bGuinea pigs were intramuscularly immunized with antigens emulsified in alum (Alhydrogel) (25% v/v) at day 0 and 1 1, and euthanized at day 21. Mice received prime and booster shots with antigens emulsified in Complete and Incomplete Freund's adjuvant (CFA/IFA) by intramuscular injections at day 0 and 15, respectively. Animals were subsequently euthanized at day 24.

cSerum IgG specific to Sbi and SpA were measured using non-toxigenic variants.

dMeans (±SEM) of serum IgG titers were measured by ELISA.

Statistical significance was calculated with the unpaired two-tailed Student's t-tests and P- values recorded.

Table 6. Amino acid sequence identity comparison between IgBDs of S A and Sbi

a%Identity

IgBDs SpA-E SpA-D SpA-A SpA-B SpA-C Sbi-I Sbi-II

SpA-E 76 76 72 64 37 40

SpA-D - - 88 80 74 37 35

SpA-A - - - 90 80 37 35

SpA-B - - - - 90 40 35

SpA-C - - - - - 44 37

Sbi-I — — — — — — 44^a Percent amino acid sequence identity was based on following amino acid sequences of individual immunoglobulin binding domains (IgBDs) of SpA and Sbi, derived from S. aureus Newman, and calculated by ClustalW.

For SpA-E:

HDE AQQN AF YQ VLNMPNLN ADQRNGFIQS LKDDP S Q S AN VLGE AQKLND S (SEQ ID NO: 14).

For SpA-D:

FNKD QQ S AF YEILNMPNLNE AQRNGFIQ S LKDDP S Q STN VLGE AKKLNES (SEQ ID NO: 15).

For SpA-A:

FNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNES (SEQ ID NO: 16).

For SpA-B: FNKEQQN AF YEILHLPNLNEEQRNGFIQ SLKDDP S Q S ANLL AE AKKLND A (SEQ ID NO: 17).

For SpA-C,

FNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAE AKKLND A (SEQ ID NO: 18).

For Sbi-I,

DQQKAFYQVLHLKGITEEQRNQYIKTLREHPERAQEVFSESLKDS (SEQ ID NO: 19). For Sbi-II:

AQQNAFYNVLKNDNLTEQEKNNYIAQIKENPDRSQQVWVESVQSS (SEQ ID NO: 20).

[00201] Monoclonal antibodies offer unique opportunities to investigate the biological attributes of humoral adaptive immune responses to microbial surface products, revealing both the molecular nature of microbial immune evasion and of protective immunity (Fischetti, 1989). For example, group A streptococcal M protein, a key virulence factor and a-helical coiled-coil surface protein (Phillips et ah, 1981), confers resistance to opsonophagocytic clearance, which may be overcome by humoral adaptive immune responses during infection (Lancefield, 1962; Scott et ah, 1986). mAbs that bind to the a- helical coiled-coil of M protein cannot induce opsonophagocytic killing of group A streptococci, which is however achieved by mAbs directed against the N-terminal, random coil domain (Jones and Fischetti, 1988; Jones et al, 1986). The N-terminal domain of M proteins is highly variable between clinical isolates, which represents the molecular basis for type-specific immunity (Hollingshead et al, 1987; Lancefield, 1962).

[00202] Similar to streptococcal M protein, protein A also functions as the protective antigen of S. aureus (Stranger- Jones et al, 2006). Virtually all clinical isolates of S. aureus express protein A, however the amino acid sequence of its IgBDs is highly conserved (McCarthy and Lindsay, 2010). Staphylococcal infections in mice or humans do not elicit protein A- specific humoral immune responses (Kim et al, 2010a), which is explained by the B cell superantigen activity of this molecule (Silverman and Goodyear, 2006). Immunization with protein A variants, in particular the SPA_KKAA molecule, elicits humoral immune responses in mice and rabbits; these antibodies crossreact with wild-type protein A and provide protection against staphylococcal disease in mice (Kim et al, 2010a). When tested in mice, S. aureus mutants lacking the structural gene for protein A (spa) display significant defects in virulence, and also permit the development of humoral immune responses against many different staphylococcal antigens as well as the development of protective immunity (Cheng et al, 2009; Kim et al, 2011). Thus, antibodies that block the immune-modulatory attributes of SpA may not only provide protection against staphylococcal challenge, these antibodies may also enable the development of humoral immune responses against many different antigens secreted by S. aureus. The inventors tested this prediction by raising mAbs against SpA_KKAA- All isolated antibodies recognized conformational epitopes of protein A and interacted with the triple-helical fold of its IgBDs. Antibodies with strong affinity and cross-reactivity for multiple or all IgBDs prevented protein A association with the Fey and the Fab domains of immunoglobulins. These antibodies, in particular mAbs 5A10, 3F6 and 3D11, triggered opsonophagocytic killing of S. aureus by phagocytes in mouse and human blood, and provided protection against S. aureus disease in mice. Further, SpA_KKAA-mAb mediated neutralization of SpA in vivo stimulated humoral immune responses against several different S. aureus antigens, supporting the hypothesis that SpA_KKAA-antibodies inhibit the B cell superantigen activities of staphylococci. These data provide insights into the mechanisms of protective immune responses against S. aureus, which involve the neutralization of the Fey and Fab binding activities of SpA, thereby triggering the opsonophagocytic killing of the invading bacteria and the production of antibodies that neutralize the virulence attributes of staphylococci. EXAMPLE 2

MATERIALS AND METHODS

[00203] Bacterial strains and growth conditions. S. aureus strains Newman and MW2 were grown in tryptic soy broth (TSB) at 37°C. Escherichia coli strains DH5a and BL21 (DE3) were grown in Luria-Bertani (LB) broth with 100 μg·ml-l ampicillin at 37°C.

[00204] Monoclonal antibodies. BALB/c mice were immunized by intramuscular injection with 10 μg SpA_KKAA emulsified with complete Freund's adjuvant and boosted fourteen days later by intramuscular injection with 10 μg SpA_KKAA emulsified with incomplete Freund's adjuvant. After four days, fusion of mouse splenocytes with myeloma cells was performed at the Frank W. Fitch Monoclonal Antibody Facility of The University of Chicago. Antibody supernatants from hybridoma cells were tested in ELISA and immunoblot assays for reactivity with SpA_KKAA-

[00205] Purification of recombinant proteins. Polypeptides derived from the amino acid sequence of the SpA-ΕκκΑΑ domain were synthesized by CPC Scientific Inc (Sunnyvale, USA). Lyophilized peptide samples were solubilized using either distilled water or dimethyl sulfoxide (DMSO), then aliquoted and frozen at -80 °C. The use of plasmids for wild-type SpA and SpA_KKAA has been previously described (Kim et ah, 2010a). Oligonucleotides for the synthesis of SpA_KK (Q⁹K, Q¹⁰K substitutions in each of the five IgBDs), SPA_AA (D³⁶A, D³⁷A substitutions in each of the five IgBDs), individual IgBDs (E, D, A, B and C) of SpA_KKAA and Sbii__4/KKAA (amino acids ranging from 33 to 255 with substitutions at Q⁵¹K, Q⁵²K, Q¹⁰³K, Q¹⁰⁴K, R²³¹A, N²³⁸A) were synthesized by Integrated DNA Technologies, Inc (USA). PCR products of SpA_KKAA variants were cloned into the pET15b vector generating N-terminal His₆ -tagged recombinant proteins. The coding sequence of Sbii_₄ was PCR amplified with two primers, 5'- AAAAAAGCTAGCTGGTCTCATCCTCAATTTGAGAAGACGCAACAAACTTCAACT AAG-3' (SEQ ID NO:8) and 5 '-AAAAAACTCGAGTTTCCAGAATGATAATAAATTAC- 3' (SEQ ID NO: 9) from S. aureus Newman chromosomal DNA with engineered N-terminal Strep tag (WSHPQFEK (SEQ ID NO: 10)). PCR products of Sbii_₄ and Sbii_₄/_KKAA were cloned into pET24b vector generating C-terminal His₆-tagged recombinant protein with engineered N-terminal Strep tag (WSHPQFEK (SEQ ID NO: 10)). All plasmids were transformed into BL21(DE3) for affinity purification. Overnight cultures of recombinant E. coli strains were diluted 1 :100 into fresh media and grown at 37°C to Aeoo 0.5, at which point cultures were induced with 1 mM isopropyl β-D-l-thiogalatopyranoside (IPTG) and grown for an additional three hours. Bacterial cells were sedimented by centrifugation, suspended in column buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl) and disrupted with a French pressure cell at 14,000 psi. Lysates were cleared of membrane and insoluble components by ultracentrifugation at 40,000^xg. Proteins in the cleared lysate were subjected to nickel- nitrilotriacetic acid (Ni-NTA) affinity chromatography. Proteins were eluted in column buffer containing successively higher concentrations of imidazole (100-500 mM). Protein concentrations were determined by bicinchonic acid (BCA) assay (Thermo Scientific).

[00206] Enzyme linked immunosorbent assay. To determine SpA or Sbi specific serum IgG, affinity purified SpA_KKAA or Sbii-ιν/κκΑΑ was used to coat ELISA plates (NUNC Maxisorp) at 1 μg·mΓ¹ in 0.1 M carbonate buffer (pH 9.5 at 4°C) overnight. The following day, plates were blocked and incubated with dilutions of hyperimmune sera and developed using OptEIA reagent (BD Biosciences). For the determination of binding affinity of SpA-specific mAbs, ELISA plates were coated with affinity purified individual immunoglobulin binding domains and synthetic peptides of SpA-ΕκκΑΑ at a concentration of 100 nM in 0.1 M carbonate buffer, pH 9.5 at 4°C overnight. The following day, plates were blocked with 1% BSA solution in PBS-T and incubated with variable concentrations of SpA- specific mAbs. To determine the avidity of specific mAbs, antibody-antigen interactions were perturbed with increasing concentration (0-4 M) of ammonium thiocyanate. For SpA and Sbi binding assays, affinity purified SpA and Sbi variants were coated onto ELISA plate at 1 μg·mΓ¹ in 0.1 M carbonate buffer (pH 9.5 at 4°C) overnight. The following day, plates were blocked and incubated with dilutions of peroxidase-conjugated human IgG, Fc and F(ab)₂ (The Jackson Laboratory) or dilutions of isotype control antibodies and SpA_KKAA-specific mAbs and developed using OptEIA reagent. For the inhibition of non-specific interaction of human IgG toward SpA, plates were incubated with either 20 μg·mΓ¹ isotype control antibodies or SpA_KKAA-specific mAbs prior to ligand binding. For competition assay, plates were coated with 10 ng-ηιΓ¹ SPA_KKAA in 0.1 M carbonate buffer (pH 9.5) at 4°C overnight. The following day, plates were blocked and incubated with 30 μg·mΓ¹ of isotype control antibodies or SpA_KKAA-specific mAbs prior to the incubation with HRP-conjugated SpA- specific mAbs (Innova Biosciences) at a final concentration of 100 ng-ml^-1. To determine the functional roles of protein A cross-reactive Sbi antibodies, affinity purified SpA was used to coat ELISA plates at 1 μg·mL^"1 in 0.1 M carbonate buffer (pH 9.5 at 4°C) overnight. On the next day, plates were blocked and incubated with 100 μΐ, of 1 :10 diluted Sbi immune sera (Sbii_4 or Sbii_4/KKAA) or SpA-specific, yet Sbi cross-reactive, MAb 3F6 at 0.5 mg-mL^"1. Finally, plates were incubated with serially diluted HRP-conjugated human IgG, Fc, or F(ab)₂ fragments and developed using OptEIA reagent. In the competitive ELISA using Sbi hyperimmune sera and MAb 3F6, microtiter plates were coated with affinity purified Sbii_ rv/KKAA or SpA_KKAA, blocked and incubated with 100 μΐ, of 1 :10 diluted Sbi immune sera (Sbii_4 or Sbii_4/KKAA) or MAb 3F6 at 0.5 mg-mL^"1. Lastly, plates were incubated with serially diluted HRP-conjugated MAb 3F6 and developed using OptEIA reagent.

[00207] Active immunization of guinea pig and mouse. Cohorts of guinea pigs and mice were actively immunized with affinity purified recombinant wild-type Sbii_iv and non-toxigenic Sbii-ιν/κκΑΑ with amino acid substitutions at Q⁵¹K, Q⁵²K, Q¹⁰³K, Q¹⁰⁴K,

231 238

R A, N A. Guinea pigs were intramuscularly immunized with 100 μg of antigens emulsified in alum (Alhydrogel) (25% v/v) at day 0 and 11, and euthanized and bled by cardiac puncture at day 21. Mice received prime and booster shots with 25 μg of antigens emulsified in Complete and Incomplete Freund's adjuvant (CFA/IFA) by intramuscular injections at day 0 and 15, respectively. Animals were subsequently euthanized and bled by cardiac puncture at day 24.

[00208] Mouse renal abscess model. Affinity purified antibodies in PBS were injected at a concentration 5, 15 or 20 mg-kg^"1 of experimental animal weight into the peritoneal cavity of BALB/c mice (6 week old, female, Charles River Laboratories) 4-24 hours prior to challenge with S. aureus. Overnight cultures of S. aureus strains were diluted 1 : 100 into fresh TSB and grown for 2 hours at 37 °C. Staphylococci were sedimented, washed and suspended in PBS to the desired bacterial concentration. Inocula were quantified by spreading sample aliquots on TSA and enumerating the colonies that formed upon incubation. BALB/c mice were anesthetized via intraperitoneal injection with 100 mg-ml^"1 ketamine and 20 mg-ml^"1 xylazine per kilogram of body weight. Mice were infected by injection with 1 x 10⁷ CFU of S. aureus Newman or 5 x 10⁶ CFU of 5. aureus MW2 into the periorbital venous sinus of the right eye. On day 4 or 15 following challenge, mice were killed by C0₂ inhalation. Both kidneys were removed, and the staphylococcal load in one organ was analyzed by homogenizing renal tissue with PBS, 0.1% Triton X-100. Serial dilutions of homogenate were spread on TSA and incubated for colony formation. The remaining organ was examined by histopathology. Briefly, kidneys were fixed in 10% formalin for 24 hours at room temperature. Tissues were embedded in paraffin, thin- sectioned, stained with hematoxylin-eosin, and inspected by light microscopy to enumerate abscess lesions. Immune serum samples collected at 15 days post infection were examined by immunob lotting against 14 affinity purified staphylococcal antigens immobilized onto nitrocellulose membrane at 2 μg. Signal intensities were quantified as previously described (Kim et al., 2010b). All mouse experiments were performed in accordance with the institutional guidelines following experimental protocol review and approval by the Institutional Biosafety Committee (IBC) and the Institutional Animal Care and Use Committee (IACUC) at the University of Chicago.

[00209] Staphylococcal survival in blood. Whole blood was collected from BALB/c mice by cardiac puncture and coagulation inhibited with 10 μg·mΓ¹ lepirudin. 50 μΐ of 5 x 10⁵ CFU-mF¹ of S. aureus Newman were mixed with 950 μΐ of mouse blood in the presence of 2 μg·mΓ¹ of mAbs. Samples were incubated at 37°C with slow rotation for 30 minutes and then incubated on ice with 1% saponin/PBS. For human blood studies, 50 μΐ of 5 x 10⁶ CFU mf¹ of S. aureus MW2 were mixed with 950 μΐ of freshly drawn human blood in the presence of 10 μg·mΓ¹ of mAbs. The tubes were incubated at 37 °C with slow rotation for 120 minutes. Aliquots were incubated on ice with 1% saponin/PBS to lyse eukaryotic cells. Dilutions of staphylococci were plated on agar for colony formation. Experiments with blood from human volunteers were performed with protocols that had been reviewed, approved, and supervised by the University of Chicago's Institutional Review Board (IRB). [00210] SpA-specific serum IgG. BALB/c mice were injected into the peritoneum with 20 μg affinity purified SpA variants in the presence of 85 μg mAb 3F6 or its isotype control at day 0 and 11. At day 21, whole blood was collected from BALB/c mice to obtain hyperimmun sera.

[00211] Measuring the abundance SpA in circulation. Passively immunized BALB/c mice were injected into the peritoneum with 200 μg affinity purified wild-type SpA. At indicated time intervals, whole blood was collected from BALB/c mice with 10 μg·mΓ¹ of lepirudin anticoagulant. All samples were kept on ice with 1% saponin/PBS for 10 minutes. Lysed samples were then diluted in 1 : 10 PBS and mixed with SDS-PAGE sample buffer in 1 : 1. Samples were boiled for 5 minutes at 90 °C prior to SDS-PAGE gel electrophoresis. Samples were transferred to PDVF and analyzed by immunob lotting with affinity-purified rabbit a-SpA_KKAA antibody. [00212] Sbi consumption assay. Overnight cultures of S. aureus Newman

Q

were diluted 1 : 100 into fresh TSB, grown for 2 hours and A600 adjusted to 0.4 (1 x 10 CFU-ml^"1) with pre-chilled TSB. Cells were washed and incubated with either 100 μΐ of isotype control or mAb 3F6 at a final concentration of 100 μg·mΓ¹ for an hour at 4°C. Following incubation, stapylococci were washed with pre-chilled TSB and incubated with 2 μg of affinity-purified wild-type Sbi for one hour at 4°C. Samples were centrifuged down at 13,000^xg for one minute, supernatants were removed and mixed with sample buffer (1 : 1). Samples were boiled for 5 minutes at 90°C prior to SDS-PAGE gel electrophoresis. Samples were transferred to PDVF and analyzed by immunoblotting with affinity-purified rabbit a- SpA_KKAA antibody.

[00213] Sequencing of monoclonal antibodies. Total RNA samples from hybridoma cells were isolated using a standardized protocol. Briefly, 1.4xl0⁷ hybridoma cells cultured in DMEM-10 medium with 10% FBS were washed with PBS, sedimented by centrifugation and lysed in TRIzol (Invitrogen). Samples were mixed with 20% chloroform and incubated at room temperature for three minutes and centrifuged at 10,000^xg for fifteen minutes at 4°C. RNAs in the aqueous layer were removed and washed with 70%> isopropanol. RNA was sedimented by centrifugation and washed with 75% diethylpyrocarbonate (DEPC)- ethanol. Pellets were dried and RNA dissolved in DEPC. cDNA was synthesized with the cDNA synthesis kit (Novagen) and PCR amplified using the PCR Reagent System (Stratagene), independent primers (5 pmol each) and a mouse variable heavy and light chain specific primer set (Novagen). PCR products were sequenced and analyzed using IMGT Vquest (available at imgt.cines.fr/IMGT_vquest).

[00214] Statistical analysis. Bacterial loads and number of abscesses in experimental animal infection model were analyzed with the two-tailed Mann- Whitney test to measure statistical significance. Unpaired two-tailed Student's t-tests were performed to analyze the statistical significance of ELISA data, immunoblotting signals, and ex vivo blood survival data. All data were analyzed by Prism (GraphPad Software, Inc.) and P values less than 0.05 were deemed significant. REFERENCES

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Claims

An isolated polypeptide comprising a variant Sbi coding sequence having (a) at least one amino acid substitution that disrupts Fc binding and (b) at least a second amino acid substitution that disrupts complement binding and (c) an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 12.

The isolated polypeptide of claim 1, comprising an amino acid sequence at least 80% identical to SEQ ID NO: 12 wherein the amino acid sequence comprises one or more of the following features:

a) an amino acid substitution or deletion at the position corresponding to Q51 of full length Sbi (SEQ ID NO: 11);

b) an amino acid substitution or deletion at the position corresponding to Q52 of full length Sbi (SEQ ID NO: 11);

c) an amino acid substitution or deletion at the position corresponding to Q103 of full length Sbi (SEQ ID NO: 11);

d) an amino acid substitution or deletion at the position corresponding to Q104 of full length Sbi (SEQ ID NO: 11);

e) an amino acid substitution or deletion at the position corresponding to R231 of full length Sbi (SEQ ID NO: 11); or

f) an amino acid substitution or deletion at the position corresponding to N238 of full length Sbi (SEQ ID NO: 11).

The isolated polypeptide of claim 1 or 2, wherein the amino acid sequence comprises one or more of the following features:

a) a K amino acid substitution at the position corresponding to Q51 of full length Sbi (SEQ ID NO: 11);

b) a K amino acid substitution at the position corresponding to Q52 of full length Sbi (SEQ ID NO: 11);

c) a K amino acid substitution at the position corresponding to Q103 of full length Sbi (SEQ ID NO: 11);

d) a K amino acid substitution at the position corresponding to Q104 of full length Sbi (SEQ ID NO: 11);

e) an A amino acid substitution at the position corresponding to R231 of full length Sbi (SEQ ID NO: 11); or f) an A amino acid substitution at the position corresponding to N238 of full length Sbi (SEQ ID NO: 11).

4. The isolated polypeptide of claim 2 or 3, comprising 2, 3, 4, 5 or 6 of the features.

5. A recombinant polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1-4.

6. A composition comprising the isolated polypeptide of any one of claims 1-4 in a pharmaceutically acceptable carrier.

7. The composition of claim 6, further comprising one or more additional staphylococcal antigens.

8. The composition of claim 7, wherein the one or more additional staphylococcal antigens are selected from the group consisting of an Emp, EsxA, EsxB, EsaC, Eap, Ebh, EsaB, Coa, vWbp, vWh, Hla, SdrC, SdrD, SdrE, SpA, IsdA, IsdB, IsdC, ClfA, ClfB, and SasF antigens.

9. The composition of any of claims 6-8, wherein the composition further comprises an adjuvant.

10. A composition of any one of claims 6-9, further defined as an immunogenic composition or a vaccine composition.

11. A method for eliciting an immune response against a staphylococcus bacterium in a subject comprising, admistering an effective amount of a composition comprising a polypeptide in accordance with any one of claims 1-4, a recombinant nucleic acid molecule of claim 5 or a composition of any one of claims 6-10.

12. The method of claim 11, wherein the staphylococcus bacterium is a S. aureus bacterium.

13. The method of claim 11 or 12, wherein the bacterium is drug resistant.

14. The method of any of claims 11-13, further comprising identifying the subject has having a staphyloccal bacteria infection.

15. The method of any of claims 11-13, wherein the subject is at risk of a staphylococcus bacteria infection

16. The method of any of claims 11-15, wherein the subject is immune deficient, is immunocompromised, is hospitalized, is undergoing an invasive medical procedure, is infected with influenza virus or is on a respirator

17. The method of any of claims 11 to 16, wherein the composition further comprises one or more other staphylococcal antigens.

18. The method of any of claims 11-17, wherein the composition is administered orally, topically, intravascularly, intrathecally, intratracheally, by inhalation, or by instillation.

19. The method of any of claims 11-18, wherein the composition is administered multiple times.

20. The method of any of claims 11-19, further comprising monitoring the patient for staphylococcal infection.

21. A method of manufacturing a polypeptide comprising:

(a) expressing one or more polynucleotide molecule(s) encoding a polypeptide according to any of claims 1-4 in a cell; and

(b) purifying the polypeptide.