured in a mouse bacteremia model. In further embodiments, the CFU concentration is reduced by 50%, 60%, 70%, 80%, 90%, or 95% (or any percentage therebetween) compared to the CFU concentration in the presence of a negative control antibody or in the absence of the antibody or its antigen-binding fragment. For example, an anti-IsdH antibody can reduce the CFU concentration by 50%, 60%, 70%, 80%, 90% or 95% (or any percentage in between). In some embodiments, an anti-surface antigen antibody can compete with haptoglobin and/or hemoglobin for binding to S. aureus, thereby inhibiting the ability of S. aureus to enter hemoglobin and utilize the iron therein. In certain embodiments, an antibody or fragment directed against a surface antigen reduces the ability of S. aureus to bind to haptoglobin and/or hemoglobin by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% (or any percentage in between) compared to S. aureus binding in the absence of the antibody. For example, an anti-IsdH antibody can reduce the ability of S. aureus to bind to haptoglobin and/or hemoglobin by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% (or any percentage in between).

As used herein, "opsonophagocytic killing assay" (OPK) refers to any assay for measuring the percentage of phagocytic killing induced in host cells in vitro after adding an antibody to a tissue sample containing a known concentration of S. aureus. The reduction in CFU is normalized to the control level of OPK observed in the presence of a control antibody. This assay measures the ability of the target antibody to induce complement activation and subsequent phagocytosis. For example, the OPK can include combining 10 μl of antibody with 10 μl of S. aureus (10 ⁶ cells/ml), followed by the addition of 10 μl of human promyelocytic leukemia (HL-60) cells (10 ⁷ cells/ml) and 10 μl of human serum pre-absorbed against S. aureus. 10 μl of the mixture can then be applied (at time T ₀ ), followed by cell lysis using 1% saponin (at time T ₆₀ ) and the S. aureus CFU concentration determined. Percent killing can be calculated as follows: 100 x (1-( _T60 / _T0 )), where _T60 refers to the CFU concentration at the end of the assay (ie, at 60 minutes) and _T0 refers to the CFU concentration at the start of the assay.

As used herein, "bacteremia model" refers to any in vivo model of S. aureus infection used to evaluate the effect of an antibody on the S. aureus bacterial load (expressed as a percentage reduction in CFU). For example, the bacteremia model can include injecting an antibody into a mouse, followed by intraperitoneal injection of 10 ⁸ CFU of S. aureus, and later collecting blood and measuring the CFU concentration, as compared to the CFU concentration after injection of a control antibody.

In certain embodiments, anti-alpha toxin antibodies or antigen-binding fragments thereof alter the biological properties of alpha toxin and/or cells expressing alpha toxin. In some embodiments, anti-alpha toxin antibodies or antigen-binding fragments thereof neutralize the biological activity of alpha toxin by binding to the polypeptide and inhibiting membrane binding and the assembly of alpha toxin monomers into transmembrane pores (e.g., alpha toxin heptamers). Neutralization assays can be performed using methods known in the art, in some cases using commercially available reagents. Neutralization of alpha toxin is typically measured as an IC50 of 1× ^10-6 M or less, 1× ^10-7 M or less, 1× ^10-8 M or less, 1× ^10-9 M or less, 1× ^10-10 M or less, and 1× ^10-11 M or less. The term "inhibitory concentration 50%" (abbreviated as "IC50") represents the concentration of an inhibitor (e.g., an anti-alpha toxin antibody or antigen-binding fragment thereof provided herein) required for 50% inhibition of a given activity of a molecule targeted by the inhibitor (e.g., oligomerization of alpha toxin to form a transmembrane pore heptamer complex). Lower IC50 values generally correspond to more potent inhibitors.

In certain embodiments, the anti-alpha toxin antibody or antigen-binding fragment thereof inhibits one or more biological activities of alpha toxin. As used herein, the term "inhibit" refers to any statistically significant reduction in biological activity, including complete blocking of activity. For example, "inhibit" can refer to a reduction in biological activity by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any percentage in between. In certain embodiments, the anti-alpha toxin antibody or antigen-binding fragment thereof inhibits one or more biological activities of alpha toxin by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any percentage in between.

In some embodiments, the anti-alpha toxin antibodies or antigen-binding fragments thereof can deplete alpha toxin secreted by pathogenic S. aureus. In some embodiments, the anti-alpha toxin antibodies or antigen-binding fragments thereof can achieve at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% depletion of alpha toxin secreted by S. aureus, or any percentage therebetween. In specific embodiments, substantially all detectable secreted alpha toxin is depleted from S. aureus-infected cells.

In certain embodiments, an anti-alpha toxin antibody or antigen-binding fragment thereof can inhibit the expression of one or more inducible genes that are directly or indirectly responsive to the environment created by S. aureus infection and/or alpha toxin expression and function. In specific embodiments, an anti-alpha toxin antibody or antigen-binding fragment thereof inhibits the expression of one or more inducible genes that are directly or indirectly responsive to the environment created by S. aureus alpha toxin expression and function by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, or any percentage therebetween.

H. Methods for producing antibodies against surface antigens and secreted toxins of Staphylococcus aureus

Exemplary techniques for producing the antibodies disclosed herein are described below. In some embodiments, recombinant or hybridoma methods can be used to produce the antibodies or fragments disclosed herein. In other embodiments, antibodies or antibody fragments can be isolated from generated antibody phage libraries using techniques known in the art. Antibodies to surface antigens and secreted toxins of Staphylococcus aureus can also be prepared using other techniques known in the art for preparing antibodies.

In some embodiments, native S. aureus IsdH, mutant IsdH, variants of IsdH, or antigenic fragments thereof can be used to generate anti-IsdH antibodies. Antibodies can also be generated using S. aureus cells expressing IsdH. IsdH can also be recombinantly produced in isolated form from bacteria or eukaryotic cells using standard recombinant DNA methods for use in generating anti-IsdH antibodies.

Polyclonal antibodies to secreted toxins or surface antigens (e.g., IsdH) can be produced by different procedures known in the art. For example, IsdH polypeptides or immunogenic fragments thereof can be administered to different host animals via subcutaneous or intraperitoneal injection of the relevant antigen to induce the production of serum containing polyclonal antibodies specific for the antigen. Host animals include, but are not limited to, rabbits, mice, and rats. In some embodiments, depending on the host species, different adjuvants can be used to increase the immune response, and these adjuvants include, but are not limited to, Freund's (complete and incomplete), mineral glues (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin), pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants (e.g., BCG (BCG)) and Corynebacterium brevis. Other adjuvants known in the art can also be used.

Monoclonal antibodies to secreted toxins or surface antigens (e.g., IsdH) can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. The term "monoclonal antibody" as used herein refers to an antibody, e.g., a single antibody, obtained from a population of substantially homologous or isolated antibodies, which antibodies comprise the same population except for possible naturally occurring mutations (which may be present in small amounts). The modifier "monoclonal" should not be construed as requiring the production of the antibody by any particular method. Monoclonal antibodies include monoclonal mammalian antibodies, chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, diabodies, vaccibodies, linear antibodies, and multispecific antibodies.

Once the antibodies disclosed herein have been produced, they can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, and size fractionation column chromatography), centrifugation, differential solubility, or by any other technique for purifying proteins. In addition, the antibodies of the present technology or fragments thereof can be fused to heterologous polypeptide sequences (including epitope "tags" and other fusion proteins, such as GST fusion proteins) to facilitate antibody purification and use in subsequent assays.

In certain embodiments, the antibodies disclosed herein are chimeric antibodies. A chimeric antibody is an antibody in which a portion of the heavy chain and/or light chain is consistent or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while another portion of the chain is consistent or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as a fragment of such an antibody, as long as they exhibit the desired biological activity. Chimeric antibodies disclosed herein include "primatized" antibodies, which comprise variable domain antigen-binding sequences and human constant region sequences derived from a non-human primate (e.g., Old World Monkeys, such as baboon, rhesus monkey, or cynomolgus monkey). Chimeric antibodies disclosed herein also include humanized antibodies produced using methods known in the art.

In other embodiments, the antibodies disclosed herein are human antibodies and are produced using methods known in the art. For example, a nucleic acid encoding a functional human antibody locus can be introduced into a rodent or other animal so that the rodent or other animal produces fully human antibodies to produce fully human antibodies. In another example, human antibodies can be derived by in vitro methods. Suitable examples include, but are not limited to, phage display, ribosome display, yeast display, and other methods known in the art. Other examples of methods for preparing human antibodies or fragments directed against Staphylococcus aureus surface antigens or secreted toxins include mouse technology (Regeneron Pharmaceuticals). See, for example, U.S. Patent No. 6,596,541 (incorporated by reference in its entirety).

In certain embodiments, it may be desirable to return the framework sequence of the antibody disclosed herein to the germline sequence, return the CDRs to the germline, and/or remove structural liability. Thus, in some embodiments, where a specific antibody disclosed herein differs from its corresponding germline sequence at the amino acid level, the antibody sequence may be mutated back to the germline sequence. By using standard molecular biology techniques, such correction mutations may occur at one, two, three, or more positions, or any combination of mutation positions.

In certain embodiments, the present disclosure encompasses antibody fragments or antibodies comprising such fragments. Antibody fragments comprise a portion of a full-length antibody, typically its antigen-binding or variable region. Examples of such antibody fragments include Fab, Fab', F(ab')2, Fd, and Fv fragments; diabodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies, which are antibodies formed from such antibody fragments.

In addition to the human, humanized and/or chimeric antibodies described above, the antibodies disclosed herein can be further modified to include one or more of the following: at least one amino acid residue and/or polypeptide substitution, addition and/or deletion in the VL domain and/or VH domain and/or Fc region, and post-translational modifications. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct possesses the desired characteristics.

Included in these modifications are antibody conjugates in which the antibody has been covalently linked to a moiety. Suitable moieties for linking to the antibody include, but are not limited to, proteins, peptides, drugs, markers, and cytotoxins. These changes can be made to the antibody to change or optimize antibody characteristics (e.g., biochemistry, binding, and/or functional characteristics) suitable for detecting, diagnosing, and/or treating Staphylococcus aureus infections and related diseases or disorders. Methods for forming conjugates, making amino acid and/or polypeptide changes, and post-translational modifications are known in the art. Also included in these modifications are fusion proteins, i.e., antibodies or fragments thereof fused to heterologous proteins, polypeptides, or peptides.

In certain embodiments, antibodies or fragments directed against surface antigens or secreted toxins of Staphylococcus aureus are generated to include an altered Fc region (also referred to herein as a "variant Fc region"), wherein one or more changes have been made in the Fc region to alter the functional properties and/or pharmacokinetic properties of the antibodies. Such changes can result in altered effector function, reduced immunogenicity, and/or increased serum half-life. In certain embodiments, the effector function of an antibody can be improved by changes in the Fc region, including but not limited to amino acid substitutions, amino acid additions, amino acid deletions, and changes in post-translational modifications (e.g., glycosylation) of Fc amino acids.

In some embodiments, Fc variant antibodies are prepared that have altered binding properties for Fc ligands (e.g., Fc receptors such as C1q) relative to native Fc antibodies. Examples of binding properties include, but are not limited to, binding specificity, equilibrium dissociation constant ( _Kd ), dissociation and association rates ( _koff and _kon , respectively), binding affinity, and/or avidity.

In certain embodiments, the antibodies disclosed herein are glycosylated to alter the effector functions of the antibody or to alter the affinity of the antibody for the target antigen. In certain embodiments, the glycosylation pattern in the variable region of the antibodies of the present invention is modified. For example, a deglycosylated antibody (i.e., the antibody lacks glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the affinity of the antibody for the target antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid mutations can be performed to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site.

In certain embodiments, the antibody disclosed herein is conjugated or covalently linked to another substance using methods known in the art. In certain embodiments, the substance connected is a detectable label (also referred to as a reporter molecule) or a solid support. Suitable substances for being connected to the antibody include but are not limited to amino acids, peptides, proteins, polysaccharides, nucleosides, nucleotides, oligonucleotides, nucleic acids, haptens, drugs, hormones, lipids, lipid assemblies, synthetic polymers, polymeric microparticles, biological cells, viruses, fluorophores, chromophores, dyes, toxins, haptens, enzymes, antibodies, antibody fragments, radioisotopes, solid matrices, semisolid matrices, and combinations thereof. Methods for combining or covalently linking another substance to an antibody are known in the art.

I. Therapeutic Methods Using Antibodies or Fragments of Staphylococcus aureus Surface Antigens or Secreted Toxins

The antibodies or fragments disclosed herein can be administered alone, in combination with each other, or in combination with another agent (such as an antibiotic) for the prevention of S. aureus infection and related symptoms and conditions (e.g., for the treatment of hyperinflammation induced by alpha toxin). These antibodies and antibody or fragment combinations can be used to treat and prevent a wide range of conditions/diseases, including both chronic and acute conditions, such as, but not limited to, bacteremia, burns, cellulitis, skin necrosis, eyelid infection, food poisoning, joint infection, neonatal conjunctivitis, osteomyelitis, pneumonia, skin infection, surgical wound infection, scalded skin syndrome, endocarditis, meningitis, abscess formation, and toxic shock syndrome. Additional details about potential diseases/conditions suitable for S. aureus therapy are provided below.

In certain embodiments, at least one antibody disclosed herein can be administered in combination with at least one additional therapeutic agent (e.g., an antibiotic). Examples of antibiotics that can be administered in combination include penicillin, oxacillin, flucloxacillin, vancomycin, and gentamicin. In certain embodiments, combined therapy using an antibiotic and at least one antibody or antigen-binding fragment thereof disclosed herein enhances therapeutic efficacy by, for example, reducing the concentration of S. aureus CFUs in host tissues, reducing the ability of S. aureus to escape opsonophagocytosis, and/or reducing the virulence of S. aureus, compared to antibody therapy alone.

Combination therapy (e.g., treatment or prevention with more than one antibody) provides benefits over monotherapy by providing multiple non-overlapping S. aureus therapeutic targets. For example, antibodies targeting secreted toxins can neutralize the harmful effects of the toxin, such as excessive inflammation induced by alpha toxin. At the same time, co-administered antibodies targeting surface antigens (e.g., IsdH) can inhibit S. aureus colony growth and opsonophagocytic escape, which cannot be altered by antibodies targeting secreted toxins. Combination therapy can also ensure that the therapy is effective for a wider range of S. aureus strains or mutants, some of which may lack the antigenic target of a specific antibody.

Specifically, the combination therapy may include one or more antibodies or antigen-binding fragments thereof that specifically bind to a surface determinant, such as SdrC, SdrD, SdrE, ClfA, ClfB, IsdA, IsdB, IsdC, IsdE, IsdH, or PNAG, and one or more antibodies or antigen-binding fragments thereof that bind to a secreted toxin, such as alpha toxin (AT). In specific embodiments, the combination therapy may include an antibody or antigen-binding fragment thereof that specifically binds to IsdH and an antibody or antigen-binding fragment thereof that specifically binds to AT; an antibody or antigen-binding fragment thereof that specifically binds to ClfA and an antibody or antigen-binding fragment thereof that specifically binds to AT; an antibody or antigen-binding fragment thereof that specifically binds to IsdH and an antibody or antigen-binding fragment thereof that specifically binds to AT; an antibody or antigen-binding fragment thereof that specifically binds to ClfA and an antibody or antigen-binding fragment thereof that specifically binds to IsdH; or an antibody or antigen-binding fragment thereof that specifically binds to IsdH, an antibody or antigen-binding fragment thereof that specifically binds to ClfA, and an antibody or antigen-binding fragment thereof that specifically binds to AT. In specific embodiments, the combination therapy may include an antibody or antigen-binding fragment thereof that specifically binds to IsdH and an antibody or antigen-binding fragment thereof that specifically binds to AT, wherein the anti-IsdH antibody or antigen-binding fragment thereof comprises the VH and/or VL, or the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3 of mAb 2F4, and wherein the anti-AT antibody or antigen-binding fragment thereof comprises the VH and/or VL, or the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3 of mAb LC10 or comprises SEQ ID NO: 130 and SEQ ID NO: 131.

In specific embodiments, the anti-AT antibody or antigen-binding fragment thereof may include the VH and/or VL, or the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3 of any one of the antibodies listed in Table 7 or 10, the anti-IsdH antibody or antigen-binding fragment thereof may include the VH and/or VL, or the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3 of any one of the antibodies listed in Table 12, and the anti-ClfA antibody or antigen-binding fragment thereof may include the VH and/or VL, or the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3 of any one of the antibodies listed in Table 14.

In various embodiments, the disclosed antibodies, antibody combinations, and/or combinations of antibodies and antibiotics can be administered therapeutically to treat S. aureus infection or as a prophylaxis to prevent infection. For example, the combination therapy can be administered prior to surgery to prevent S. aureus complications, or after surgery to treat S. aureus infection acquired during surgery.

Also provided herein are pharmaceutical compositions for use in treating Staphylococcus aureus infections or as a preventive. In several embodiments, the pharmaceutical composition comprises at least one antibody disclosed herein and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means one or more non-toxic materials that do not interfere with the effectiveness or biological activity of the active ingredient. Such preparations may contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may also contain compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans. The term "carrier" refers to a natural or synthetic organic or inorganic ingredient with which the active ingredient is combined to facilitate drug administration.

The therapeutic composition of the present technology can be formulated into a specific dosage. The dosage regimen can be adjusted to provide the desired reaction (e.g., therapeutic response) of the best. For example, as indicated by the emergency state of the treatment situation, a single bolus can be given, several divided doses can be given over time, or the dosage can be proportionally reduced or increased. In certain embodiments, the selected dosage is suitable for intravenous, intramuscular, intranasal, oral, topical or subcutaneous delivery. The amount of the active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and a specific mode of administration.

Also disclosed herein are pharmaceutical kits for use in treating Staphylococcus aureus infections or as a prophylactic for such infections. In some embodiments, the kit includes one or more containers filled with a sterile therapeutic liquid formulation or lyophilized formulation comprising at least one antibody or antigen-binding fragment thereof disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the container filled with the liquid formulation is a prefilled syringe. In other embodiments, the container filled with a sterile lyophilized powder formulation is suitable for reconstitution and subsequent administration. In certain embodiments, these formulations include antibodies and antigen-binding fragments thereof recombinantly fused or chemically bound to at least one other moiety, including but not limited to a heterologous protein, heterologous polypeptide, heterologous peptide, macromolecule, small molecule, marker sequence, diagnostic or detectable agent, therapeutic moiety, drug moiety, radioactive metal ion, secondary antibody, and solid support. In certain embodiments, these formulations are formulated as sterile liquids in single-dose vials. In some embodiments, the formulations are supplied in prefilled syringes.

J. Diseases associated with Staphylococcus aureus infection

The antibodies and antigen-binding fragments thereof disclosed herein can be used to detect, diagnose, prevent and/or treat diseases associated with Staphylococcus aureus infection. These antibodies can also be used to alleviate and/or prevent one or more symptoms of diseases associated with Staphylococcus aureus infection.

Also provided herein are methods for preventing, treating, or managing pneumonia in a subject, comprising administering to a subject in need thereof an amount effective for preventing, treating, or managing pneumonia, the composition comprising an isolated antibody or antigen-binding fragment thereof, or a combination thereof, that immunospecifically binds to a Staphylococcus aureus toxin or surface determinant.

As used herein, the terms "treat," "treating," or "treatment" refer to therapeutic treatments and prophylactic or preventative measures, wherein the purpose is to prevent or slow (mitigate) an undesirable physiological change or disorder, such as the development of a disease. Beneficial or desired clinical results include, but are not limited to, relief of symptoms, alleviation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delayed or slowed progression of the disease, improvement or alleviation of the disease state. "Treatment" may also mean prolonging survival, as compared to expected survival if not receiving treatment. Those in need of treatment include those already suffering from the condition or disorder, as well as those susceptible to the condition or disorder, or those for which the condition or disorder is to be prevented.

In some embodiments, methods for preventing, treating, or managing a skin infection in a subject are provided, comprising administering to a subject in need thereof an amount effective for preventing, treating, or managing the skin infection, the composition comprising an isolated antibody or antigen-binding fragment thereof, or a combination thereof, that immunospecifically binds to a Staphylococcus aureus toxin or surface determinant according to the present invention. In certain embodiments, the skin infection is skin necrosis. In some embodiments, the skin infection comprises a Staphylococcus aureus infection of the skin. In certain embodiments, the method prevents a skin infection.

In some embodiments, methods are provided for preventing, treating, or managing S. aureus infection associated with dialysis treatment, high-risk surgery, pneumonia, ventilator-associated pneumonia (VAP), or reinfection following discharge from a hospital for a previous prior treatment or surgery, the method comprising administering to a subject in need thereof a composition comprising an isolated antibody or antigen-binding fragment thereof, or a combination thereof, that immunospecifically binds to a S. aureus toxin or surface determinant.

In some embodiments, methods for preventing, treating, or managing conditions associated with Staphylococcus aureus infection are also provided, comprising administering to a subject in need thereof a composition comprising an isolated antibody or antigen-binding fragment thereof that immunospecifically binds to a Staphylococcus aureus toxin or surface determinant in an amount effective to reduce cell lysis. In certain embodiments, the method prevents conditions associated with Staphylococcus aureus infection. In some embodiments, the cell is a red blood cell from the blood or lung.

Provided herein are methods for preventing or reducing the severity of Staphylococcus aureus-associated sepsis in a mammalian subject, comprising administering to the subject an effective amount of an isolated antibody or antigen-binding fragment thereof that immunospecifically binds to a Staphylococcus aureus toxin or surface determinant, or a combination thereof. Also provided are methods for reducing the Staphylococcus aureus bacterial load in the bloodstream or heart of a mammalian subject, comprising administering to the subject an effective amount of an isolated antibody or antigen-binding fragment thereof that immunospecifically binds to a Staphylococcus aureus toxin or surface determinant, or a combination thereof. Also provided are methods for reducing Staphylococcus aureus bacterial agglutination and/or thromboembolic lesion formation in a mammalian subject, comprising administering to the subject an effective amount of an isolated antibody or antigen-binding fragment thereof that immunospecifically binds to a Staphylococcus aureus toxin or surface determinant, or a combination thereof.

The method of preventing S. aureus associated sepsis in a mammalian subject suitably comprises administering to the subject an effective amount of an isolated antibody or antigen-binding fragment thereof that immunospecifically binds to a S. aureus toxin or surface determinant, or a combination thereof, prior to an infectious event. As used herein, an "infectious event" refers to an event in which a subject is exposed or can be exposed to a S. aureus infection. Exemplary infectious events include, but are not limited to, surgery on any part of the body, including the head, mouth, hands, arms, legs, trunk, internal organs (e.g., heart, brain, intestines, kidneys, stomach, lungs, liver, spleen, pancreas, etc.), bones, and skin. Surgery causes conditions that can easily become infected with S. aureus, such as open surgical wounds and organs. Additional infectious events include trauma to any part of the body that provides an open wound or otherwise allows S. aureus infection to enter the body and approach the bloodstream. Additional infectious events include blood transfusions, injection of drugs, either illicit or legal, needle sticks, tattoo needles, insertion and maintenance of intravenous (IV) lines, insertion and maintenance of surgical drains, and skin breaks such as at the site of pressure sores (decubitus ulcers).

In embodiments where these methods provide for the prevention of S. aureus-associated sepsis, the isolated antibody or antigen-binding fragment thereof, or combination thereof, that immunospecifically binds to a S. aureus toxin or surface determinant is suitably administered at least 1 hour prior to the infectious event. For example, at least 1 hour prior to surgery (infectious event). Suitably, the isolated antibody or antigen-binding fragment thereof, or combination thereof, that immunospecifically binds to a S. aureus toxin or surface determinant is administered at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, or more prior to the infectious event. In embodiments, the isolated antibody or antigen-binding fragment thereof, or a combination thereof, that immunospecifically binds to a S. aureus toxin or surface determinant is suitably administered about 6 hours to about 36 hours, about 6 hours to about 36 hours, about 12 hours to about 36 hours, about 12 hours to about 24 hours, about 24 hours to about 36 hours, about 20 hours to about 30 hours, about 20 hours to about 28 hours, about 22 hours to about 26 hours, or about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours, or about 31 hours, or about 32 hours, or about 33 hours, or about 34 hours, or about 35 hours, or about 36 hours before the infectious event.

As used herein, "prevention" of S. aureus-associated sepsis refers to reducing the risk of a subject developing S. aureus-associated sepsis during an infectious event. Suitably, the risk of a subject developing S. aureus-associated sepsis is reduced by at least 30% compared to a subject who was not administered an isolated antibody, antigen-binding fragment thereof, or combination thereof that immunospecifically binds to a S. aureus toxin or surface determinant prior to the infectious event. More suitably, the risk is reduced by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or the risk is completely eliminated compared to a subject who was not administered an isolated antibody, antigen-binding fragment thereof, or combination thereof that immunospecifically binds to a S. aureus toxin or surface determinant prior to the infectious event.

In a method of reducing the severity of S. aureus-associated sepsis in a mammalian subject, such method suitably comprises administering to a subject exhibiting symptoms of S. aureus-associated sepsis an effective amount of an isolated antibody or antigen-binding fragment thereof, or a combination thereof, that immunospecifically binds to a S. aureus toxin or surface determinant. Such symptoms may include, for example, chills, confusion or delirium, fever or low body temperature (hypothermia), dizziness due to low blood pressure, rapid heartbeat, shaking, skin rash, and warm skin.

As used herein, "reducing severity" when used in conjunction with sepsis refers to reducing the symptoms currently exhibited by a subject who has developed S. aureus-associated sepsis. Suitably, these symptoms are reduced by at least 30% compared to the symptoms currently exhibited by a subject who has also acquired S. aureus-associated sepsis, but to whom the subject has not been administered an isolated antibody, antigen-binding fragment thereof, or combination thereof that immunospecifically binds to a S. aureus toxin or surface determinant. More suitably, these symptoms are reduced by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or the symptoms are completely eliminated (i.e., the subject is cured of infection and sepsis) compared to a subject who has not been administered an isolated antibody, antigen-binding fragment thereof, or combination thereof that immunospecifically binds to a S. aureus toxin or surface determinant prior to the infectious event.

Non-limiting examples of some common conditions caused by S. aureus infection include burns, cellulitis, skin necrosis, eyelid infection, food poisoning, joint infection, pneumonia, skin infection, surgical wound infection, scalded skin syndrome and toxic shock syndrome. In addition, it is a common pathogen in foreign body infections, such as intravascular lines, pacemakers, artificial heart valves and joint implants. Some conditions or diseases caused by S. aureus infection are further described below. Some or all of the conditions and diseases described below may involve the direct action of secreted toxins as infectious components or mediators of the condition or disease state, while some or all of the conditions may involve the indirect or secondary action of secreted toxins (e.g., as primary virulence factors that cause the main symptoms or most symptoms associated with the condition, or as reagents that further promote the disease by disrupting cellular function and cell lysis).

a) Burns

Burn wounds are often initially sterile. However, moderate and severe burns often damage the physical and immune barriers to infection (e.g., blistering, cracking, or peeling of the skin), leading to loss of fluids and electrolytes, and causing local or systemic physiological dysfunction. Contact of damaged skin with live bacteria can lead to mixed colonization at the site of injury. Infection can be limited to inert debris ("eschar") on the burn surface, or colonization can progress to full skin infection and invade the viable tissue under the eschar. More serious infections can reach under the skin, enter the lymphatic system and/or blood circulation, and develop into sepsis. Typically, Staphylococcus aureus is found among the pathogens that colonize burn wound infections. Staphylococcus aureus can destroy granulation tissue and produce severe sepsis.

b) Cellulitis

Cellulitis is an acute infection of the skin that often begins as a surface infection that can spread beneath the skin's layers. Cellulitis is most commonly caused by a combination of Staphylococcus aureus and Streptococcus pyogenes. Cellulitis can lead to systemic infection.

c) Skin necrosis

Cutaneous necrosis is an infection of the skin and subcutaneous tissue that spreads easily across the fascial plane within the subcutaneous tissue. The condition causes the top and/or bottom layers of the skin to become necrotic and can spread to underlying and surrounding tissues.

d) Necrotizing fasciitis

Necrotizing fasciitis has been referred to as "flesh-eating disease" or "flesh-eating bacteria syndrome." Necrotizing fasciitis can be caused by a polymicrobial infection (e.g., Type I, caused by a mixed bacterial infection) or by a monomicrobial infection (e.g., Type II, caused by a single pathogenic strain of bacteria). Many types of bacteria can cause necrotizing fasciitis, non-limiting examples of which include Group A Streptococci (e.g., Streptococcus pyogenes), Staphylococcus aureus, Vibrio vulnificus, Clostridium perfringens, and Bacteroides fragilis. Individuals with suppressed or compromised immune systems are more likely to suffer from skin necrosis (e.g., necrotizing fasciitis).

Historically, group A Streptococcus has been diagnosed as the cause of most cases of type II cutaneous necrotizing fasciitis. However, since 2001, methicillin-resistant Staphylococcus aureus (MRSA) has been observed with increasing frequency as a cause of monomicrobial necrotizing fasciitis. The infection begins locally (sometimes at the site of trauma) and can be severe (e.g., due to surgery), mild, or even inapparent. Patients often complain of severe pain that may be excessive for the particular appearance of the skin. As the disease progresses, the tissue often becomes swollen within a few hours. Diarrhea and vomiting are also common symptoms.

If the bacteria are deep in the tissues, signs of inflammation may not be apparent in the early stages of infection. If the bacteria are not deep, signs of inflammation such as redness and swelling or burning of the skin quickly develop. The skin color may progress to purple, and blisters may form, with subsequent necrosis (e.g., death) of the subcutaneous tissue. Patients with necrotizing fasciitis typically have a fever and appear very ill. If left untreated, a mortality rate of up to 73% has been recorded.

e) Pneumonia

Staphylococcus aureus has also been identified as a cause of staphylococcal pneumonia. Staphylococcal pneumonia causes inflammation and swelling of the lungs, which in turn causes fluid to accumulate in the lungs. The fluid that accumulates in the lungs can prevent oxygen from entering the bloodstream. Those who suffer from influenza are at risk of developing bacterial pneumonia. Among those who have already suffered from influenza, Staphylococcus aureus is the most common cause of bacterial pneumonia. Common symptoms of staphylococcal pneumonia include coughing, difficulty breathing, and fever. Additional symptoms include fatigue, yellow or bloody mucus, and chest pain that worsens with breathing. Methicillin-resistant Staphylococcus aureus (MRSA) is increasingly being diagnosed as a strain identified in staphylococcal pneumonia.

f) Surgical wound infection

Surgical wounds often penetrate deep into the body. Therefore, if a wound becomes infected, infection of such wounds poses a significant risk to the patient. Staphylococcus aureus is often the causative agent of infection in surgical wounds. Staphylococcus aureus is very good at invading surgical wounds, and sutured wounds can be infected by Staphylococcus aureus cells that cause far fewer infections in normal skin. Invasion of surgical wounds can lead to severe Staphylococcus aureus sepsis. Invasion of the bloodstream by Staphylococcus aureus can lead to seeding and infection of internal organs (particularly heart valves and bones), causing systemic diseases such as endocarditis and osteomyelitis.

g) Scalded skin syndrome

S. aureus may be the primary causative agent of "scalded skin syndrome" (also known as "staphylococcal scalded skin syndrome"), "toxic epidermal necrolysis," "localized bullous impetigo," "Ritter's disease," and "Lyell's disease." Scalded skin syndrome frequently occurs in older children, typically in outbreaks caused by a flowering of S. aureus strains that produce epidermolytic exotoxins (e.g., exfoliative toxins A and B, sometimes referred to as scalded skin syndrome toxins) that cause separations within the epidermal layer. One of the exotoxins is encoded by the bacterial chromosome, and the other is plasmid-encoded. The exotoxins are proteases that cleave desmoglein-1, a protein that normally holds the stratum granulosum and stratum spinosum layers of the skin together.

The bacteria may initially infect only a small lesion. However, toxins destroy the intercellular junctions, spreading through the epidermal layer and allowing the infection to penetrate the outer layer of the skin, producing the scaling that characterizes the disease. This shedding of the outer layer of skin usually exposes the normal skin beneath, but the fluid loss during this process can cause severe damage in young children if not properly treated.

h) Toxic shock syndrome

Toxic shock syndrome (TSS) is caused by strains of Staphylococcus aureus that produce the so-called "toxic shock syndrome toxin." The disease can be caused by infection with S. aureus anywhere, but is often mistakenly thought to be exclusive to women who use tampons. The disease involves toxemia and sepsis and can be fatal.

The symptoms of toxic shock syndrome vary depending on the underlying cause. TSS, caused by infection with the bacterium Staphylococcus aureus, typically presents in otherwise healthy individuals with a high fever, accompanied by low blood pressure, malaise, and confusion, and can rapidly progress to stupor, coma, and multiple organ failure. The characteristic rash often seen in the early course of the disease resembles a sunburn and can affect any area of the body, including the lips, mouth, eyes, palms, and soles of the feet. In patients who survive the initial onslaught of infection, the rash desquamates, or peels off, after 10-14 days.

As noted above, due to the increase in multidrug-resistant strains of Staphylococcus aureus, an increasing number of antibiotics commonly used to treat Staphylococcus aureus infections no longer control or eliminate infections with methicillin-resistant and multidrug-resistant Staphylococcus aureus. Antibodies to surface determinants and secreted toxins of Staphylococcus aureus as described herein can help reduce the severity of infection and can also assist in clearing, preventing (prophylactically) or reducing pathogenic Staphylococcus aureus from an infected host. These antibodies can also be used to detect Staphylococcus aureus and, when present in patient samples, diagnose Staphylococcus aureus infections.

K. Methods for detecting Staphylococcus aureus using antibodies or fragments directed against surface antigens or secreted toxins of Staphylococcus aureus

In various embodiments, the antibodies disclosed herein can be used alone or in combination to detect the presence of S. aureus in a sample.

In certain embodiments, the method comprises contacting a test sample with one of the isolated antibodies or fragments disclosed herein. The antibody or antigen-binding fragment thereof then binds to a surface antigen or secreted toxin of Staphylococcus aureus to form an antigen-antibody complex. In further embodiments, the method comprises contacting the antigen-antibody complex with a detectable label, wherein the signal generated by the detectable label is directly correlated with the presence of Staphylococcus aureus in the sample. For example, the detectable label may comprise one or more fluorescent labels that bind to the antibody or antigen in the antibody-antigen complex such that an increase in fluorescence is correlated with an increased concentration of Staphylococcus aureus or secreted toxin in the sample.

In other embodiments, the detectable label competes with a S. aureus surface antigen or secreted toxin for binding to the antibody or antigen-binding fragment thereof, wherein the signal produced by the detectable label is indirectly correlated with the concentration of S. aureus or secreted toxin in the sample. For example, the detectable label can include one or more fluorescent labels that compete with the surface antigen or secreted toxin for antibody binding, such that a decrease in fluorescence is correlated with an increased concentration of S. aureus or secreted toxin in the sample.

In certain embodiments, the detectable signal produced by the detectable label in the test sample is compared to the signal from at least one control sample having known concentrations of antigen and antibody. In embodiments where a control sample is used, the detectable label can be used to detect antibody-antigen complexes in both the control and test samples, and any statistically significant difference in the detectable signal between the samples indicates the concentration, presence, or absence of S. aureus and/or secreted toxin in the test sample.

In other embodiments, a combination of antibodies is used to detect S. aureus in a sample. In various embodiments, the method comprises contacting a test sample with an isolated antibody or antigen-binding fragment thereof directed against a surface antigen of S. aureus and an isolated antibody or antigen-binding fragment thereof directed against a toxin secreted by S. aureus. The antibody or fragment combination then binds to the surface antigen of S. aureus and the secreted toxin to form two antigen-antibody complexes. In further embodiments, the method comprises contacting a test sample comprising the antigen-antibody complex with at least one detectable label, wherein the signal generated by the detectable label(s) is directly correlated with the presence of S. aureus in the sample. For example, the detectable label(s) may comprise one or more fluorescent labels that bind to the antibody or antigen in at least one of the antibody-antigen complexes, such that an increase in fluorescence is correlated with an increased concentration of S. aureus or secreted toxin in the sample.

In other embodiments, the at least one detectable label competes with a surface antigen and/or secreted toxin of S. aureus for binding to the antibody or fragment combination. Thus, the signal generated by the detectable label(s) is indirectly correlated with the concentration of S. aureus in the sample. For example, the detectable label(s) can include one or more fluorescent labels that compete with the surface antigen and/or secreted toxin for antibody binding, such that a decrease in fluorescence is correlated with an increased concentration of S. aureus or secreted toxin in the sample.

In certain embodiments, the detectable signal produced by the detectable label in the test sample is compared to the signal from at least one control sample having known concentrations of antigen and antibody. In embodiments where a control sample is used, the detectable labels are used to detect antibody-antigen complexes in the control and test samples, and any statistically significant difference in the detectable signal between the samples is indicative of the concentration, presence, or absence of S. aureus and/or secreted toxin in the test sample.

In certain embodiments, the detection method is used to detect the presence of S. aureus in a patient sample, and the method further comprises diagnosing the patient with a S. aureus infection. In some embodiments, the method is suitable for use in an automated or semi-automated system.

In certain embodiments, kits for various research and diagnostic purposes are also provided, comprising at least one antibody or antigen-binding fragment thereof disclosed herein. For example, these kits can be used to detect Staphylococcus aureus in a sample or to immunoprecipitate toxins secreted by Staphylococcus aureus. For separation and purification purposes, the kits can include the antibody or antigen-binding fragment thereof coupled to beads (e.g., agarose beads).

In this application, the use of the singular includes the plural, unless otherwise expressly stated. Also in this application, the use of "or" means "and/or" unless otherwise stated. In addition, the use of the term "including" as well as other forms (e.g., "includes" and "included") is not limiting. Any ranges described herein should be understood to include the endpoints and all values between the endpoints.

The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, papers, books, and monographs, are hereby expressly incorporated herein by reference in their entirety for any purpose. To the extent that the applications and patents or patent applications incorporated by reference are inconsistent with the invention contained in this specification, this specification supersedes any contradictory material.

Examples

The following examples are provided to illustrate but in no way limit the present disclosure.

Example 1 - Materials and Methods

The materials and methods used in Examples 2 to 9 are provided below.

Neutralization of hemolytic activity

In a 96-well plate, 50 microliters of each B cell hybridoma culture supernatant was mixed with recombinant α-toxin-His (rAT-his, 0.1 μg/ml final concentration) and subsequently added to 50 μl of 5% rabbit red blood cells (RBC) in PBS. Control wells contained only RBC and culture medium with or without AT. The plate was incubated at 37°C for 1 hour, and the intact cells were precipitated by centrifugation. 50 μl of supernatant was transferred to a new 96-well plate and A ₄₉₀ was measured in a spectrophotometer. Neutralization activity was calculated relative to the dissolution with RBC and individual rAT-his and calculated: % inhibition = 100x [100-(A ₄₉₀ nAT+Ab)/(A ₄₉₀ nAT without Ab)].

Inhibition using purified mAb was also tested. Anti-AT mAb was added to a 96-well plate at approximately 80 μg/mL in PBS, and the sample was serially diluted (twice) in PBS to a final volume of 50 μL. Non-specific IgG1 (R347) was included as an isotype control. Twenty-five microliters of mAb dilutions were mixed with 25 μL of nAT (natural alpha toxin) at approximately 0.1 μg/mL in a 96-well round-bottom plate, followed by the addition of 50 μL of 5% RBC. Inhibition of hemolytic activity was calculated as above.

Neutralization of A549 dissolution

A549 cells were maintained in RMPI supplemented with non-essential amino acid glutamine and 10% fetal bovine serum in a 5% CO ₂ 37 ℃ incubator. The cells were washed once with Hank's balanced media and plated at 10 ⁴ / well in RPMI, 5% FBS below 50 μl, then incubated at 37 ℃ with 5% CO ₂ for 20 hours. Anti-AT mAb was added to 96-well plates at approximately 80 μg/mL in RPMI, and the samples were serially diluted (twice) in RPMI. Unrelated IgG1 (R347) was included as an isotype control. In a separate 96-well plate, 30 μl of diluted antibody was mixed with 30 μl of nAT (final concentration, 5 μg/ml). Fifty microliters from each well were transferred to the plate containing adherent A549 cells. Control wells of A549 cells with or without nAT were included. The plate was incubated at 37°C with 5% _CO2 for 3 hours, centrifuged, and 50 μl of supernatant was transferred to a new 96-well plate. Cell lysis was measured as the release of lactate dehydrogenase (LDH) using the Cytotox 96 non-radioactive assay kit (Promega) according to the manufacturer's protocol. Background LDH was subtracted from each well and inhibition of LDH release was calculated: % inhibition = 100 x [100 - ( _A590 nAT + Ab) / ( _A590 nAT without Ab)].

Neutralization of THP-1 dissolution

THP-1 cells were maintained in a 5% CO ₂ 37°C incubator in RPMI medium (Invitrogen) supplemented with non-essential amino acids (Invitrogen), 2mM glutamine (Invitrogen), and 10% fetal bovine serum (Invitrogen). Anti-AT mAb was added to a 96-well plate at approximately 80 μg/ml in RPMI, and samples were serially diluted (two-fold) in RPMI to a final volume of 50 μL. An irrelevant IgG1 (R347) was included as an isotype control. Twenty-five microliters of the mAb dilution was mixed with 25 μl of native alpha toxin (nAT) to a final of 1.5 μg/ml, followed by addition of 50 μl of RMPI-washed THP-1 cells (10 ⁶ cells/ml in RPMI with 10% FBS) to a 96-well plate. Control wells consisted of THP-1 cells alone or with nAT. The plate was incubated in a 5% CO ₂ 37° C. incubator for 3 h, centrifuged, and 50 μl of the supernatant was transferred to a new 96-well plate. Cell lysis was measured as the release of lactate dehydrogenase (LDH) using the Cytotox 96 non-radioactive assay kit (Promega) according to the manufacturer's instructions. Inhibition of LDH release was calculated as described above.

Murine pneumonia model

Twenty-four hours prior to infection, groups of 10 7-9 week old C57BL/6J mice (Harlan) received 0.5 ml of mAb at the indicated concentrations via intraperitoneal injection (ip). The animals were then anesthetized with isoflurane, held upright, and inoculated into the left and right nostrils with 0.05 ml of a Staphylococcus aureus bacterial suspension (1 x 10 ⁸ CFU to 3 x 10 ⁸ CFU) in sterile PBS. The animals were placed in a supine position in a cage for recovery and observed twice daily for the duration of the study. Animal survival was monitored for a maximum of 6 days.

Alternatively, animals were euthanized by _CO inhalation 48 h after bacterial infection. Lungs and kidneys were removed to sterile PBS, homogenized, diluted, and plated for bacterial counts. The statistical significance of mortality studies was determined using the log-rank test. The significance of bacterial recovery from organs was calculated using analysis of variance and Dunnett's post hoc test.

Mouse model of skin necrosis

Groups of five 6-8 week old female BALB/c mice (Harlan) were shaved on their backs and given 0.5 ml of IgG at the concentration indicated on the chart by intraperitoneal injection. Twenty-four hours later, mice were infected by subcutaneous injection of 50 μL of a bacterial suspension (1×10 ⁸ Staphylococcus aureus). Animals were monitored twice daily for signs of infection, and the size of abscesses was measured daily. The area of the lesion was calculated using the formula A=L×W. Statistical significance was determined using analysis of variance and Dunnett's post hoc test.

Rat model of sepsis

Preparation of bacterial challenge dose: Staphylococcus aureus SF8300 (USA300) was provided by BinhDiep (University of California, San Francisco). The bacteria were cultured overnight in 50 mL of tryptic soy broth (TSB) at 37°C with shaking at 250 rpm. Ten mL was taken from the overnight culture and added to 1 L of fresh TSB and grown at 37°C with shaking to an optical density at 600 nm (OD600) of 0.8. The bacteria were recovered by centrifugation at 8000 rpm for 15 minutes at 4°C and washed in phosphate-buffered saline (PBS). The bacteria were collected by centrifugation and resuspended in PBS with 10% glycerol to a final bacterial stock concentration of ~2 x ¹⁰¹⁰ cfu/mL.

Mouse Challenge and Survival: Groups of ten 8-9 week old female BALB/c mice were injected intraperitoneally (IP) with the indicated concentrations of LC10 or R347 (45 mg/kg) in 500 μL PBS. Twenty-four hours later, the animals were challenged intravenously (IV) in the tail vein with 200 μL of a bacterial suspension (5 × 10 ⁷ cfu diluted from a frozen stock in PBS (pH 7.2). Survival of the mice was monitored for 14 days after challenge. Statistical analysis was evaluated using the log-rank test: R347 (control) versus LC10 (anti-ATAb)-immunized animals.

Bacterial Burden in Heart: 14 h post-infection, infected mice were euthanized with CO . Hearts were removed, homogenized in 1 mL of cold PBS in a Dissolved Matrix A tube, and plated on TSA plates for bacterial counts. Bacterial burden in cardiac tissue was analyzed using an unpaired, two-tailed Student's t-test for pairwise comparisons between R347 and LC10 mAbs. Data were considered significant if p < 0.05.

Bacterial Burden in Blood: Animals were euthanized with CO2 at 8, 24, 48, 72, and 144 h post-infection. Blood was collected by cardiac puncture, and 100 μL was immediately plated on TSB plates for cfu enumeration. Data were analyzed using an unpaired Student's t-test. Values between LC10 and R347 mAb were considered statistically different if p < 0.05.

Receptor binding assay

Red blood cell ghosts were prepared by incubating 5 mL of washed and concentrated rabbit red blood cells (RBCs) in 500 mL of lysis buffer (5 mM phosphate, 1 mM EDTA, pH 7.4) at approximately 4°C with constant stirring. The ghosts were then removed by centrifugation at 15,000 x g and washed three times with lysis buffer. They were then washed in PBS and resuspended in a final volume of 3 mL.

To assess the binding of nAT to the cell membrane, RBC ghosts were diluted in PBS to an OD ₆₀₀ of approximately 0.2, and 50 μL were coated onto a 1/2-well 96-well plate (Costar) and incubated overnight at 4°C. The liquid was then removed from the plate, and the wells were blocked with 100 μL of 1% BSA in PBS (pH 7.4) for 2 hours at 4°C and washed 3 times with PBS. A 20-molar excess of IgG was mixed with 3 μg/mL of nAT, and 50 μL were added to the blocked plate. The plate was incubated at 4°C for 2 hours and washed 3 times with PBS. Biotinylated rabbit anti-AT IgG was added to the wells at 1 mg/mL and incubated at 4°C for 1 hour, washed 3 times, and then incubated with streptavidin peroxidase conjugate (1:30,000, Jackson Immunoresearch). The wells were washed three times and developed with Sure Blue Reserve (KPL). A ₄₅₀ was read using a microplate reader (Molecular Devices) and the % bound AT was calculated. % bound AT = 100 x (A ₄₅₀ -AT+IgG/A ₄₅₀ -AT alone).

Measurement of kinetic rates and binding constants ( _KD )

The kinetic rate constants (k _on , k _off ) of anti-AT IgG antibodies bound to purified nAT were measured using the IgG-capture assay on a BIAcore 3000 instrument (BIAcore). In brief, rat anti-mouse IgG was immobilized on a CM5 sensor chip according to the manufacturer's instructions. The final surface density of the capture reagent on the sensor chip was approximately 2500 response units (RU) as described herein. A reference flow cell surface was prepared on this sensor chip using the same immobilization scheme and omitting nAT. Anti-AT IgG antibodies were prepared in instrument buffer (HBS-EP buffer, comprising 0.01M HEPES, pH 7.4, 0.15M NaCl, 3mM EDTA, and 0.005% P-20) with two-fold serial dilutions of nAT at 20nM. Serial dilutions of nAT in instrument buffer were obtained in the range of approximately 0.78nM to approximately 50nM.

A sequential approach was used for kinetic measurements. First, each anti-AT IgG was injected onto the capture and reference surfaces at a flow rate of 50 μL/min. Once the binding of the captured IgG was stabilized, a single concentration of nAT protein was injected onto the two surfaces at a flow rate of 50 μL/min. The generated binding response curve was used to determine the association phase data. After injecting nAT, the flow was then switched back to the instrument buffer for 10 minutes, allowing the collection of dissociation phase data, followed by a 1-minute pulse of 10 mM glycine at pH 1.5 to regenerate the IgG capture surface on the chip. For all anti-AT IgGs, the binding response from the dual injection of nAT at each concentration was recorded.

In addition, throughout the injection series, several buffer injections are interspersed. Use selection buffer injection together with reference cell response (commonly referred to as " dual reference ") to correct for injection artifacts (injection artifacts) and/or the original data set of non-specific binding interactions (DGMyszka (Mishka), Improving biosensor analysis (improving biosensor analysis), J.Mol.Recognit. (" molecular recognition magazine ") 12 (1999), 279-284 pages). Then the fully corrected binding data are integrally fitted to a 1: 1 binding model (BIAevaluation4.1 software, BIAcore, Uppsala (Uppsala, Sweden), which includes the entry for correcting mass transport-limited binding, which should be detected. These analyses determine kinetic rate (on, off) constants, and apparent _K is then calculated as k _off / _kon accordingly.

Measurement of cytokine levels in Staphylococcus aureus-infected lungs

Seven to nine-week-old C57BL/6J mice were treated with 2A3.1hu (fully human 2A3.1) or R347 (45 mg/kg) by intraperitoneal injection 24 hours prior to nasal infection with 1.5 x 10 ⁸ cfu USA300 (BAA-1556, ATCC). Four and twenty-four hours after infection, mice were euthanized and the lungs were flushed three times with 1 ml of PBS. Bronchoalveolar lavage (BAL) fluid was stored at -70°C. Proinflammatory cytokines were quantified using a 7-proinflammatory type II mouse cytokine kit (Mesoscale, Gaithersburg, Maryland (MD)) according to the manufacturer's instructions. Cytokine levels are expressed as pg/ml.

Dot blot assay

Chemical synthesis spans overlapping peptides of amino acid 40 to 293 (New England Peptide). The synthesis of AT _1-50 was attempted but unsuccessful. Alpha toxin (AT), AT peptide and AT fragment (1 μg) were spotted on nitrocellulose and blocked with the blocking agent casein in PBS for 10 min. Then, at room temperature, the blot was probed with a single IgG of 2 μg/mL for 3 hours. These blots were washed and incubated with alkaline phosphatase-bound goat anti-mouse or goat anti-rabbit IgG (1:1000, Caltag Laboratories) for 1 hour, and developed using BCIP/NBT membrane phosphatase substrate system (KPL).

Example 2-Target Selection and Validation

Based on its conservative and/or disclosed vaccine potential across clinical isolates, 13 kinds of surface antigens and four kinds of secreted toxins are selected as antibody targets for verification. Included in this group are alpha toxins and three soluble regulatory proteins (PSMs). Also included are 8 kinds of staphylococcal cell wall-anchored antigens/adhesins. Five of the selected targets have homologs in Staphylococcus aureus and Staphylococcus epidermidis. These targets are related to nutrient acquisition, biofilm formation and cell division. Antibody targeting for alpha toxin is a hypothetical method for reducing or neutralizing toxin activity (such as tissue damage and immune regulation abnormalities). Also targeting Staphylococcus aureus surface determinants (IsdH, SdrC, ClfB, ClfA and IsdB), which are important for Staphylococcus aureus colonization, immune escape and fitness. It is considered that the possible approach for enhancing antibody therapy involves combining opsonins and toxin-neutralizing monoclonal antibodies.

Antibodies against identified targets were evaluated for reduced virulence and/or reduced colonization and immune evasion in both in vitro and in vivo assays. Target fitness was also validated via active/passive immunization in a murine infection model.

Example 3 - Identification of anti-IsdH antibodies

The primary target identified was the Staphylococcus aureus iron-regulated surface determinant H (IsdH). IsdH contains a 7-amino acid loop between the B1b and B2 β-sheets, and this 7-amino acid loop is conserved across several members of the iron-regulated surface determinant family, including IsdA and IsdB. Mutations in this 7-amino acid loop reduce the ability of S. aureus to bind hemoglobin by more than 100-fold and also impair the ability of S. aureus to evade phagocytic killing. Visai et al., J. Microbiology, 155(3): 667-679 (2008).

Anti-IsdH monoclonal antibodies (mABs) were identified using VelocImmune (Regeneron Pharmaceuticals) and phage panning (Dyax or CAT libraries). 59 IgG antibodies were purified (29 from the Dyax library, 16 from the CAT library, and 14 from VelocImmune mice).

Example 4 - Anti-IsdH mAB Screening Cascade

The anti-IsdH mAB identified was evaluated for in vitro whole-cell S. aureus binding by ELISA. The antibodies were also screened for inhibition of S. aureus haptoglobin binding by ELISA. The antibodies were then evaluated in an opsonophagocytic killing assay (OPK) (described below). Eleven anti-IsdH IgG antibodies were identified that were opsonogenic against four S. aureus isolates. Five anti-IsdH antibodies effectively bound to S. aureus after passage in vivo in a mouse infection model (described below). The top five anti-IsdH mABs (3 from the Dyax library and 2 from the CAT library) were selected for scale-up antibody production, affinity testing, and subsequent in vivo testing. In vivo testing included studies in a bacteremia model (described below). Antibody 2F4 significantly reduced CFU in the bacteremia model. These five antibodies were then characterized and evaluated for use in combination therapy.

The opsonophagocytic killing (OPK) assay involves combining 10 μl of S. aureus ( ¹⁰ cells/ml), 10 μl of monoclonal antibody, and 60 μl of DMEM plus 0.1% gelatin. The solution is incubated at 4°C for 30 minutes. After 30 minutes, 10 μl of human promyelocytic leukemia (HL-60) cells at ¹⁰ cells/ml are added, along with 10 μl of human serum pre-absorbed against S. aureus. At time _T0 , 10 μl of the solution is plated and then incubated at 37°C for 60 minutes with shaking at 1500 rpm. At time _T60 , the HL-60 cells are solubilized with 1% saponin, replated, and the CFU concentration is determined. The OPK percentage was calculated as follows: 100 x (1-( _T60 / _T0 )), where _T60 refers to the CFU concentration at the end of the assay (ie, at 60 minutes) and _T0 refers to the CFU concentration at the start of the assay.

Eleven monoclonal anti-IsdH antibodies were identified that were sufficiently opsonizing against the four S. aureus isolates for further study. Figure 19 illustrates that antibodies B11, 2F4, and A7 had increased OPK percentages compared to control antibody R347 cells when tested in S. aureus strains Newman and USA300.

To determine whether the antigens targeted by these antibodies are expressed in vivo by S. aureus, antibody binding was assessed after in vivo passage in mice. Mice were challenged intraperitoneally with approximately 5 x 10 ⁸ CFU of S. aureus. After 1 to 6 hours, the mice were bled and the blood was collected in ice-cold citrate. Eukaryotic cells were lysed with 1% NP-40. The lysed cells were washed three times with phosphate-buffered saline (PBS) and sonicated, and then the S. aureus bacteria were resuspended in buffer (after lysis and resuspension, approximately 0.5-10 x 10 ⁶ CFU were recovered). Anti-IsdH antibodies were administered to the cell lysates, and antibody binding was evaluated by staining and FACs sorting.

Five of the eleven anti-IsdH mAbs (designated 1C1, 2F4, A7, IsdH003, and IsdH0016) bound to S. aureus after passage in vivo. Figure 2 shows the binding of antibodies B11, 2F4, A7, and 1C1 to S. aureus strains ARC2081 and USA300 compared to control antibody R347. The figure shows that antibodies 2F4, A7, and 1C1 bind to S. aureus ex vivo.

Two of the five anti-IsdH mAbs (1C1 and 2F4) also compete with haptoglobin (Hp) for binding to IsdH, while the remaining three do not. Figure 3 shows that antibody 1C1 competes with Hp for binding to the subunit Neat-1 on IsdH. Compared to Hp binding in the presence of control antibody R347, an increase in the concentration of 1C1 (in μg/ml) correlates with a decrease in Hp binding to IsdH. Similarly, Figure 3 shows that antibody 2F4 competes with Hp for binding to the subunit Neat-2 on IsdH. Compared to Hp binding in the presence of control antibody R347, an increase in the concentration of 2F4 (in μg/ml) correlates with a decrease in Hp binding to IsdH.

To evaluate whether antibodies 1C1, 2F4, A7, IsdH003, and IsdH0016 are effective when administered in vivo, a mouse bacteremia model was utilized. Mice were injected intraperitoneally with 45, 15, or 5 mg/kg of monoclonal antibody and then allowed to recover overnight. The next day, mice were intraperitoneally infected with approximately 10^⁸ CFU of Staphylococcus aureus (Newman strain). Approximately 4 hours later, blood was collected and CFU concentrations were assessed (measured as log[CFU/ml]). Figure 4 shows that antibodies 1C1, A7, IsdH003, and IsdH0016 did not reduce CFU concentrations in the bacteremia model. However, Figure 5 shows that antibody 2F4 did reduce CFU concentrations in the mouse bacteremia model.

Antibody 2F4 was further evaluated for binding to different S. aureus strains in vitro. The antibody bound to 23 of 25 S. aureus isolates after in vivo passage and extraction in mice. Figure 6 illustrates 2F4 binding to strains ARC2379 (USA100), ARC2081 (USA200), and BAA-1556 (USA300). The table below illustrates the results of binding experiments with 25 S. aureus strains after in vivo passage in mice.

Binding of antibody 2F4 after in vivo passage in Staphylococcus aureus strains

Antibody 2F4 was also evaluated in the OPK assay against S. aureus clinical isolates - Newman, ARC634 (USA100), ARC2081 (USA200), and BAA-1556 (USA300). Figure 7 shows that 2F4 is opsonogenic to major S. aureus clinical isolates.

2F4 was subsequently evaluated for affinity to IsdH and to Neat-2 subunits in a hu lgGFc capture assay. The average affinity averaged across three experiments revealed a _KD of 3.66 nM for IsdH and a _KD of 2.57 nM for Neat-2. Figure 8.

Example 5 - Anti-IsdH & Anti-AT Antibody Combination Therapy

Alpha toxin and IsdH play different roles in the pathogenesis of Staphylococcus aureus infection. The former is a secreted toxin, while the latter is a surface protein important for colonization, immune evasion, and bacterial fitness. Both can be differentially expressed by Staphylococcus aureus during infection. Combining monoclonal antibodies with different modes of action can potentially produce additive or synergistic effects while reducing the risk that the strain will evade therapy.

2A3 (anti-alpha toxin antibody) was evaluated for use in combination with 2F4 (anti-IsdH antibody). When administered in combination, antibodies 2A3 and 2F4 exhibited a synergistic effect in an organ burden model. Figure 9 shows that the renal distribution of S. aureus strain USA300 was reduced in the presence of both antibodies compared to either the individual antibodies or the control antibody R347.

The results of these combination therapy experiments suggest that a combinatorial approach for preventing or treating S. aureus may be effective.

Example 6—Anti-ClfA mAb inhibits ClfA binding

Anti-ClfA mAb inhibits ClfA binding to immobilized fibrinogen in vitro. ClfA, a virulence factor, has been reported to promote the binding of Staphylococcus aureus to fibrinogen present in plasma. This leads to bacterial agglutination in the blood.

Three anti-ClfA mAbs generated using B-cell hybridoma technology were evaluated for their ability to inhibit ClfA binding to immobilized fibrinogen. Antibody R347 was used as a negative control. The activity of each anti-ClfA mAb in this assay was calculated at the IC50, which is the concentration required to induce 50% inhibition of binding. As shown in Figure 10, along with the IC50 for each antibody, the anti-ClfA antibodies inhibited ClfA binding to immobilized fibrinogen.

Example 7 - Anti-ClfA mAb 11H10 inhibits agglutination of S. aureus with three different clinical isolates in human plasma.

To evaluate the agglutination of S. aureus in human plasma, bacteria were incubated with each anti-CIfA mAb and visually examined for bacterial aggregation after 3 min of incubation at 37°C. For a more accurate comparison, the activity of the mAbs in this assay was compared at the lowest concentration required to inhibit agglutination. 11H10 was more effective than either 27H4 or 23D6 ( FIG. 11 ). In addition, agglutination experiments were performed with three different clinical isolates, and 11H10 exhibited inhibition against all three isolates compared to the other two anti-CIfA mAbs that covered one or both strains.

Example 8-Epitope Binding of 11H10

Given the distinct characteristics of 11H10 compared to 23D6 and 27H4 discussed above, its binding characteristics were further explored. Epitope competition binding was run by Octet to assess whether 11H10 binds to a different epitope than 23D6 and 27H4. As shown in Figure 12, 23D6 and 27H4 compete for binding to CIfA, suggesting that they may share a common region on CIfA for binding. However, there was no competition between 11H10 and 23D6 or between 11H10 and 27H4, demonstrating that the 11H10 epitope on CIfA is different from that of 23D6 and 27H4.

Example 9 - Passive immunization with anti-ClfA mAb 11H10 demonstrates efficacy in a lethal IV challenge model

Bacterial burden in the heart

To test whether staphylococcal agglutination occurs in vivo, mice were first challenged with the USA300 isolate in the tail vein and the number of bacteria in the heart was counted 14 h post-infection. As shown in Figure 13, intraperitoneal prophylactic administration of 45 mg/kg of the anti-ClfA mAb 11H10 resulted in a significant decrease in bacterial cfu in the heart (p = 0.031). This was dose-dependent, as 15 mg/kg of 11H10 only slightly reduced the bacterial load in the heart compared to the negative control R347.

Survival

The USA300 challenge dose used for IV challenge was determined to induce 20% survival after 2 weeks. The ability of the anti-ClfA mAb 11H10 to increase animal survival was investigated in this model. Figure 14 shows that 11H10 injection resulted in a significant increase in survival within 2 weeks after infection (p = 0.0114 at 45 mg/kg and p = 0.0239 at 15 mg/kg).

Example 10 - Efficacy of the combination of anti-ClfA mAB 11H10 and anti-AT Ab LC10 in a lethal IV challenge model

Six-week-old BALB/c female mice were passively immunized intraperitoneally (IP) with the indicated concentrations of mAb (diluted in 500 μl PBS) and challenged intravenously (IV) in the tail vein with an LD20 dose of bacteria (in 200 μl PBS) 24 h later. Survival was monitored up to 14 days after infection.

Data were analyzed using the log-rank (mantel-cox) test, and p values were considered statistically significant if < 0.05. To test whether staphylococcal agglutination occurs in vivo, mice were first challenged in the tail vein with CA-MRSA USA300, HA-MRSA-100, or HA-MSSA USA200 isolates, and 14 h post-infection, the number of bacteria in the heart and kidneys was counted.

The efficacy of the combination of anti-ClfA mAB 11H10 and anti-AT Ab LC10 was tested in a lethal IV challenge model. As shown in Figure 15, prophylactic administration of anti-ClfA mAb 11H10, anti-AT LC10 mAb, and the combination of both anti-ClfA mAb and anti-AT LC10 mAb resulted in a significant reduction in bacterial cfu in the heart (Figure 15b) and kidney (Figure 15c).

Figure 15 also demonstrates the ability of anti-ClfA mAb 11H10, anti-AT LC10 mAb, and the combination of anti-ClfA mAb and anti-ATLC10 mAb to increase animal survival as studied in an IV challenge model using a USA300 challenge dose. Figure 15a shows that the combination of anti-ClfA mAb and anti-AT mAb resulted in a significant increase in the response relative to survival within 2 weeks post-infection compared to controls and compared to either anti-ClfA mAb or anti-AT mAb alone.

Figures 16 and 17 further demonstrate the ability of the combination of anti-ClfA mAb 11H10 and anti-At mAb LC10 to increase animal survival as studied in an IV challenge model using a challenge dose of HA-MRSA USA100 (Figure 16) and a challenge dose of HA-MSSA USA200 (Figure 17).

Example 11 - Efficacy of the combination of anti-IsdH mAb 2F4 and anti-AT Ab LC10 in a lethal IV challenge model

The experiment was performed as described above in Example 10.

The efficacy of the combination of anti-IsdH mAb 2F4 and anti-AT Ab LC10 was tested in a lethal IV challenge model. As shown in Figure 18, the combination of anti-IsdH mAb 2F4 and anti-AT Ab LC10 increased animal survival as studied in an IV challenge model using a challenge dose of HA-MRSA USA100. Figure 18 shows that the combination resulted in a significant increase in survival response within 6 days after infection compared to the R347 control.

Sequence Listing

Table 1: VL CDR sequences of mAbs 2A3.1, 10A7.5, 12B8.19 and 25E9.1

SEQ ID NO: describe sequence SEQ ID NO: 1 VL CDR1 RASQSISSWLA SEQ ID NO: 2 VL CDR2 KASSLES SEQ ID NO: 3 VL CDR3 QQYNSYWT

Table 2: VL CDR sequences of mAB 28F6.1

SEQ ID NO: describe sequence SEQ ID NO: 4 mAb 28F6.1 VL CDR1 RASQGIRNDLG SEQ ID NO: 5 mAb 28F6.1 VL CDR2 DASSLQS

SEQ ID NO: 6 mAb 28F6.1 VL CDR3 LQDYNYPWT

Table 3: VH CDR sequences of mAb 2A3.1

SEQ ID NO: describe sequence SEQ ID NO: 7 VH CDR1 SYDMH SEQ ID NO: 8 VH CDR2 GIGTAGDTYYPGSVKG SEQ ID NO: 9 VH CDR3 DNYSSTGGYYGMDV

Table 4: VH CDR sequences of mAbs 10A7.5 and 12B8.19

SEQ ID NO: describe sequence SEQ ID NO: 10 VH CDR1 RYDMH SEQ ID NO: 11 VH CDR2 VIGTDGDTYYPGSVKG SEQ ID NO: 12 VH CDR3 DRYSSSNHYNGMDV

Table 5: VH CDR sequences of mAb 28F6.1

SEQ ID NO: describe sequence SEQ ID NO: 13 mAb 28F6.1 VH CDR1 SYAMT SEQ ID NO: 14 mAb 28F6.1 VH CDR2 VISGSGGSTYYADSVKG SEQ ID NO: 15 mAb 28F6.1 VH CDR3 DGRQVEDYYYYYGMDV

Table 6: VH CDR sequences of mAb 25E9.1

SEQ ID NO: describe sequence SEQ ID NO: 7 mAb 25E9.1 VH CDR1 SYDMH SEQ ID NO: 17 mAb 25E9.1 VH CDR2 VIDTAGDTYYPGSVKG SEQ ID NO: 18 mAb 25E9.1 VH CDR3 DRYSGNFHYNGMDV

Table 7: VL and VH amino acid sequences of anti-α toxin mAbs

Table 8: VL and VH nucleotide sequences of anti-alpha toxin mAbs

Table 9: Summary of alpha toxin VL and VH CDRs

describe SEQ ID NO VL CDR 1 1, 4

describe SEQ ID NO VL CDR 2 2, 5, 73, 77 VL CDR 3 3, 6, 64, 68, 74 VH CDR 1 7, 10, 13, 69 VH CDR 2 8, 11, 14, 17, 70, 75 VH CDR 3 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76, 78

Table 10: VL and VH amino acid sequences of anti-alpha toxin mAbs with Fc variant regions

Table 11: Alpha toxin amino acid sequence

Table 12 - Representative amino acid sequences of antibodies that specifically bind to the Staphylococcus aureus surface antigen IsdH

Table 13: Nucleotide sequences encoding the VH and VL amino acid sequences of mAbs against the surface antigens IsdH and ClfA of Staphylococcus aureus

Table 14 - Representative amino acid sequences of antibodies that specifically bind to the surface antigen ClfA of Staphylococcus aureus

The foregoing examples and tables are intended to illustrate but in no way limit the present disclosure. Other embodiments of the disclosed apparatus and methods will be apparent to those skilled in the art from consideration of the specification and practice of the apparatus and methods disclosed herein.

The following are embodiments of the present invention:

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to an IsdH surface determinant antigen of Staphylococcus aureus (S. aureus),

wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VH), each of which comprises three complementarity determining regions (CDR1, CDR2, and CDR3)

And among them:

a. VH CDR1 is identical to the amino acid sequence of SEQ ID NO: 90, 96, 102, 108, or 114, or comprises 1, 2, or 3 amino acid residue mutations relative thereto;

b. VH CDR2 is identical to the amino acid sequence of SEQ ID NO: 91, 97, 103, 109, or 115, or comprises 1, 2, or 3 amino acid residue mutations relative thereto; and

c. VH CDR3 is identical to the amino acid sequence of SEQ ID NO: 92, 98, 104, 110, or 116, or comprises 1, 2, or 3 amino acid residue mutations relative thereto;

and/or

d. VL CDR1 is identical to the amino acid sequence of SEQ ID NO: 93, 99, 105, 111, or 117, or comprises 1, 2, or 3 amino acid residue mutations relative thereto;

e. VL CDR2 is identical to the amino acid sequence of SEQ ID NO: 94, 100, 106, 112, or 118, or comprises 1, 2, or 3 amino acid residue mutations relative thereto; and

f. VL CDR3 is identical to the amino acid sequence of SEQ ID NO: 95, 101, 107, 113, or 119, or comprises 1, 2, or 3 amino acid residue mutations relative thereto.

2. The isolated antibody or antigen-binding fragment thereof of embodiment 1, wherein the isolated antibody or antigen-binding fragment thereof comprises:

a. a VH CDR1 that is identical to the amino acid sequence of SEQ ID NO: 90, 96, 102, 108, or 114, or comprises 1, 2, or 3 amino acid residue mutations relative thereto;

b. a VH CDR2 identical to the amino acid sequence of SEQ ID NO: 91, 97, 103, 109, or 115, or comprising 1, 2, or 3 amino acid residue mutations therein;

c. a VH CDR3 identical to the amino acid sequence of SEQ ID NO: 92, 98, 104, 110, or 116, or comprising 1, 2, or 3 amino acid residue mutations therein;

d. a VL CDR1 identical to the amino acid sequence of SEQ ID NO: 93, 99, 105, 111, or 117, or comprising 1, 2, or 3 amino acid residue mutations relative thereto;

e. a VL CDR2 identical to the amino acid sequence of SEQ ID NO: 94, 100, 106, 112, or 118, or comprising 1, 2, or 3 amino acid residue mutations therein; and

f. A VL CDR3 that is identical to the amino acid sequence of SEQ ID NO: 95, 101, 107, 113, or 119, or comprises 1, 2, or 3 amino acid residue mutations relative thereto.

3. The isolated antibody or antigen-binding fragment thereof of embodiment 1 or 2, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 correspond to a set of amino acid sequences selected from the group consisting of SEQ ID NOs: 90, 91, 92, 93, 94, and 95; SEQ ID NOs: 96, 97, 98, 99, 100, and 101; SEQ ID NOs: 102, 103, 104, 105, 106, and 107; SEQ ID NOs: 108, 109, 110, 111, 112, and 113; and SEQ ID NOs: 114, 115, 116, 117, 118, and 119.

4. An isolated antibody or antigen-binding fragment thereof that specifically binds to a surface determinant antigen of Staphylococcus aureus (S. aureus) IsdH, wherein the VH amino acid sequence is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 80, 82, 84, 86, or 88.

5. The isolated antibody or antigen-binding fragment thereof of embodiment 4, wherein the VH amino acid sequence is identical to the amino acid sequence of SEQ ID NO: 80, 82, 84, 86, or 88, or comprises 1 to 10 amino acid residue mutations relative thereto.

6. An isolated antibody or antigen-binding fragment thereof that specifically binds to a surface determinant antigen of Staphylococcus aureus (S. aureus), wherein the VL amino acid sequence is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 81, 83, 85, 87, or 89.

7. The isolated antibody or antigen-binding fragment thereof of embodiment 6, wherein the VL amino acid sequence is identical to the amino acid sequence of SEQ ID NO: 81, 83, 85, 87, or 89, or comprises 1 to 10 amino acid residue mutations relative thereto.

8. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-7, wherein the VH amino acid sequence is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 80, 82, 84, 86, or 88 and the VL amino acid sequence is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 81, 83, 85, 87, or 89.

9. The isolated antibody or antigen-binding fragment thereof of embodiment 8, wherein the VH amino acid sequence corresponds to the amino acid sequence of SEQ ID NO: 80, 82, 84, 86, or 88 and the VL amino acid sequence corresponds to the amino acid sequence of SEQ ID NO: 81, 83, 85, 87, or 89.

10. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-8, wherein the VH and VL are selected from the group consisting of SEQ ID NOs: 80 and 81; SEQ ID NOs: 82 and 83; SEQ ID NOs: 84 and 85; SEQ ID NOs: 86 and 87; and SEQ ID NOs: 88 and 89.

11. The isolated antibody or fragment of any one of embodiments 1-3, wherein the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3 correspond to a set of amino acid sequences selected from the group consisting of SEQ ID NOs: 90, 91, 92, 93, 94, and 95.

12. The isolated antibody or antigen-binding fragment thereof of embodiment 1 or 2, wherein the VH amino acid sequence comprises four VH framework regions (FR1, FR2, FR3, FR4), and wherein the four VH framework regions have an amino acid sequence that is at least about 80%, 85%, 90%, 95% or 100% identical to the corresponding amino acid sequences of the four VH framework regions in SEQ ID NO: 80, 82, 84, 86, or 88.

13. The isolated antibody or antigen-binding fragment thereof of embodiment 1 or 2, wherein the VL amino acid sequence comprises four VL framework regions (FR1, FR2, FR3, FR4), and wherein the four VL framework regions have an amino acid sequence that is at least about 80%, 85%, 90%, 95% or 100% identical to the corresponding amino acid sequences of the four VL framework regions in SEQ ID NO: 81, 83, 85, 87, or 89.

14. The isolated antibody or antigen-binding fragment thereof of embodiment 1 or 2, wherein the four VH framework regions have amino acid sequences that are at least about 80%, 85%, 90%, 95% or 100% identical to the corresponding amino acid sequences of four VH framework regions in SEQ ID NO: 80, 82, 84, 86, or 88, and wherein the four VL framework regions have amino acid sequences that are at least about 80%, 85%, 90%, 95% or 100% identical to the corresponding amino acid sequences of four VL framework regions in SEQ ID NO: 81, 83, 85, 87, or 89.

15. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-14, wherein the VH and VL correspond to SEQ ID NOs 80 and 81.

16. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-15, wherein the antibody or antigen-binding fragment thereof has at least one of the following:

a. The dissociation constant (KD) for Staphylococcus aureus surface antigen is about 70 nM or less;

b. the ability of S. aureus to evade opsonophagocytosis by at least 50% as measured by an opsonophagocytic killing assay; or

c. Ability to reduce the number of S. aureus colony forming units (CFU) by at least 50%, as measured by a bacteremia model.

17. An isolated antibody or antigen-binding fragment thereof that specifically binds to a Staphylococcus aureus (S. aureus) IsdH surface determinant antigen, and wherein the antibody or antigen-binding fragment thereof has at least one of the following:

a. The dissociation constant (KD) for Staphylococcus aureus surface antigen is about 70 nM or less;

b. the ability of S. aureus to evade opsonophagocytosis by at least 50% as measured by an opsonophagocytic killing assay;

c. The ability to reduce the number of Staphylococcus aureus colony-forming units (CFU) by at least 50%, as measured by a bacteremia model;

d. Ability to reduce immune cell infiltration, bacterial load, and pro-inflammatory cytokine release as measured in animal organ burden models.

18. The isolated antibody or antigen-binding fragment thereof of embodiment 17, having the amino acid sequence of the antibody or antigen-binding fragment thereof of any one of embodiments 1-16.

19. A composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1-18 and a pharmaceutically acceptable excipient.

20. An isolated nucleic acid encoding the amino acid sequence of any one of embodiments 1-15.

21. An isolated antibody or antigen-binding fragment thereof that specifically binds to a surface determinant antigen of Staphylococcus aureus (S. aureus) ClfA.

wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VH), each of which comprises three complementarity determining regions (CDR1, CDR2, and CDR3)

And among them:

a. VH CDR1 is identical to the amino acid sequence of SEQ ID NO: 133 or 141, or comprises 1, 2, or 3 amino acid residue mutations relative thereto;

b. VH CDR2 is identical to the amino acid sequence of SEQ ID NO: 134 or 142, or comprises 1, 2, or 3 amino acid residue mutations relative thereto; and

c. VH CDR3 is identical to the amino acid sequence of SEQ ID NO: 135 or 143, or comprises 1, 2, or 3 amino acid residue mutations relative thereto;

and/or

d. VL CDR1 is identical to the amino acid sequence of SEQ ID NO: 137 or 145, or comprises 1, 2, or 3 amino acid residue mutations relative thereto;

e. VL CDR2 is identical to the amino acid sequence of SEQ ID NO: 138 or 146, or comprises 1, 2, or 3 amino acid residue mutations relative thereto; and

f. VL CDR3 is identical to the amino acid sequence of SEQ ID NO: 139 or 147, or comprises 1, 2, or 3 amino acid residue mutations relative thereto.

22. The isolated antibody or antigen-binding fragment thereof of embodiment 21, wherein the isolated antibody or antigen-binding fragment thereof comprises:

a. VH CDR1 identical to the amino acid sequence of SEQ ID NO: 133 or 141, or comprising 1, 2, or 3 amino acid residue mutations relative thereto

b. a VH CDR2 identical to the amino acid sequence of SEQ ID NO: 134 or 142, or comprising 1, 2, or 3 amino acid residue mutations relative thereto;

c. a VH CDR3 having an amino acid sequence identical to that of SEQ ID NO: 135 or 143, or comprising 1, 2, or 3 amino acid residue mutations relative thereto;

d. a VL CDR1 identical to the amino acid sequence of SEQ ID NO: 137 or 145, or comprising 1, 2, or 3 amino acid residue mutations relative thereto;

e. a VL CDR2 identical to the amino acid sequence of SEQ ID NO: 138 or 146, or comprising 1, 2, or 3 amino acid residue mutations relative thereto; and

f. A VL CDR3 that is identical to the amino acid sequence of SEQ ID NO: 139 or 147, or comprises 1, 2, or 3 amino acid residue mutations relative thereto.

23. The isolated antibody or antigen-binding fragment thereof of embodiment 21 or 22, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 correspond to a set of amino acid sequences selected from the group consisting of SEQ ID NOs: 133, 134, 135, 137, 138, and 139; and SEQ ID NOs: 137, 138, 139, 145, 146, and 147.

24. An isolated antibody or antigen-binding fragment thereof that specifically binds to a surface determinant antigen of Staphylococcus aureus (S. aureus) CIfA, wherein the VH amino acid sequence is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 132 or 140.

25. The isolated antibody or antigen-binding fragment thereof of embodiment 24, wherein the VH amino acid sequence is identical to the amino acid sequence of SEQ ID NO: 132 or 140, or comprises 1 to 10 amino acid residue mutations relative thereto.

26. An isolated antibody or antigen-binding fragment thereof that specifically binds to a surface determinant antigen of Staphylococcus aureus (S. aureus) CIfA, wherein the VL amino acid sequence is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 136 or 144.

27. The isolated antibody or antigen-binding fragment thereof of embodiment 26, wherein the VL amino acid sequence is identical to the amino acid sequence of SEQ ID NO: 136 or 144, or comprises 1 to 10 amino acid residue mutations relative thereto.

28. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 21-27, wherein the VH amino acid sequence is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 132 or 140 and the VL amino acid sequence is at least 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 136 or 144.

29. The isolated antibody or antigen-binding fragment thereof of embodiment 28, wherein the VH amino acid sequence corresponds to the amino acid sequence of SEQ ID NO: 132 or 140; and the VL amino acid sequence corresponds to the amino acid sequence of SEQ ID NO: 136 or 144.

30. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 21-28, wherein the VH and VL are selected from the group consisting of SEQ ID NOs: 132 and 136; and SEQ ID NOs: 140 and 144.

31. The isolated antibody or fragment of any one of embodiments 21-23, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 correspond to a set of amino acid sequences selected from the group consisting of SEQ ID NOs: 141, 142, 143, 144, 145, 146, and 147.

32. The isolated antibody or antigen-binding fragment thereof of embodiment 21 or 22, wherein the VH amino acid sequence comprises four VH framework regions (FR1, FR2, FR3, FR4), and wherein the four VH framework regions have an amino acid sequence that is at least about 80%, 85%, 90%, 95% or 100% identical to the corresponding amino acid sequences of the four VH framework regions in SEQ ID NO: 132 or 140.

33. The isolated antibody or antigen-binding fragment thereof of embodiment 21 or 22, wherein the VL amino acid sequence comprises four VL framework regions (FR1, FR2, FR3, FR4), and wherein the four VL framework regions have an amino acid sequence that is at least about 80%, 85%, 90%, 95% or 100% identical to the corresponding amino acid sequences of the four VL framework regions in SEQ ID NO: 136 or 144.

34. The isolated antibody or antigen-binding fragment thereof of embodiment 21 or 22, wherein the four VH framework regions have an amino acid sequence that is at least about 80%, 85%, 90%, 95% or 100% identical to the corresponding amino acid sequences of the four VH framework regions in SEQ ID NO: 132 or 144, and wherein the four VL framework regions have an amino acid sequence that is at least about 80%, 85%, 90%, 95% or 100% identical to the corresponding amino acid sequences of the four VL framework regions in SEQ ID NO: 136 or 144.

35. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 21-34, wherein the VH and VL correspond to SEQ ID NOs 140 and 144.

36. A composition comprising the isolated antibody or antigen-binding fragment thereof as described in any one of embodiments 21-35 and a pharmaceutically acceptable excipient.

37. An isolated nucleic acid encoding the amino acid sequence of any one of embodiments 21-35.

38. A composition comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to Staphylococcus aureus alpha toxin (AT) and an isolated antibody or antigen-binding fragment thereof that specifically binds to a surface determinant antigen of Staphylococcus aureus.

39. The composition of embodiment 38, wherein the S. aureus surface determinant antigen is selected from the group consisting of SdrC, SdrD, SdrE, ClfA, ClfB, IsdA, IsdB, IsdC, IsdE, IsdH, SpA, FnbA, and PNAG.

40. The composition of embodiment 38 or 39, wherein the surface determinant antigen is IsdH.

41. The composition of embodiment 40, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds IsdH is the antibody or antigen-binding fragment thereof according to any one of embodiments 1-18.

42. The composition of embodiment 38 or 39, wherein the surface determinant antigen is CIfA.

43. The composition of embodiment 42, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds CIfA is the antibody or antigen-binding fragment thereof according to any one of embodiments 21-35.

44. The composition of any one of embodiments 38-43, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds to AT comprises:

a. VH CDR1 comprising an amino acid sequence identical to SEQ ID NO 7, 10, 13, or 69 or comprising 1, 2, or 3 amino acid residue mutations relative thereto;

b. VH CDR2 comprising an amino acid sequence identical to SEQ ID NO 8, 11, 14, 17, 70, or 75 or comprising 1, 2, or 3 amino acid residue mutations relative thereto; and

c. a VH CDR3 comprising an amino acid sequence identical to SEQ ID NO 9, 12, 15, 16, 18, 65, 66, 67, 71, 72, 76, or 78 or comprising 1, 2, or 3 amino acid residue mutations relative thereto;

and/or

d. VL CDR1, comprising the amino acid sequence of SEQ ID NO: 1 or 4;

e. VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73 or 77; and

f. VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68 or 74.

45. The composition of any one of embodiments 38-43, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds to AT comprises:

a. VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 10, 13, or 69;

b. VH CDR2 comprising the amino acid sequence of SEQ ID NO: 8, 11, 14, 17, 70 or 75;

c. a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 9, 12, 15, 18, 16, 65, 66, 67, 71, 72, 76, or 78;

d. VL CDR1, comprising the amino acid sequence of SEQ ID NO: 1 or 4;

e. VL CDR2 comprising the amino acid sequence of SEQ ID NO: 2, 5, 73 or 77; and

f. VL CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 6, 64, 68 or 74.

46. The composition of any of embodiments 38-45, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the isolated antibody or antigen-binding fragment thereof that specifically binds to AT correspond to SEQ ID NOs: 7, 8, 9, 1, 2, and 3; SEQ ID NOs: 10, 11, 12, 1, 2, and 3; SEQ ID NOs: 13, 14, 15, 4, 5, and 6; SEQ ID NOs: 7, 17, 18, 1, 2, and 3; SEQ ID NOs: 7, 8, 16, 1, 2, and 64; SEQ ID NOs: 7, 8, 65, 1, 2, and 64; SEQ ID NOs: 7, 8, 66, 1, 2, and 64; SEQ ID NOs: 7, 8, 67, 1, 2, and 68; SEQ ID NOs: 7, 8, 67, 1, 2, and 64; NO: 7, 8, 65, 1, 2 and 68; SEQ ID NO: 69, 70, 71, 1, 2 and 68; SEQ ID NO: 7, 8, 72, 1, 73 and 74; SEQ ID NO: 69, 75, 71, 1, 2 and 68; SEQ ID NO: 69, 75, 76, 1, 2 and 68; SEQ ID NO: 69, 75, 76, 1, 77 and 74; amino acid sequence of SEQ ID NO: 69, 70, 71, 1, 77 and 74.

47. The composition of any of embodiments 38-43, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds to AT comprises a heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and/or (iii) comprises a light chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 63.

48. The composition of any of embodiments 38-43, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds to AT comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 20, 22, 24, 26, 28, 41, 43, 45, 47, 49, 51, 53, 55, 57, 79, 59, 61, or 62 and (iii) comprises a light chain variable domain having the amino acid sequence of SEQ ID NO: 19, 21, 23, 25, 27, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 63.

49. The composition of any one of embodiments 38-48, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the isolated antibody or antigen-binding fragment thereof that specifically binds to AT correspond to the amino acid sequences of SEQ ID NOs: 69, 70, 71, 1, 2, and 68.

50. The composition of any of embodiments 38-49, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds to AT comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 57 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 58.

51. The composition of any of embodiments 38-50, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds to AT further comprises an Fc variant region.

52. The composition of embodiment 51, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds to AT comprises SEQ ID NO: 130 and SEQ ID NO: 131.

53. The composition of any one of embodiments 30 or 40-41, further comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to CIfA.

54. The composition of embodiment 53, wherein the isolated antibody or antigen-binding fragment thereof that specifically binds CIfA is the antibody or antigen-binding fragment thereof according to any one of embodiments 21-35.

55. A method of determining the presence of Staphylococcus aureus in a test sample, the method comprising contacting the test sample with the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-15 or 21-34 and a detectable label.

56. The method of embodiment 55, comprising:

a. contacting the test sample with the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to a surface determinant antigen of Staphylococcus aureus to form a first complex;

b. contacting the complex with the detectable label, wherein the detectable label is bound to the antibody or antigen-binding fragment thereof, or to the antigen, so as to form a second complex; and

c. detecting the presence of Staphylococcus aureus in the test sample based on the signal produced by the detectable label in the second complex, wherein the presence of Staphylococcus aureus is directly correlated with the signal produced by the detectable label.

57. The method of embodiment 55, comprising:

a. contacting the test sample with the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to a surface determinant antigen of Staphylococcus aureus to form a first complex;

b. contacting the complex with the detectable label, wherein the detectable label competes with the antigen for binding to the antibody or antigen-binding fragment thereof to form a second complex; and

c. detecting the presence of Staphylococcus aureus in the test sample based on the signal produced by the detectable label in the second complex, wherein the presence of Staphylococcus aureus is indirectly related to the signal produced by the detectable label.

58. The method of any one of embodiments 55-57, wherein the sample is a patient sample and the method further comprises diagnosing the patient with a Staphylococcus aureus infection.

59. The method of any one of embodiments 55-58, wherein the method is suitable for use in an automated system or a semi-automated system.

60. A method of determining the presence of Staphylococcus aureus in a test sample, the method comprising contacting the test sample with the composition of embodiment 19 or 36 and at least one detectable label.

61. The method of embodiment 60, comprising:

a. contacting the test sample with a composition as described in Example 13, wherein the composition binds to a surface determinant antigen of Staphylococcus aureus and a toxin polypeptide secreted by Staphylococcus aureus;

b. contacting the test sample with the at least one detectable label, wherein the at least one detectable label binds to the surface determinant antigen, the secreted toxin, or an antibody or antigen-binding fragment thereof that binds to the surface determinant antigen or the secreted toxin; and

c. detecting the presence of Staphylococcus aureus in the test sample based on the signal produced by the at least one detectable label, wherein the presence of Staphylococcus aureus is directly correlated with the signal produced by the at least one detectable label.

62. The method of embodiment 60, comprising:

a. contacting the test sample with a composition as described in Example 13, wherein the composition binds to a surface determinant antigen of Staphylococcus aureus and a toxin polypeptide secreted by Staphylococcus aureus;

b. contacting the test sample with the at least one detectable label, wherein the at least one detectable label competes with the surface determinant antigen or the secreted toxin for binding to the antibody in the composition of embodiment 13; and

c. detecting the presence of Staphylococcus aureus in the test sample based on the signal produced by the at least one detectable label, wherein the presence of Staphylococcus aureus is indirectly correlated with the signal produced by the at least one detectable label.

63. The method of any one of embodiments 60-62, wherein the sample is a patient sample and the method further comprises diagnosing the patient with a Staphylococcus aureus infection.

64. The method of any one of embodiments 60-63, wherein the method is suitable for use in an automated system or a semi-automated system.

65. A method of treating a Staphylococcus aureus infection in a patient, the method comprising administering to the patient the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-18 or 21-35, or the composition of any one of embodiments 19, 36, or 38-54.

66. A method for preventing or reducing the severity of Staphylococcus aureus-associated sepsis in a patient, the method comprising administering to the patient the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-18 or 21-35, or the composition of any one of embodiments 19, 36, or 38-54.

67. A method of delaying the onset of sepsis associated with a Staphylococcus aureus infection in a patient, the method comprising administering to the patient the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-18 or 21-35, or the composition of any one of embodiments 19, 36, or 38-54.

68. A method of preventing the onset of sepsis associated with a Staphylococcus aureus infection in a patient, the method comprising administering to the patient the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-18 or 21-35, or the composition of any one of embodiments 19, 36, or 38-54.

69. A method of reducing the Staphylococcus aureus bacterial load in the bloodstream or heart of a patient, the method comprising administering to the patient an effective amount of the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-18.

70. A method of reducing Staphylococcus aureus bacterial agglutination and/or thromboembolic lesion formation in a patient, the method comprising administering to the patient an effective amount of the isolated antibody or antigen-binding fragment thereof of any one of Examples 1-18.

71. The method of any one of embodiments 65-70, further comprising administering an antibiotic to the patient.

72. The method of embodiment 69, wherein the antibiotic is penicillin, oxacillin, flucloxacillin, or vancomycin.

Claims (6)

1. An isolated antibody or its antigen-binding fragment that specifically binds to the surface determinant antigen of Staphylococcus aureus (S. aureus) IsdH, wherein the antigen-binding fragment is selected from Fab, Fab’, F(ab’)2, Fd and Fv fragments; The isolated antibody or its antigen-binding fragment includes a heavy chain variable region (VH) and a light chain variable region (VH), each of which includes three complementarity-determining regions (CDR1, CDR2 and CDR3). And among them: a. VH CDR1 consists of the amino acid sequence of SEQ ID NO: 90; b. VH CDR2 consists of the amino acid sequence of SEQ ID NO: 91; c.VH CDR3 consists of the amino acid sequence of SEQ ID NO: 92; d.VL CDR1 consists of the amino acid sequence of SEQ ID NO: 93; e.VL CDR2 consists of the amino acid sequence of SEQ ID NO: 94; and f.VL CDR3 consists of the amino acid sequence of SEQ ID NO: 95. 2. The isolated antibody or its antigen-binding fragment as claimed in claim 1, wherein the amino acid sequence of VH is as shown in SEQ ID NO: 80, and the amino acid sequence of VL is as shown in SEQ ID NO: 81. 3. A composition comprising the isolated antibody or antigen-binding fragment thereof as described in claim 1 or 2 and a pharmaceutically acceptable excipient. 4. A composition comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to Staphylococcus aureus α-toxin (AT) and an isolated antibody or antigen-binding fragment thereof that specifically binds to Staphylococcus aureus surface determinant antigen, wherein the Staphylococcus aureus surface determinant antigen is IsdH, and the isolated antibody or antigen-binding fragment thereof that specifically binds to IsdH is the antibody or antigen-binding fragment thereof as described in claim 1 or 2. 5. The use of the isolated antibody or its antigen-binding fragment as described in claim 1 or 2, or the composition as described in claim 3 or 4, in the preparation of a product for treating patients with Staphylococcus aureus infection, preventing patients with Staphylococcus aureus-associated sepsis or reducing its severity, and/or delaying the onset of patients with Staphylococcus aureus-associated sepsis. 6. The use of the isolated antibody or its antigen-binding fragment as described in claim 1 or 2 in the preparation of a product that reduces Staphylococcus aureus bacterial aggregation and/or thromboembolic lesion formation in patients.