Composition for preventing or treating novel staphylococcal infectious diseases

Abstract

The present invention relates to a composition for preventing or treating staphylococcal infections and thrombotic diseases caused by staphylococcal infections. The present invention utilizes the cross-reactivity of antibodies against staphylococcal toxins to comprehensively inhibit cell lysis caused by 11 staphylococcal toxins with only a minimal combination of antigens. The present invention also effectively controls blood coagulation in infected subjects by inducing opsonophagocytosis, making it useful as an efficient therapeutic composition that can comprehensively eliminate or alleviate the overall pathological conditions caused by staphylococcal infection, rather than simply alleviating individual symptoms.

Description

Technical Field

[0001] The present invention relates to a composition for preventing or treating staphylococcal infectious diseases, which contains a specific combination of staphylococcal toxins as an active ingredient.

Background Art

[0002] Staphylococcus aureus is a Gram-positive bacterium that causes severe infections in human skin, soft tissues, and bloodstream, and can be transformed into a methicillin-resistant strain (methicillin-resistant S. aureus; MRSA) that is resistant to the beta-lactam antibiotic methicillin through various routes. Such MRSA infections are difficult to treat, have a poor prognosis, and induce a large social cost.

[0003] The two-component leukocidin (BCL) of S. aureus is an important virulence factor belonging to the pore-forming toxin (PFT) family. BCL has two subunits: an S-component (LukS-PV, LukE, HlgA, HlgC, and LukA that migrate slowly on a chromatography column) that targets host cells, and a polymerized F-component (LukF-PV, LukD, HlgB, and LukB that migrate rapidly on a chromatography column). In contrast, LukAB is secreted in a soluble heterodimeric form that has already bound, and S. aureus α-toxin (Hla) is a single component that forms a β-barrel PFT.

[0004] S. aureus BCL toxin and Hla induce lysis of host cells such as neutrophils, monocytes / macrophages, and red blood cells (RBCs) by binding to receptors specifically expressed on the surface of host cells. LukSF-PV and HlgCB use C5aR1 and C5aR2, LukED uses CCR5, CXCR1, and CXCR2, and HlgAB and LukED both use CXCR1 and CXCR2 as receptors, but may also use CCR2 and the DARC (Duffy antigen receptor for chemokines) receptor. On the other hand, LukAB binds to CD11b. Hla recognizes the ADAM10 protein expressed on the cell membranes of RBCs, epithelial cells, endothelial cells, and immune cells such as neutrophils, monocytes / macrophages, and T cells.

[0005] To date, all human clinical trials of vaccines developed for the purpose of preventing invasive S. aureus infections have failed. Although most vaccines generated high-titer opsonizing antibodies against surface antigens of S. aureus, they ultimately could not be completed as effective vaccines, which is due to an incomplete understanding of host immune mechanisms and the S. aureus infection mechanism and the absence of means to enable sustainable long-term immune induction against S. aureus in humans.

[0006] Recently, two new approaches have been made for S. aureus vaccine development. One is to use surface antigens of S. aureus to induce opsonophagocytosis via antibodies generated by immunization. The generated antibodies were expected to bind to the bacterial surface and kill it, but these opsonin antibody-based vaccine candidates could not demonstrate efficacy in clinical trials, and some actually showed harmful results when S. aureus infections occurred (Fowler VG, et al., 2013, Jama 309:1368-78). The other is to use S. aureus toxoids as vaccine candidates, and such a strategy is to induce neutralizing antibodies by immunization with multiple toxoid antigens.

[0007] It has been reported that 11 components, including 5 S. aureus BCLs and Hla (α-toxin), specifically recognize 5 different receptor families expressed in host cells and RBCs, namely chemokine receptors, complement receptors, CD11b receptors, DARC receptors, and ADAM10 receptors (Wilke GA et al., 2010, Proc Natl Acad Sci USA 107:13473-8). Immunization with the active toxin components is difficult to use as a vaccine due to these toxicities, and there are also manufacturing difficulties in immunizing all 11 toxoid proteins.

[0008] Therefore, the present inventors attempted to discover the most efficient combination of toxin candidate substances for broadly blocking 11 toxin-mediated cytotoxicities with antibodies generated by immunization. At the same time, the present inventors explored protein fragments and their combinations that best induce opsonophagocytosis and can most effectively control blood coagulation in the infected body, in order to break out of the fragmentary improvement of individual symptoms caused by staphylococcal infection and develop a substantial preventive vaccine or therapeutic agent that can comprehensively remove or alleviate the overall pathological condition caused by the infection.

[0009] Numerous papers and patent documents are referenced throughout this specification and their citations are represented. The disclosures of the cited papers and patent documents are hereby incorporated by reference in their entirety to more clearly explain the level of the technical field to which the present invention pertains and the content of the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0010] The inventors have made intensive research efforts to develop a prophylactic or therapeutic agent composition for staphylococcal infectious diseases that can significantly neutralize the cytotoxicity caused by staphylococcal infection and efficiently block the lysis of host cells. For this purpose, the inventors examined the cross-reactivity of antibodies against each toxin derived from Staphylococcus aureus, and searched for an optimal combination that exhibits the broadest neutralizing activity based on this. As a result, when combining three or more of the single toxins of Hla, LukS, LukAB, and HlgA, more specifically, when combining all four toxins, all staphylococcal toxin-mediated cell lysis activities generated in the subject by inoculating these are evenly blocked by each antibody generated in the subject, and it was found that the hemolytic action can be significantly suppressed, thereby completing the present invention.

[0011] Accordingly, an object of the present invention is to provide a composition for preventing or treating staphylococcal infectious diseases. Another object of the present invention is to provide a composition for preventing or treating thrombotic disorders caused by staphylococcal infection. Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

Means for Solving the Problems

[0012] According to one aspect of the present invention, the present invention provides a prophylactic or therapeutic composition for staphylococcal infectious diseases containing, as an active ingredient, three or more staphylococcal-derived toxins selected from the group consisting of Hla (alpha-hemolysin), LukS (Leukocidal toxin S), LukAB (Leukocidal toxin AB), and HlgA (gamma-hemolysin); an antibody or an antigen-binding fragment thereof that specifically recognizes the toxin; or a nucleotide encoding the toxin.

[0013] The inventors have made intensive research efforts to develop a prophylactic or therapeutic agent composition for staphylococcal infectious diseases that can significantly neutralize the cytotoxicity caused by staphylococcal infection and efficiently block the lysis of host cells. For this purpose, the inventors examined the cross-reactivity of antibodies against each toxin derived from Staphylococcus, and searched for an optimal combination that exhibits the broadest neutralizing activity. As a result, when combining three or more toxins of Hla, LukS, LukAB, and Hlg, more specifically, when combining all four toxins, the 11 toxin-mediated cell lysis activities of Staphylococcus are evenly blocked by each antibody generated in the subject by inoculating these, and it was found that the hemolytic action in the blood can be significantly suppressed.

[0014] The present invention may be in the form of a vaccine that administers each toxin protein or a nucleotide encoding the same to a subject to form an antibody against each toxin in the subject, or may be used in the form of an antibody therapeutic agent containing a separated and purified antibody against each toxin as a pharmacological component. Therefore, in the former case, the term "composition for preventing or treating staphylococcal infectious diseases" has the same meaning as "staphylococcal vaccine composition".

[0015] In the present specification, the term "antibody" means an immunoglobulin protein that specifically recognizes an antigen by including one or more variable domains that bind to an epitope of the antigen and are generated by the mammalian immune system. As an antibody that specifically recognizes each staphylococcal toxin used in the present invention, all polyclonal or monoclonal antibodies can be used, and specifically, a polyclonal antibody having cross-reactivity can be used.

[0016] The antibodies of the present invention can be produced by methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), the recombinant DNA method (U.S. Patent No. 4,816,567), or the phage antibody library method (Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:581-597 (1991)). General procedures for antibody production are described in detail in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; and Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984.

[0017] As used herein, the term "antigen binding fragment" means a portion of a polypeptide within the overall immunoglobulin structure to which an antigen can bind, including, but not limited to, F(ab’)2, Fab’, Fab, Fv, and scFv.

[0018] As used herein, the term "specifically binding" has the same meaning as "specifically recognizing", and means that an antigen and an antibody (or a fragment thereof) specifically interact through an immunological reaction.

[0019] The present invention may also be used in the form of a DNA vaccine or an mRNA vaccine containing, as an active ingredient, nucleotides encoding the amino acids of the four toxins described above.

[0020] As used herein, the term "nucleotide" refers to deoxyribonucleotides or ribonucleotides that exist in single-stranded or double-stranded forms, and includes analogs of natural nucleotides unless otherwise specifically mentioned (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)). When the present invention is used in the form of a DNA vaccine or an mRNA vaccine, the three or four toxin genes of the present invention may be included in respective gene delivery systems, or a plurality of toxin antigen genes may be simultaneously inserted into one gene delivery system and expressed in a subject.

[0021] As used herein, the term "express" means that a gene becomes replicable as an episomal factor in a subject cell or by completion of integration into a chromosome by artificially introducing it using a gene delivery system in order for the subject to express an exogenous gene or increase the natural expression level of an endogenous gene. Therefore, the term "expression" has the same meaning as "transformation", "transfection" or "transduction".

[0022] As used herein, the term "gene delivery system" means a mediator for introducing and expressing a desired target gene into a target cell. An ideal gene delivery system must be harmless to the human body, easy to mass-produce, and capable of efficiently delivering genes.

[0023] As used herein, the term "gene delivery" means that a gene is transported into a cell and has the same meaning as intracellular transduction of a gene. At the tissue level, gene delivery in the above terms has the same meaning as gene spread. Therefore, the gene delivery system of the present invention may be described as a gene transduction system and a gene spread system.

[0024] For the production of the gene delivery agent of the present invention, the nucleotide sequence of the present invention is present within a suitable expression construct and is preferably operably linked to expression regulatory sequences (e.g., promoters, signal sequences, arrays of transcription factor binding sites). As used herein, the term "operably linked" means a functional linkage between a nucleic acid expression regulatory sequence and another nucleic acid sequence, whereby the regulatory sequence regulates the transcription and / or translation of the other nucleic acid sequence.

[0025] The gene delivery agent of the present invention can be prepared in various forms, which can be prepared in the form of (i) a naked recombinant DNA molecule, (ii) a plasmid, (iii) a viral vector, (iv) a liposome or niosome encapsulating the naked recombinant DNA molecule or plasmid, or (v) a liposome encapsulating mRNA.

[0026] According to a specific embodiment of the present invention, the Hla is a variant in which the 35th amino acid residue in the amino acid sequence of the Hla (alpha-hemolysin) protein is substituted. More specifically, the 35th amino acid His residue is substituted with Leu.

[0027] According to a specific embodiment of the present invention, the LukAB is a variant in which the 323rd Glu amino acid residue in the amino acid sequence of the LukAB (Leukocidin AB) protein is substituted. More specifically, the 323rd amino acid Glu residue is substituted with Ala.

[0028] As used herein, the term "prevention" means suppressing the occurrence of a disease or illness in a subject who has not been diagnosed as having the disease or illness but is at risk of developing such a disease or illness.

[0029] As used herein, the term "treatment" means (a) inhibiting the development of a disease, illness or symptom; (b) alleviating a disease, illness or symptom; or (c) eliminating a disease, illness or symptom. Administration of the composition of the present invention to a subject can play a role in inhibiting, eliminating or alleviating the development of symptoms caused by staphylococcal infection by efficiently inhibiting the broad range of cytolytic activities mediated by 11 toxins derived from staphylococci. Therefore, the composition of the present invention may itself be a composition for treating these diseases, or may be administered together with other pharmacological components and applied as an adjuvant for treating the diseases. For this reason, as used herein, the term "treatment" or "therapeutic agent" includes the meaning of "adjuvant treatment" or "adjuvant therapeutic agent".

[0030] As used herein, the terms "administer" or "administering" refer to directly administering a therapeutically effective amount of the composition of the present invention to a subject such that the same amount is formed in the subject's body.

[0031] In the present invention, the term "therapeutically effective amount" means the content of the composition in which the pharmacological component in the composition is contained to such an extent that it provides a therapeutic or prophylactic effect to the individual to whom the pharmaceutical composition of the present invention is to be administered, and thus includes the meaning of "prophylactically effective amount".

[0032] As used herein, the term "subject" includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or macaques. Specifically, the subject of the present invention is a human.

[0033] According to a specific embodiment of the present invention, the staphylococcus may be methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA) or pathogenic staphylococcus, and more specifically, methicillin-resistant Staphylococcus aureus.

[0034] Examples of staphylococcal infections include, but are not limited to, soft tissue infections, septic arthritis, osteomyelitis, otitis media, pneumonia, sepsis, acute respiratory tract infection, infections due to catheter use, postoperative wound infections, bacteremia, endocarditis, and food poisoning.

[0035] According to a specific embodiment of the present invention, the composition of the present invention may additionally contain, as an active ingredient, a glycopeptide antibiotic in addition to the aforementioned staphylococcal-derived toxin.

[0036] As used herein, the term "glycopeptide antibiotic" means a hydrophilic antibiotic having a molecular weight of about 1,400 Da or more and having a glycopeptide core which is a fused ring structure to which monosaccharides are bound. Glycopeptide antibiotics inhibit the synthesis of bacterial peptidoglycan and include, for example, Ramoplanin, dalbavancin, oritavancin, telavancin, vancomycin, and teicoplanin, but are not limited thereto, and any glycopeptide antibiotic known in the art can be used.

[0037] Specifically, the glycopeptide antibiotic used in the present invention is selected from the group consisting of vancomycin, teicoplanin, and combinations thereof.

[0038] As shown in the examples described below, when the combination of toxins discovered in the present invention or a separated and purified antibody against these toxins is administered in combination with a glycopeptide antibiotic such as vancomycin or teicoplanin, it was confirmed that the residual bacteria in the kidney can be completely removed, the survival rate of infected individuals is further improved, and a multifaceted and synergistic protective effect on patients can be exerted.

[0039] Combined administration is carried out such that all of the toxin of the present invention and the glycopeptide antibiotic are contained in one dosage form, or separate dosage forms each containing the toxin and the glycopeptide antibiotic are administered simultaneously, or are administered sequentially with an appropriate time difference in any order. When administered sequentially, for example, the toxin of the present invention or a separated and purified antibody thereto is administered first, followed by administration of the glycopeptide antibiotic.

[0040] According to another aspect of the present invention, the present invention provides a method for preventing or treating a staphylococcal infection disease, which includes the step of administering the composition of the present invention described above to a subject.

[0041] According to still another aspect of the present invention, the present invention provides a composition for preventing or treating a staphylococcal infection disease, which contains as an active ingredient one or more proteins selected from the group consisting of ClfA (Clumping factor A), FnbpA (fibrinectin-binding protein A), FnbpB (fibrinectin-binding protein B), and functional portions thereof; an antibody or an antigen-binding fragment thereof that specifically recognizes the protein; or a nucleotide encoding the protein.

[0042] Since the antibody or its antigen-binding fragment, nucleotide, and the composition of the present invention used in the present invention for preventing or treating the staphylococcal infection disease have already been described above, the description thereof is omitted to avoid excessive duplication.

[0043] The present inventors have also diligently conducted research efforts to explore a composition that can prevent or treat staphylococcal infectious diseases or improve the therapeutic sensitivity of staphylococcal therapeutic compositions such as vaccines by disabling the host immune system evasion system specific to Staphylococcus. As a result, when administering to a subject one or more combinations selected from the group consisting of ClfA, FnbpA, FnbpB belonging to MSCRAMM (microbial surface components recognizing adhesive matrix molecules) and functional portions of each of these proteins, it was found that the protein competitively binds to Factor H (human complement factor H, FH) in host serum instead of MSCRAMM on the surface of live bacteria, thereby blocking the hydrolysis of complement C3b to iC3b and promoting opsonophagocytosis against bacteria by the maintained active C3b.

[0044] As used herein, the term "functional portion" includes any length of partial segment that maintains the biological activity of the ClfA, FnbpA, and FnbpB proteins. Therefore, the functional portion of the ClfA, FnbpA, and FnbpB proteins means a partial segment of each protein that can specifically interact with FH in serum or function as an antigen that can induce the production of antibodies that can specifically bind to MSCRAMM on the surface of live bacteria.

[0045] According to a specific embodiment of the present invention, the functional portion is a segment containing the N2-N3 domain of the protein, and more specifically, the N2-N3 domain of the protein (ClfAN2N3, FnbpAN2N3, and FnbpBN2N3). The interaction between ClfAN2N3, FnbpAN2N3, FnbpBN2N3 and FH is known, and the present inventors have for the first time elucidated that these segments can competitively bind to FH and the antibodies against them can competitively bind to MSCRAMM on the surface of live bacteria, thereby efficiently blocking the interaction between FH in host serum and MSCRAMM on the bacterial surface.

[0046] According to a specific embodiment of the present invention, the composition of the present invention includes some sections containing the N2-N3 domain of ClfA and some sections containing the N2-N3 domain of FnbpB; an antibody or an antigen-binding fragment thereof that specifically recognizes the sections; or a nucleotide encoding the sections as an active ingredient.

[0047] When the composition of the present invention is manufactured into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.

[0048] The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention is one commonly used in formulation, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention can additionally include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington’s Pharmaceutical Sciences (19th ed., 1995).

[0049] The pharmaceutical composition of the present invention can be administered orally or parenterally, specifically, it may be administered intravenously, subcutaneously, or intraperitoneally.

[0050] The suitable dosage of the pharmaceutical composition of the present invention can be variously formulated depending on factors such as the formulation method, administration method, patient's age, weight, gender, pathological condition, diet, administration time, administration route, excretion rate, and reactivity. The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 - 100 mg / kg based on an adult.

[0051] The pharmaceutical composition of the present invention can be manufactured in unit dosage form or in a multi-dose container by formulating it with a pharmaceutically acceptable carrier and / or excipient by a method that can be easily implemented by a person having ordinary knowledge in the technical field to which the present invention pertains. At this time, the dosage form may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and a dispersant or stabilizer can be additionally included.

[0052] When the pharmaceutical composition of the present invention is manufactured into a vaccine composition, it is packaged in one vial or prefilled syringe, etc., in which a plurality of antigens of the present invention and various suitable adjuvants are selectively combined, or each antigen and adjuvant are packaged in separate vials and can be mixed immediately before use (prepared at the time of use, bed side mixing) for use.

[0053] According to still another aspect of the present invention, the present invention provides a method for preventing or treating a staphylococcal infection disease, which includes the step of administering the composition of the present invention described above to a subject.

[0054] According to still another aspect of the present invention, the present invention provides a composition for preventing or treating a thrombotic disorder caused by staphylococcal infection, which contains as an active ingredient one or more proteins selected from the group consisting of Coa (coagulase), vWbp (von Willebrand factor binding protein) and functional portions thereof; an antibody or an antigen-binding fragment thereof that specifically recognizes the protein; or a nucleotide encoding the protein.

[0055] The inventors have made intensive research efforts to develop a method for minimizing systemic damage to patients caused by blood coagulation during infection and further improving viability by efficiently blocking a specific Staphylococcus aureus host immune system evasion mechanism that induces fibrin coagulation in host blood vessels. As a result, it was confirmed that coagulase (Coa) and vWF-binding domain protein (vWbp), known as major virulence factors of Staphylococcus aureus, specifically, when using specific functional segments of these, blood coagulation is significantly inhibited by the antibodies generated by their inoculation.

[0056] As used herein, the term "functional portion" when referring to the Coa or vWbp protein encompasses any length of segment that maintains the biological activity of the Coa or vWbp protein. Thus, it means a segment of each protein that can be recognized by a Staphylococcus aureus surface protein (e.g., Fnbp) or function as an antigen that induces the production of antibodies that significantly inhibit blood coagulation due to Staphylococcus aureus infection.

[0057] According to a specific embodiment of the present invention, the functional portion of the Coa is the N-terminal segment of the Coa protein, and more specifically, a segment containing 284 consecutive amino acid residues from the N-terminus.

[0058] According to a specific embodiment of the present invention, the functional portion of the vWbp is the N-terminal segment of the vWbp protein, and more specifically, a segment containing 253 consecutive amino acid residues from the N-terminus of the vWbp protein.

[0059] As used herein, the term "thrombotic disease" means a systemic disease in which blood flow is reduced or blocked by a thrombus formed by the aggregation of platelets and fibrin proteins in the microcirculation system of blood vessels, thereby inducing ischemic damage to various organs such as the kidney, heart, and brain.

[0060] Specifically, the thrombotic diseases caused by staphylococcal infections to be prevented or treated by the composition of the present invention are one or more diseases selected from the group consisting of stroke, cerebral infarction, cerebral thrombosis, cerebral embolism, lacunar infarction, acute coronary syndrome, angina pectoris, aortic stenosis, myocardial infarction, leg block, cerebral ischemia, acute ischemic arteriovascular event, thrombophlebitis, venous thromboembolism, deep vein thrombosis, pulmonary embolism, peripheral vascular disease, atherosclerosis, vasospasm and restenosis caused by staphylococcal infections.

[0061] According to still another aspect of the present invention, the present invention provides a method for preventing or treating a thrombotic disorder caused by a staphylococcal infection, which includes the step of administering the composition of the present invention described above to a subject.

Advantages of the Invention

[0062] Summarizing the features and advantages of the present invention, they are as follows: (a) The present invention provides a composition for preventing or treating staphylococcal infection diseases and a composition for preventing or treating thrombotic diseases caused by staphylococcal infections. (b) The present invention can uniformly suppress the cell lysis caused by 11 toxins of staphylococci with only a minimum combination of toxin antigens by utilizing the cross-reactivity of antibodies against staphylococcal toxins. (c) The present invention can also be usefully utilized as an efficient therapeutic composition that can induce opsonophagocytosis and effectively control blood coagulation in the infected body, thereby getting out of the fragmented improvement of individual symptoms caused by staphylococcal infections and comprehensively removing or reducing the overall pathological state caused by the infection.

Brief Description of the Drawings

[0063]

Figure 1

[0064]

Figure 2

[0065]

Figure 3

[0066]

Figure 4

[0067]

Figure 5

[0068]

Figure 6

[0069]

Figure 7

[0070]

Figure 8

[0071]

Figure 9

[0072]

Figure 10

[0073]

Figure 11

[0074]

Figure 12

[0075]

Figure 13

[0076]

Figure 14

[0077]

Figure 15

[0078]

Figure 16

[0079]

Figure 17

[0080]

Figure 18

[0081]

Figure 19

[0082]

Figure 20

[0083]

Figure 21

[0084]

Figure 22

[0085]

Figure 23

[0086]

Figure 24

[0087]

Figure 25

[0088]

Figure 26

[0089]

Figure 27

[0090]

Figure 28

[0091]

Figure 29

[0092]

Figure 30

[0093]

Figure 31

[0094]

Figure 32

[0095]

Figure 33

[0096]

Figure 34

[0097]

Figure 35

[0098]

Figure 36

[0099]

Figure 37

[0100]

Figure 38

[0101]

Figure 39

[0102]

Figure 40

[0103]

Figure 41

Mode for Carrying Out the Invention

[0104] Hereinafter, the present invention will be described in more detail through examples. These examples are merely for explaining the present invention more specifically, and it will be apparent to those having ordinary knowledge in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Examples

[0105] Example 1: Selection of Staphylococcus Toxins Experimental Method Experimental Ethics Human blood was obtained from four healthy volunteers, and all experiments were conducted under the approval of the IRB of Pusan National University in Korea (PNU IRB / 2019_59_BR). Written consent forms were received from all study participants.

[0106] Bacteria The S. aureus USA300 LAC strain was cultured in TSB (tryptic soy broth) at 37°C with stirring until the mid-logarithmic phase (OD600 0.8 - 1.5). The E. coli DH5α strain was cultured in LB (Luria Broth) supplemented with 10 μg / ml kanamycin (Km) at 37°C with stirring.

[0107] Purification of Recombinant BCL Toxin and Production of Toxoid The polyhistidine-tagged recombinant toxin was cloned and expressed by an E. coli expression system. From the USA300 genomic sequence, the target genes LukS-PV, LukF-PV, LukE, LukD, HlgA, HlgB, HlgC, LukAB, Hla, HlaH35L, LukST244A-PV, LukAE323AB were amplified by PCR using Q5 High Fidelity DNA polymerase (Thermo Fisher Scientific). The PCR products were cloned into the pET28a-vector to obtain proteins tagged with 6x His at the N-terminus or C-terminus. To purify LukAB and LukAE323AB, 6x His-tags were inserted on both sides of the N- and C-termini, and the resulting clones were sequenced to confirm the structure. The recombinant proteins were expressed in BL21 pLysS E. coli using 0.2 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG, at a concentration of 0.2 mM). The expressed proteins were purified using Ni-sepharose 6 fast resin (GE healthcare, 17-5318-01, 5 ml) and loading buffer (20 mM sodium phosphate, 0.1% triton X-100, 150 mM NaCl, 5 mM imidazole, pH 7.4), washing buffer (20 mM sodium phosphate, 150 mM NaCl, 5 mM imidazole, pH 7.4) and elution buffer (20 mM sodium phosphate, 150 mM NaCl, 500 mM imidazole, pH 7.4). The primer sequences used in Example 1 are shown in Table 1 below.

Table 1

[0108] Immunization of rabbits to obtain toxin antibodies 500 μg of each of six S. aureus toxins dissolved in 500 μl of PBS and purified single components of these toxoid proteins were mixed with 500 μl of Freund's complete adjuvant (Sigma-Aldrich) to produce an emulsion. After subcutaneous injection of each emulsion into rabbits, the same amount of antigen was mixed with 500 μl of Freund's incomplete adjuvant (Sigma-Aldrich) and subcutaneously injected 2 weeks later. The presence or absence of IgG formation of each antigen was confirmed by Western blot from 500 μl of blood collected from the rabbit ear vein 2 weeks later. When antibodies were formed, whole rabbit blood was collected to obtain serum, which was stored at -80 °C until use.

[0109] Coupling of the purified protein to CNBr-activated Sepharose beads The coupling between the recombinant single-component toxin and CNBr-activated Sepharose beads was performed according to the manufacturer's manual (GE HealthCare). Briefly, 1 g of CNBr-activated Sepharose beads was washed three times with coupling buffer (3 ml / time, 0.1 M NaHCO3, pH 8.5). 5 mg of the recombinant protein dissolved in 1 ml of coupling buffer was added to the CNBr-activated beads and suspended in 4 ml of coupling buffer. After incubation at room temperature for 2 hours, the beads were washed three times with coupling buffer. Thereafter, the beads were incubated with 4 ml of 1 M methanolamine (pH 8.0) at room temperature for 4 hours. After incubation, the beads were washed with 0.1 M sodium phosphate (pH 7.4) until no UV absorbance appeared at 280 nm and stored at 4 °C until use.

[0110] Purification of rabbit anti-single component recognizing IgG derived from rabbit serum After equilibrating the antigen-coupled Sepharose column with a washing buffer (0.1 M sodium phosphate containing 0.1 M NaCl, pH 7.4), the antigen-immunized rabbit serum (1 ml, 60 mg of protein) was loaded onto the column. After strongly washing the column with the washing buffer, the bound IgG was eluted with an elution buffer (0.15 M glycine / HCl, pH 2.2). The collected IgG was neutralized with a neutralization buffer (1 M Tris / HCl buffer, pH 9.0), then the buffer was replaced with PBS, and the collected IgG was analyzed by SDS-PAGE under reducing and non-reducing conditions.

[0111] Dot blot immunoassay The PVDF-membrane strip (Immobilon P, pore size 0.22 μm, Millipore) was activated with methanol for 30 seconds. Thereafter, the strip was washed with H2O for 5 minutes while gently stirring. After washing, 1 μg of recombinant single-component toxin (LukS-PV, LukF-PV, HlgA, HlgB, HlgC, LukE, LukD and LukAB) was loaded onto the strip. Each single-component and LukAB protein (1 μg each), and S. aureus FnbpAN2N3 (N2-N3 domain of fibronectin-binding protein) as a negative control group were spotted. After drying the strip at room temperature for 2 hours, the protein was fixed with methanol for 15 seconds and washed 3 times with H2O and 3 times with 1X TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl). The strip was blocked with 5% skim milk for 2 hours and incubated with IgG immunized with single-component-toxin (diluted 1:1600 with 5% skim milk) at 4°C for 1 hour. After incubation, the membrane was washed 3 times with 1X TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Tween 20). Mouse anti-rabbit IgG-HRP (diluted 1:3000, Santacruz) was added to the membrane and incubated at 4°C for 1 hour. Thereafter, the membrane was washed 3 times with 1X TBST and applied to the Pico EPD Western blotting detection kit [ELPIS-Biotech].

[0112] Isolation of human PMNs Using the collected whole blood (2 ml) and PolymorphprepTM (2 ml, Axis-shield), polymorphonuclear cells (PMNs) were separated in a round-bottom tube at room temperature. The samples were centrifuged at 450 x g for 30 minutes at 20 °C. After removing the supernatant, two leukocyte bands containing mononuclear cells (upper band) and PMNs (lower band) were obtained. Thereafter, the collected PMNs were washed twice with RPMI (Gibco), and collected after centrifugation at 400 x g for 10 minutes at 20 °C. The collected cells were gently resuspended in 2 ml RPMI + 0.02% HSA buffer, and the cell count was calculated using a hemocytometer (EKDS, Tokyo).

[0113] Separation of Rabbit RBCs 1 ml of rabbit blood was collected into a hirudin-coated vacuum tube (BD, Becton Drive) to block aggregation through the ear vein. Thereafter, the tube was centrifuged at 9,100 x g for 10 minutes at 4 °C to obtain red blood cells (RBCs). The RBCs were washed twice with 0.02% BSA containing PBS, and collected after centrifugation at 500 x g for 5 minutes at 4 °C. The RBCs were diluted to 2% with 0.02% BSA containing PBS to prepare a preservation solution.

[0114] Cell Lysis and Hemolysis Assays To evaluate the in vitro viability of primary human PMNs depending on the presence or absence of S. aureus BCL or Hla, PMNs were seeded at 2 x 106 cells per well in a 48-well plate (SPL) and mixed with the same amount of S- and F-components (65 ng each) (LukAB was 130 ng). This mixture was cultured at 37 °C under 5% CO2 conditions for 1 hour, and the number of surviving cells was measured using a hemocytometer.

[0115] Toxin-Mediated RBC Hemolysis Assay To perform a hemolysis assay of rabbit RBCs in the presence or absence of HlaWT toxin in vitro, 2% RBCs were seeded at 100 μl per well in a 96-well plate. Subsequently, 0.25 μg of purified recombinant HlaWT toxin was added to the 96-well plate and cultured at 37 °C under 5% CO2 conditions for 1 hour. Subsequently, centrifuged at 500 xg for 5 minutes to collect the supernatant, and the absorbance at 405 nm was measured using a spectrophotometer (Eppendorf BioPhotomter).

[0116] Western blot analysis Purified BCL toxin, Hla and toxoid were applied to 15% SDS-PAGE gel electrophoresis and transferred to a 0.45 μm PVDF membrane at 100 V, 400 mA for 75 minutes at 4 °C using a transfer buffer (192 mM Glycin, 25 mM Tris, 0.02% SDS, 20% MtOH). Subsequently, the membrane was blocked with 5% skim milk for 1 hour. After culturing the membrane with immunized rabbit serum (1:6000), it was blocked with 5% skim milk at 4 °C for 1 hour. After culturing, the membrane was washed 3 times with 1X TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Tween20). Mouse anti-rabbit IgG-HRP (Santacruz) was added and cultured at 4 °C for 1 hour. Subsequently, the membrane was washed 3 times with 1X TBST and analyzed using a Pico EPD Western blotting detection kit [ELPIS-Biotech].

[0117] Experimental results Five purified recombinant S. aureus two-component leukocidin (BCL) and Hla hydrolyzed human PMN and RBC respectively, but the toxoid did not hydrolyze. To investigate the relationship between S. aureus toxins and toxin-mediated cytotoxicity of host cells, the inventors first cloned five BCLs and Hla (A-C in Figure 1). For the purification of the six expressed recombinant toxins, a His x 6-tag was inserted into the N-terminal or C-terminal site of the target toxin (Figure 2). As shown in Figure 3, recombinant proteins of four S-components and three F-components of S. aureus BCL were purified and homogenized. However, since the monomer of LukAB was expressed slowly, LukABWT was purified in the form of a dimer (Figure 3A). Also, recombinant HlaWT and HlaH35L, LukST244A and LukAE323AB toxoids were also purified homogeneously (Figure 3B). The Hla toxoid (HlaH35L) with a single active amino acid substitution cannot form a cytolytic pore because the His-35 residue is replaced by Leu, but it is known to maintain the ability to bind to receptors on host cells (Menzies BE et al., 1994. Infect Immun 62:1843-7; Menzies BE et al., 1996. Infect Immun 64:1839-41). When the Thr-244 residue of LukS-PV is replaced by Ala (LukST244A-PV), the binding affinity with all human leukocytes and C5a receptor-expressing undifferentiated U937 cells is greatly reduced (Laventie BJ et al., 2014. PLoS One 9:e92094). Therefore, in the present invention, the LukST244A mutation was purified. Also, since the Glu-323 residue of LukA was reported to be important for LukAB cytotoxicity and essential for the interaction between LukAB and its target CD11b (DuMont AL et al., 2014. Infect Immun 82:1268-76), LukAE323AB was prepared and used. To confirm whether wild-type leukocidin and toxoid exhibit cytolytic activity against human PMNs, the same amount of S-component and F-component were mixed to prepare wild-type leukocidins such as LukSF-PV, LukED, HlgAB, HlgCB, LukAB and inactive leukocidins such as LukST244A-PV and LukAE323AB.

[0118] After individually expressing and purifying the S- and F-components of BCL, Hla, and these toxoid proteins, their toxicities were tested using human PMN and RBC. When the same amounts of the S- and F-components (0.25 μg / ml each) were mixed, and LukAB was mixed at 0.5 μg / ml, all five homologous BCLs (LukSF-PV, LukED, LukAB, HlgAB, HlgCB) completely lysed human polymorphonuclear leukocytes (PMN, 2 × 106 cells) within 30 minutes. However, the mixture of LukST244A-PV toxoid (0.25 μg / ml) and LukF-PV (0.25 μg / ml) showed no toxicity to human PMN (Figure 3C). Also, the LukAE323AB dimer toxoid (0.5 μg / ml) showed no cytotoxicity to human PMN (Figure 3C). Together with this, the purified HlaWT (2.5 μg / ml) lysed 2% rabbit RBC within 30 minutes, but its inactive toxoid, HlaH35L, was unable to show hemolytic activity against rabbit RBC (Figure 3D). Summing this up, it can be seen that the purified S. aureus BCL and these toxoid proteins were successfully cloned and purified uniformly.

[0119] Anti-rabbit IgG against the single S- or F-component has cross-reactivity against other BCL components. Since S. aureus BCL has a high sequence homology between S-S or F-F single components, it was expected that the single-component-immunized rabbit antibodies would bind to other single components of the S. aureus BCL toxin. However, since these recognition patterns by single-component-IgGs are important for exploring BCL-antibodies that comprehensively neutralize five BCL-mediated cytotoxicities, the inventors obtained rabbit polyclonal antibodies against eight different single components by affinity purification using single-component-conjugated sepharose columns (Figure 4). Also, since the recombinant toxins and toxoids of the present invention have a 6x His-tag at the N- or C-terminus, His-tag-derived IgG was removed using a 6x His-tagged S. aureus fibronectin-binding protein A (FnbpAN2-N3)-conjugated sepharose column (data not shown). Using these purified rabbit single-component-recognition IgGs, the inventors examined the cross-reactivity between BCL single components and the purified BCL-recognition IgGs by dot blot immunoassay (Figure 5). All the IgGs used did not bind to the FnbpAN2-N3, which is a negative control group protein. Anti-LukS-PV-IgG recognized all four S-components (LukS-PV > HlgC > HlgA > LukE), but could not recognize the three F-components and LukAB (columns a in Figure 5, the dark and light boxes indicate homologous and non-homologous antigens, respectively). Anti-LukE-IgG also recognized four S-components (LukE > LukS > HlgC > HlgA) (column f in Figure 5). Anti-HlgA-IgG recognized Hlg > HlgC > LukF (column c). Anti-HlgC-IgG recognized HlgC ≧ LukS > HlgA (column e in Figure 5). In the case of F-component-IgGs, anti-LukF-PV-IgG showed a positive signal for LukF > LukD (column b in Figure 5). Anti-HlgB-IgG recognized three F-components of LukF-PV > HlgB > LukD (column d in Figure 5). Anti-LukD-IgG showed a binding affinity for LukD > LukF-PV (column g in Figure 5). As expected, LukAB-IgG showed binding specificity only for its homologous antigen, the LukAB antigen (column h in Figure 5).In summary, it can be seen that LukS-PV and LukE-IgG have the broadest binding ability to the four BCL S-components. However, interestingly, although anti-HlgA-IgG is an S-component that recognizes IgG, it also recognized the LukF-PV F-component (columns c and g in Fig. 5). In the case of anti-HlgB-IgG, it recognized three F-components (HlgB, LukD, LukF), while the other two F-components (LukF-PV and LukD) recognized only these homologous F-components and one non-homologous F-component. In summary, it can be seen that the BCL S- and F-components have different recognition patterns for homologous and non-homologous antigens.

[0120] The four anti-S-component-IgGs show a partial neutralizing effect on the cell-lysing activity of the four BCL-mediated PMNs. The inventors sought to examine how in vitro BCL-mediated neutrophil cytolytic activity is neutralized by the addition of anti-S- or F-component-IgG. Co-culturing human PMNs with each BCL and anti-LukS-PV-IgG (Figure 6A), anti-LukS-PV-IgG suppresses LukSF-PV-(98.5 ± 0.5%), LukED-(74 ± 1%) and HlgCB-(68.1 ± 1%)-mediated PMN cytolytic activity, but the PMN cytolytic activity by HlgAB and LukAB cannot be completely neutralized by anti-LukS-PV-IgG. In contrast, anti-HlgA-IgG neutralizes only HlgAB-induced (90.3 ± 3%) and LukED-mediated PMN lysis (27.5 ± 4%), and the cytotoxicity of LukSF-PV, HlgCB and LukAB-mediated PMNs could hardly be blocked (Figure 6B). Anti-HlgC-IgG suppressed HlgAB-(43.9 ± 0.5%), LukED-(58.5 ± 1%) and LukSF-PV-(73.2 ± 0.5%), HlgCB-(85.4 ± 1%) and LukAB-(0%)-mediated PMN lysis (Figure 6C). As expected, anti-LukE-IgG suppressed more than 70% of the four BCL-mediated PMN lysis, but LukAB-induced lysis (Figure 6D) could not be completely suppressed. In the dot blot experiment (Figure 5), anti-LukE-IgG strongly recognized LukE > LukS-PV, but weakly recognized HlgC > HlgA. However, it was found that more than 75 ± 3% of the four BCL-mediated PMN lysis was blocked by anti-LukE-IgG, and there was a difference between the dot blot immunoassay and the neutralizing ability of single S-component IgG in the cytolytic activity of BCL-mediated human PMNs.

[0121] Three anti-F-component-IgGs neutralized the HlgCB-mediated PMN cytolytic activity As shown in Fig. 7, when anti-F-component-IgG was co-cultured with human PMN and BCL, anti-LukF-PV IgG blocked the LukSF-PV-mediated PMN lysis activity and the HlgCB-induced PMN lysis activity at 85±0.5% and 74±1%, respectively, while the cell lysis activities mediated by HlgAB, LukED, and LukAB were hardly neutralized by anti-LukF-IgG (Fig. 7A). Also, anti-HlgB-IgG suppressed the HlgAB- and HlgCB-mediated PMN lysis activities at 85.8±1% and 65±0.5%, respectively, but the inhibition rates of the lysis activities mediated by LukED-(10±0.5%), LukSF-PV-(10±1%), and LukAB-(2±1%) were low (Fig. 7B). In the case of anti-LukD-IgG, the lysis activities of PMN mediated by LukED-(84±4%) and HlgCB(77±1%) were also suppressed, but the lysis activities mediated by HlgAB-(18.4±3%), LukSF-(20.8±0.2%), and LukAB(3±0.1%) could not be effectively neutralized (Fig. 7C). Summing this up, in the dot blot immunoassay, two F-component-IgGs, anti-LukF-IgG and anti-LukD-IgG, could not recognize HlgB (Fig. 5), but these two F-component IgGs generally neutralized the HlgCB-mediated PMN lysis activity, and it was found that by recognizing the specific sequence of non-homologous HlgB, they suppressed the polymerization of HlgCB BCL.

[0122] Different combinations of anti-BCL-component-IgG cause differences in the neutralizing ability against BCL-mediated cytotoxicity The inventors hypothesized that screening for the minimal combination of BCL anti-S- or anti-F-component IgG that can comprehensively neutralize five BCL-mediated cytotoxicities would provide important information for designing new therapeutic agents against S. aureus infection. LukSF-PV and HlgCB have been reported to recognize the C5aR1 and C5aR2 receptors that are generally expressed on host cells (Spaan AN et al., 2013. Cell Host Microbe 13:584-594). However, LukED recognizes CCR5, CXCR1, and CXCR2 (Alonzo F, et al., 2013. Nature 493:51-5), and while both HlgAB and LukED have CXCR1 and CXCR2 as receptors and also recognize CCR2 (Spaan AN et al., 2017. Nat Rev Microbiol 15:435-447), LukAB binds to CD11b (DuMont AL et al., 2014. Infect Immun 82:1268-76). Based on the experimental results in Figures 6 and 7, the inventors examined which combinations of anti-single-component-IgG or anti-toxoid-IgG could efficiently neutralize the cytotoxicity induced by the five BCLs. As shown in Figure 8, when anti-LukE-IgG (5 μg) and anti-LukD-IgG (5 μg) were co-cultured with human PMNs and the five BCLs for 60 minutes, the cytotoxicity of the four BCL-mediated PMNs except LukAB was neutralized by these two IgGs by more than 70% (Figure 8A). However, the mixture of anti-HlgA- and anti-HlgB-IgG only neutralized HlgAB-(79.8±5%) and HlgCB-(60±1%)-mediated cytotoxicity (Figure 8B), and no inhibitory effect was observed against LukED, LukSF-PV, and LukAB. From these results, although HlgAB and LukED have been reported to bind to CXCR1, CXCR2, and CCR2, it can be seen that the IgG mixture of HlgA and HlgB can hardly neutralize LukED-mediated cytotoxicity. Contrary to expectations, the mixture of anti-HlgA-IgG and anti-HlgB-IgG inhibited the cytolytic activity of C5aR-recognizing HlgCB.

[0123] The mixture of anti-HlgC-IgG and anti-HlgB-IgG neutralizes the lytic activities of HlgCB (93.4 ± 0.5%) > LukSF-PV (70 ± 1%) > HlgAB (57.8 ± 1%), and it was confirmed that the C5aR-recognizing BCL-rabbit polyclonal IgG mixture also inhibits the cytotoxicity of chemokine-receptor-recognizing HlgAB (Figure 8C). However, the mixture of anti-LukS-PV-IgG and anti-LukF-PV-IgG neutralizes the LukSF-PV- (93.5 ± 1%) and HlgCB- (78.7 ± 1%) mediated lytic activities and also inhibits the LukED (68 ± 0.5%)-induced PMN lytic activity (Figure 8D). In summary, as expected, it was confirmed that anti-LukAB-IgG only neutralizes the cytotoxicity of LukAB and cannot inhibit the other four BCL-mediated toxicities (Figure 8E). Thus, different combinations of anti-BCL-IgG appear with differences in the neutralizing ability against BCL-mediated cytotoxicity and have no effect on the toxicity of LukAB.

[0124] Among the combinations of anti-S-component-IgG, the mixture of anti-LukS-PV- and anti-HlgA-IgG showed the highest neutralizing activity against BCL-mediated cytotoxicity. Next, the neutralizing ability of the homologous BCL S-IgG combination against the cytolytic activity of five BCL-mediated PMNs was examined. For this purpose, the neutralizing ability of six different combinations of anti-S-component-IgGs against the cytolytic activity of five BCLs was measured (Figure 9). Six possible combinations of anti-S-component-IgGs were co-cultured with human PMNs with five different BCLs to calculate the survival rate of each PMN. Each survival rate was LukS-PV+HlgC, average survival rate 59.7% (Figure 9A); LukS-PV+HlgA, 85.8% (Figure 9B); LukS-PV+LukE, 45.2% (Figure 9C); HlgA+LukE, 45.9% (Figure 9D); HlgA+HlgC, 31.3% (Figure 9E); HlgC+LukE, 36.8% (Figure 9F). Among these, the mixture of anti-LukS-PV and anti-HlgA-IgG most efficiently suppressed the cytolytic activity of four BCLs compared to the remaining five combinations (Figure 9B). This confirmed that the combination could suppress the toxicity of four BCLs, although it still had no effect on the toxicity of LukAB.

[0125] Among the combinations of anti-F-component-IgGs, the mixture of anti-LukF- and anti-LukD-IgG showed the highest neutralizing activity against the cytolytic toxicity of four BCLs. Furthermore, the inventors examined the neutralizing ability of the isogenic BCL F-IgG combinations against the cytolytic activity of five BCL-mediated PMNs. To this end, the neutralizing ability of four different combinations of anti-F-component-IgGs against BCL-mediated cytolytic activity was measured (Figure 10). The mean survival rates of PMNs following treatment with each mixture were as follows: anti-LukF-PV- and anti-HlgB-IgG, 52.9% (Figure 10A); anti-LukD and anti-HlgB-IgG, 42.6% (Figure 10B); anti-LukF-PV- and anti-LukD-IgG, 68.6% (Figure 10C); anti-LukF-PV-, anti-LukD and anti-HlgB-IgG, 69.0% (Figure 10D). Among these, the combination of anti-LukF-PV- and anti-HlgB-IgG showed the highest PMN survival rate compared to the other three combinations. This indicates that the three anti-F-component-IgGs exhibit good neutralizing activity against the four BCL toxicities, although they still had no effect on the toxicity of LukAB.

[0126] A mixture of anti-LukS-PV-, HlgA- and LukAE323AB-IgGs neutralizes the cytolytic activity of five BCL-mediated PMNs As shown in FIGS. 6 to 10, no combination of any BCL-component-IgG and any IgG was able to suppress LukAB-induced cytolytic activity. Therefore, in order to neutralize all five BCL-induced toxicities using anti-BCL-component-IgG, the inventors had to use anti-LukAB toxoid-IgG and other selected anti-BCL-component-IgGs together. For this purpose, purified recombinant LukAE323AB toxoid and its rabbit anti-LukAE323AB IgG were purified (FIG. 4). Since it was confirmed that a mixture of anti-LukS-PV- and HlgA-IgG neutralized four BCL-mediated cytolytic activities with high efficiency (FIG. 9B), it was hypothesized that a three-IgG mixture of anti-LukS-PV-, anti-HlgA and anti-LukAE323AB toxoid-IgG would neutralize five BCL-mediated cytolytic activities. Also, a mixture of three anti-F-component IgGs and anti-LukAE323AB toxoid-IgG could be a promising candidate (FIG. 10D).

[0127] To test such a possibility, human PMNs were cultured with five BCLs and two different IgG combinations (FIG. 11). The measurement results of the average survival rate of PMNs showed 94.7% and 68.0% respectively when using the combination of anti-LukS-PV-, anti-HlgA and anti-LukAE323AB toxoid-IgG (FIG. 11A) and the combination of anti-LukF-PV, anti-HlgB- and anti-LukAE323AB toxoid-IgG (FIG. 11B). It was found that when two S-component IgGs and one LukAB toxoid IgG were mixed, the cytolytic activity of five S. aureus BCL-mediated PMNs was significantly neutralized.

[0128] The mixture of anti-LukS-PV-, HlgA-, HlaH35L and LukAE323AB-IgG suppresses all of the native S. aureus toxin-mediated cytolytic and hemolytic activities of human PMNs So far, the cytolytic activity of human PMN has been examined in vitro using five recombinant BCLs. Therefore, the present inventors attempted to confirm whether purified anti-BCL IgG could also act on the native BCL secreted from pathogenic S. aureus USA300. For this purpose, first, the amount of BCL was measured at different time points during USA300 culture. As shown in Figure 12, it was confirmed that all five BCLs were secreted in large amounts at 1.5 to 3 hours of culture (Figure 12A). Under the same conditions, Hla was highly secreted between 1.5 and 9 hours. Based on such results, it was found that the five BCLs and Hla toxin generally accumulated at 3 hours of culture. Therefore, the supernatant cultured for 3 hours in the S. aureus USA3000 culture medium was collected and concentrated to a protein concentration of 1 mg / ml and used as a stock solution. When the secreted BCL and Hla in the stock solution were quantified by dot blot analysis, the average protein amounts of LukSF-PV, HlgAB, LukED, HlgCB, LukAB, and Hla were 0.13, 0.26, 0.32, 0.37, 0.24, and 1.50 μg / ml, respectively (Table 2).

Table 2

[0129] Next, it was investigated whether human PMNs could be protected in vitro by a mixture of four antibodies, anti-LukS-PV-, HlgA, HlaH35L, and LukAE323AB toxoid-IgG (Figure 13). When human PMNs were prepared from human whole blood using PolymorphprepTM solution, the PMN band clearly appeared without any damage (left in Figure 13A). However, when six recombinant toxins (Hla, LukSF-PV, HlgAB, LukED, HlgCB, LukAB 4 μg) were added to human whole blood, the white PMN band could not be recovered after centrifugation (middle in Figure 13A). When a mixture of six toxins and four IgGs (a total of 64 μg of anti-HlaH35L, LukS-PV-, HlgA, and LukAE323AB-IgG) was added to human whole blood, the PMN band reappeared after centrifugation (right in Figure 13A). Furthermore, as a result of measuring the cell count of the recovered PMNs with a hemocytometer (Figure 13B), only 20% of PMNs were recovered from the six-toxin-treated whole blood (column 2) compared to the control group (column 1). However, 95% of PMNs were recovered from the four-IgG-treated human whole blood (column 3). This confirmed that the four IgGs selected in the present invention could efficiently block the cell-lysing activity of six toxin-mediated PMNs in vitro.

[0130] Also, S. aureus Hla and BCL were known to induce hemolytic activity during infection (Seilie ES, et al., 2017. Semin Cell Dev Biol 72:101-116). When six toxins were cultured with rabbit whole blood, higher hemolytic activity was observed compared to PBS-treated blood (second tube in Figure 13C). However, when the four IgGs selected in the present invention and six toxins were administered to rabbit whole blood, the hemolytic activity was suppressed to 50% compared to the control group (Figures 13C and 13D). This confirmed that the four toxin antibodies of the present invention could suppress the hemolytic effect of S. aureus toxin-mediated human blood.

[0131] Finally, the inventors sought to examine the protective effect of the four selected IgGs against S. aureus culture medium-mediated PMN cytolysis. First, to determine the amount of protein required in the S. aureus culture medium to exhibit toxicity to human PMNs (2 × 106 cells), various doses of the culture medium were incubated with PMNs (Figure 14A). When 16 μg / ml of the culture medium was added, most of the PMNs were hydrolyzed. Based on this, when human PMNs (2 × 106 cells) were cultured in 250 μl of RPMI at 37 °C for 1 hour, 100% of the PMNs survived (Figure 14B, column 1). When the PMNs were co-cultured with USA300 culture medium (final concentration 16 μg / ml) and a mixture of anti-LukS-PV-, HlgA-, LukAE323AB, and HlaH35L-IgG (total final concentration 160 μg / ml), approximately 90% of the PMNs survived (column 2). As expected, when cultured only with USA300 culture medium (final concentration 16 μg / ml), only 5% of the PMNs survived (column 3). This shows that damage due to six toxin-mediated PMN cytolysis can be protected against in vitro by adding the four toxins and toxoid-IgGs selected in the present invention.

[0132] Four rabbit anti-toxin-IgGs exhibit a bactericidal effect when co-cultured with human whole blood and S. aureus USA300 Serum antibodies formed by immunization with an antigen are known to induce host-phagocyte-mediated opsonophagocytosis to remove infected pathogens (Miller LS et al., 2020. FEMS Microbiol Rev 44:123-153). Since it was confirmed that the four anti-rabbit toxin IgG selected in the present invention have inhibitory effects on S. aureus BCL and Hla toxin-mediated PMN cell lysis and RBC hemolytic activity, it was attempted to examine whether these four antibodies have a bactericidal effect when co-cultured with S. aureus USA300 cells and human whole blood. For this purpose, USA300 cells (2×106 cells) were co-cultured with native purified IgG (40 μg) or a mixture of four toxin-derived IgG (total 40 μg) and human whole blood (100 μl) for 3 hours, and the remaining CFU (colony forming unit) was measured (Figure 15). When the control group sample reached 2×106 CFU, the samples treated with IgG and the four toxin-IgG mixture showed 25% and 6% CFU of the control group, respectively, indicating that the four anti-toxin-IgG mixture has a bactericidal effect on bacteria in human whole blood.

[0133] Example 2: Search for CWAP proteins that bind to human complement factor H (FH) Experimental method Purification of pET28a vector containing clfA, clfB, sdrC, sdrD, sdrE, fnbpA and fnbpB N2-N3 domains Cloning was performed using the Gibson assembly master mix (New England Biolabs, USA) according to the manufacturer's protocol. The pET28a plasmid (Novagen, Germany) with a 6-histidine-containing motif (6-his-tag) was used as the vector. To express the CWAPN2N3 domain, approximately 1 kbp of each gene of clfA (215-575), clfB (200-555), sdrC (166-507), sdrD (224-570), sdrE (261-610), fnbpA (180-559), and fnbpB containing the N2 and N3 domains of the S. aureus USA300 LAC strain was used. The coding regions were amplified by PCR using Q5 high-fidelity polymerase (primer sequences are summarized in Table 3). The cloned pET28a-clfA, clfB, sdrC, sdrD, sdrE, fnbpA, and fnbpB were transformed into E. coli DH5α (Enzynomics, Korea), spread on LB agar plates containing 10 μg / ml kanamycin (Km) (Sigma-Aldrich, USA), and then cultured overnight at 37°C.

Table 3

[0134] Transformation of pET28a-CWAP plasmid DNA into E. coli BL21(DE3)pLySs 100 ng of DNA of pET28a-clfA, clfB, sdrC, sdrD, sdrE, fnbpA, and fnbpB was transformed into E. coli BL21(DE3)pLySs cells using the heat shock method, spread on LB agar plates containing 10 μg / ml kanamycin (Km) and 50 μg / ml chloramphenicol (Cm), and then cultured overnight at 37°C.

[0135] Expression and purification of the CWAPN2N3 domain E. coli BL21(DE3)pLySs inserted with pET28a-clfA, clfB, sdrC, sdrD, sdrE, fnbpA and fnbpB was cultured overnight at 37 °C in LB with 10 μg / ml Km and 50 μg / ml Cm. 10 ml of the culture sample was inoculated into 1 L of LB containing 10 μg / ml of Km and 50 μg / ml of Cm and cultured at 37 °C for one and a half to two hours. When the OD600 value reached 0.3 - 0.5, 200 μl of 1 M IPTG was added to the 1 L sample and the sample was cultured at 37 °C for 3 hours. To purify the ClfAN2N3, ClfBN2N3, SdrCN2N3, SdrDN2N3, SdrEN2N3, FnbpAN2N3, FnbpBN2N3 domains, the culture solution was centrifuged at 7,000 xg for 20 minutes at 4 °C. After freezing the pellet at -80 °C for 30 minutes, the pellet was suspended in 40 ml of 20 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl, 5 mM imidazole, 0.1% triton X-100, and 5 mg lysozyme (Bioshop, Canada) and completeTM EDTA-free protease inhibitor cocktail (Merck, USA) and cultured at room temperature for 15 minutes. To break the bacterial cell wall, the sample was sonicated on ice for 30 minutes. After centrifuging the mixture at 20,400 xg for 20 minutes at 4 °C, the supernatant was collected and filtered through a 0.45 μm syringe membrane filter. The expressed recombinant protein was purified using a Ni-sepharose 6 fast column (GELifeScience, USA). Briefly, before applying the sample, the Ni-sepharose column was equilibrated with 20 mM sodium phosphate (pH 7.4) containing 150 mM NaCl and 5 mM imidazole (buffer A). The filtered solution was loaded onto the column, and after washing the column with buffer A, the protein was eluted stepwise with 20 mM sodium phosphate (pH 7.4) containing 150 mM NaCl and 500 mM imidazole, respectively. The eluted fractions were collected and subjected to SDS-PAGE analysis under reducing conditions.

[0136] Preparation of CWAPN2N3 domain-coupled sepharose affinity column The coupling between the recombinant CWAPN2N3 domain and CNBr-activated Sepharose beads (GE Health Care) was performed according to the manufacturer's protocol. Briefly, 1 g of CNBr-activated Sepharose beads was washed three times with coupling buffer (0.1 M NaHCO3, pH 8.5), dissolved in 1 ml of coupling buffer, and 5 mg of CWAPN2N3 protein dissolved in 4 ml of coupling buffer was added and suspended. After incubation at room temperature for 2 hours, the beads were washed three times with coupling buffer. Subsequently, the beads were incubated with 4 ml of 1 M ethanolamine (pH 8.0) at room temperature for 4 hours, then washed with 0.1 M sodium phosphate (pH 7.2) until no UV absorbance appeared at 280 nm, and stored at 4°C.

[0137] Production of anti-CWAPN2N3 domain recognition antibody in rabbits 500 μg of purified CWAPN2N3 domain dissolved in 500 μl of PBS was mixed with 500 μl of complete Freund's adjuvant (CFA, Sigma-Aldrich) to obtain an emulsion. After subcutaneous injection of the emulsion into rabbits, the same amount of antigen was mixed and injected together with 500 μl of incomplete Freund's adjuvant (IFA, Sigma-Aldrich) 7 days later. One week later, 500 μl of blood was collected from the rabbit ear vein, and the production of IgG was examined by Western blot. When the formation of IgG was confirmed, the rabbit serum was collected and stored at -80°C.

[0138] Purification of anti-CWAPN2N3 recognition IgG from rabbit serum After washing the Sepharose column (Sigma-Aldrich) coupled with S. aureus protein with elution buffer (0.15 M glycine / HCl, pH 2.2), the column was equilibrated with washing buffer (0.1 M sodium phosphate containing 0.1 M NaCl, pH 7.4). After loading antigen-immunized rabbit serum (2 ml, about 100 mg of protein) onto the column, the column was washed with washing buffer and IgG was eluted with elution buffer. The collected IgG was rapidly neutralized with neutralization buffer (1 M Tris / HCl buffer, pH 9.0). Thereafter, it was replaced with a CWAPN2N3 domain-coupled Sepharose column. After washing the column with elution buffer (0.15 M glycine / HCl, pH 2.2), the column was equilibrated with washing buffer (0.1 M sodium phosphate containing 0.1 M NaCl, pH 7.4). IgG (about 6 mg of protein) collected from the protein A column was loaded onto the column. After washing the column with washing buffer, IgG was eluted with elution buffer. The collected IgG was rapidly neutralized with neutralization buffer (1 M Tris / HCl buffer, pH 9.0). After replacing the buffer with PBS, the collected IgG was analyzed by SDS-PAGE under reducing and non-reducing conditions.

[0139] Preparation of F(ab’)2 Fragments from Rabbit IgG The F(ab’)2 fragments used in the present invention were produced by a previously reported method (Hymes AJ et al., 1979. J Biol Chem 254:3148-51). The method for producing anti-human FH-IgG F(ab’)2 fragments is typically applicable to the production of other F(ab’)2 fragments as well. First, goat anti-human FH-IgG was purified using a S. aureus protein-coupled sepharose column (Sigma-Aldrich). Briefly, 500 μl (40 mg of protein) of goat-anti-human FH (Complement Technology, Inc.) was loaded onto a protein column (1.5×8 cm, 10 ml) equilibrated with buffer A (0.1 M sodium phosphate containing 0.1 M NaCl, pH 7.4). After washing the column with buffer A, the bound IgG was eluted with buffer B (0.15 M glycine / HCl, pH 2.2) while monitoring the UV absorbance at 280 nm. The collected IgG was immediately neutralized to pH 7.5 with 1 M Tris / HCl (pH 9.0), concentrated on Centricon (Merck Millipore Ltd.), and then 2.5 mg of goat FH IgG and 50 μg of pepsin (Sigma-Aldrich) were mixed with 500 μl of 0.1 M acetate buffer (pH 4.2) and incubated at 37°C for 18 hours. After terminating the reaction while performing buffer exchange with 100 mM cold phosphate buffer (pH 7.4) containing 100 mM NaCl, it was confirmed whether the IgG was appropriately cleaved under reducing and non-reducing conditions. During the progress of the reaction, the digestion solution was concentrated and exchanged with buffer A, and the fluid fraction of the protein column containing the F(ab’)2 fragments was collected.

[0140] Purification of Human CWAPN2N3-Recognizing IgG from Human IVIg Commercially available intravenous IgG (IVIg, 500 mg of protein) in 5 ml was loaded onto a Sepharose column coupled with CWAPN2N3 (SdrE, FnbpA: 1 ml of beads; ClfA, FnbpB: 3 ml of beads) and washed by the method described above. Human anti-CWAPN2N3 recognizing IgG was eluted and neutralized by the method described above. The obtained human IgG was analyzed by SDS-PAGE under reducing and non-reducing conditions.

[0141] Dot blot immunoassay 3 μg of the N2-N3 domain of CWAPN2N3 (ClfAN2N3, ClfBN2N3, SdrCN2N3, SdrDN2N3, SdrEN2N3, FnbpAN2N3, FnbpBN2N3) and BSA as a negative control group were spotted onto a PVDF membrane (Immobilon-PSQ Transfer Membrane, Merck, USA) and dried. The membrane was blocked with 5% skim milk in TBS at room temperature for 2 hours and then incubated with 7 μg each of purified human fibrinogen (Sigma-Aldrich, USA), purified human fibronectin (Sigma-aldrich, USA), human FH (Complement Technology, USA), and human FI (Complement Technology, USA) dissolved in 700 μl of 5% skim milk. Subsequently, the membrane was washed 3 times with TBST for 10 minutes each and then incubated with primary antibodies diluted 6000-fold [rabbit-anti-human fibrinogen-IgG (Sigma-Aldrich, USA), rabbit-anti-human fibronectin-IgG (Sigma-Aldrich, USA), goat anti-human FH-IgG (Complement Technology, USA), and goat anti-human factor I-IgG (cat. No. A238, Complement Technology, USA)] at room temperature for 1 hour. The membrane was washed 3 times with TBST for 10 minutes each and then incubated with secondary antibodies diluted 3000-fold (mouse anti-rabbit IgG HRP or donkey anti-goat-IgG-HRP, Santacruz, USA) at room temperature for 1 hour. Subsequently, the membrane was washed 3 times for 10 minutes each and then incubated with enhanced chemiluminescence (ECL) reagent (pico EPD, Elpis-Biotec, Korea) for 1 minute.

[0142] Flow cytometry analysis FACS analysis was performed to quantify the binding of FH to S. aureus. Bacteria were cultured overnight on blood plates and suspended in PBS. After washing the bacteria twice with PBS, the number of bacteria was counted by measuring the absorbance at 600 nm. 20 μg of the CWAPN2N3 domain of ClfAN2N3, SdrCN2N3, SdrEN2N3, FnbpAN2N3, FnbpBN2N3 rabbit F(ab’)2 fragments was cultured with USA300 (2×107 cells) at 4°C for 1 hour. Thereafter, the sample was centrifuged at 1,500 x g for 3 minutes at 4°C, and the bacterial pellet was washed twice with 200 μl of PBS. Then, the bacteria were suspended in PBS, 30 μg of FH was added, and the mixture was cultured at 4°C for 1 hour. Thereafter, the sample was centrifuged at 1,500 x g for 3 minutes at 4°C, and the bacterial pellet was washed twice with 200 μl of PBS. Then, the bacteria were resuspended in 20 μl of PBS together with a polyclonal goat anti-human FH Fab’2-FITC conjugate and cultured on ice for 1 hour. S. aureus cells were sonicated for 30 seconds for flow cytometry analysis, and the measurement results of 10,000 times were recorded.

[0143] Cleavage of C3b to iC3b by human FH / FI complex in the presence of CWAPN2N3 domain 20 μl of coating buffer consisting of 25 mM sodium carbonate and 25 mM sodium bicarbonate (pH 9.6) containing 3 μg of CWAPN2N3 domain, BSA, and goat anti-human FH-IgG (1:1000 dilution) was used to coat a microtiter plate. After washing three times with PBST, the wells were incubated at room temperature for 2 hours with 100 μl of 0.2% BSA in PBS and then washed three times with PBST containing 0.05% Tween 20 (pH 7.4) in PBS. Subsequently, 0.37 μg of purified human FH was added. PBS was added instead of human FH as a negative control group. After incubation for 30 minutes, the wells were washed three times with PBST, and 75 μl of a mixture containing 1 μg of purified C3b and 1 μg of purified human FI was added to each well and incubated at 37°C for 3 hours. After incubation, the supernatant was extracted, and the amount of iC3b produced was measured by Western blot using goat anti-human C3-IgG (Complement Technology, USA) diluted 1:6000 as the primary antibody. Subsequently, the membrane was washed three times with TBST and incubated with donkey anti-goat-IgG-HRP (Santacruz, USA), the secondary antibody. After treatment with ECL reagent for 1 minute, images were obtained by the method described above.

[0144] Detection of human FH on live S. aureus USA300 LAC Bacteria (1×105 CFU) were prepared in 100 μl of PBS, and 5 μg of the CWAPN2N3 domain and 0.5 μg of FH were added, followed by incubation at 4°C for 1 hour. Subsequently, the sample was centrifuged at 1,500 xg for 3 minutes at 4°C, 90 μl of the supernatant was collected, and the bacterial pellet was washed twice with 200 μl of PBS. Finally, the bacterial pellet was resuspended in 90 μl of PBS. Each resuspended bacterial pellet and the collected supernatant were loaded onto a 6-15% gradient SDS-PAGE gel. After gel electrophoresis, the proteins were transferred to a 0.45 μm PVDF membrane (Immobilon-P Transfer Membrane, Merck, USA) at 4°C. The membrane was blocked with 5% non-fat milk in TBST at room temperature for 1 hour. Subsequently, the membrane was incubated with the primary antibody, goat anti-human FH-IgG, washed three times with TBST, and then incubated with the secondary antibody, donkey anti-goat-IgG-HRP (Santacruz, USA). After treatment with ECL reagent for 1 minute, images were obtained by the method described above.

[0145] Measurement of the amount of iC3b in the presence of the CWAPN2N3 domain S. aureus USA300 LAC strain (1×105 CFU) was prepared in 100 μl of GVB++. After adding 10 μg of CWAPN2N3 and 0.1 μg of human FH, the mixture was incubated at 4°C for 1 hour. Then, the sample was centrifuged at 1,500 xg for 3 minutes at 4°C, and 90 μl of the supernatant was collected. The bacterial pellet was washed twice with 200 μl of PBS, and 1.5 μg of purified C3b and 0.25 μg of purified human FI in GVB++ were added to the supernatant and the pellet, followed by incubation at 37°C for 3 hours. Each pellet and the collected supernatant were loaded onto a 6-15% gradient SDS-PAGE gel. After gel electrophoresis, the bands were transferred to a PVDF membrane at 4°C. The membrane was blocked with 5% non-fat milk in TBST at room temperature for 1 hour, and then incubated with the primary antibody, goat anti-human C3-IgG. Subsequently, the membrane was washed three times with TBST and incubated with the secondary antibody, donkey anti-goat-IgG-HRP (Santacruz, USA). After treatment with ECL reagent for 1 minute, images were obtained by the method described above.

[0146] Investigation of the binding specificity of rabbit anti-CWAP-F(ab’)2 to other CWAPN2N3 domains 1 μg of CWAPN2N3 domains (ClfAN2N3, ClfBN2N3, SdrCN2N3, SdrDN2N3, SdrEN2N3, FnbpAN2N3, FnbpBN2N3) was spotted onto a PVDF membrane and dried. The membrane was blocked with 5% non-fat milk in TBS for 2 hours at room temperature, and then incubated with a primary antibody (rabbit Fab’2 recognizing CWAPN2N3 domain) diluted 6,000:1 in 5% non-fat milk for 1 hour at room temperature. The membrane was washed three times with TBST for 10 minutes each, and then incubated with a secondary antibody (mouse anti-rabbit IgG HRP, Santacruz, USA) diluted 3,000:1 in 5% non-fat milk for 1 hour at room temperature. Thereafter, the membrane was washed three times with TBST for 10 minutes each, incubated with ECL reagent (pico EPD, Elpis-Biotec, Korea) for 1 minute, and then images were obtained by the method described above.

[0147] Investigation of the binding specificity of rabbit and human anti-CWAP-IgG to other CWAPN2N3 domains 200 ng of CWAPN2N3 domains (ClfAN2N3, ClfBN2N3, SdrCN2N3, SdrDN2N3, SdrEN2N3, FnbpAN2N3, FnbpBN2N3) was loaded onto an SDS-PAGE gel. After gel electrophoresis, the bands were transferred to a PVDF membrane at 4°C, blocked with 5% non-fat milk in TBS for 1 hour at room temperature, and then incubated with a primary antibody (rabbit and human IgG recognizing CWAPN2N3 domain) diluted 6,000:1 for 1 hour at room temperature. The membrane was washed three times with TBST for 10 minutes each, and then incubated with a secondary antibody (mouse anti-rabbit IgG HRP, goat anti-human IgG HRP, Santacruz, USA) diluted 3,000:1 in 5% non-fat milk for 1 hour at room temperature. Thereafter, the membrane was washed three times with TBST for 10 minutes each, incubated with ECL reagent (pico EPD, Elpis-Biotec, Korea) for 1 minute, and then images were obtained by the method described above.

[0148] Bactericidal activity of CWAPN2N3 domains and purified human or rabbit CWAPN2N3 - recognizing IgG against S. aureus in human whole blood The bactericidal assay by opsonophagocytosis using human whole blood was performed by the previously reported method (van der Maten E et al., 2017. Sci Rep 7:42137). Briefly, purified CWAPN2N3 protein, human and rabbit anti - CWAPN2N3 - IgG were diluted to 0.5 mg / ml with PBS. After adding 100 μl of fresh whole blood into a 1.5 - ml tube, 10 μl of the mixture of protein or IgG (5 μg) was added to the same tube and vortexed. Finally, after adding 100 μl of USA300 cells (1×105 cells / well), the mixture was suspended 10 times and vortexed for 10 - 20 seconds. After culturing the mixture at 37°C for 3 hours, the bacteria were serially diluted and spread on TSB agar plates or sheep blood plates to measure the CFU.

[0149] Data processing and statistical analysis Unless otherwise stated, each experiment was performed at least 3 times independently, and the experimental results were expressed as mean ± standard deviation. Other experimental results are representative values of at least 3 independent experiments with similar result values. Statistical significance was measured by the independent Student's t - test or the log - rank method using GraphPad Prism software. A P - value of 0.05 or less was considered to be statistically significant.

[0150] Experimental results Three N2 - N3 domains of the cell wall - associated protein (CWAP) bind to human complement factor H (FH). To investigate whether the N2-N3 domain of staphylococcal CWAP (referred to as "CWAPN2N3") recognizes novel ligand molecules in human serum, the coding regions of seven CWAPN2N3s (approximately 350-370 amino acids tagged with 6x His, Figures 16A and 16B) were amplified by PCR, and the PCR products (Figure S1) were cloned into the pET28a vector to produce expression constructs (pET28a-CWAPN2N3). To overproduce His-tagged recombinant CWAPN2N3, E. coli BL21(DE3) cells carrying pET28a-CWAPN2N3 were stimulated with 0.2 mM IPTG at 37 °C for 3 hours. The protein purified on a nickel-affinity column migrated on SDS-PAGE to match the expected molecular weight of 35-45 kDa (Figure 16C). The yield of purified CWAPN2N3 was calculated to be 15-20 mg per liter of culture medium.

[0151] Since CWAPN2N3 has a similar three-dimensional structure (Arora S, et al., 2016. Front Microbiol 7:540), the inventors attempted to examine whether four human proteins, fibrinogen (Fg), fibronectin (Fn), complement factor H (FH), and complement factor I (FI), recognize seven CWAPN2N3s in vitro. After confirming that the same amount of protein was blotted onto the PVDF membrane by Ponceau S staining, each PVDF membrane strip was cultured with each 7 μg of human Fg, Fn, FH, and FI proteins to perform a dot blot immunoassay. After washing the unbound proteins, the bound proteins on the membrane were tested using anti-Fg-, Fn-, FH-, and FI-IgG (Figure 17A). Under the above conditions, human Fg showed a high specific binding affinity with FnbpAN2N3 as previously reported (Deivanayagam CC et al., 2002. Embo j 21:6660-72.), while Fn specifically bound to FnbpBN2N3 (Broker BM et al, 2014. Int J Med Microbiol 304:204-14), and it was found that the two FnbpN2N3 proteins had a high binding affinity with human Fg and Fn proteins. In addition, human FH was recognized by four CWAPN2N3s, ClfAN2N3, SdrEN2N3, FnbpAN2N3, and FnbpBN2N3. Under the same conditions, five CWAPN2N3 proteins, ClfAN2N3, SdrCN2N3, SdrEN2N3, FnbpAN2N3, and FnbpBN2N3, showed binding ability to FI. Interestingly, ClfBN2N3 and SdrDN2N3 did not show binding ability to these four human proteins.Interactions between FnbpA and Fg, FnbpB N2N3 and Fn, and SdrE N2N3 and FH or FI have been reported (Sharp JA, et al., 2012. PLoS One 7: e38407; Bingham RJ et al., 2008. Proc Natl Acad Sci USA 105: 12254 - 8; Burke FM e al., 2011. FEBS J 278: 2359 - 71; Zhang Y et al., 2017. Biochem J 474: 1619 - 163130), but the interactions between ClfA N2N3, FnbpA N2N3, FnbpB N2N3 and FH, FI are not known.

[0152] If these CWAPN2N3 domains can bind to human FH, the FH-binding CWAPN2N3 domains can induce the conversion of complement C3b to iC3b in the presence of FI. To prove such a possibility, SdrEN2N3 and anti-FH-IgG proteins were used as positive control groups, because SdrEN2N3 is known to bind to human FH (Sharp JA et al., 2012. PLoS One 7: e38407; Zhang Y et al., 2017. Biochem J 474: 1619-1631), and anti-FH-IgG binds to FH. As shown in Figure 17B, seven CWAPN2N3 proteins were coated on microplates together with positive control group proteins, and BSA was also coated as a negative control group. After culturing with FH, unbound FH was washed away. After adding C3b and FI, the plates were cultured at 37 °C for 3 hours. The formed iC3b was detected by Western blot using anti-C3-mAb (Figure 17B). As expected, the BSA-coated fraction could hardly form the 42 kDa band of iC3b even after adding FH / C3b / FI (lane 1), indicating that BSA could not attract FH. However, the SdrEN2N3- and anti-FH-IgG-coated fractions clearly formed the 42 kDa band in the presence of FH / C3b / FI (lanes 11 and 17), but not when adding FI / C3b only (lanes 12 and 18), indicating that SdrEN2N3 and anti-FH-IgG attracted FH and caused the conversion of C3b to iC3b by FI. Under the same conditions, the ClfAN2N3-, FnbpAN2N3- and FnbpBN2N3-coated fractions generated the 42 kDa band by adding FH, C3b and FI (lanes 3, 13 and 15 respectively), but not when FH was absent (lanes 4, 14 and 16). Through such results, it was reconfirmed that the ClfAN2N3, FnbpAN2N3, FnbpBN2N3 and SdrEN2N3 domains can bind to human FH, and FH attracts FI to enable the hydrolysis of complement C3b to iC3b in vitro.

[0153] The addition of the FH-binding CWAPN2N3 protein prevents FH from accumulating on the S. aureus cell surface, thereby reducing the conversion of C3b to C3b. Since it was confirmed that four S. aureus CWAPs can bind to FH, we sought to determine whether the addition of the FH-binding CWAPN2N3 protein can inhibit the movement of FH to live S. aureus cells expressing CWAP. For this purpose, S. aureus USA300 LAC live cells were cultured with FH and five different proteins (ClfAN2N3, SdrEN2N3, FnbpAN2N3, FnbpBN2N3, and SdrC) (Figure 18A). Under PBS, all added FH proteins migrated to the bacterial surface (pellet) (lane 2) and were not present in the supernatant (lane 1). In the case of ClfAN2N3, SdrEN2N3, and FnbpAN2N3, approximately half of the added FH was present in the supernatant (lanes 3, 5, and 7), and the other half was present in the pellet (lanes 4, 6, and 8). As expected, the addition of FnbpBN2N3 completely inhibited the increase in FH on the bacterial surface (lane 10), and most of the added FH accumulated in the supernatant (lane 9). Conversely, in the case of SdrCN2N3, which was shown to have no binding ability to FH (Figure 17A), all added FH migrated to the bacterial surface that was not in the supernatant (lane 12), confirming that the SdrCN2N3 protein has no inhibitory effect on the binding of FH to S. aureus (lane 11). Collectively, these results indicate that the four FH-binding proteins, FnbpAN2N3, FnbpBN2N3, SdrEN2N3, and ClfAN2N3, function as competitive inhibitors of the CWAP protein expressed on the bacterial surface by binding to FH. Among the four proteins, FnbpBN2N3 significantly inhibited the movement of FH to the S. aureus surface.

[0154] To additionally verify such observation results, the inventors attempted to examine whether the FH-recognizing CWAPN2N3 protein blocks the formation of FH-mediated iC3b on the S. aureus USA300 cell surface. When S. aureus cells are cultured with FH / C3b / FI in the absence of the FH-binding CWAPN2N3 protein, it is expected that the FH transferred to the bacterial surface generates a larger amount of 43 kDa iC3b fragments from 100 kDa C3b. When the FH-binding CWAPN2N3 protein is added, since the CWAPsN2N3 protein captures FH, only a small amount of FH is transferred to the bacterial surface and a smaller amount of 43 kDa iC3b accumulates on the bacterial surface. To confirm such a possibility, when S. aureus USA300 cells are cultured with FH / C3b / FI and each of the five proteins (Figure 18B), the amount of iC3b generated in the PBS and SdrC-added fractions was higher in the four FH-binding CWAPN2N3 proteins (lanes 1 and 3 vs lanes 2, 4-6, respectively). Such results indicate that the FH-binding CWAPN2N3 protein suppresses the conversion from C3b to iC3b by capturing FH, thereby blocking the FI-mediated conversion of C3b to iC3b on the bacterial surface.

[0155] Rabbit anti-CWAPN2N3 F(ab’)2 fragments suppress the transfer of FH to the S. aureus surface. Next, we sought to examine whether rabbit anti-FH-binding CWAPN2N3-IgG could also inhibit the movement of FH to the surface of live S. aureus USA300 cells. To this end, there was a difficulty in using rabbit anti-human FH-IgG or rabbit anti-CWAPN2N3-IgG in that S. aureus proteins non-specifically captured IgG by binding to the Fc domain of IgG. To solve this, pepsin was used to treat the native FH-IgG to produce six different F(ab’)2 fragments (Figure 23). As shown in Figure 24A, the purified anti-FH-Fab’2 and five anti-CWAPN2N3 F(ab’)2 fragments showed a size of 100 kDa under non-reducing conditions and 25 kDa under reducing conditions, confirming that all the anti-F(ab’)2 fragments were properly produced. As a result of examining the binding specificity of the six purified anti-F(ab’)2 fragments to homologous antigens, four F(ab’)2 fragments of ClfAN2N3, FnbpAN2N3, FnbpBN2N3 and FH specifically recognized these homologous antigens (Figures 24B and 24C), while two F(ab’)2 fragments of SdrCN2N3 and SdrEN2N3 weakly recognized SdrDN2N3 and FnbpBN2N3, respectively (Figure 24C), confirming that the six F(ab’)2 fragments produced had the desired properties.

[0156] As shown in Fig. 19A, when USA300 cells were co-cultured with FH, FITC-conjugated anti-FH-Fab’2 sections successfully recognized the FH that had migrated to the bacterial surface, and it was found that FH bound to the CWAP protein of live USA300 bacterial cells. Furthermore, to examine the binding specificity between the four CWAPN2-N3 proteins of live bacteria and FH, S. aureus USA300 cells (2×107 cells) were first cultured with five different anti-CWAP F(ab’)2 sections (each with a protein amount of 20 μg), and the bacterial cells were washed to remove unbound anti-CWAP F(ab’)2 sections. Thereafter, S. aureus USA300 cells were cultured with purified FH (each with a protein amount of 30 μg), and then the bacterial cells were washed to remove unbound FH. Thereafter, the amount of FH that had migrated to the surface of each anti-CWAP F(ab’)2-section-treated bacterial cell was examined by FACS analysis using FITC-conjugated anti-FH-Fab’2 sections (Fig. 19A). Bacterial cells treated with three anti-CWAPN2N3-F(ab’)2 sections (SdrE, FnbpA, and FnbpB) showed specifically suppressed binding of FH compared to anti-F(ab’)2-section-untreated cells (c-e vs a in Fig. 19A), but treatment with anti-ClfA F(ab’)2 sections showed only a low level of inhibitory effect (b in Fig. 19A). As expected, treatment with 20 μg of anti-SdrC-F(ab’)2 sections also had no effect on the binding of FH to USA300 cells (f in Fig. 19A). In summary, these results reconfirm that the four purified anti-CWAPN2N3 antibodies can block the binding between the CWAP protein expressed on the surface of live bacteria and FH.

[0157] Based on FACS data, the amount of FH that migrated to the bacterial surface was quantified (Figure 19B). As expected, the anti-FnbpB-F(ab’)2 fragment inhibited the binding of FH to the S. aureus cell surface by 60% compared to the control group (column 6 vs 1). The inhibitory ability decreased gradually in the order of anti-SdrE (50%, column 4) > FnbpA (40%, column 5) > ClfA-Fab’2 (30%, column 6). The anti-SdrC-F(ab’)2 fragment showed little inhibitory effect (10%, column 3), and these results are consistent with those in Figure 2A.

[0158] Purified rabbit FH-binding CWAPN2N3 proteins and these antibodies enhance the human whole blood-mediated bactericidal activity against S. aureus USA300 cells If the inventors obtain human and rabbit anti-CWAPN2N3 domains that recognize IgG from human intravenous injection IgG (IVIg) and rabbit serum, respectively, these anti-FH-binding CWAPN2N3-recognizing IgGs should bind to the S. aureus CWAP protein. By such binding, the movement of FH to the S. aureus surface is blocked, and ultimately, the C3b-mediated opsonophagocytosis by host phagocytes can be enhanced. To prove this, purified human and rabbit anti-CWAPN2N3-recognizing IgGs such as anti-ClfAN2N3, anti-SdrEN2N3, anti-FnbpAN2N3, and anti-FnbpBN2N3-IgG were purified. The purification of these four IgGs was performed using these specific protein-conjugated sepharose columns. Since healthy human serum is known to contain anti-S. aureus CWAP-specific antibodies (Dryla A et al., 2005. Clin Diagn Lab Immunol 12:387-98), commercially available human IVIg was used as the human serum source. Rabbit serum was obtained by inoculating the CWAPN2N3 domain into rabbits three times. After loading human IVIg or anti-CWAPN2N3 domain-inoculated rabbit serum onto the CWAPN2N3-conjugated column, rabbit anti-CWAPN2N3 domain-recognizing-IgG (Figure 24) and human anti-CWAPN2N3 domain-recognizing-IgG (Figure 25) were eluted. As a result, human IVIg contained approximately 0.1% of anti-CWAPsN2N3 domain-recognizing IgG, and rabbit serum contained approximately 1% of anti-CWAPN2N3 domain-recognizing IgG, respectively. As a result of examining the purity of these four IgGs under reducing and non-reducing conditions by SDS-PAGE (Figure 26), it was found that the purified IgGs form tetramers under non-reducing conditions.

[0159] Next, the specificities of the purified rabbit and human IgG against each antigen (FnbpAN2N3, FnbpBN2N3, ClfAN2N3, and SdrEN2N3 domains) were examined by Western blot or dot blot, respectively. As shown in Figure 20A, the four rabbit anti-CWAPN2N3-recognizing IgG specifically recognized these homologous antigens against the seven CWAPN2N3 domains, while the purified human anti-CWAP-IgG showed a broad recognition pattern excluding ClfA (Figure 20B). For example, anti-SdrEN2N3-IgG recognized ClfA and four antigens of three Sdr families. Also, the anti-FnbpAN2N3 domain recognized five antigens of three Sdr and two FNBP families. Anti-FnbpBN2N3-IgG also recognized four antigens of ClfBN2N3, SdrEN2N3, and two FnbpN2N3 proteins (Figure 20B). Such results indicate that human anti-CWAPN2N3-IgG has a broader recognition pattern than rabbit anti-CWAPN2N3-IgG.

[0160] Finally, the inventors sought to examine the opsonophagocytosis-mediated bacterial killing activity of the CWAPN2N3 domain or their purified antibodies. S. aureus USA300 cells (1×105 cells) were cultured with the CWAPN2N3 domain protein (5 μg) in 100 μl of human whole blood at 37 °C for 3 hours, and then the CFU (colony-forming unit) of S. aureus USA300 LAC cells was measured. When each CWAPN2N3 domain was added, the CFU decreased significantly compared to the control group (Figure 21A). Such results support that the CWAPN2N3 domain can suppress the movement of FH to the S. aureus cell surface and increase the accumulation of C3b, inducing C3b-mediated opsonophagocytosis by host phagocytic cells. Similarly, the killing effect of human and rabbit anti-CWAPN2N3-IgG-mediated bacteria on S. aureus USA300 LAC cells was examined using human whole blood (Figures 21B and 21C). When human anti-CWAPN2N3-IgG (5 μg) was cultured with S. aureus USA300 LAC cells (1×105 cells) in 100 μl of human whole blood at 37 °C for 3 hours, the CFU decreased 2- to 4-fold compared to the control groups such as PBS, human IVIg, and pure rabbit serum (Figures 21B and 21C). Furthermore, when a mixture of anti-ClfAN2N3 and FnbpBN2N3 IgG was added to the mixture of bacteria and whole blood, the CFU also decreased compared to the control group (Figure 21B). Also, when a mixture of 5 μg of rabbit anti-ClfA, anti-FnbpB, and anti-FNBPAN2N3-IgG was added to the mixture of bacteria and whole blood, the CFU decreased 2-fold compared to pure rabbit IgG (Figure 21C). In the case of rabbit IgG, the four IgG mixtures showed higher bacterial killing activity than one IgG. In summary, such results indicate that anti-CWAPN2N3 domain IgG has specific bacterial killing activity against the S. aureus USA300 LAC strain when cultured with human whole blood.

[0161] Example 3: Selection of Blood Coagulation Inhibitors Experimental Method Purification of Recombinant Coa and vWbp Proteins Six polyhistidine-tagged recombinant proteins (Coawhole, CoaN, CoaC, vWbpwhole, vWbpN, and vWbpC) were cloned and expressed using an E. coli expression system. These genes were amplified from the USA300 genomic sequence by PCR using Q5 High Fidelity DNA polymerase (Thermo Fisher Scientific). The PCR products were cloned into the pET28a-vector to express proteins tagged with 6x His at the N-terminus or C-terminus. To purify the cloned recombinant proteins, 6x His-tags were inserted on both sides of the N- and C-termini, and the resulting clones were sequenced to confirm the structure. The recombinant proteins were expressed in BL21 pLysS E. coli using 0.2 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG, at a concentration of 0.2 mM). The expressed proteins were purified using Ni-Sepharose 6 Fast resin (GE healthcare, 17-5318-01, 5 ml) and loading buffer (20 mM sodium phosphate, 0.1% triton X-100, 150 mM NaCl, 5 mM imidazole, pH 7.4), washing buffer (20 mM sodium phosphate, 150 mM NaCl, 5 mM imidazole, pH 7.4), and elution buffer (20 mM sodium phosphate, 150 mM NaCl, 500 mM imidazole, pH 7.4).

[0162] SDS-PAGE analysis of purified recombinant Coa and vWbp domain proteins 3 μg of each domain protein (Coawhole, CoaN, CoaC, vWbpwhole, vWbpN, vWbpC) was mixed with 4X loading sampling buffer (LSB, SH+) and loaded onto a 15% SDS-PAGE gel for 1 hour (35 mA, 600 V, 100 W).

[0163] Rabbit inoculation to obtain Coa and vWbp protein antibodies 500 μg of the purified single components of S. aureus Coa and vWbp proteins dissolved in 500 μl of PBS were mixed with 500 μl of complete Freund's adjuvant (Sigma-Aldrich) to prepare an emulsion. After subcutaneous injection of each emulsion into rabbits, the same amount of antigen was mixed with 500 μl of incomplete Freund's adjuvant (Sigma-Aldrich) and subcutaneously injected 2 weeks later. Two weeks later, 500 μl of blood was taken from the rabbit ear vein, and the presence or absence of IgG formation for each antigen was examined by Western blot. When antibodies were formed, rabbit whole blood was collected to obtain serum, which was stored at -80 °C until use.

[0164] Investigation of antibody specificity against different domain proteins of Coa and vWbp 3 μg of each domain protein (Coawhole, CoaN, CoaC, vWbpwhole, vWbpN, vWbpC) and negative control group protein (BSA) were spotted onto a PVDF membrane (0.22 μm, Immobilon-PSQ Transfer Membrane, Merck, USA) and dried. The membrane was blocked with 5% skim milk in TBS at room temperature for 2 hours and then incubated with primary antibodies (anti-Coawhole, CoaN, -vWbpwhole, -vWbpN) diluted 3,000:1 with 5% skim milk at room temperature for 1 hour. The membrane was washed 3 times with TBST for 10 minutes each and then incubated with secondary antibody (mouse anti-rabbit IgG HRP, Santacruz, USA) diluted 6,000:1 with 5% skim milk at room temperature for 1 hour. Thereafter, the membrane was washed 3 times with TBST for 10 minutes each and incubated with ECL reagent (pico EPD, Elpis-Biotec, Korea) for 1 minute.

[0165] Expression patterns of Coa and vWbp proteins during S. aureus USA300 culture USA300 was cultured overnight at 37 °C and 180 rpm in 10 ml of RPMI. Thereafter, 1 ml of bacterial seed culture solution was added to 50 ml of RPMI, and while culturing at 37 °C and 180 rpm, 1 ml of the bacterial culture solution was taken at each time point (0 h, 1.5 h, 3 h, 6 h, 9 h, 12 h). Thereafter, the absorbance (OD600) of the collected bacterial culture solution was measured, and it was centrifuged at 4,400 xg for 3 minutes at room temperature. 20 μl of the bacterial supernatant was loaded onto a 15% SDS-PAGE gel, and after electrophoresis, the bands were transferred to a PVDF membrane at 4 °C. The membrane was blocked with 5% skim milk in TBST for 1 hour at room temperature. Thereafter, the membrane was cultured with the primary antibodies anti-rabbit-Coawhole- and anti-rabbit-rvWbpwhole-IgG. The membrane was washed 3 times with TBST and cultured with the secondary antibody mouse anti-rabbit-IgG-HRP (Santacruz, USA). After treating with ECL reagent for 1 minute, an image was obtained.

[0166] Identification of Coa and vWbp-recognizing proteins expressed on the cell surface of S. aureus 3 μg of the N2-N3 domains of 7 recombinant MSCRAMMs (ClfAN2N3, ClfBN2N3, SdrCN2N3, SdrDN2N3, SdrEN2N3, FnbpAN2N3, FnbpBN2N3) and BSA as a negative control group were spotted onto a PVDF membrane (Immobilon-PSQ Transfer Membrane, Merck, USA) and dried. The membrane was blocked with 5% skim milk in TBS at room temperature for 2 hours and incubated with 7 μg each of the purified recombinant proteins Coawhole, CoaN, CoaC, vWbpwhole, vWbpN, vWbpC dissolved in 700 μl of 5% skim milk. Subsequently, the membrane was washed 3 times with TBST for 10 minutes each and incubated with primary antibodies (anti-rabbit-Coawhole-IgG, anti-rabbit-CoaN-IgG, anti-rabbit-CoaC-IgG, anti-rabbit-vWbpwhole-IgG, anti-rabbit-vWbpN-IgG, anti-rabbit-vWbpC-IgG) diluted 3,000:1 at room temperature for 1 hour. The membrane was washed 3 times with TBST for 10 minutes each and incubated with a secondary antibody (mouse anti-rabbit IgG HRP, Santacruz, USA) diluted 6,000:1 in 5% skim milk at room temperature for 1 hour. Subsequently, the membrane was washed 3 times with TBST for 10 minutes each and incubated with an enhanced chemiluminescence (ECL) reagent (pico EPD, Elpis-Biotec, Korea) for 1 minute.

[0167] Confirmation of the binding specificity between Coa and Fnbp proteins using anti-Fnbp-F(ab’)2 sections Bacteria (USA300, 1×105 CFU) were cultured at 4°C for 1 hour with 3 μg of CoaC protein or 20 μg of anti-FnBp-F(ab’)2 and anti-SdrC-F(ab’)2 sections as the negative control group. Subsequently, the samples were centrifuged at 4,400 xg for 3 minutes at 4°C, and the bacterial pellet was washed twice with 500 μl of PBS. Each resuspended bacterial pellet was loaded onto a 15% gradient SDS-PAGE gel, and after electrophoresis, the bands were transferred to a 0.45 μm PVDF membrane (Immobilon-P Transfer Membrane, Merck, USA) at 4°C. The membrane was blocked with 5% skim milk in TBST at room temperature for 1 hour and cultured with the primary antibody, anti-rabbit-CoaC-IgG. Subsequently, it was washed three times with TBST and cultured with the secondary antibody, mouse anti-rabbit IgG HRP (Santacruz, USA). After treating with ECL reagent for 1 minute, an image was obtained.

[0168] Induction of blood coagulation by each dose of purified Coawhole or vWbpwhole 200 μl of hirudin-treated human blood was placed in a 5 ml round-bottom tube, and various doses of Coawhole and vWbpwhole (1 μg, 5 μg, 10 μg, 20 μg, 40 μg, 50 μg) or PBS were added. The samples were cultured at room temperature, and blood coagulation was confirmed while tilting the tubes at a 45° angle at 1.5-hour intervals.

[0169] Measurement of blood coagulation blockade by each dose of purified anti-Coawhole-IgG or anti-vWbpwhole-IgG 200 μl of hirudin-treated human blood was placed in a 5 ml round-bottom tube, and various doses of anti-Coawhole-IgG and anti-vWbpwhole-IgG (10 μg, 20 μg, 40 μg, 80 μg, 100 μg) were added. Subsequently, 5 μg of Coawhole and vWbpwhole or PBS were added. The samples were cultured at room temperature, and blood coagulation was confirmed while tilting the tubes at a 45° angle at 1.5-hour intervals.

[0170] Inhibitory effect of Coa and vWbp-mediated blood coagulation by anti-Coawhole IgG or anti-vWbpwhole IgG S. aureus USA300 LAC cells were cultured in 50 ml of RPMI at 37 °C for 2 hours at 180 rpm. After centrifuging the bacteria at 4,400 x g for 3 minutes at 4 °C, 100 μl of the bacterial supernatant was added to 200 μl of hirudin-treated human blood. 5 μg of vWbpwhole and 1 μg of Coawhole were added to 200 μl of hirudin-treated human blood and used as a positive control group. 80 μg of anti-Coawhole-IgG and 80 μg of anti-vWbpwhole-IgG or a mixture of 40 μg each of anti-Coawhole-IgG and anti-vWbpwhole-IgG were first added to 200 μl of hirudin-treated human blood, and then 100 μl of the bacterial supernatant was added. The samples were cultured at room temperature, and blood coagulation was confirmed while tilting the tubes at an angle of 45° at 1.5-hour intervals.

[0171] Inhibitory effect of anti-Coawhole-IgG or vWbpwhole-IgG on blood coagulation in vivo To verify the in vivo effects of Coawhole and vWbpwhole, an in vivo passive immunity experiment was conducted with the antibodies obtained by injecting these into rabbits. First, 4 rabbits were divided into 2 groups of 2 each (Figure 37). The first group was injected with PBS and then 1.6 × 108 CFU of S. aureus USA300 bacteria into the auricular vein. The second group was injected with a mixture of 1 mg of anti-coawhole-IgG purified by a coawhole protein-conjugated sepharose column and 1 mg of anti-vWbpwhole-IgG purified by a vWbpwhole protein-conjugated sepharose column into the left auricular vein, and then 1 hour later, 1.6 × 108 CFU of USA300 bacteria was injected into the right auricular vein. Thereafter, the body weight changes and survival rates of each rabbit were examined.

[0172] Experimental results Cloning and purification of recombinant S. aureus coagulase and von Willebrand Factor (vWF)-binding domain protein (vWbp) Since S. aureus Coa and vWbp proteins have been proposed to act as major virulence factors of S. aureus, the inventors hypothesized that these factors bind to the bacterial surface via specific receptors in order to avoid the host immune system by forming fibrin clots in blood vessels rather than being simply secreted during the infection process. This suggests the presence of some uncharacterized receptor proteins on the bacterial surface. If such receptors on the bacterial surface could be discovered, antibodies against them could be used to block the binding between Coa or vWbp and the receptors, thereby preventing fibrin clot formation during the S. aureus infection process. To identify these receptors expressed on the bacterial surface, attempts were made to produce recombinant proteins containing Coa and vWbp domains and antibodies against them.

[0173] Investigation of antibody - specificity against different domain proteins of Coa and vWbp using dot blot As shown in Fig. 27, six proteins were prepared, namely the full-length Coa and vWbp proteins, the N-terminal D1-D2 domains of Coa and vWbp, and the C-terminal repetitive domains of Coa and vWbp, based on the genomic sequence of the S. aureus USA300 LAC strain. The full-length Coa and vWbp proteins consist of 607 and 508 amino acids (aa), respectively. The N-terminal domains of Coa and vWbp are composed of 284 and 253 aa, and the C-terminal repetitive domains are composed of 297 and 233 aa, respectively. After cloning these genes, six different recombinant proteins (Coa-whole, Coa-N, Coa-C, vWbp-whole, vWbp-N, and vWbp-C) were expressed and then purified uniformly as shown in Fig. 28. These proteins were used to generate rabbit polyclonal anti-Coa and vWbp-recognizing IgG by two subcutaneous inoculations. As a result of examining the antigen recognition specificity of IgG for each purified protein, anti-Coawhole-IgG recognized Coawhole, CoaN, and CoaC, whereas anti-CoaN-IgG recognized only Coawhole and CoaN and could not recognize CoaC (Fig. 29A). In addition, anti-vWbp-IgG also showed specific binding to these antigens (Fig. 29B). Such results indicate that the four generated Coa and vWbp-IgGs have appropriate specificity for these antigens.

[0174] Expression patterns of Coa and vWbp proteins during S. aureus USA300 culture Since all six proteins and their rabbit polyclonal antibodies were obtained, the expression patterns of these two secretory enzymes were examined by Western blotting using RPMI medium in which the S. aureus USA300 strain was cultured at each time point. As shown in Fig. 30A, the Coawhole protein (60 kDa) was secreted at the 1.5-hour culture time point, and the maximum amount was detected at the 3-hour culture time point, and then gradually decreased until the 9-hour time point. As expected, S. aureus protein A (45 kDa) accumulated in the culture supernatant until the 3-hour culture time point (red arrow). However, vWbp (50 kDa) was detected at the maximum amount at the 1.5-hour culture time point and then gradually decreased until the 12-hour time point (Fig. 30B). Protein secretion showed a pattern similar to that of Coa (Fig. 30B). It can be seen from this that the maximum amount of Coawhole and vWbpwhole proteins is secreted between 1.5 and 3 hours.

[0175] Identification of Coa- and vWbp-recognizing proteins expressed on the S. aureus cell surface The present inventors attempted to identify proteins that recognize Coa and vWbp, which are expressed on the surface of bacteria during bacterial growth. First, the S. aureus cell wall-associated protein (CWAP) was focused on as a molecule that might recognize these two secreted proteins. The present inventors applied seven recombinant CWAP proteins to the screening of Coa- and vWbp-recognizing proteins using a dot blot immunoassay (Figure 31). After spotting these seven CWAP proteins and the control BSA on PVDF membrane strips, each strip was co-cultured with six purified recombinant Coa and vWbp domain proteins, and the strip was cultured with each domain-specific IgG to examine which domains of Coa or vWbp bind to the CWAP N2N3 protein. As shown in Figure 31A, the Coawhole, CoaN, and CoaC proteins specifically recognized the FnbpA N2N3 and FnbpB N2N3 domains. Under the same conditions, the vWbpwhole protein showed binding ability to five different N2-N3 domains, namely the ClfB N2N3, SdrC N2N3, SdrE N2N3, FnbpA N2N3, and FnbpB N2N3 domains. The vWbp N domain strongly bound to FnbpA N2N3 and weakly bound to the remaining three CWAP proteins (Figure 31B). However, the vWbp-C protein showed a broader binding ability to the remaining N2-N3 domains except for ClfA N2N3 and SdrD N2N3. Such results suggest that the Coa protein recognizes the Fnbp protein expressed on the bacterial surface, and thus, the two S. aureus Fnbp CWAP proteins can function as Coa-recognizing proteins. However, the vWbp protein is widely recognized by some S. aureus CWAPs.

[0176] Confirmation of the binding specificity between Coa and Fnbp proteins using anti-Fnbp F(ab’)2 fragments Since specific binding between the Coa protein and the two FnbpN2N3 proteins was observed, we attempted to confirm this again using live S. aureus USA300 bacteria, Coawhole, and anti-Fnbp-F(ab’)2 fragments. The reason for preparing the anti-Fnbp-F(ab’)2 fragments was that it was known that S. aureus protein A binds to the IgG Fc domain and depletes the added IgG, so it was predicted that anti-Fnbp-IgG would not be able to effectively suppress the interaction between Coawhole and the Fnbp proteins of USA300 cells. We hypothesized that by using the anti-Fnbp-F(ab’)2 fragments, we would be able to confirm whether the binding between Coa and the two FnbpN2N3 proteins was specific. As shown in Figure 32, when S. aureus cells were cultured with the CoaC protein or a mixture of CoaC and anti-SdrC-F(ab’)2 fragments, the CoaC-protein migrated to the bacterial pellet (lanes 1 and 2). However, when the bacterial cells were cultured with CoaC and anti-FnbpAN2N3-F(ab’)2 fragments or anti-FnbpBN2N3-F(ab’)2 fragments, the added CoaC did not migrate to the bacterial pellet (lanes 3 and 4), indicating that the interaction between the Fnbp expressed on the bacterial surface and the CoaC protein was specifically inhibited by the anti-Fnbp-F(ab’)2 fragments. From these results, we confirmed that the Coa protein recognizes the Fnbp proteins expressed on the bacterial surface.

[0177] Blood coagulation induction by Coawhole or vWbpwhole and blood coagulation inhibition effect by anti-Coawhole-IgG or vWbpwhole-IgG Next, the inventors sought to examine the biological functions of recombinant Coawhole and vWbpwhole using human whole blood. When human blood was cultured with various doses of Coawhole protein, blood coagulated when 1 μg of Coawhole protein was added, and blood coagulated when 5 μg of vWbpwhole was added (Figure 33A), confirming that the purified recombinant Coawhole and vWbpwhole proteins are functionally active proteins. Next, the inventors sought to examine the effects of anti-Coawhole-IgG and anti-vWbpwhole-IgG on Coa- and vWbp-mediated blood coagulation (Figure 33B). When 80 μg of anti-Coawhole-IgG or 80 μg of anti-vWbpwhole-IgG was added to a mixture of human blood and 5 μg of Coawhole or vWbpwhole protein, all enzyme-mediated blood coagulation was suppressed. This confirmed that the generated anti-Coawhole- and vWbpwhole-IgG inhibit Coawhole- or vWbpwhole protein-mediated blood coagulation reactions in vitro.

[0178] Anti-Coawhole-IgG and anti-vWbpwhole-IgG inhibit USA300 culture medium-mediated human blood coagulation. As shown in Figure 30, since Coa and vWbp that had already been secreted during S. aureus 300 LAC cell culture were detected, an attempt was made to examine whether secreted Coa- and vWbp-mediated blood coagulation was blocked by anti-Coawhole-IgG and anti-vWbpwhole-IgG. When 200 μl of human whole blood was cultured with 100 μl of USA300 culture medium (containing approximately 0.5 μg of Coa and 0.5 μg of vWbp protein), the blood coagulated after 1.5 hours of culture (tube 4 in Figure 34). When 80 μg of purified anti-Coawhole-IgG or anti-vWbpwhole-IgG was added under the same conditions, the blood did not coagulate (tubes 6 and 7). Also, when 40 μg each of anti-Coawhole-IgG or anti-vWbpwhole-IgG was added to Coa- or vWbp-containing blood, coagulation was induced (tube 8). Through such results, it can be seen that Coa or vWbp secreted by live bacteria is suppressed by the added IgG.

[0179] The CoaN and vWbpN domains induced human blood coagulation, but the CoaC and vWbpC proteins did not. To examine which domains of the Coa and vWbp proteins are involved in human blood coagulation, whole blood was cultured with four proteins: CoaN, vWbpN, CoaC, and vWbpC. After 1.5 hours of culture, only the two N-terminal domains, CoaN and vWbpN, induced blood coagulation, while the two C-terminal domains did not (Figure 35). From such results, it was found that the prothrombin-binding N-terminal domain, rather than the fibrinogen-binding C-terminal domain, is essential for human blood coagulation.

[0180] CoaN- and vWbpN-mediated human blood coagulation or blood coagulation by Coa and vWbp secreted by USA300 is suppressed by anti-CoaN-IgG and anti-vWbpN-IgG, rather than anti-CoaC and anti-vWbpC IgG. Since it was observed that CoaN or vWbpN is important for blood coagulation, two trigger proteins were used to examine whether anti-CoaN- or vWbpN-specific IgG can block these protein-mediated coagulations. One was Coa and vWbp contained in the USA300 culture medium (Figure 36A), and the other was purified recombinant CoaN or vWbpN (Figure 36B). When anti-CoaN-IgG or anti-vWbpN-IgG was added to tubes (tube 2) containing culture medium and blood, coagulation was not induced (tubes 3 and 4), but when anti-CoaC-IgG or anti-vWbpC-IgG was added, coagulation was induced (tubes 5 and 6). Also, CoaN- and vWbpN-mediated human blood coagulation was inhibited by anti-CoaN-IgG and anti-vWbpN-IgG (Figure 36B, tubes 4 and 5). Such results indicate that anti-CoaN-IgG and anti-vWbpN-IgG are important molecules for preventing Coa- or vWbp-mediated blood coagulation due to S. aureus infection.

[0181] Inhibitory effect of anti-Coawhole-IgG or vWbpwhole-IgG on blood coagulation in vivo Since the inventors observed the fact that Coawhole, vWbpwhole, CoaN, and vWbpN domains have blood coagulation-inducing activity in human blood and that each polyclonal antibody obtained by inoculating these four proteins into rabbits selectively inhibits the blood coagulation reaction caused by these proteins, a passive immunity experiment in vivo was conducted using the antibodies obtained by injecting Coawhole and vWbpwhole proteins into rabbits.

[0182] Among 4 rabbits, after injecting 2 rabbits in the first group with PBS, 1.6×108 CFU of S. aureus USA300 bacteria were injected into the auricular vein. After injecting 2 rabbits in the second group with a mixture of 1 mg of anti - coa whole - protein - conjugated sepharose column - purified anti - coa whole - IgG and 1 mg of vWbp whole - protein - conjugated sepharose column - purified anti - vWbp whole - IgG into the left auricular vein, 1.6×108 CFU of USA300 bacteria were injected into the right auricular vein 1 hour later.

Table 4

[0183] As a result of measuring the body weights of the rabbits at 12 - hour intervals, the first rabbit in the first group died after 138 hours, while all rabbits in the second group survived until 167 hours and the least weight loss was observed (Figure 38). Based on the calculation of the survival rate, the rabbits in the second group (No. 2 and No. 3) injected with 1 mg each of anti - vWbp whole - IgG and anti - i - vWbp whole - IgG had the result of surviving for 7 days with the least weight loss (Figure 39). Through the above results, the blood - coagulation inhibitory effects of anti - coa - IgG and anti - vWbp - IgG confirmed in vitro were confirmed again in vivo.

[0184] Example 4: Confirmation of the combined administration effect with antibiotics In order to maximize the therapeutic effect of the combination of 4 kinds of staphylococcal toxins discovered in Example 1 on MRSA - infected diseases, the inventors explored the optimal antibiotic that can completely remove the remaining MRSA strains in the kidneys of infected individuals by combined administration with the toxins. As a result, as described below, it was confirmed that there is a significant synergistic effect when vancomycin and teicoplanin are administered in combination with the vaccine composition of the present invention.

[0185] 4 - 1: Vancomycin Experimental method First, 20 μg each of four types of antigens (HlgA, LukS, HlaH35L, and LukAE323AB), for a total of 80 μg, was dissolved in 250 μl of saline, then mixed with the same volume of alum adjuvant, cultured for 1 hour, and then intramuscularly injected three times at two-week intervals. Thereafter, sera from five rabbits inoculated two weeks later were collected, and total IgG in the sera was separated and purified using a protein A column. After the purified total IgG was intravenously injected into rabbits at a concentration of 10 mg / kg / rabbit, vancomycin (7.5 mg / kg / rabbit) was intramuscularly injected, and then the rabbits were infected with S. aureus USA300 MRSA bacteria (1×109 cells / rabbit), and the survival rate of the rabbits, the number of residual bacteria in the kidneys, and the appearance of abscesses formed in the kidneys were examined for 15 days.

[0186] A total of 20 rabbits (New Zealand White) were classified into four groups of 5 rabbits each: The first group (G1 - Alum - IgG injection) was sera from rabbits injected with alum only, and the polyclonal IgG was purified by a protein - A column and intravenously injected (I.V.) at a concentration of 10 mg / kg, and then infected with S. aureus USA300 bacteria at 1×109 CFU / rabbit.

[0187] The second group (G2 - anti - 4 antigen - IgG - mixture injection) was sera from five rabbits inoculated with 20 μg each of four antigens (HlgA, LukS, HlaH35L, and LukAE323A), for a total of 80 μg, dissolved in 250 μl of saline, then mixed with the same volume of alum and inoculated by intramuscular injection three times at two - week intervals. The polyclonal IgG obtained by purifying one IgG against these antigens using a protein - A column was intravenously injected at a concentration of 10 mg / kg, and then the rabbits were infected with S. aureus USA300 bacteria at 1×109 CFU / rabbit.

[0188] Group 3 (injected with vancomycin only) was injected intramuscularly into 5 rabbits with vancomycin at a concentration of 7.5 mg / kg twice a day for 5 days, 10 times in total. After that, the rabbits were infected with S. aureus USA300 at 1×109 CFU / rabbit.

[0189] Group 4 (injected with anti-4 antigen-IgG-mixture-vancomycin) was inoculated with the antigen in the same manner as Group 2. After that, IgG was purified from the obtained serum using a protein-A column, and polyclonal IgG was injected intravenously at a concentration of 10 mg / kg. Then, vancomycin at 7.5 mg / kg was injected intramuscularly twice a day for 5 days, 10 times in total. After that, the rabbits were infected with S. aureus USA300 at 1×109 CFU / rabbit.

[0190] After inoculating the above four groups with S. aureus USA300 MRSA, the body weight of the rabbits was measured daily, and the weight change was observed for 14 days. On the 15th day, the rabbits were sacrificed and the kidneys were removed, and the number of generated abscesses and residual bacteria was measured in CFU (colony forming unit).

[0191] Experimental Results After infecting with MRSA, the survival rates of the rabbits in each group were examined for 2 weeks. As shown in Figure 40A, all 5 rabbits in Group 4 (injected with anti-4 antigen-IgG-mixture-vancomycin) survived, while in Group 3 (injected with vancomycin only), 1 rabbit died on the 3rd day and 1 rabbit died on the 5th day, showing a survival rate of 60%. In contrast, all 5 rabbits in Group 1 (injected with alum only, G1) died by the 3rd day, with a survival rate of 0%. In Group 2 (injected with anti-4 antigen-IgG-mixture), which was injected with only the antibody purified from the rabbits inoculated with the four antigens, 2 rabbits died on the 3rd day and 2 rabbits died on the 4th day, showing a survival rate of 20% until the 14th day. Summing up the above results, it was found that when the antibody mixture induced by the four antigens discovered in the present invention was administered in combination with vancomycin, a significantly improved therapeutic effect was exerted.

[0192] As a result of collecting and observing the kidneys of the rabbits in Group 2 and Group 4, abscesses were observed in the kidneys of 4 out of 5 dead rabbits in Group 2, while no abscesses were observed in the kidneys of the 5 rabbits in Group 4 (Figure 40B). From the above results, it was confirmed that the antibodies against the four staphylococcal toxins antigens neutralize the toxins secreted during MRSA infection to protect immune cells, and that the combined administration of vancomycin can more perfectly remove bacteria while protecting the host in multiple aspects.

[0193] 4-2: Teicoplanin Experimental method A total of 20 rabbits (New Zealand white) were classified into 4 groups of 5 rabbits each: 10 rabbits inoculated with 4 types of vaccines at 2-week intervals 3 times were divided into 2 groups. Five rabbits in Group 2 (G2) were inoculated with USA300 MRSA bacteria, and then the survival rate for 7 days, the number of residual bacteria in the kidneys, and the appearance of abscesses generated in the kidneys were examined. The remaining 5 rabbits in Group 4 (G4) were inoculated with USA300 MRSA bacteria in the same way as G2, and 3 hours later, teicoplanin was intramuscularly injected. Five of the remaining 10 rabbits were classified into Group 1 (G1) injected with only saline and Group 3 (G3) injected with only teicoplanin: Group 1 (G1 - inoculated with only saline) was inoculated with only saline 3 times at 2-week intervals and infected with S. aureus USA300 bacteria at a concentration of 8.5×107 CFU / kg. Group 2 (G2 - inoculated with 4 antigen-mixture 3 times) was prepared by mixing 4 antigens [HlgA (40 μg / rabbit), LukS (30 μg / rabbit), HlaH35L (25 μg / rabbit), and LukAE323A (40 μg / rabbit)] in 135 μl of saline, then mixing with 135 μl of alum, and inoculating by intramuscular injection 3 times at 2-week intervals. After 7 days when the serum IgG titer had sufficiently increased, it was infected with S. aureus USA300 bacteria at a concentration of 8.5×107 CFU / kg. Group 3 (injected with teicoplanin only) was injected intramuscularly with S. aureus USA300 bacteria at a concentration of 8.5×107 CFU / kg into 5 rabbits, and 3 hours later, teicoplanin at 7.5 mg / kg was injected intramuscularly once. Group 4 (G4 - anti-4 antigen-IgG-mixture-teicoplanin injection) was inoculated with 4 antigens (HlgA, LukS, HlaH35L, and LukAE323A) 3 times at 2-week intervals in the same manner as Group 2. After 7 days, if the serum IgG titer increased sufficiently, rabbits were inoculated with S. aureus USA300 bacteria at a concentration of 8.5×107 CFU / kg, and 3 hours later, teicoplanin at 7.5 mg / kg was injected intramuscularly once.

[0194] After inoculating the rabbits in Group 2 and Group 4 with the antigen, blood was collected weekly to quantitatively measure the IgG titer against the 4 antigens by ELISA. After 3 inoculations, 7 days later, S. aureus USA300 MRSA bacteria were inoculated, and the body weight of the rabbits was measured daily to observe the weight change up to 7 days. On the 7th day, the rabbits were sacrificed and the kidneys were removed, and the number of generated abscesses and residual bacteria was measured in CFU (colony forming unit).

[0195] Experimental results Blood was collected from the rabbits weekly while inoculating the 4-antigen mixture 3 times to measure the IgG titer against each antigen in the serum (Figure 41A). As shown in the antibody titer of Group 2 (Figure 41A(i)) and the antibody titer of Group 4 (Figure 41A(i)), each antigen uniformly induced antibody production and the IgG titer was maintained at a constant level. On the 42nd day after 2 inoculations, the antibody titer decreased slightly, but after 3 inoculations, it increased again on the 49th day, so the rabbits were infected with USA300 MRSA bacteria. The ELISA results shown in Figure 2a-ii represent the average value of 5 rabbits for the antibody titer generated in the sera of 5 rabbits in Group 4. The IgG values were measured in a manner almost similar to that of Group 2, and it was found that the IgG values against each antigen increased after 3 inoculations.

[0196] After infecting the rabbits with MRSA and examining the survival rates of the rabbits in each group for 7 days, as a result, all 10 rabbits in the third group (G3 - only injected with teicoplanin) and the fourth group (G4 - after injecting the four - antigen mixture three times at two - week intervals and then infecting with USA300 MRSA and subsequently injecting teicoplanin intramuscularly) survived until the 7th day with a 100% survival rate. In contrast, in the second group (G2 - inoculated with the four - antigen mixture three times), 1 rabbit died on the 1st day and 1 rabbit died on the 5th day after USA300 MRSA infection, showing a survival rate of 60%. For the first group (G1) injected with only PBS, 1 rabbit died on the 1st day, 2 rabbits died on the 4th day, and 1 rabbit died on the 7th day, remaining at a survival rate of 20%. Summarizing the above results, it was found that when teicoplanin is administered in combination in addition to the four - antigen mixture of the present invention that induces efficient active immunity, the survival rate of individuals infected with MRSA is significantly improved.

[0197] Figure 41C shows the result of calculating the number of remaining bacteria in the crushed kidneys of living rabbits as CFU values. The second group injected with the four - antigen mixture three times at two - week intervals and the third group injected with only teicoplanin showed a statistically significant decrease in the number of remaining bacteria in the kidneys compared to the first group injected with only PBS. The fourth group (G4) administered the four antigens and teicoplanin in combination had a reduction in the number of remaining bacteria by approximately 100 - fold on average compared to the first group.

[0198] The results of collecting and observing the kidneys of the surviving rabbits in each group until the 7th day are shown in Figure 41D. In the kidneys of the rabbits in the third and fourth groups, which showed similar survival rates, abscesses were observed in the kidney of rabbit No. 12 among the 6 rabbits in the third group, and the color of the kidney of rabbit No. 13 had changed to black. In contrast, in the kidneys of the 6 rabbits in the fourth group, no abscesses were observed except for rabbit No. 6. On the other hand, in the first group injected with only PBS, the kidney of rabbit No. 2 was collected from the dead rabbit, and the kidney of rabbit No. 3 was from a surviving rabbit, but it was found that all had abscesses.

[0199] As described above, the four antigens discovered in the present invention can uniformly suppress the cell lysis caused by 11 toxins of Staphylococcus as a combination of minimum unit antigens. In addition to the excellent therapeutic effect by such a combination of antigens, the present inventors, in order to completely remove the bacteria remaining in the kidneys of infected individuals, found that when vancomycin, teicoplanin, or a combination thereof is administered in combination, not only are the remaining bacteria almost completely removed, but the survival rate of infected individuals is also further improved.

[0200] As described above, specific parts of the present invention have been described in detail. However, for those with ordinary knowledge in the art, such specific descriptions are merely preferred embodiments, and thus it is obvious that the scope of the present invention is not limited. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Hla (alpha-hemolysin) Ｈ３５Ｌ , LukS (Leukocidal toxin S), LukA Ｅ３２３Ａ B (Leukocidal toxin A Ｅ３２３Ａ B) and Staphylococcus aureus-derived toxins containing HlgA (gamma-hemolysin); or a vaccine composition for Staphylococcus aureus containing, as an active ingredient, a nucleotide encoding the toxin containing Hla H35L, LukS, LukA E323A B and HlgA.

2. The Staphylococcus aureus vaccine composition according to claim 1, wherein the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA).

3. The Staphylococcus aureus vaccine composition according to claim 1, further comprising one or more proteins selected from the group consisting of ClfA (Clumping factor A), FnbpA (fibronectin-binding protein A), FnbpB (fibronectin-binding protein B), and functional portions thereof; or nucleotides encoding said proteins.

4. The Staphylococcus aureus vaccine composition according to claim 3, wherein the functional portion comprises the N2-N3 domain of said protein.

5. The Staphylococcus aureus vaccine composition according to claim 4, wherein the composition comprises partial sections containing the N2-N3 domain of ClfA and partial sections containing the N2-N3 domain of FnbpB; or nucleotides encoding said sections as an active ingredient.

6. The Staphylococcus aureus vaccine composition according to claim 1, further comprising one or more proteins selected from the group consisting of Coa (Coagulase), vWbp (von Willebrand factor binding protein), and functional portions thereof; or nucleotides encoding said proteins.

7. The Staphylococcus aureus vaccine composition according to claim 6, wherein the functional portion of Coa comprises 284 consecutive amino acid residues starting from the N-terminus of the Coa protein.

8. The Staphylococcus aureus vaccine composition according to claim 6, wherein the functional portion of vWbp comprises 253 consecutive amino acid residues starting from the N-terminus of the vWbp protein.

Images