STAPHYLOCOCCUS AUREUS VACCINE COMPOSITION

Abstract

The present disclosure relates to immunogenic compositions for inducing an immune response in a subject for the treatment and / or prevention of Staphylococcus aureus infection. The immunogenic compositions disclosed herein comprise S. aureus Protein A (SpA) polypeptides and S. aureus Leukocidin A (LukA) and / or Leukocidin B (LukB) variant polypeptides. The present disclosure further relates to methods of generating an immune response against S. aureus in a subject comprising administering the disclosed immunogenic compositions.

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 170,089, filed April 2, 2021, and U.S. Provisional Patent Application No. 63 / 249,452, filed September 28, 2021, which are incorporated by reference in their entireties herein.

[0002] The present disclosure relates to Staphylococcus aureus immunogenic compositions and uses of the described compositions for inducing an immune response in a subject for the treatment and / or prevention of Staphylococcus infection. [Background technology]

[0003] Staphylococcus aureus causes a wide range of invasive diseases, including sepsis, infective endocarditis, and toxic shock, as well as less severe skin and soft tissue infections (Tong et al., “Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestation, and Management” Clin. Microbiol. Rev. 28(3):603-661 (2015)). Currently, there is no approved vaccine to combat S. aureus, and emerging antibiotic resistance further limits treatment options (Sause et al., “Antibody-Based Biologics and Their Promise to Combat Staphylococcus aureus Infections,” Trends Pharmacol. Sci. 37(3):231-241 (2016)). The ability of S. aureus to cause diverse clinical syndromes is often associated with large changes in genome content (Copin et al., “After the Deluge: Mining Staphylococcus aureus Genomic Data for Clinical Associations and Host-Pathogen Interactions,” Curr. Opin. Microbiol. 41:43-50 (2018) and Recker et al., “Clonal Differences in Staphylococcus aureus Bacteraemia-Associated Mortality,” Nat. Microbiol. 2(10):1381-1388 (2017)).Notably, approximately 40% of the genome is not shared by all S. aureus isolates (Bosi et al., “Comparative Genome-Scale Modelling of Staphylococcus aureus Strains Identifies Strain-Specific Metabolic Capabilities Linked to Pathogenicity,” Proc. Natl. Acad. Sci. USA 113(26):E3801-3809 (2016)), further complicating the identification of conserved targets for the generation of vaccines and biologics.

[0004] The present disclosure is directed to overcoming these and other limitations in the art. Summary of the Invention

[0005] A first aspect of the disclosure relates to an immunogenic composition comprising (i) a Staphylococcus aureus Protein A (SpA) polypeptide and (ii) a Staphylococcus aureus Leukocidin A (LukA) variant polypeptide. In an alternative aspect, the invention provides a combination of two or more compositions comprising together (i) a Staphylococcus aureus Protein A (SpA) polypeptide and (ii) a Staphylococcus aureus Leukocidin A (LukA) variant polypeptide.

[0006] In one aspect, the LukA variant polypeptide comprises an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Lys83, Ser141, Val113, and Val193 of SEQ ID NO:25.

[0007] Additional aspects of the present disclosure relate to immunogenic compositions or combinations of two or more immunogenic compositions comprising LukA variant polypeptides that include one or more additional amino acid substitutions, deletions, and / or additions relative to those described above.

[0008] Another aspect of the present disclosure relates to an immunogenic composition or a combination of two or more immunogenic compositions comprising together: (i) a Staphylococcus aureus protein A (SpA) polypeptide; (ii) a Staphylococcus aureus leukocidin A (LukA) variant polypeptide; and (iii) a Staphylococcus aureus leukocidin B (LukB) polypeptide or a variant thereof.

[0009] Additional aspects of the present disclosure relate to an immunogenic composition or a combination of two or more immunogenic compositions comprising one or more nucleic acid molecules encoding a S. aureus protein A (SpA) polypeptide or variant thereof, a LukA variant polypeptide, and a LukB polypeptide or variant thereof of the immunogenic composition described herein.

[0010] Another aspect of the present disclosure is directed to an immunogenic composition or a combination of two or more immunogenic compositions comprising one or more vectors comprising one or more nucleic acid molecules encoding a S. aureus protein A (SpA) polypeptide or variant thereof, a LukA variant polypeptide, and a LukB polypeptide or variant thereof of the immunogenic composition described herein.

[0011] Another aspect of the present disclosure is directed to an immunogenic composition comprising a host cell, the host cell comprising one or more of the nucleic acid molecules or vectors described herein.

[0012] Another aspect of the present disclosure relates to a method of treating or preventing a Staphylococcal infection in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunogenic composition or combination of immunogenic compositions described herein under conditions effective to treat or prevent a Staphylococcal infection in the subject.

[0013] Another aspect of the present disclosure relates to a method of eliciting an immune response against Staphylococcus aureus in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunogenic composition or combination of immunogenic compositions described herein under conditions effective to elicit said immune response against S. aureus in said subject.

[0014] Another aspect of the present disclosure relates to a method of decolonizing or preventing colonization or recolonization of Staphylococcus bacteria in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunogenic composition or combination of immunogenic compositions described herein under conditions effective to decolonize or prevent colonization or recolonization of Staphylococcus bacteria in the subject.

[0015] Another aspect of the present disclosure relates to the use of an immunogenic composition or a combination of immunogenic compositions described herein in a method of generating an immune response against S. aureus in a subject.

[0016] Staphylococcus aureus (S. aureus) is responsible for a large number of hospital-acquired and community-acquired infections. To evade clearance by the immune system, S. aureus employs a wide range of strategies. Staphylococcus protein A (SpA), a surface protein, is one major virulence factor of S. aureus that exhibits at least two functions relevant for promoting infection. First, cell-wall-anchored SpA on the bacterial surface binds to the Fcγ domain of IgG, neutralizing the effector function of antibodies. Antibodies bind nonspecifically "upside-down", thereby protecting staphylococci from phagocytic killing (OPK) by host immune cells and preventing proper clearance. Second, SpA functions as a major immune evasion determinant that prevents the development of protective immunity during S. aureus colonization and infection. During colonization and invasive disease, released SpA crosslinks VH3 clonal B cell receptors and induces the secretion of non-S. aureus specific antibodies that are unable to recognize staphylococcal determinants as antigens. This B cell superantigen activity (i.e., VH3 binding activity of released SpA) is involved in preventing the development of protective immunity against S. aureus during colonization or invasive disease. The use of SpA variants as vaccine antigens that have lost their immunoglobulin binding activity (1) neutralizes its ability to bind IgG via Fcγ, (2) neutralizes its ability to bind IgG via the VH3 idiotypic heavy chain, allowing anti-staphylococcal immunity to develop, and (3) induces SpA specific antibodies that induce opsonophagocytic clearance via surface-bound SpA.

[0017] Staphylococcal leukocidins A and B form a bicomponent toxin (LukAB) that has a different mechanism of action in promoting S. aureus infection. LukAB is a secreted toxin that upon binding to phagocytes assembles into pores, enters the membrane, and lyses the host cell. This allows S. aureus to evade attack from neutrophils and avoid clearance by the host. Antibodies induced by immunization with LukA, LukB, or LukAB toxoids neutralize LukAB toxin activity, resulting in viable phagocytes that can eliminate S. aureus.

[0018] Immunogenic compositions containing combinations of these antigens, i.e., SpA, LukA, LukB, and LukAB, induce antibodies that neutralize two S. aureus virulence factors, preventing two independent major evasion mechanisms of S. aureus and allowing antibody-mediated opsonophagocytosis to become effective. [Brief description of the drawings]

[0019] [Figure 1-1]Alignment of 15 different Staphylococcus aureus LukA amino acid sequences, including clonal complex (CC) 8 (SEQ ID NO: 1), CC45 (SEQ ID NO: 2), HMPREF0772_044 (TCH60) from CC30 (SEQ ID NO: 27), SAR2108 (MRSA252) from CC30 (SEQ ID NO: 36), SALG_02329 (A9635) from CC45 (SEQ ID NO: 34), SAPIG2061 (ST398) from CC398 (SEQ ID NO: 35), and No. 35), SATG_01930 (D139) (SEQ ID NO: 37) from CC10, NEWMAN (SEQ ID NO: 26) from CC8, SAB1876C (RF122) (SEQ ID NO: 32) from CC151, SAV2005 (Mu50) (SEQ ID NO: 38) from CC5, SA1813 (N315) (SEQ ID NO: 31) from CC5, SACOL2006 (SEQ ID NO: 33) from CC8, HMPRE0776_0173 USA300 (TCH959) (SEQ ID NO: 29) from CC7, HMPREF0774_2356 TCH130 (SEQ ID NO: 28) from CC72, and MW1942 (MW2) (SEQ ID NO: 30) from CC1. The amino acid sequence of the majority of the LukA sequence generated from comparison of the aligned sequences is provided as SEQ ID NO: 25. The positions of the amino acid substitutions described herein are also identified within each of the LukA sequences. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 1-3] This is a continuation of Figure 1-1. [Figure 2-1]1 is an alignment of 14 different Staphylococcus aureus LukB amino acid sequences, including LukB CC8 (SEQ ID NO: 15), CC45 (SEQ ID NO: 16), A9635 from CC45 (SEQ ID NO: 40), E1410 from CC30 (SEQ ID NO: 43), MRSA252 from CC30 (SEQ ID NO: 45), D139 from CC10 (SEQ ID NO: 42), Mu.50 from CC5 (SEQ ID NO: 46), JKD6008 from CC239 (SEQ ID NO: 44), COL from CC8 (SEQ ID NO: 41), USA300_FPR3757 from CC8 (SEQ ID NO: 115), NEWMAN from CC8 (SEQ ID NO: 116), RF122 from CC151 (SEQ ID NO: 98), MW2 from CC1 (SEQ ID NO: 47), and TCH130 from CC72 (SEQ ID NO: 99). The amino acid sequence of most of the LukB sequences generated from comparison of the aligned sequences is provided as SEQ ID NO: 39. The positions of the amino acid substitutions described herein are also identified within each of the LukB sequences. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 2-3] This is a continuation of Figure 2-1. [Diagram 3] Cytotoxicity of the different LukAB variants used for immunization. Intoxication of primary human polymorphonuclear leukocytes ("PMNs") (n=4) with a titration of the different LukAB variants was performed for 1 h. Cell viability was assessed with CellTiter. Data are obtained in two separate experiments and are the mean ± SEM from four donors. [Figure 4A]Antibody titers against LukAB CC8 or CC45 in mice immunized with different LukAB variants. Envigo Hsd:ND4 (4 weeks old) mice (n=5 / antigen) were subcutaneously administered 20 μg of LukAB in 50 μl of 10% glycerol 1× TBS mixed with 50 μl of adjuvant TiterMax® Gold. Cohorts of 5 mice were also mock immunized with the same volume of 10% glycerol 1× TBS and TiterMax® Gold. After two boosts of the same antigen-adjuvant cocktail (with a 2-week interval between boosts), mice were bled via cardiac puncture to obtain serum. Sera from mice immunized with the indicated immunizing antigens were pooled and serially diluted to determine antibody titers against CC8 LukAB (FIG. 4A) or CC45 LukAB (FIG. 4B). Plates were coated with 2 μg / ml CC8 or CC45 LukAB. Heat maps show mean absorbance values ​​from duplicate measurements. [Figure 4B] Antibody titers against LukAB CC8 or CC45 in mice immunized with different LukAB variants. Envigo Hsd:ND4 (4 weeks old) mice (n=5 / antigen) were subcutaneously administered 20 μg of LukAB in 50 μl of 10% glycerol 1× TBS mixed with 50 μl of adjuvant TiterMax® Gold. Cohorts of 5 mice were also mock immunized with the same volume of 10% glycerol 1× TBS and TiterMax® Gold. After two boosts of the same antigen-adjuvant cocktail (with a 2-week interval between boosts), mice were bled via cardiac puncture to obtain serum. Sera from mice immunized with the indicated immunizing antigens were pooled and serially diluted to determine antibody titers against CC8 LukAB (FIG. 4A) or CC45 LukAB (FIG. 4B). Plates were coated with 2 μg / ml CC8 or CC45 LukAB. Heat maps show mean absorbance values ​​from duplicate measurements. [Diagram 5]Neutralization profiles for sera from mice immunized with different LukAB variants against various LukAB toxins are provided. Intoxication of human PMN (n=4) for 1 h with 0.156 μg / ml (LD90) of the indicated LukAB variants in the presence of 4–0.031% serum from mice immunized with the indicated antigens. Sera from mice immunized with the indicated immunizing antigens were pooled and heat inactivated before use. Cell viability was determined with CellTiter. Heatmap displays the average percentage of dead cells for four donors, with black representing no cell death and white representing 100% cell death. [Figure 6A] 6A-6C are tables showing the percentage of dead human polymorphonuclear leukocytes following intoxication with LD90 LukAB toxin sequence variants in the absence or presence of 2% (FIG. 6A), 1% (FIG. 6B), and 0.5% (FIG. 6C) mouse serum from mice immunized with the indicated antigens. Data are presented as the percentage of dead cells. Unshaded cells indicate the lowest cell death, while cells shaded in the darkest grey indicate the highest cell death. [Figure 6B] 6A-6C are tables showing the percentage of dead human polymorphonuclear leukocytes following intoxication with LD90 LukAB toxin sequence variants in the absence or presence of 2% (FIG. 6A), 1% (FIG. 6B), and 0.5% (FIG. 6C) mouse serum from mice immunized with the indicated antigens. Data are presented as the percentage of dead cells. Unshaded cells indicate the lowest cell death, while cells shaded in the darkest grey indicate the highest cell death. [Figure 6C] 6A-6C are tables showing the percentage of dead human polymorphonuclear leukocytes following intoxication with LD90 LukAB toxin sequence variants in the absence or presence of 2% (FIG. 6A), 1% (FIG. 6B), and 0.5% (FIG. 6C) mouse serum from mice immunized with the indicated antigens. Data are presented as the percentage of dead cells. Unshaded cells indicate the lowest cell death, while cells shaded in the darkest grey indicate the highest cell death. [Figure 7A]LukAB RARPR-33 intoxication at high concentrations is not cytotoxic. Freshly isolated human PMNs from healthy donors (n=4-6) were incubated with different concentrations of LukAB variants for 1 h. Cell viability was determined by absorbance on CellTiter (Figures 7A and 7B). The percentage of dead cells was calculated by subtracting the background (healthy cells + PBS) and normalizing to Triton X100-treated cells, which were set to 100% dead. Mean ± SEM is shown. Human PMNs isolated from healthy donors (n=4-6) were incubated with different concentrations of LukAB variants for 2 h. Cell viability was determined by LDH release (Figures 7C and 7D). Mean ± SEM is shown. [Figure 7B] LukAB RARPR-33 intoxication at high concentrations is not cytotoxic. Freshly isolated human PMNs from healthy donors (n=4-6) were incubated with different concentrations of LukAB variants for 1 h. Cell viability was determined by absorbance on CellTiter (Figures 7A and 7B). The percentage of dead cells was calculated by subtracting the background (healthy cells + PBS) and normalizing to Triton X100-treated cells, which were set to 100% dead. Mean ± SEM is shown. Human PMNs isolated from healthy donors (n=4-6) were incubated with different concentrations of LukAB variants for 2 h. Cell viability was determined by LDH release (Figures 7C and 7D). Mean ± SEM is shown. [Figure 7C]LukAB RARPR-33 intoxication at high concentrations is not cytotoxic. Freshly isolated human PMNs from healthy donors (n=4-6) were incubated with different concentrations of LukAB variants for 1 h. Cell viability was determined by absorbance on CellTiter (Figures 7A and 7B). The percentage of dead cells was calculated by subtracting the background (healthy cells + PBS) and normalizing to Triton X100-treated cells, which were set to 100% dead. Mean ± SEM is shown. Human PMNs isolated from healthy donors (n=4-6) were incubated with different concentrations of LukAB variants for 2 h. Cell viability was determined by LDH release (Figures 7C and 7D). Mean ± SEM is shown. [Figure 7D] LukAB RARPR-33 intoxication at high concentrations is not cytotoxic. Freshly isolated human PMNs from healthy donors (n=4-6) were incubated with different concentrations of LukAB variants for 1 h. Cell viability was determined by absorbance on CellTiter (Figures 7A and 7B). The percentage of dead cells was calculated by subtracting the background (healthy cells + PBS) and normalizing to Triton X100-treated cells, which were set to 100% dead. Mean ± SEM is shown. Human PMNs isolated from healthy donors (n=4-6) were incubated with different concentrations of LukAB variants for 2 h. Cell viability was determined by LDH release (Figures 7C and 7D). Mean ± SEM is shown. [Figure 8A]Figure 8 shows intoxication of LukAB RARPR-33 and D39A / R23E toxoids at high concentrations. Freshly isolated human PMNs from healthy donors (n=5) were incubated with different concentrations of LukAB variants for 2 hours. Cell viability was determined by absorbance on CellTiter (Figures 8A and 8B). The percentage of dead cells was calculated by subtracting background (healthy cells + PBS) and normalizing to Triton X100-treated cells, which were set to 100% dead. Mean ± SEM is shown. Human PMNs isolated from healthy donors (n=5) were incubated with different concentrations of LukAB variants for 2 hours. Cell viability was determined by LDH release (Figures 8C and 8D). Mean ± SEM is shown. [Figure 8B] Figure 8 shows intoxication of LukAB RARPR-33 and D39A / R23E toxoids at high concentrations. Freshly isolated human PMNs from healthy donors (n=5) were incubated with different concentrations of LukAB variants for 2 hours. Cell viability was determined by absorbance on CellTiter (Figures 8A and 8B). The percentage of dead cells was calculated by subtracting background (healthy cells + PBS) and normalizing to Triton X100-treated cells, which were set to 100% dead. Mean ± SEM is shown. Human PMNs isolated from healthy donors (n=5) were incubated with different concentrations of LukAB variants for 2 hours. Cell viability was determined by LDH release (Figures 8C and 8D). Mean ± SEM is shown. [Figure 8C]Figure 8 shows intoxication of LukAB RARPR-33 and D39A / R23E toxoids at high concentrations. Freshly isolated human PMNs from healthy donors (n=5) were incubated with different concentrations of LukAB variants for 2 hours. Cell viability was determined by absorbance on CellTiter (Figures 8A and 8B). The percentage of dead cells was calculated by subtracting background (healthy cells + PBS) and normalizing to Triton X100-treated cells, which were set to 100% dead. Mean ± SEM is shown. Human PMNs isolated from healthy donors (n=5) were incubated with different concentrations of LukAB variants for 2 hours. Cell viability was determined by LDH release (Figures 8C and 8D). Mean ± SEM is shown. [Figure 8D] Figure 8 shows intoxication of LukAB RARPR-33 and D39A / R23E toxoids at high concentrations. Freshly isolated human PMNs from healthy donors (n=5) were incubated with different concentrations of LukAB variants for 2 hours. Cell viability was determined by absorbance on CellTiter (Figures 8A and 8B). The percentage of dead cells was calculated by subtracting background (healthy cells + PBS) and normalizing to Triton X100-treated cells, which were set to 100% dead. Mean ± SEM is shown. Human PMNs isolated from healthy donors (n=5) were incubated with different concentrations of LukAB variants for 2 hours. Cell viability was determined by LDH release (Figures 8C and 8D). Mean ± SEM is shown. [Figure 9]Neutralization profiles of sera from mice immunized with two different LukAB toxoids against various LukAB toxins. Intoxication of human PMNs (n=4) for 1 h with 0.156 μg / ml (LD90) of the indicated LukAB toxin variants in the presence of 0.125% serum from mice immunized with the two different antigens. Sera from mice immunized with both indicated immunizing antigens were pooled and heat inactivated before use. Cell viability was determined with CellTiter. Bar graphs show the mean + SEM of four different donors. Statistical significance was determined using an unpaired t-test, with P<0.05 considered significant. *P<0.05, **P<0.001, ***P<0.0001. [Figure 10] 14B is a schematic diagram of the immunization schedule for male Göttingen minipigs. Göttingen minipigs were intramuscularly immunized three separate times at three-week intervals. Three weeks after the final immunization, the minipigs were challenged with S. aureus in the SSI model. Eight days later, bacterial loads were determined. The table in FIG. 14B provides an overview of the experimental groups tested. [Figure 11A] Figure 15 shows the efficacy of LukAB RARPR-33 and Spa*+ / -GLA-SE in a minipig SSI model. Minipigs were immunized intramuscularly three separate times at 3-week intervals. Three weeks after the final immunization, minipigs were challenged with S. aureus in the SSI model. Eight days later, bacterial load was determined. Bacterial load in the mid muscle (Figure 15A) and deep muscle (Figure 15B) is shown 8 days after challenge with S. aureus. Each point represents one minipig and the geometric mean is shown. The dotted line represents the limit of detection. Statistical significance was determined using ANOVA with Dunnett's post-hoc test to correct for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001. [Figure 11B]Figure 15 shows the efficacy of LukAB RARPR-33 and Spa*+ / -GLA-SE in a minipig SSI model. Minipigs were immunized intramuscularly three separate times at 3-week intervals. Three weeks after the final immunization, minipigs were challenged with S. aureus in the SSI model. Eight days later, bacterial load was determined. Bacterial load in the mid muscle (Figure 15A) and deep muscle (Figure 15B) is shown 8 days after challenge with S. aureus. Each point represents one minipig and the geometric mean is shown. The dotted line represents the limit of detection. Statistical significance was determined using ANOVA with Dunnett's post-hoc test to correct for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001. [Figure 12] Schematic diagram of the immunization schedule for male Göttingen minipigs. Minipigs received three intramuscular immunizations at three-week intervals. Three weeks after the third immunization, animals were challenged with 106 CFU of S. aureus in a surgical site infection model. Eight days after challenge (day 71), bacterial loads were determined in the surgical site and in the spleen. At several time points, blood was drawn and serum was collected. The table shows details of the three experimental groups. [Figure 13A]13A-C are graphs showing the immunogenicity of LukAB RARPR-33 and SpA*. Göttingen minipigs (n=3) were immunized with 100 μg LukAB RARPR-33 + 100 μg SpA* in combination with AS01b (25 μg MPL and 25 μg QS-21) or 10 μg GLA-SE. The control group received the antigen formulation buffer. Serum was collected before each immunization and 3 weeks after the third immunization. Specificity against LukAB CC8 (FIG. 13A), LukAB CC45 (FIG. 13B), or SpA* (FIG. 13C) was determined by ELISA. EC50 titers are shown. Each point represents a single animal. The geometric mean ± geometric standard deviation of each group is shown. The dotted line indicates the limit of detection and is set at 30. Samples below this value are censored at 30. Statistical significance between animals immunized with LukAB RARPR-33+SpA* in combination with AS01b or GLA-SE and the buffer control group was determined after three immunizations using a one-way Tobit model with Bonferroni correction to correct for multiple comparisons. **P<0.01, ***P<0.001, ****<0.0001. [Figure 13B]13A-C are graphs showing the immunogenicity of LukAB RARPR-33 and SpA*. Göttingen minipigs (n=3) were immunized with 100 μg LukAB RARPR-33 + 100 μg SpA* in combination with AS01b (25 μg MPL and 25 μg QS-21) or 10 μg GLA-SE. The control group received the antigen formulation buffer. Serum was collected before each immunization and 3 weeks after the third immunization. Specificity against LukAB CC8 (FIG. 13A), LukAB CC45 (FIG. 13B), or SpA* (FIG. 13C) was determined by ELISA. EC50 titers are shown. Each point represents a single animal. The geometric mean ± geometric standard deviation of each group is shown. The dotted line indicates the limit of detection and is set at 30. Samples below this value are censored at 30. Statistical significance between animals immunized with LukAB RARPR-33+SpA* in combination with AS01b or GLA-SE and the buffer control group was determined after three immunizations using a one-way Tobit model with Bonferroni correction to correct for multiple comparisons. **P<0.01, ***P<0.001, ****<0.0001. [Figure 13C]13A-C are graphs showing the immunogenicity of LukAB RARPR-33 and SpA*. Göttingen minipigs (n=3) were immunized with 100 μg LukAB RARPR-33 + 100 μg SpA* in combination with AS01b (25 μg MPL and 25 μg QS-21) or 10 μg GLA-SE. The control group received the antigen formulation buffer. Serum was collected before each immunization and 3 weeks after the third immunization. Specificity against LukAB CC8 (FIG. 13A), LukAB CC45 (FIG. 13B), or SpA* (FIG. 13C) was determined by ELISA. EC50 titers are shown. Each point represents a single animal. The geometric mean ± geometric standard deviation of each group is shown. The dotted line indicates the limit of detection and is set at 30. Samples below this value are censored at 30. Statistical significance between animals immunized with LukAB RARPR-33+SpA* in combination with AS01b or GLA-SE and the buffer control group was determined after three immunizations using a one-way Tobit model with Bonferroni correction to correct for multiple comparisons. **P<0.01, ***P<0.001, ****<0.0001. [Figure 14A]Figure 14 shows cross-neutralization of LukAB by vaccine-induced antibodies. Göttingen minipigs (n=3) were immunized with 100 μg LukAB RARPR-33 + 100 μg SpA* in combination with AS01b (25 μg MPL and 25 μg QS-21) or 10 μg GLA-SE. The control group received antigen formulation buffer. Serum was collected before each immunization and 3 weeks after the third immunization. THP-1 cells were incubated with different sequence variants of LukAB toxins (CC8 (Figure 14A), CC45 (Figure 14B), CC22a (Figure 14C), CC398 (Figure 14D)) in the presence of serially diluted sera from minipigs before and after immunization. Relative potencies are shown, representing the difference in IC50 potencies (serum dilution at which 50% of cytotoxicity was observed) between serum samples and a reference LukAB monoclonal antibody. Graphs show the geometric mean ± geometric standard deviation. Each point represents one animal. Statistical significance was determined for samples from animals after three immunizations with LukAB RARPR-33+SpA* in combination with AS01b or GLA-SE and compared to the buffer control group. One-way ANOVA with Dunnett's post-hoc test was used to correct for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001. [Figure 14B]Figure 14 shows cross-neutralization of LukAB by vaccine-induced antibodies. Göttingen minipigs (n=3) were immunized with 100 μg LukAB RARPR-33 + 100 μg SpA* in combination with AS01b (25 μg MPL and 25 μg QS-21) or 10 μg GLA-SE. The control group received antigen formulation buffer. Serum was collected before each immunization and 3 weeks after the third immunization. THP-1 cells were incubated with different sequence variants of LukAB toxins (CC8 (Figure 14A), CC45 (Figure 14B), CC22a (Figure 14C), CC398 (Figure 14D)) in the presence of serially diluted sera from minipigs before and after immunization. Relative potencies are shown, representing the difference in IC50 potencies (serum dilution at which 50% of cytotoxicity was observed) between serum samples and a reference LukAB monoclonal antibody. Graphs show the geometric mean ± geometric standard deviation. Each point represents one animal. Statistical significance was determined for samples from animals after three immunizations with LukAB RARPR-33+SpA* in combination with AS01b or GLA-SE and compared to the buffer control group. One-way ANOVA with Dunnett's post-hoc test was used to correct for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001. [Figure 14C]Figure 14 shows cross-neutralization of LukAB by vaccine-induced antibodies. Göttingen minipigs (n=3) were immunized with 100 μg LukAB RARPR-33 + 100 μg SpA* in combination with AS01b (25 μg MPL and 25 μg QS-21) or 10 μg GLA-SE. The control group received antigen formulation buffer. Serum was collected before each immunization and 3 weeks after the third immunization. THP-1 cells were incubated with different sequence variants of LukAB toxins (CC8 (Figure 14A), CC45 (Figure 14B), CC22a (Figure 14C), CC398 (Figure 14D)) in the presence of serially diluted sera from minipigs before and after immunization. Relative potencies are shown, representing the difference in IC50 potencies (serum dilution at which 50% of cytotoxicity was observed) between serum samples and a reference LukAB monoclonal antibody. Graphs show the geometric mean ± geometric standard deviation. Each point represents one animal. Statistical significance was determined for samples from animals after three immunizations with LukAB RARPR-33+SpA* in combination with AS01b or GLA-SE and compared to the buffer control group. One-way ANOVA with Dunnett's post-hoc test was used to correct for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001. [Figure 14D]Figure 14 shows cross-neutralization of LukAB by vaccine-induced antibodies. Göttingen minipigs (n=3) were immunized with 100 μg LukAB RARPR-33 + 100 μg SpA* in combination with AS01b (25 μg MPL and 25 μg QS-21) or 10 μg GLA-SE. The control group received antigen formulation buffer. Serum was collected before each immunization and 3 weeks after the third immunization. THP-1 cells were incubated with different sequence variants of LukAB toxins (CC8 (Figure 14A), CC45 (Figure 14B), CC22a (Figure 14C), CC398 (Figure 14D)) in the presence of serially diluted sera from minipigs before and after immunization. Relative potencies are shown, representing the difference in IC50 potencies (serum dilution at which 50% of cytotoxicity was observed) between serum samples and a reference LukAB monoclonal antibody. Graphs show the geometric mean ± geometric standard deviation. Each point represents one animal. Statistical significance was determined for samples from animals after three immunizations with LukAB RARPR-33+SpA* in combination with AS01b or GLA-SE and compared to the buffer control group. One-way ANOVA with Dunnett's post-hoc test was used to correct for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001. [Figure 15A] Figure 15 shows the efficacy of immune responses at the surgical site and spleen in animals immunized with LukAB RARPR-33+SpA* combined with different adjuvants and challenged with S. aureus. Göttingen minipigs (n=3) were immunized with 100 μg LukAB RARPR-33 and 100 μg SpA* adjuvanted with AS01b (25 μg MPL and 25 μg QS-21) or 10 μg GLA-SE. The control group received buffer only. Three weeks after the third immunization, animals were challenged with 106 CFU of S. aureus CC398 in the SSI model. Eight days after challenge, bacterial load (Log10 CFU / gram of tissue) was determined at the surgical site in the mid muscle (Figure 15A), deep muscle (Figure 15B), and spleen (Figure 15C). Each point represents a single animal. The geometric mean of each group is shown. Statistical significance was determined using ANOVA with Dunnett's post-hoc test to correct for multiple comparisons. *P<0.05, **P<0.01. [Figure 15B] Figure 15 shows the efficacy of immune responses at the surgical site and spleen in animals immunized with LukAB RARPR-33+SpA* combined with different adjuvants and challenged with S. aureus. Göttingen minipigs (n=3) were immunized with 100 μg LukAB RARPR-33 and 100 μg SpA* adjuvanted with AS01b (25 μg MPL and 25 μg QS-21) or 10 μg GLA-SE. The control group received buffer only. Three weeks after the third immunization, animals were challenged with 106 CFU of S. aureus CC398 in the SSI model. Eight days after challenge, bacterial load (Log10 CFU / gram of tissue) was determined at the surgical site in the mid muscle (Figure 15A), deep muscle (Figure 15B), and spleen (Figure 15C). Each point represents a single animal. The geometric mean of each group is shown. Statistical significance was determined using ANOVA with Dunnett's post-hoc test to correct for multiple comparisons. *P<0.05, **P<0.01. [Figure 15C] Figure 15 shows the efficacy of immune responses at the surgical site and spleen in animals immunized with LukAB RARPR-33+SpA* combined with different adjuvants and challenged with S. aureus. Göttingen minipigs (n=3) were immunized with 100 μg LukAB RARPR-33 and 100 μg SpA* adjuvanted with AS01b (25 μg MPL and 25 μg QS-21) or 10 μg GLA-SE. The control group received buffer only. Three weeks after the third immunization, animals were challenged with 106 CFU of S. aureus CC398 in the SSI model. Eight days after challenge, bacterial load (Log10 CFU / gram of tissue) was determined at the surgical site in the mid muscle (Figure 15A), deep muscle (Figure 15B), and spleen (Figure 15C). Each point represents a single animal. The geometric mean of each group is shown. Statistical significance was determined using ANOVA with Dunnett's post-hoc test to correct for multiple comparisons. *P<0.05, **P<0.01. [Figure 16A]Schematic diagram of the immunization schedule for male Gottingen minipigs. Gottingen minipigs were intramuscularly immunized three separate times at three-week intervals. Three weeks after the final immunization, the minipigs were challenged with S. aureus in the SSI model. Eight days later, the bacterial load was determined. [Figure 16B] The table provides an overview of the experimental groups tested. [Figure 16C] Figure 16 shows the efficacy of LukAB RARPR-33 and Spa* in a minipig SSI model. Minipigs were immunized intramuscularly three separate times at 3-week intervals. Three weeks after the final immunization, minipigs were challenged with S. aureus in the SSI model. Eight days later, bacterial load was determined. Bacterial load in the mid muscle (Figure 16C) and deep muscle (Figure 16D) is shown 8 days after challenge with S. aureus. Each point represents one minipig and the geometric mean is shown. The dotted line represents the limit of detection. Statistical significance was determined using ANOVA with Dunnett's post-hoc test to correct for multiple comparisons. *P<0.05. [Figure 16D] Figure 16 shows the efficacy of LukAB RARPR-33 and Spa* in a minipig SSI model. Minipigs were immunized intramuscularly three separate times at 3-week intervals. Three weeks after the final immunization, minipigs were challenged with S. aureus in the SSI model. Eight days later, bacterial load was determined. Bacterial load in the mid muscle (Figure 16C) and deep muscle (Figure 16D) is shown 8 days after challenge with S. aureus. Each point represents one minipig and the geometric mean is shown. The dotted line represents the limit of detection. Statistical significance was determined using ANOVA with Dunnett's post-hoc test to correct for multiple comparisons. *P<0.05. [Figure 17A] Schematic diagram of the immunization schedule for male Gottingen minipigs. Gottingen minipigs were intramuscularly immunized three separate times at three-week intervals. Three weeks after the final immunization, the minipigs were challenged with S. aureus in the SSI model. Eight days later, the bacterial load was determined. [Figure 17B] The table provides an overview of the experimental groups tested. [Figure 17C]Figure 17 shows the efficacy of LukAB RARPR-33 and SpA* in a minipig SSI model. Minipigs were immunized intramuscularly three separate times at 3-week intervals. Three weeks after the final immunization, minipigs were challenged with S. aureus in the SSI model. Eight days later, bacterial load was determined. Bacterial load in the mid muscle (Figure 17C) and deep muscle (Figure 17D) is shown 8 days after challenge with S. aureus. Each point represents one minipig and the geometric mean is shown. The dotted line represents the limit of detection. Statistical significance was determined using ANOVA with Dunnett's post-hoc test to correct for multiple comparisons. **P<0.01, ****P<0.0001. [Figure 17D] Figure 17 shows the efficacy of LukAB RARPR-33 and SpA* in a minipig SSI model. Minipigs were immunized intramuscularly three separate times at 3-week intervals. Three weeks after the final immunization, minipigs were challenged with S. aureus in the SSI model. Eight days later, bacterial load was determined. Bacterial load in the mid muscle (Figure 17C) and deep muscle (Figure 17D) is shown 8 days after challenge with S. aureus. Each point represents one minipig and the geometric mean is shown. The dotted line represents the limit of detection. Statistical significance was determined using ANOVA with Dunnett's post-hoc test to correct for multiple comparisons. **P<0.01, ****P<0.0001. [Figure 18A] Immunogenicity of LukAB RARPR-33 and SpA* in combination with different adjuvants. The experimental setup is shown in Figure 18A, Swiss Webster mice were subcutaneously immunized three separate times at 2-week intervals and then bled at the indicated time points. Figure 18B is an overview of the groups included. Antibody specificity against LukAB CC8 (Figure 18C), LukAB CC45 (Figure 18D) or SpA* (Figure 18E) in serum was determined by ELISA. EC50 titers of are shown. Each point represents a single animal. Geometric mean ± geometric standard deviation of each group is shown. The dotted line indicates the limit of detection and is set at 30. Samples below this value are censored at 30. [Figure 18B]Immunogenicity of LukAB RARPR-33 and SpA* in combination with different adjuvants. The experimental setup is shown in Figure 18A, Swiss Webster mice were subcutaneously immunized three separate times at 2-week intervals and then bled at the indicated time points. Figure 18B is an overview of the groups included. Antibody specificity against LukAB CC8 (Figure 18C), LukAB CC45 (Figure 18D) or SpA* (Figure 18E) in serum was determined by ELISA. EC50 titers of are shown. Each point represents a single animal. Geometric mean ± geometric standard deviation of each group is shown. The dotted line indicates the limit of detection and is set at 30. Samples below this value are censored at 30. [Figure 18C] Immunogenicity of LukAB RARPR-33 and SpA* in combination with different adjuvants. The experimental setup is shown in Figure 18A, Swiss Webster mice were subcutaneously immunized three separate times at 2-week intervals and then bled at the indicated time points. Figure 18B is an overview of the groups included. Antibody specificity against LukAB CC8 (Figure 18C), LukAB CC45 (Figure 18D) or SpA* (Figure 18E) in serum was determined by ELISA. EC50 titers of are shown. Each point represents a single animal. Geometric mean ± geometric standard deviation of each group is shown. The dotted line indicates the limit of detection and is set at 30. Samples below this value are censored at 30. [Figure 18D] Immunogenicity of LukAB RARPR-33 and SpA* in combination with different adjuvants. The experimental setup is shown in Figure 18A, Swiss Webster mice were subcutaneously immunized three separate times at 2-week intervals and then bled at the indicated time points. Figure 18B is an overview of the groups included. Antibody specificity against LukAB CC8 (Figure 18C), LukAB CC45 (Figure 18D) or SpA* (Figure 18E) in serum was determined by ELISA. EC50 titers of are shown. Each point represents a single animal. Geometric mean ± geometric standard deviation of each group is shown. The dotted line indicates the limit of detection and is set at 30. Samples below this value are censored at 30. [Figure 18E]Immunogenicity of LukAB RARPR-33 and SpA* in combination with different adjuvants. The experimental setup is shown in Figure 18A, Swiss Webster mice were subcutaneously immunized three separate times at 2-week intervals and then bled at the indicated time points. Figure 18B is an overview of the groups included. Antibody specificity against LukAB CC8 (Figure 18C), LukAB CC45 (Figure 18D) or SpA* (Figure 18E) in serum was determined by ELISA. EC50 titers of are shown. Each point represents a single animal. Geometric mean ± geometric standard deviation of each group is shown. The dotted line indicates the limit of detection and is set at 30. Samples below this value are censored at 30. [Figure 19A] Shown are the results of LukAB CC8 and CC45 toxin neutralization assays, respectively, performed with serum samples from five mice from groups 1-5 (listed in FIG. 18B), isolated two weeks after the last immunization. Relative potencies are shown, representing the difference in IC50 potency (serum dilution at which 50% of cytotoxicity was observed) between serum samples and a reference LukAB monoclonal antibody. Graphs show the geometric mean ± geometric standard deviation. Each point represents one animal. [Figure 19B] Shown are the results of LukAB CC8 and CC45 toxin neutralization assays, respectively, performed with serum samples from five mice from groups 1-5 (listed in FIG. 18B), isolated two weeks after the last immunization. Relative potencies are shown, representing the difference in IC50 potency (serum dilution at which 50% of cytotoxicity was observed) between serum samples and a reference LukAB monoclonal antibody. Graphs show the geometric mean ± geometric standard deviation. Each point represents one animal. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition Before the compositions and methods are described, it should be understood that the present invention is not limited to the specific compositions or methodologies described, as these may vary. It should also be understood that the terms used herein are for the purpose of describing only specific versions or embodiments, and are not intended to limit the scope of the embodiments herein, which are limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments of the present invention, the preferred methods, devices, and materials are described herein. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein should be construed as an admission that the embodiments of the present invention are not entitled to antedate such disclosure by prior invention.

[0021] It should be noted that as used in this specification and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0022] Unless otherwise stated, any numerical value, such as a concentration or concentration range described herein, should be understood in all cases to be modified by the term "about". Thus, numerical values ​​typically include ±10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all individual numerical values ​​within that range, including all possible subranges, integers within that range, and fractions of values, unless the context clearly indicates otherwise.

[0023] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0024] As used herein, the terms "comprise," "includes," "included," "including," "having," "having," "containing," "containing," or any other variation thereof, are understood to imply the inclusion of a specified integer or group of integers but not the exclusion of any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or device. Furthermore, unless expressly stated to the contrary, "or" refers to an inclusive or, not an exclusive or. For example, condition A or condition B is satisfied by any one of the following: A is true (or present) and B is false (or absent), A is false (or absent), B is true (or present), and both A and B are true (or present). As used herein, the conjunction term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to be within the meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also understood to be within the meaning and thus meets the requirements of the term "and / or".

[0025] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," as used throughout this specification and claims, indicates the inclusion of any recited integer or group of integers, but indicates that additional integers or groups of integers cannot be added to the specified method, structure, or composition.

[0026] As used herein, the term "consists essentially of," or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, refers to the inclusion of any recited integer or group of integers, and optionally, any recited integer or group of integers that do not substantially change the basic or novel characteristics of the specified method, structure, or composition.

[0027] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like, more preferably humans.

[0028] It should also be understood that the terms "about," "approximately," "generally," "substantially," and the like, used herein when referring to dimensions or characteristics of preferred inventive components, indicate that the described dimensions / characteristics are not precise boundaries or parameters, as would be understood by one of ordinary skill in the art, but do not exclude minor variations that are functionally the same or similar. At a minimum, such references involving numerical parameters include variations that do not alter the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement, or other systematic errors, manufacturing tolerances, and the like).

[0029] The terms "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences (e.g., Staphylococcus LukA, LukB, SpA polypeptides and the polynucleotides encoding them) refer to two or more sequences or subsequences that are identical, or have a specified percentage of identical amino acid residues or nucleotides, when compared and aligned for maximum correspondence, as determined using one of the following sequence comparison algorithms or by visual inspection.

[0030] For sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on designated program parameters.

[0031] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, (1995 Supplement)).

[0032] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0033] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0034] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0035] As used herein, the term "polynucleotide", interchangeably referred to as "nucleic acid molecule", "nucleotide", or "nucleic acid", refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. Furthermore, "polynucleotide" refers to triple-stranded regions that include RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contain one or more modified bases, as well as DNA or RNA with backbones modified for stability or for other reasons. "Modified bases" include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA, and thus "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides as they are typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also encompasses relatively short nucleic acid strands often referred to as oligonucleotides.

[0036] As used herein, the term "vector" refers to any number of nucleic acids into which a desired sequence can be inserted, e.g., restricted and ligated, for transport between genetic environments or for expression in a host cell. Nucleic acid vectors can be DNA or RNA. Vectors include, but are not limited to, plasmids, phages, phagemids, bacterial genomes, viral genomes, self-amplifying RNA, and replicons.

[0037] As used herein, the term "host cell" refers to a cell that contains a nucleic acid molecule of the invention. A "host cell" may be any type of cell, for example, a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a "host cell" is a cell that has been transfected or transduced with a nucleic acid molecule of the invention. In another embodiment, a "host cell" is the progeny or potential progeny of such a transfected or transduced cell. The progeny of a cell may or may not be identical to the parent cell, for example, due to mutations or environmental influences that may occur in subsequent generations or upon integration of the nucleic acid molecule into the host cell genome.

[0038] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes the transcription of a gene into RNA. The term also includes the translation of RNA into one or more polypeptides, and further includes all naturally occurring post-transcriptional and post-translational modifications. The expressed polypeptide can be present in the cytoplasm of a host cell, in the extracellular environment, such as the growth medium of a cell culture, or anchored in the cell membrane.

[0039] As used herein, the term "peptide", "polypeptide", or "protein" may refer to a molecule composed of amino acids and may be recognized as a protein by those of skill in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" may be used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0040] The polypeptide sequences described herein are written according to the usual convention that the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of amino acids are known, it is the L-form of the amino acid that is represented unless expressly indicated otherwise.

[0041] The term "isolated" can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium of the source of its origin (if produced by recombinant DNA technology), or chemical precursors or other chemicals (if chemically synthesized). Additionally, an isolated polypeptide refers to a polypeptide that can be administered to a subject as an isolated polypeptide; in other words, a polypeptide may not be considered "isolated" simply because it is attached to a column or embedded in a gel. Additionally, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that does not naturally occur as a fragment and / or is not typically in a functional state.

[0042] As used herein, the phrase "immune response" or its equivalent "immunological response" refers to the development of a humoral (antibody-mediated), cellular (mediated by antigen-specific T cells or their secretory products), or both humoral and cellular response directed against a protein, peptide, carbohydrate, or polypeptide of the present disclosure in a recipient subject. Such a response can be an active response induced by administration of an immunogen, or a passive response induced by administration of an antibody, an antibody-containing substance, or primed T cells. A cellular immune response is elicited by presentation of a polypeptide epitope in association with class I or class II MHC molecules, activating antigen-specific CD4(+) T helper cells and / or CD8(+) cytotoxic T cells. The response also includes activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, or other components of innate immunity. As used herein, "active immunity" refers to any immunity conferred to a subject by administration of an antigen.

[0043] The present disclosure is directed to immunogenic compositions suitable for eliciting an immune response against Staphylococcus aureus. As described herein, in some embodiments, the immunogenic compositions comprise a Staphylococcus aureus protein A (SpA) polypeptide and a S. aureus leukocidin A (LukA) variant polypeptide. In some embodiments, the immunogenic compositions further comprise a S. aureus leukocidin B (LukB) polypeptide or a variant polypeptide thereof. In some embodiments, the immunogenic compositions comprise a S. aureus SpA protein and a S. aureus LukB variant polypeptide. The present disclosure is further directed to uses and methods of use of the immunogenic compositions in the treatment and / or prevention of S. aureus infection.

[0044] Thus, in a general aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (i) a Staphylococcus aureus protein A (SpA) polypeptide; (ii) a S. aureus LukA variant polypeptide, wherein the LukA variant polypeptide comprises amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Lys83, Ser141, Val113, and Val193 of SEQ ID NO:25.

[0045] In certain embodiments, the composition further comprises (iii) a S. aureus leukocidin B (LukB) polypeptide or a variant polypeptide thereof. In certain embodiments, the composition further comprises (iv) an adjuvant.

[0046] Components (i), (ii), (iii), and (iv) of the composition may be formulated as a single product, i.e., as a single composition. Alternatively, components (i), (ii), (iii), and (iv) may each be formulated in a single composition, or in a composition comprising a combination of two or more of the components together. Thus, in a further aspect, the present invention provides a method for the preparation of a pharmaceutical composition comprising: (i) a Staphylococcus aureus protein A (SpA) polypeptide; (ii) a S. aureus LukA variant polypeptide, wherein the LukA variant polypeptide comprises amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Lys83, Ser141, Val113, and Val193 of SEQ ID NO:25.

[0047] In certain embodiments, the combination of two or more compositions further comprises (iii) a S. aureus leukocidin B (LukB) polypeptide or a variant thereof. In certain embodiments, the combination of two or more compositions further comprises (iv) an adjuvant.

[0048] In certain embodiments, the combination of compositions can be combined into a single composition prior to use, while in other embodiments, the combination of compositions are used as separate compositions that are administered in combination with each other.

[0049] S. aureus leukocidin A (LukA) polypeptides of immunogenic compositions In one aspect, the immunogenic composition of the present disclosure comprises an S. aureus LukA variant polypeptide. Suitable LukA variant polypeptides include one or more amino acid residue insertions, substitutions, and / or deletions that render LukAB binary complexes containing such LukA variants non-cytotoxic. LukA variant polypeptides also stabilize LukAB heterodimers, increase the melting temperature, and / or increase the solubility of the heterodimer.

[0050] In all embodiments, the LukA variant polypeptide of the immunogenic composition may be a variant of a full-length LukA protein that includes all of the amino acid residues corresponding to the full-length mature LukA protein sequence. As referred to herein, a "mature" leukocidin protein sequence is a sequence of a leukocidin protein that lacks an amino-terminal secretion signal, and typically includes the first 27-28 amino acid residues on the amino terminus.

[0051] In any embodiment, the LukA variant polypeptide of the immunogenic composition can be a variant shorter than the full-length mature LukA protein. In any embodiment, the variant LukA polypeptide is at least 100 amino acid residues in length. In any embodiment, the variant LukA polypeptide is at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300 amino acid residues in length.

[0052] Exemplary LukA variant proteins and polypeptides of the immunogenic compositions described herein are LukA variant proteins of clonal complexes CC8 (SEQ ID NO:1) and CC45 (SEQ ID NO:2) (see Table 1 below), and one of skill in the art will readily appreciate that the amino acid substitutions and / or deletions of LukA identified in the context of SEQ ID NO:1 and SEQ ID NO:2 are conserved amino acid residues across various clonal complexes or within regions of LukA that are highly conserved across various clonal complexes. Indeed, an alignment of LukA protein sequences from 15 different strains of S. aureus (see FIG. 1) shows that the amino acid residues identified herein as residues subject to mutation are residues that are conserved across all 15 of the aligned LukA amino acid sequences. While the residue positions of the identified mutations may vary between individual LukA sequences, the sequence alignment shows the correspondence between these positions. For clarity, a LukA consensus sequence having the amino acid sequence of SEQ ID NO:25 was generated from the sequence alignment and utilized for the purposes of assigning the locations of specific amino acid mutations. For example, the amino acid substitution at lysine residue 83 in SEQ ID NO: 25 corresponds to the lysine residue at position 80 in the LukA sequence of SEQ ID NO: 1, the lysine residue at position 81 in the LukA sequence of SEQ ID NO: 2, and the lysine residue at position 83 in the LukA sequences of SEQ ID NOs: 26 to 38. Thus, the identified amino acid mutations described herein can be universally applied to the corresponding amino acid residues in any LukA amino acid sequence known now or in the future.

[0053] According to this aspect of the disclosure, in any embodiment, the LukA variant polypeptide of the immunogenic composition comprises an amino acid residue insertion, substitution, and / or deletion at one or more amino acid residues corresponding to Lys83, Ser141, Val113, Val193 of SEQ ID NO: 25. In any embodiment, the LukA variant polypeptide, in addition to the insertion, substitution, and / or deletion of one or more amino acid residues described above, further comprises an amino acid substitution or deletion at the amino acid residue corresponding to Glu323 of SEQ ID NO: 25. In any embodiment, the amino acid substitution or deletion at Glu323 comprises a glutamic acid to alanine substitution at position 323 (Glu323Ala) of SEQ ID NO: 25.

[0054] In any embodiment, the amino acid substitutions at one or more of the identified positions of LukA (and other S. aureus proteins described herein) are conservative substitutions. Such conservative substitutions involve the replacement of one amino acid residue with another that is a member of the same class that acts as a functional equivalent, resulting in a silent modification; that is, the change to the native sequence will not significantly reduce the basic properties of LukA. These classes of amino acid residues include non-polar (hydrophobic) amino acids (e.g., alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine), polar neutral amino acids (e.g., glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine), positively charged (basic) amino acids, (e.g., arginine, lysine, histidine, and negatively charged (acidic) amino acids (e.g., aspartic acid and glutamic acid).

[0055] In other embodiments, the amino acid substitutions at one or more of the identified positions of the variant leukocidin or SpA polypeptides described herein are non-conservative modifications (i.e., substitutions that disrupt the sequence, structure, function, or activity of the identified region). Such substitutions may be desirable to reduce or mitigate the cytotoxicity of the protein. Non-conservative substitutions include the replacement of one particular class of amino acid residue with a different class of amino acid residue. For example, the replacement of a non-polar (hydrophobic) amino acid residue with a polar neutral amino acid, or vice versa. In another embodiment, non-conservative substitutions include the replacement of a positively charged (basic) amino acid residue with a negatively charged (acidic) amino acid residue, such as aspartic acid and glutamic acid, or vice versa. Such molecular modifications can be accomplished by methods well known in the art, including primer extension methods on plasmid templates using single-stranded templates (Kunkel et al., Proc. Acad. Sci., USA 82:488-492 (1985), which is incorporated herein by reference in its entirety), double-stranded DNA templates (Papworth, et al., Strategies 9(3):3-4 (1996), which is incorporated herein by reference in its entirety), and by PCR cloning (Braman, J. (ed.), IN VITRO MUTAGENESIS PROTOCOLS, 2nd ed. Humana Press, Totowa, NJ (2002), which is incorporated herein by reference in its entirety).

[0056] In any embodiment, the LukA variant polypeptide of the immunogenic composition comprises a lysine to methionine substitution at a residue corresponding to the lysine at position 83 of SEQ ID NO:25 (Lys83Met). In any embodiment, the LukA variant polypeptide of the immunogenic composition comprises a serine to alanine substitution at a residue corresponding to the serine at position 141 of SEQ ID NO:25 (Ser141Ala). In any embodiment, the LukA variant polypeptide of the immunogenic composition comprises a valine to isoleucine substitution at a residue corresponding to the valine at position 113 of SEQ ID NO:25 (Val113Ile). In any embodiment, the LukA variant polypeptide of the immunogenic composition comprises a valine to isoleucine substitution at a residue corresponding to the valine at position 193 of SEQ ID NO:25 (Val193Ile).

[0057] In any embodiment, the LukA variant polypeptide of the immunogenic composition comprises any one or more substitutions at residues corresponding to Lys83, Ser141, Val113, Val193 of SEQ ID NO:25, as well as a glutamic acid to alanine substitution at the residue corresponding to glutamic acid residue 323 of SEQ ID NO:25 (Glu323Ala).

[0058] In any embodiment, the LukA variant polypeptide of the immunogenic composition comprises a protein or polypeptide thereof having an amino acid residue insertion, substitution, and / or deletion at two of the aforementioned amino acid residues corresponding to Lys83, Ser141, Val113, and Val193 of SEQ ID NO:25. In any embodiment, the LukA variant polypeptide comprises an amino acid residue insertion, substitution, and / or deletion at three of the aforementioned amino acid residues. In any embodiment, the LukA variant polypeptide comprises an amino acid residue insertion, substitution, and / or deletion at all four of the aforementioned amino acid residues. In any embodiment, the LukA variant polypeptide of the present disclosure comprises lysine to methionine, serine to alanine, and valine to isoleucine amino acid substitutions at the aforementioned amino acid residues corresponding to Lys83Met, Ser141Ala, Val113Ile, and Val193Ile of SEQ ID NO:25. In any embodiment, the variant LukA protein or polypeptide thereof further comprises an amino acid substitution corresponding to Glu323Ala of SEQ ID NO:25, i.e., the variant LukA comprises substitutions corresponding to Lys83Met, Ser141Ala, Val113Ile, Val193Ile, and Glu323Ala of SEQ ID NO:25.

[0059] Exemplary LukA variant polypeptides of the immunogenic compositions described herein have amino acid substitutions corresponding to Lys83Met, Ser141Ala, Val113Ile, Val193Ile, and Glu323Ala of SEQ ID NO: 25. In any embodiment, the LukA variant polypeptide of the immunogenic composition is a CC8 LukA variant that includes any one or more amino acid substitutions selected from Lys80Met, Ser138Ala, Val110Ile, Val190Ile, and Glu320Ala of SEQ ID NO: 1. In any embodiment, the LukA variant polypeptide of the immunogenic composition is a CC8 LukA variant that includes amino acid substitutions corresponding to each of Lys80Met, Ser138Ala, Val110Ile, Val190Ile, and Glu320Ala of SEQ ID NO: 1. In any embodiment, the LukA variant polypeptide has an amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:3.

[0060] In any embodiment, the LukA variant polypeptide of the immunogenic composition is a CC45 LukA variant polypeptide that includes any one or more amino acid substitutions corresponding to Lys81Met, Ser139Ala, Val111Ile, Val191Ile, and Glu321Ala of SEQ ID NO: 2. In any embodiment, the LukA variant polypeptide of the immunogenic composition is a CC45 LukA variant polypeptide that includes an amino acid substitution corresponding to each of Lys81Met, Ser139Ala, Val111Ile, Val191Ile, and Glu321Ala of SEQ ID NO: 2. In some embodiments, the LukA variant polypeptide has an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 4. Other exemplary variant LukA proteins include any one of the LukA proteins of SEQ ID NOs:26-38, which contain amino acid substitutions corresponding to the Lys83Met, Ser141Ala, Val113Ile, Val193Ile, and Glu323Ala substitutions of SEQ ID NO:25.

[0061] In any embodiment, the LukA variant polypeptide of the immunogenic composition described herein comprises an amino acid substitution at one or more of the amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO: 25. In one embodiment, the amino acid substitution at one or more of the foregoing residues introduces a cysteine ​​residue that can form a disulfide bond to stabilize the conformation of the LukAB heterodimer structure. For example, in one embodiment, the LukA variant polypeptide described herein comprises a tyrosine to cysteine ​​substitution at the amino acid residue corresponding to Tyr74 of SEQ ID NO: 25 (Tyr74Cys) and an asparagine to cysteine ​​substitution at the amino acid residue corresponding to Asp140 of SEQ ID NO: 25 (Asp140Cys). These cysteine ​​residues at positions 74 and 140 form disulfide bonds, thereby increasing the thermal stability of the variant LukA compared to wild-type LukA or compared to other variant LukA proteins or polypeptides described herein that do not contain paired cysteine ​​residues capable of forming disulfide bonds.

[0062] In another embodiment, the LukA variant polypeptide of the immunogenic compositions described herein comprises a glycine to cysteine ​​substitution (Gly149Cys) at the amino acid residue corresponding to Gly149 of SEQ ID NO: 25, and a glycine to cysteine ​​substitution (Gly156Cys) at the amino acid residue corresponding to Gly156 of SEQ ID NO: 25. These introduced cysteine ​​residues at positions 149 and 156 form disulfide bonds, thereby increasing the thermal stability of the variant LukA compared to wild-type LukA or compared to other variant LukA polypeptides described herein that do not contain paired cysteine ​​residues capable of forming disulfide bonds.

[0063] In any embodiment, the variant LukA polypeptide of the immunogenic composition comprises an amino acid substitution at each of the amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO:25. In any embodiment, the amino acid substitution at each of these amino acid residues is accompanied by the introduction of a cysteine ​​residue as described above. In any embodiment, the variant LukA polypeptide of the immunogenic composition comprises an amino acid substitution at each of the amino acid residues corresponding to amino acid residues Tyr71, Asp137, Gly146, and Gly153 of SEQ ID NO:1. In any embodiment, the amino acid substitution at each of these amino acid residues is accompanied by the introduction of a cysteine ​​residue as described above. In any embodiment, the variant LukA polypeptide of the immunogenic composition comprises an amino acid substitution at each of the amino acid residues corresponding to amino acid residues Tyr72, Asp138, Gly147, and Gly154 of SEQ ID NO:2. In any embodiment, the amino acid substitution at each of these amino acid residues is accompanied by the introduction of a cysteine ​​residue as described above.

[0064] In any embodiment, a variant LukA protein or polypeptide of the immunogenic composition comprises amino acid substitutions at one or more amino acid residues corresponding to Lys83, Ser141, Val113, Val193, and Glu323 in combination with amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO: 25. In any embodiment, a variant LukA polypeptide comprises amino acid substitutions at amino acid residues corresponding to residues Lys83, Ser141, Val113, Val193, and Glu323 as well as residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO: 25.

[0065] In any embodiment, an exemplary LukA variant polypeptide of the immunogenic composition is a CC8 LukA variant polypeptide having an amino acid substitution at a residue corresponding to each of Lys80, Ser138, Val110, Val190, Glu320, Tyr71, Asp137, Gly146, and Gly153 of SEQ ID NO: 1. In any embodiment, an exemplary LukA variant polypeptide is a CC8 LukA variant polypeptide having an amino acid substitution at a residue corresponding to each of Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Glu320Ala, Tyr71Cys, Asp137Cys, Gly146Cys, and Gly153Cys of SEQ ID NO: 1. In any embodiment, the CC8 LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:5.

[0066] In any embodiment, an exemplary LukA variant polypeptide of the immunogenic composition is a CC45 LukA variant polypeptide having an amino acid substitution at a residue corresponding to each of Lys81, Ser139, Val111, Val191, Glu321, Tyr72, Asp138, Gly147, and Gly154 of SEQ ID NO: 2. In any embodiment, an exemplary LukA variant polypeptide is a CC45 LukA variant polypeptide having an amino acid substitution at a residue corresponding to each of Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Glu321Ala, Tyr72Cys, Asp138Cys, Gly147Cys, and Gly154Cys of SEQ ID NO: 2. In some embodiments, the CC45 LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:6.

[0067] Other exemplary LukA variant polypeptides of the immunogenic composition include any one of the LukA proteins of SEQ ID NOs: 26-38, which contain amino acid substitutions corresponding to Lys83Met, Ser141Ala, Val113Ile, Val193Ile, Glu323Ala, Tyr74Cys, Asp140Cys, Gly149Cys, and Gly156Cys of SEQ ID NO: 25.

[0068] In any embodiment, the LukA variant polypeptide of the immunogenic compositions described herein comprises an amino acid substitution or deletion at the amino acid residue corresponding to amino acid residue Thr249 of SEQ ID NO: 25. In any embodiment, the LukA variant comprises a substitution at the residue corresponding to Thr249, which is a threonine to valine substitution at this residue (Thr249Val).

[0069] In any embodiment, a LukA variant protein or polypeptide of the immunogenic compositions described herein comprises an amino acid substitution at the amino acid residue corresponding to Thr249 of SEQ ID NO: 25 in combination with any one of the other amino acid residue substitutions described herein, i.e., substitutions at residues corresponding to Lys83, Ser141, Val113, Val193, Glu323, Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO: 25. In any embodiment, a LukA variant protein or polypeptide described herein comprises an amino acid substitution at the amino acid residue corresponding to Thr249 of SEQ ID NO: 25 in combination with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all nine of the other amino acid residue substitutions described herein. In any embodiment, the variant LukA protein or polypeptide comprises amino acid substitutions at each of the residues corresponding to Lys83, Ser141, Val113, Val193, Glu323, and Thr249 of SEQ ID NO:25.

[0070] In any embodiment, an exemplary LukA variant polypeptide of the immunogenic composition is a CC8 LukA variant polypeptide having an amino acid substitution at residue Thr246 alone or in combination with any one or more amino acid substitutions corresponding to each of Lys80, Ser138, Val110, Val190, and Glu320 of SEQ ID NO: 1. In any embodiment, an exemplary LukA variant polypeptide of the immunogenic composition is a CC8 LukA variant polypeptide having an amino acid substitution at residues corresponding to each of Lys80, Ser138, Val110, Val190, Glu320, and Thr246 of SEQ ID NO: 1. In any embodiment, an exemplary LukA variant polypeptide is a CC8 LukA variant polypeptide having an amino acid substitution at residues corresponding to each of Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Glu320Ala, and Thr246Val of SEQ ID NO: 1. In one embodiment, an exemplary LukA variant polypeptide having amino acid substitutions at residues corresponding to each of the aforementioned positions has an amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:7.

[0071] In any embodiment, an exemplary LukA variant polypeptide of the immunogenic composition is a CC45 LukA variant polypeptide having an amino acid substitution at residue Thr247 alone or in combination with any one or more amino acid substitutions corresponding to each of Lys81, Ser139, Val111, Val191, and Glu321 of SEQ ID NO: 2. In any embodiment, an exemplary LukA variant polypeptide of the immunogenic composition is a CC45 LukA variant polypeptide having an amino acid substitution at residues corresponding to each of Lys81, Ser139, Val111, Val191, Glu321, and Thr247 of SEQ ID NO: 2. In any embodiment, an exemplary LukA variant polypeptide is a CC45 LukA variant polypeptide having an amino acid substitution at residues corresponding to each of Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Glu321Ala, and Thr247Val of SEQ ID NO: 2. In one embodiment, an exemplary LukA variant polypeptide having amino acid substitutions at residues corresponding to each of the aforementioned positions has an amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:8.

[0072] Other exemplary variant LukA proteins of the immunogenic composition include any one of the LukA proteins of SEQ ID NOs: 26-38, containing the indicated amino acid substitutions at amino acid residues corresponding to Lys83, Ser141, Val113, Val193, Glu323, and Thr249 of SEQ ID NO: 25.

[0073] In any embodiment, the variant LukA protein or polypeptide of the immunogenic composition comprises amino acid substitutions at each of the residues corresponding to Lys83, Ser141, Val113, Val193, Glu323, Thr249, Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO:25.

[0074] In any embodiment, an exemplary LukA variant polypeptide of the immunogenic composition is a CC8 LukA variant polypeptide having an amino acid substitution at a residue corresponding to each of Lys80, Ser138, Val110, Val190, Glu320, Tyr71, Asp137, Gly146, Gly153, and Thr246 of SEQ ID NO: 1. In any embodiment, an exemplary LukA variant polypeptide is a CC8 LukA variant polypeptide having an amino acid substitution at a residue corresponding to each of Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Glu320Ala, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys, and Thr246Val of SEQ ID NO: 1. In one embodiment, an exemplary LukA variant polypeptide having amino acid substitutions at residues corresponding to each of the aforementioned positions has an amino acid sequence of SEQ ID NO:9, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:9.

[0075] In any embodiment, an exemplary LukA variant polypeptide of the immunogenic composition is a CC45 LukA variant polypeptide having an amino acid substitution at a residue corresponding to each of Lys81, Ser139, Val111, Val191, Glu321, Tyr72, Asp138, Gly147, Gly154, and Thr247 of SEQ ID NO: 2. In any embodiment, an exemplary LukA variant polypeptide is a CC45 LukA variant polypeptide having an amino acid substitution at a residue corresponding to each of Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Glu321Ala, Tyr72Cys, Asp138Cys, Gly147Cys, Gly154Cys, and Thr247Ala of SEQ ID NO: 2. In one embodiment, an exemplary LukA variant polypeptide having amino acid substitutions at residues corresponding to each of the aforementioned positions has an amino acid sequence of SEQ ID NO:10, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:10.

[0076] Other exemplary variant LukA proteins of the immunogenic composition include any one of the LukA proteins of SEQ ID NOs:26-38, containing the indicated amino acid substitutions at residues corresponding to Lys83, Ser141, Val113, Val193, Glu323, Thr249, Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO:25.

[0077] Table 1 below provides exemplary variant LukA amino acid sequences of the immunogenic compositions disclosed herein. [Table 1] TIFF2024512751000002.tif208170TIFF2024512751000003.tif163170

[0078] S. aureus leukocidin B (LukB) polypeptides of immunogenic compositions In some aspects, the immunogenic compositions of the disclosure comprise a S. aureus leukocidin B (LukB) protein or polypeptide. In any embodiment, the S. aureus LukB protein or polypeptide is a wild-type protein or polypeptide. Suitable LukB polypeptides include any one of the LukB polypeptides disclosed herein, e.g., a polypeptide having any amino acid sequence selected from SEQ ID NOs: 15, 16, and 39-51. In any embodiment, the LukB polypeptide is a CC8 LukB polypeptide. A suitable CC8 LukB polypeptide comprises the amino acid sequence of SEQ ID NO: 15. In any embodiment, the LukB polypeptide is a CC45 LukB polypeptide. A suitable CC45 LukB polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

[0079] In any embodiment, the LukB polypeptide of the immunogenic composition disclosed herein comprises a LukB variant polypeptide. A suitable LukB variant polypeptide comprises an insertion, substitution, and / or deletion of one or more amino acid residues that improves the stability of LukB, thereby contributing to the toxoid stability of LukAB. As described herein, these variant LukB proteins and polypeptides are ideal vaccine antigen candidates, and they can be included in immunogenic compositions that comprise SpA polypeptides alone or in combination with leukocidin A (LukA) variant proteins or polypeptides. When an immunogenic composition comprises a combination of LukB and LukA polypeptides, the resulting toxoid mimics the structure of the S. aureus LukAB toxin, thereby facilitating the generation of a robust immune response against one of the most potent toxins of S. aureus.

[0080] In any embodiment, the LukB variant polypeptide of the immunogenic composition is a variant of the full-length LukB protein, which comprises all of the amino acid residues corresponding to the full-length mature LukB protein sequence. In any embodiment, the LukB variant polypeptide is a variant shorter than the full-length mature LukB protein. In any embodiment, the variant LukB polypeptide is at least 100 amino acid residues in length. In any embodiment, the variant LukB polypeptide is at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300 amino acid residues in length.

[0081] Exemplary LukB variant proteins and polypeptides described herein are variant LukBs of clonal complexes CC8 (SEQ ID NO: 15) and CC45 (SEQ ID NO: 16) (see Table 2 below), and one of skill in the art will readily appreciate that the amino acid substitutions and / or deletions of LukB identified in the context of SEQ ID NO: 15 and SEQ ID NO: 16 are conserved amino acid residues across various clonal complexes or within regions of LukB that are highly conserved across various clonal complexes. Alignment of LukB protein sequences from 14 different strains of S. aureus (see FIG. 2) shows that the amino acid residues identified herein as residues subject to mutation are conserved residues across all 14 of the aligned LukB amino acid sequences. Although the positions of the residues of the identified mutations may vary between individual LukB-sequences, the sequence alignment shows the correspondence between these positions. For clarity, a LukB consensus sequence having the amino acid sequence of SEQ ID NO: 39 was generated from the sequence alignment and utilized for the purpose of assigning the locations of specific amino acid mutations. For example, the amino acid substitution of glutamic acid residue 109 of SEQ ID NO: 39 corresponds to the glutamic acid residue at position 109 of the LukB sequences of SEQ ID NOs: 15, 42, 44, and 46-51, the glutamic acid residue at position 110 of the LukB sequences of SEQ ID NOs: 16, 40, 43, and 45, and the glutamic acid residue at position 60 of the LukB sequence of SEQ ID NO: 41. Thus, the identified amino acid mutations described herein can be universally applied to the corresponding amino acid residues of any LukB amino acid sequence known now or in the future.

[0082] In any embodiment, a suitable LukB variant polypeptide of the immunogenic compositions disclosed herein comprises an amino acid substitution or deletion at an amino acid residue corresponding to amino acid residue Val53 of SEQ ID NO: 39. In any embodiment, the amino acid substitution at Val53 comprises a valine to leucine (Val53Leu) substitution. In any embodiment, an exemplary LukB variant polypeptide comprises a substitution corresponding to Val53Leu of SEQ ID NO: 39.

[0083] In any embodiment, an exemplary LukB variant polypeptide of the immunogenic composition is a CC8 LukB variant polypeptide having an amino acid substitution at an amino acid position corresponding to position 53 of SEQ ID NO: 15. In any embodiment, an exemplary LukB variant polypeptide is a CC8 LukB variant polypeptide having an amino acid substitution from valine to leucine at a position corresponding to position 53 of SEQ ID NO: 15. In any embodiment, an exemplary CC8 LukB sequence having a valine to leucine substitution at position 53 comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 17.

[0084] In any embodiment, an exemplary LukB variant polypeptide of the immunogenic composition is a CC45 LukB variant polypeptide having an amino acid substitution at an amino acid position corresponding to position 53 of SEQ ID NO: 16. In any embodiment, an exemplary LukB variant polypeptide is a CC45 LukB variant polypeptide having an amino acid substitution from valine to leucine at a position corresponding to position 53 of SEQ ID NO: 16. An exemplary LukB variant polypeptide comprising a valine to leucine substitution comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 18.

[0085] Other exemplary variant LukB proteins include any one of the LukB proteins of SEQ ID NOs: 40-51 that contain an amino acid substitution corresponding to Val53Leu.

[0086] In any embodiment, the LukB variant polypeptide of the immunogenic composition described herein comprises an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO: 39. In any embodiment, the amino acid substitution at one or more of the foregoing residues introduces a cysteine ​​residue that can form a disulfide bond to stabilize the conformation of the LukAB heterodimeric structure. For example, in one embodiment, a LukB variant protein or polypeptide described herein comprises a glutamic acid to cysteine ​​substitution (Glu45Cys) at the amino acid residue corresponding to Glu45 of SEQ ID NO: 39 and a threonine to cysteine ​​substitution (Thr121Cys) at the amino acid residue corresponding to Thr121 of SEQ ID NO: 39. These introduced cysteine ​​residues at positions 45 and 121 form disulfide bonds, thereby increasing the thermal stability of the variant LukB compared to wild-type LukB or compared to other variant LukB proteins or polypeptides described herein that do not contain paired cysteine ​​residues capable of forming disulfide bonds.

[0087] In any embodiment, the LukB variant proteins or polypeptides of the immunogenic compositions described herein comprise a glutamic acid to cysteine ​​substitution (Glu109Cys) at the amino acid residue corresponding to Glu109 of SEQ ID NO: 39, and an arginine to cysteine ​​substitution (Arg154Cys) at the amino acid residue corresponding to Arg154 of SEQ ID NO: 39. These introduced cysteine ​​residues at positions 109 and 154 form disulfide bonds, thereby increasing the thermal stability of the variant LukB compared to wild-type LukB or compared to other variant LukB proteins and polypeptides described herein that do not contain paired cysteine ​​residues capable of forming disulfide bonds.

[0088] In any embodiment, the LukB variant polypeptide of the immunogenic composition is a CC8 LukB variant polypeptide comprising an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO: 15. In any embodiment, the LukB variant polypeptide of the immunogenic composition is a CC8 LukB variant polypeptide comprising an amino acid substitution at each amino acid residue corresponding to amino acid residues Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO: 15. In any embodiment, the amino acid substitution at each of these amino acid residues involves the introduction of a cysteine ​​residue as described above. In any embodiment, an exemplary LukB variant polypeptide comprising a cysteine ​​amino acid substitution at residues corresponding to Glu45, Glu109, Thr121, and Arg154 comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 21.

[0089] In any embodiment, the LukB variant polypeptide of the immunogenic compositions described herein comprises an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Glu45, Glu110, Thr122, and Arg155 of SEQ ID NO: 16. In any embodiment, the LukB variant polypeptide of the immunogenic compositions is a CC45 LukB variant polypeptide comprising an amino acid substitution at each amino acid residue corresponding to amino acid residues Glu45, Glu110, Thr122, and Arg155 of SEQ ID NO: 16. In any embodiment, the amino acid substitution at each of these amino acid residues is accompanied by the introduction of a cysteine ​​residue as described above. In any embodiment, an exemplary LukB variant polypeptide comprising a cysteine ​​amino acid substitution at residues corresponding to Glu45, Glu110, Thr122, and Arg155 comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 22.

[0090] In any embodiment, the LukB variant polypeptide of the immunogenic compositions disclosed herein comprises an amino acid substitution at an amino acid residue corresponding to Val53 of SEQ ID NO: 39 in combination with an amino acid substitution at one or more amino acid residues corresponding to Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO: 39. In any embodiment, the LukB variant polypeptide is a CC8 LukB variant polypeptide comprising an amino acid substitution at amino acid residues corresponding to Val53, Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO: 15. In any embodiment, the LukB variant polypeptide is a CC8 LukB variant polypeptide comprising an amino acid substitution at amino acid residues corresponding to Val53Leu, Glu45Cys, Glu109Cys, Thr121Cys, and Arg154Cys of SEQ ID NO: 15. In any embodiment, an exemplary CC8 LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:19, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:19.

[0091] In any embodiment, the LukB variant polypeptide of the immunogenic composition is a CC45 LukB variant polypeptide comprising amino acid substitutions at amino acid residues corresponding to Val53, Glu45, Glu110, Thr122, and Arg155 of SEQ ID NO: 16. In any embodiment, the LukB variant polypeptide is a CC45 LukB variant polypeptide comprising amino acid substitutions at amino acid residues corresponding to Val53Leu, Glu45Cys, Glu110Cys, Thr123Cys, and Arg155Cys of SEQ ID NO: 16. In any embodiment, an exemplary CC45 LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:20.

[0092] Other exemplary LukB variant polypeptides of the immunogenic compositions include any one of the LukB proteins of SEQ ID NOs: 40-51, containing the indicated amino acid substitutions of residues corresponding to Val53, Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO:39.

[0093] Table 2 below provides exemplary variant LukB amino acid sequences of the immunogenic compositions disclosed herein. [Table 2] TIFF2024512751000005.tif86170

[0094] Staphylococcal Protein A (SpA) Polypeptides of Immunogenic Compositions - Patent application The immunogenic compositions described herein contain S. aureus Protein A polypeptide. "Protein A" or "SpA" are used interchangeably herein and refer to a cell wall-anchored surface protein of S. aureus that functions to provide bacterial evasion from the innate and adaptive immune responses of an infected host. Protein A can bind immunoglobulins at their Fc portion and binds to the V receptor of the B cell receptor in appropriate stimulating B cell proliferation and apoptosis. H It can interact with the β-amyloid β-blocker domain, can bind to the von Willebrand factor A1 domain to activate intracellular coagulation, and can bind to TNF receptor-1 and contribute to the pathogenesis of staphylococcal pneumonia.

[0095] The majority of S. aureus strains express the structural gene for protein A (SpA), a well-characterized virulence factor whose cell wall-anchored surface protein product (SpA) contains five highly homologous immunoglobulin-binding domains designated E, D, A, B, and C. The immunoglobulin domains, which show approximately 80% identity at the amino acid level, are 56 to 61 residues in length and are organized as tandem repeats. Each of the immunoglobulin-binding domains assembles into a three-helix bundle that is associated with the Fc domain of immunoglobulin G (IgG), the V domain of IgM, and the V domain of IgM. H It is composed of a triplet chain (Fab), an antiparallel α-helix that binds to von Willebrand factor in its A1 domain, and to tumor necrosis factor α (TNF-α) receptor 1 (TNFR1).

[0096] SpA prevents neutrophil phagocytosis of staphylococci by binding to the Fc component of IgG. In addition, SpA can activate intravascular coagulation through binding to the von Willebrand factor A1 domain. Plasma proteins such as fibrinogen and fibronectin act as bridges between staphylococci (ClfA and ClfB) and the platelet integrin GPIIb / IIIa, an activity that is complemented through SpA association with vWF A1, allowing staphylococci to capture platelets via the GPIb-α platelet receptor. SpA also binds to TNFR1, and this interaction contributes to the pathogenesis of staphylococcal pneumonia. SpA activates proinflammatory signaling through TNFR1-mediated activation of TRAF2, p38 / c-Jun kinase, mitogen-activated protein kinase (MAPK), and Rel-transcription factor NF-κB. SpA binding further induces TNFR1 release, an activity that appears to require TNF-converting enzyme (TACE). Each of the disclosed activities is mediated through the five IgG-binding domains and can be disrupted by the same amino acid substitutions that are initially defined by their requirement for interaction between Protein A and human IgG1 (Cedergren et al., (1993)).

[0097] SpA also targets the B cell receptor V H It functions as a B cell superantigen by capturing the Fab region of IgM containing 3. Following intravenous challenge, Staphylococcus aureus SpA mutants show reduced staphylococcal burden in organ tissues and a dramatically reduced ability to form abscesses.

[0098] In any embodiment, the SpA polypeptide of the immunogenic composition is a wild-type (non-variant) SpA polypeptide. In any embodiment, the SpA polypeptide comprises at least one SpA A, B, C, D, or E IgG domain. In any embodiment, the SpA polypeptide comprises at least an SpA A domain. In any embodiment, the SpA A domain comprises the amino acid sequence of SEQ ID NO: 55 or 48. In any embodiment, the SpA polypeptide comprises at least an SpA B domain. In any embodiment, the SpA B domain comprises the amino acid sequence of SEQ ID NO: 56 or 49. In any embodiment, the SpA polypeptide comprises at least an SpA C domain. In any embodiment, the SpA C domain comprises the amino acid sequence of SEQ ID NO: 57 or 50. In any embodiment, the SpA polypeptide comprises at least an SpA D domain. In any embodiment, the SpA D domain comprises the amino acid sequence of SEQ ID NO: 58 or 51. In any embodiment, the SpA polypeptide comprises at least an SpA E domain. In any embodiment, the SpA E domain comprises the amino acid sequence of SEQ ID NO: 59 or 52. In any embodiment, the SpA polypeptide comprises at least two of the SpA IgG domains, at least three of the SpA IgG domains, at least four of the SpA IgG domains, or all five of the SpA IgG domains. In any embodiment, the SpA polypeptide comprises the amino acid sequence of SEQ ID NO: 53, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 53. Exemplary SpA domains and full length sequences are provided in Table 3 below.

[0099] In any embodiment, the SpA polypeptide of the immunogenic composition is an SpA variant polypeptide. As referred to herein, the terms "Protein A variant," "SpA variant," "Protein A variant polypeptide," and "SpA variant polypeptide" refer to an SpA variant polypeptide that is an SpA variant polypeptide that is an SpA variant polypeptide. HSpA variant refers to a polypeptide comprising an SpA IgG domain having at least one amino acid substitution that disrupts binding to staphylococcal protein A and protein A-like proteins. In certain embodiments, the SpA variant polypeptide comprises a variant A domain, a variant B domain, a variant C domain, a variant D domain, and / or a variant E domain. Suitable SpA variant polypeptides include variants thereof and fragments thereof that are non-toxic and stimulate an immune response against staphylococcal protein A and protein A-like proteins and / or bacteria expressing same.

[0100] Described herein are SpA variant polypeptides that do not bind immunoglobulins and are therefore non-cytotoxic variants of wild-type SpA polypeptides. The SpA variant polypeptides are non-toxic and stimulate a humoral immune response to protect against staphylococcal infection and disease.

[0101] In any embodiment, the SpA variant polypeptide of the immunogenic composition is a full-length SpA variant comprising at least one variant E, D, A, B, or C domain. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:60 or 61. In any embodiment, the SpA variant polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:54.

[0102] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises a fragment of a full-length SpA polypeptide. The SpA variant polypeptide fragment may comprise one, two, three, four, five or more IgG binding domains. The IgG binding domains may be, for example, one, two, three, four, five or more variant A, B, C, D and / or E domains.

[0103] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises one, two, three, four, five, or more variant A domains. In any embodiment, the SpA variant polypeptide comprises one, two, three, four, five, or more variant B domains. In any embodiment, the SpA variant polypeptide comprises one, two, three, four, five, or more variant C domains. In any embodiment, the SpA variant polypeptide comprises one, two, three, four, five, or more variant D domains. In any embodiment, the SpA variant polypeptide comprises one, two, three, four, five, or more variant E domains.

[0104] In any embodiment, the variant A domain of the SpA variant polypeptide comprises one or more amino acid substitutions, for example, within the amino acid sequence of SEQ ID NO: 55 or 48. The variant B domain comprises one or more amino acid substitutions, for example, within the amino acid sequence of SEQ ID NO: 56 or 49. The variant C domain comprises one or more amino acid substitutions, for example, within the amino acid sequence of SEQ ID NO: 57 or 50. The variant D domain comprises one or more amino acid substitutions, for example, within the amino acid sequence of SEQ ID NO: 58 or 51. The variant E domain comprises one or more amino acid substitutions, for example, within the amino acid sequence of SEQ ID NO: 59 or 52.

[0105] In certain embodiments, the SpA variant polypeptide of the immunogenic composition comprises variant E, D, A, B, and / or C domains, which comprise amino acid sequences having 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:59 or 52, SEQ ID NO:58 or 51, SEQ ID NO:55 or 48, SEQ ID NO:56 or 49, and SEQ ID NO:57 or 50, respectively.

[0106] In any embodiment, the SpA variant polypeptide comprises a variant E domain comprising substitutions at amino acid positions 6, 7, 33, and / or 34 of SEQ ID NO:59. In any embodiment, the SpA variant polypeptide comprises a variant D domain comprising substitutions at amino acid positions 9, 10, 36, and / or 37 of SEQ ID NO:58. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises a variant A domain comprising substitutions at amino acid positions 7, 8, 34, and / or 35 of SEQ ID NO:55. In any embodiment, the SpA variant polypeptide comprises a variant B domain comprising substitutions at amino acid positions 7, 8, 34, and / or 35 of SEQ ID NO:56. In any embodiment, the SpA variant polypeptide comprises a variant C domain comprising substitutions at amino acid positions 7, 8, 34, and / or 35 of SEQ ID NO:57. Amino acid substitutions in the variant E, D, A, B, and / or C domains are described in WO2011 / 005341 and WO2020 / 232471, which are incorporated by reference in their entireties.

[0107] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises one or more amino acid substitutions in the IgG Fc binding subdomain of SpA domain D and / or at corresponding amino acid positions in other IgG domains. The one or more amino acid substitutions may disrupt or reduce binding of the SpA variant polypeptide to IgG Fc. In any embodiment, the SpA variant polypeptide comprises one or more amino acid substitutions in the IgG Fc binding subdomain of SpA domain D and / or at corresponding amino acid positions in other IgG domains. HThe one or more amino acid substitutions may further comprise one or more amino acid substitutions in the V3 binding subdomain and / or at corresponding amino acid positions in other IgG domains. H Binding to 3 may be disrupted or reduced.

[0108] The aforementioned amino acid substitutions in SpA domain D (i.e., the IgG Fc subdomain binding region or V H 3 binding subdomain region) may be incorporated into the SpA A, B, C, and / or E domains at the corresponding positions in each domain. The corresponding positions are defined by aligning SpA domain D with SpA domains A, B, C, and / or E to determine which residues in SpA domains A, B, C, and / or E correspond to the variant SpA D residues. For example, an amino acid substitution at glutamine residue position 9 in SEQ ID NO: 58 of SpA domain D corresponds to glutamine residue position 7 in SEQ ID NO: 55 of SpA domain A, glutamine residue position 7 in SEQ ID NO: 56 of SpA domain B, glutamine residue position 7 in SEQ ID NO: 57 of SpA domain C, and glutamine residue position 6 in SEQ ID NO: 59 of SpA domain E. Thus, the identified amino acid mutations described herein may be universally applied to the corresponding amino acid residues of any SpA domain amino acid sequence known now or in the future.

[0109] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises (a) one or more amino acid substitutions in the IgG Fc binding subdomain of SpA domain D and / or at corresponding amino acid positions in other IgG domains, and (b) one or more amino acid substitutions in the V H The one or more amino acid substitutions in the IgG Fc and V3 binding subdomains and / or at corresponding amino acid positions in other IgG domains may be used to modify the IgG Fc and V3 binding subdomains of the SpA variant polypeptide such that the SpA variant polypeptide has reduced or eliminated toxicity in the host organism. H Reduces binding to 3.

[0110] In any embodiment, amino acid residues F5, Q9, Q10, S11, F13, Y14, L17, N28, I31, and / or K35 of the IgG Fc binding subdomain of the SpA D domain of SEQ ID NO:58 are modified or substituted to reduce or eliminate binding to IgG Fc. In any embodiment, the corresponding modifications are incorporated into the SpA A, B, C, and / or E domains. The corresponding positions are defined by aligning SpA domain D with SpA domains A, B, C, and / or E to determine the residues in SpA domains A, B, C, and / or E that correspond to the residues of interest in SpA domain D.

[0111] In any embodiment, amino acid residues Q26, G29, F30, S33, D36, D37, Q40, N43, and / or E47 of the VH3 binding subdomain of the SpA D domain of SEQ ID NO:58 are H 3. Corresponding modifications can be incorporated into the SpA A, B, C, and / or E domains.

[0112] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises one, two, three, four, five, six, seven, eight, nine, ten or more variant D domains. The variant D domain may comprise one, two, three, four, five, six, seven, eight, nine, ten or more amino acid residue substitutions or modifications. The amino acid residue substitutions or modifications may occur, for example, at amino acid residues F5, Q9, Q10, S11, F13, Y14, L17, N28, I31, and / or K35 of the IgG Fc binding subdomain of SpA domain D (SEQ ID NO:58), and / or at amino acid residues Q26, G29, F30, S33, D36, D37, Q40, N43, and / or E47 of the VH3 binding subdomain of SpA domain D (SEQ ID NO:58). In any embodiment, the amino acid residue substitution or modification is at amino acid residues Q9 and Q10 of SEQ ID NO: 58. In any embodiment, the amino acid residue substitution or modification is at amino acid residues D36 and D37 of SEQ ID NO: 58. Amino acid substitutions in the variant A, B, C, D, and / or E domains are described in WO2011 / 005341, which is incorporated herein by reference in its entirety.

[0113] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90% (but not 100%) sequence identity to SEQ ID NO: 53 or 72. In any embodiment, the SpA variant polypeptide comprises an amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 53 or 72, or a fragment of at least n consecutive amino acids of SEQ ID NO: 53 or 72, where n comprises at least 7, at least 8, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, or at least 425 amino acids. In any embodiment, the SpA variant polypeptide may comprise one or more amino acid deletions from the carboxy (C) terminus (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, or 35 amino acids) and / or one or more amino acid deletions from the amino (N) terminus (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, or 35 amino acids) of SEQ ID NO: 72. In any embodiment, the last 35 C-terminal amino acids are deleted. In certain embodiments, the first 36 N-terminal amino acids are deleted. In any embodiment, the SpA variant polypeptide comprises amino acid residues 37-327 of SEQ ID NO: 72.

[0114] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises all five SpA IgG binding domains, arranged from N-terminus to C-terminus, in the following order: E, D, A, B, and C domains. In any embodiment, the SpA variant polypeptide comprises the E, D, A, B, and C domains of SpA, consecutively. In any embodiment, the SpA variant polypeptide comprises one, two, three, four, or five of the native E, D, A, B, and / or C domains. In embodiments where one, two, three, four, or five of the native domains are deleted, the SpA variant polypeptide can prevent excessive B cell proliferation and apoptosis that may occur when SpA functions as a B cell superantigen. In any embodiment, the SpA variant polypeptide comprises only the SpA E domain. In any embodiment, the SpA variant polypeptide comprises only the SpA D domain. In any embodiment, the SpA variant polypeptide comprises only the SpA A domain. In any embodiment, the SpA variant polypeptide comprises only the SpA B domain. In any embodiment, the SpA variant polypeptide comprises only the SpA C domain.

[0115] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises a mutation of at least one of eleven dipeptide sequence repeats (e.g., a QQ dipeptide repeat and / or a DD dipeptide repeat) compared to SEQ ID NO: 72. As an example, the SpA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 73, in which the XX dipeptide repeats at amino acid positions 7 and 8, 34 and 35, 60 and 61, 68 and 69, 95 and 96, 126 and 127, 153 and 154, 184 and 185, 211 and 212, 242 and 243, and 269 and 270 are substituted to reduce the affinity of the SpA variant polypeptide for immunoglobulins. Useful dipeptide substitutions of Gln-Gln (QQ) dipeptide may include, but are not limited to, Lys-Lys (KK), Arg-Arg (RR), Arg-Lys (RK), Lys-Arg (KR), Ala-Ala (AA), Ser-Ser (SS), Ser-Thr (ST), and Thr-Thr (TT) dipeptides. The QQ dipeptide is preferably replaced with a KR dipeptide. Useful dipeptide substitutions of Asp-Asp (DD) dipeptide may include, but are not limited to, Ala-Ala (AA), Lys-Lys (KK), Arg-Arg (RR), Lys-Arg (KR), His-His (HH), and Val-Val (VV) dipeptides. Dipeptide substitutions may, for example, decrease the affinity of the SpA variant polypeptide for the Fc portion of human IgG and the Fab portion of VH3-containing human B cell receptors.

[0116] Thus, in any embodiment, the SpA variant polypeptide of the immunogenic composition may comprise SEQ ID NO: 78, in which one or more, preferably all eleven, of the XX dipeptide repeats are substituted with an amino acid different from the corresponding dipeptide of SEQ ID NO: 72. In any embodiment, the SpA variant polypeptide comprises SEQ ID NO: 79, in which the amino acid doublets at positions 60 and 61 are Lys and Arg (K and R), respectively. In any embodiment, the SpA variant polypeptide comprises SEQ ID NO: 80 or SEQ ID NO: 81. In certain embodiments, the SpA variant polypeptide comprises SEQ ID NO: 75, with preferred examples of SEQ ID NO: 75 being SEQ ID NO: 76 or SEQ ID NO: 77 (SEQ ID NO: 76 with an N-terminal methionine).

[0117] In any embodiment, the SpA variant polypeptide N-terminus comprises a deletion of the first 36 amino acids of SEQ ID NO:72 and the C-terminus comprises a deletion of the last 35 amino acids of SEQ ID NO:72. An SpA variant polypeptide comprising an N-terminal deletion of 36 amino acids of SEQ ID NO:72 and a C-terminal deletion of 35 amino acids of SEQ ID NO:72 may further comprise a deletion of the fifth Ig binding domain (i.e., downstream of Lys-327 of SEQ ID NO:72). This SpA variant comprises the amino acid sequence of SEQ ID NO:73, where the XX dipeptide may be replaced with an amino acid such that the amino acid differs from the corresponding dipeptide sequence of SEQ ID NO:72. In any embodiment, the SpA variant polypeptide comprises SEQ ID NO:74.

[0118] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises one, two, three, or four of the natural A, B, C, D, and / or E domains, as described above. For example, the SpA variant polypeptide may comprise only the SpA E domain, but not the D, A, B, or C domains. Thus, the SpA variant polypeptide may comprise a variant SpA E domain, which comprises a substitution at at least one amino acid residue of SEQ ID NO:83. The substitution may be, for example, at amino acid positions 60 and 61 of SEQ ID NO:83. In any embodiment, the SpA variant polypeptide may comprise SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO:82. In any embodiment, the SpA variant polypeptide may comprise SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO:82 with at least one amino acid substitution. SpA variant polypeptides are described in WO2015 / 144653, which is incorporated herein by reference in its entirety.

[0119] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises amino acid substitutions at amino acids 43Q, 44Q, 96Q, 97Q, 162Q, 163Q, 220Q, 221Q, 278Q, and 279Q of SEQ ID NO: 84. The amino acid substitutions at amino acids 43Q, 44Q, 96Q, 97Q, 162Q, 163Q, 220Q, 221Q, 278Q, and 279Q of SEQ ID NO: 84 can be, for example, lysine (K) or arginine (R) substitutions. In certain embodiments, the SpA variant polypeptide comprises amino acid substitutions at amino acids 70D, 71D, 131D, 132D, 189D, 190D, 247D, 248D, 305D, and 306D of SEQ ID NO: 84. The amino acid substitutions at amino acids 70D, 71D, 131D, 132D, 189D, 190D, 247D, 248D, 305D, and 306D of SEQ ID NO: 84 can be, for example, alanine (A) or valine (V) substitutions. In certain embodiments, the SpA variant polypeptide can be selected from SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, and SEQ ID NO: 100. SpA variant polypeptides are described in US2016 / 0304566, which is incorporated herein by reference in its entirety.

[0120] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises a variant A domain, e.g., a variant A domain comprising an amino acid sequence of SEQ ID NO: 62, 67, 88, or 93, or an amino acid sequence having at least 90% identity to any one of the amino acid sequences of SEQ ID NO: 62, 67, 88, or 93. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises a variant B domain, e.g., a variant B domain comprising an amino acid sequence of SEQ ID NO: 63, 68, 89, or 94, or an amino acid sequence having at least 90% identity to any one of the amino acid sequences of SEQ ID NO: 63, 68, 89, or 94. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises a variant C domain, e.g., a variant C domain comprising an amino acid sequence of SEQ ID NO: 64, 69, 90, or 95, or an amino acid sequence having at least 90% identity to any one of the amino acid sequences of SEQ ID NO: 64, 69, 90, or 95. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises a variant D domain, e.g., a variant D domain comprising an amino acid sequence of SEQ ID NO: 66, 71, 91, or 96, or an amino acid sequence having at least 90% identity to any one of the amino acid sequences of SEQ ID NO: 66, 71, 91, or 96. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises a variant E domain, e.g., a variant E domain comprising an amino acid sequence of SEQ ID NO: 65, 70, 92, or 97, or an amino acid sequence having at least 90% identity to any one of the amino acid sequences of SEQ ID NO: 65, 70, 92, or 97.

[0121] In any embodiment, the variant A domain of the SpA variant polypeptide of the immunogenic composition can comprise, for example, the amino acid sequence of SEQ ID NO: 62. The variant B domain can comprise, for example, the amino acid sequence of SEQ ID NO: 63. The variant C domain can comprise, for example, the amino acid sequence of SEQ ID NO: 64. The variant D domain can comprise, for example, the amino acid sequence of SEQ ID NO: 66. The variant E domain can comprise, for example, the amino acid sequence of SEQ ID NO: 65.

[0122] In any embodiment, the SpA variant polypeptide of the immunogenic composition can comprise variant A, B, C, D, and E domains, which can comprise amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:62 or 67, SEQ ID NO:63 or 68, SEQ ID NO:64 or 69, SEQ ID NO:66 or 71, and SEQ ID NO:65 or 70, respectively.

[0123] In any embodiment, the SpA variant polypeptide of the immunogenic composition can comprise variant A, B, C, D, and E domains, which can comprise amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:88 or 93, SEQ ID NO:89 or 94, SEQ ID NO:90 or 95, SEQ ID NO:91 or 96, and SEQ ID NO:92 or 97, respectively.

[0124] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises a variant D domain, wherein the variant D domain comprises substitutions at amino acid positions corresponding to 9, 10, and / or 33 of SEQ ID NO:58.

[0125] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises (i) a lysine substitution for a glutamine amino acid residue in each of the SpA A-E domains at amino acid positions corresponding to positions 9 and 10 of the SpA D domain (SEQ ID NO: 58), and (ii) a glutamic acid substitution for a serine amino acid residue in each of the SpA A-E domains at amino acid position corresponding to position 33 of the SpA D domain (SEQ ID NO: 58). The SpA variant polypeptide does not detectably cross-link IgG and IgE in the blood and / or activate basophils compared to a negative control. By not detectably cross-linking IgG and IgE in the blood and / or by not activating basophils, the SpA variant polypeptide does not pose significant safety or toxicity concerns for human patients and / or does not pose a significant risk of anaphylactic shock in human patients.

[0126] In any embodiment, the V from human IgG H K of the SpA variant polypeptides described herein relative to 3 A The binding affinity was measured using an SpA variant polypeptide (SpA KKAA ) is reduced compared to the SpA variant polypeptide consisting of a glutamine to lysine substitution in each of domains A to E at amino acid positions corresponding to positions 9 and 10 of domain D (SEQ ID NO: 58) and an aspartic acid to alanine substitution in each of domains A to E at amino acid positions corresponding to positions 36 and 37 of domain D, was used as a comparison standard and KKAA It is named SpA. KKAA The variant polypeptide has the amino acid sequence of SEQ ID NO: 54. In certain embodiments, the SpA variant polypeptide is KKAAV from human IgG was reduced by at least 2-fold compared to H K for 3 A In certain embodiments, the SpA variant polypeptide of the immunogenic composition has binding affinity to SpA KKAA V from human IgG reduced by at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3-fold or more, or any value therebetween, compared to H K for 3 A In certain embodiments, the SpA variant polypeptide of the immunogenic composition has binding affinity to SpA KKAA V from human IgG that is reduced by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300% or more, or any value therebetween, compared to H K for 3 A In certain embodiments, the SpA variant polypeptide of the immunogenic composition has a binding affinity of about 1×10 5 M -1 V from human IgG is less than H K for 3 A In certain embodiments, the SpA variant polypeptide of the immunogenic composition has a binding affinity of about 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 x 10 5 M -1 V from human IgG that is less than or any value therebetween H K for 3 AIn any embodiment, the SpA variant polypeptide of the immunogenic composition does not have substitutions in any of the SpA A-E domains corresponding to positions 36 and 37 of the SpA D domain (SEQ ID NO:58).

[0127] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises (i) a lysine substitution for a glutamine amino acid residue in each of the SpA A-E domains at positions corresponding to positions 9 and 10 of the SpA D domain (SEQ ID NO:58), and (ii) a glutamic acid substitution for a serine amino acid residue in each of the SpA A-E domains at a position corresponding to position 33 of the SpA D domain (SEQ ID NO:58). In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an SpA E domain having an amino acid sequence of SEQ ID NO:65, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:65. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an SpA D domain having an amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 66. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an SpA A domain having an amino acid sequence of SEQ ID NO: 62, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 62. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an SpA B domain having an amino acid sequence of SEQ ID NO:63, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:63.In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an SpA C domain having an amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 64. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an amino acid sequence of SEQ ID NO: 60, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 60. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises the amino acid sequence of SEQ ID NO: 60.

[0128] In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises (i) a lysine substitution for a glutamine amino acid residue in each of the SpA A-E domains at positions corresponding to positions 9 and 10 of the SpA D domain (SEQ ID NO:58), and (ii) a threonine substitution for a serine amino acid residue in each of the SpA A-E domains at a position corresponding to position 33 of the SpA D domain (SEQ ID NO:58). In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an SpA E domain having an amino acid sequence of SEQ ID NO:70, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:70. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an SpA D domain having an amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 71. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an SpA A domain having an amino acid sequence of SEQ ID NO: 67, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 67. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an SpA B domain having an amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:68.In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an SpA C domain having an amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 69. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises an amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 61. In any embodiment, the SpA variant polypeptide of the immunogenic composition comprises the amino acid sequence of SEQ ID NO: 61.

[0129] The SpA variant polypeptides do not detectably cross-link IgG and IgE in the blood and / or activate basophils compared to negative controls. By not detectably cross-linking IgG and IgE in the blood and / or activating basophils, the SpA variant polypeptides do not pose significant safety or toxicity issues for human patients or pose significant risk of anaphylactic shock in human patients. SpA variant polypeptides suitable for use in the compositions and methods disclosed herein are described in WO2020 / 232471, which is incorporated herein by reference in its entirety.

[0130] Table 3 below provides exemplary SpA polypeptide amino acid sequences of the immunogenic compositions disclosed herein. [Table 3] TIFF2024512751000007.tif219170TIFF2024512751000008.tif210170TIFF2024512751000009.tif225170TIFF2024512751000010.tif68170

[0131] According to all aspects of the disclosure herein, the LukA variant polypeptides, LukB polypeptides, and SpA polypeptides of the immunogenic compositions disclosed herein may further comprise one or more heterologous amino acid sequences. Suitable heterologous amino acid sequences include, but are not limited to, tag sequences, immunogens, signal sequences, and the like. Suitable tag sequences include, but are not limited to, polyhistidine tags, polyarginine tags, FLAG tags, step tag II, ubiquitin tags, NusA tags, chitin-binding domains, calmodulin-binding peptides, cellulose-binding domains, Hat-tags, S-tags, SBPs, maltose-binding proteins, glutathione S-transferases (see Terpe K., "Overview of Tag Protein Fusions: From Molecular and Biochemical Fundamentals to Commercial Systems," Appl. Microbiol. Biotechnol. 60:523-33 (2003), incorporated herein by reference). Suitable immunogens include, but are not limited to, T cell epitopes, B cell epitopes. Suitable signal sequences include, but are not limited to, PelB signal sequence, Sec signal sequence, Tat signal sequence, AmyE signal sequence (see Freudl R., "Signal Peptides for Recombinant Protein Secretion in Bacterial Expression Systems", Microbial Cell Factories 17:52 (2018), incorporated herein by reference). In some embodiments, the LukA, LukB, and SpA polypeptides described herein comprise a PelB sequence (MKYLLPTAAAGLLLLAAQPAMA, SEQ ID NO: 23). In some embodiments, the LukA, LukB, and SpA polypeptides described herein comprise a His-tag (e.g., NSAHHHHHHGS, SEQ ID NO: 24). Thus, in some embodiments, the SpA, LukA, and / or LukB polypeptides described herein comprise both the PelB sequence and a His-tag as described above.

[0132] S. aureus LukA, LukB, and SpA polynucleotides and constructs Another aspect of the present disclosure is directed to nucleic acid molecules encoding the LukA variant polypeptides, LukB polypeptides, and SpA polypeptides described herein, as well as immunogenic compositions comprising one or more of these nucleic acid molecules. The nucleic acid molecules described herein include isolated polynucleotides, recombinant polynucleotide sequences, portions of expression vectors, or portions of linear DNA sequences, including linear DNA sequences used for in vitro or in vivo transcription / translation, as well as vectors suitable for prokaryotic and eukaryotic expression and secretion of the variant LukA, LukB, and SpA polypeptides described herein. The polynucleotides of the present disclosure may be produced by chemical synthesis, such as solid-phase polynucleotide synthesis on an automated nucleic acid synthesizer, and assembled into complete single-stranded or double-stranded molecules. Alternatively, the polynucleotides of the present disclosure may be produced by other techniques, such as PCR followed by conventional cloning. Techniques for producing or obtaining polynucleotides of a given sequence are well known in the art.

[0133] In any embodiment, the immunogenic composition disclosed herein comprises a polynucleotide encoding a LukA variant polypeptide. In any embodiment, the polynucleotide encodes a LukA variant comprising a lysine to methionine substitution (Lys83Met) at a residue corresponding to lysine at position 83 of SEQ ID NO:25. In any embodiment, the polypeptide of the present disclosure encodes a variant LukA polypeptide comprising a serine to alanine substitution (Ser141Ala) at a residue corresponding to serine at position 141 of SEQ ID NO:25. In any embodiment, the polypeptide of the present disclosure encodes a variant LukA polypeptide comprising a valine to isoleucine substitution (Val113Ile) at a residue corresponding to valine at position 113 of SEQ ID NO:25. In any embodiment, the polypeptide of the present disclosure encodes a LukA polypeptide comprising a valine to isoleucine substitution (Val193Ile) at a residue corresponding to valine at position 193 of SEQ ID NO:25. In any embodiment, a polynucleotide of the disclosure encodes a variant LukA polypeptide thereof that includes lysine to methionine, serine to alanine, and valine to isoleucine amino acid substitutions at residues corresponding to the aforementioned amino acid residues, i.e., Lys803Met, Ser141Ala, Val113Ile, and Val193Ile of SEQ ID NO: 25. In any embodiment, a polynucleotide of the disclosure encodes a variant LukA polypeptide thereof that further includes an amino acid substitution corresponding to Glu323Ala, i.e., the polynucleotide encodes a variant LukA that includes substitutions corresponding to the Lys83Met, Ser141Ala, Val113Ile, Val193Ile, and Glu323Ala substitutions of SEQ ID NO: 25.

[0134] In one embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC8 LukA variant sequence, such as a nucleic acid molecule encoding a variant of SEQ ID NO: 1 that includes amino acid substitutions corresponding to Lys80Met, Ser138Ala, Val110Ile, Val190Ile, and Glu320Ala in SEQ ID NO: 1. An exemplary nucleic acid molecule encoding CC8 LukA is provided herein as SEQ ID NO: 101. Thus, in any embodiment, an exemplary nucleic acid molecule is a variant of SEQ ID NO: 101, which variant comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 101.

[0135] In any embodiment, an exemplary nucleic acid molecule of the immunogenic composition is a nucleic acid molecule encoding a LukA variant sequence of SEQ ID NO: 3 (LukA CC8 Glu320Ala, Lys80Met, Ser138Ala, Val110Ile, Val190Ile), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 3. An exemplary nucleic acid molecule encoding this LukA CC8 variant comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to SEQ ID NO: 103. In any embodiment, a nucleic acid molecule encoding this LukA CC8 variant comprises the nucleotide sequence of SEQ ID NO: 103.

[0136] In another embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a CC45 LukA variant sequence, e.g., a nucleic acid molecule encoding a variant of SEQ ID NO: 2 that includes amino acid substitutions corresponding to Lys81Met, Ser139Ala, Val111Ile, Val191Ile, and Glu321Ala of SEQ ID NO: 2. An exemplary nucleic acid molecule encoding CC45 LukA is provided herein as SEQ ID NO: 102. Thus, in any embodiment, an exemplary nucleic acid molecule is a variant of SEQ ID NO: 102, which variant comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 102.

[0137] In another embodiment, an exemplary nucleic acid molecule of the immunogenic composition is a nucleic acid molecule encoding a LukA variant sequence of SEQ ID NO: 4 (LukA CC45 Glu321Ala, Lys81Met, Ser139Ala, Val111Ile, Val191Ile), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 4. An exemplary nucleic acid molecule encoding this LukA CC45 variant comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to SEQ ID NO: 104. In any embodiment, a nucleic acid molecule encoding this LukA CC8 variant comprises the nucleotide sequence of SEQ ID NO: 104.

[0138] In any embodiment, one or more polynucleotides of the immunogenic composition encode a LukA variant protein or polypeptide comprising an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO: 25. In any embodiment, the polynucleotide encodes a LukA variant protein or polypeptide comprising a tyrosine to cysteine ​​substitution at amino acid residue corresponding to Tyr74 of SEQ ID NO: 25 (Tyr74Cys) and an asparagine to cysteine ​​substitution at amino acid residue corresponding to Asp140 of SEQ ID NO: 25 (Asp140Cys). In any embodiment, the polynucleotide encodes a LukA variant protein or polypeptide comprising a glycine to cysteine ​​substitution at amino acid residue corresponding to Gly149 of SEQ ID NO: 25 (Gly149Cys) and a glycine to cysteine ​​substitution at amino acid residue corresponding to Gly156 of SEQ ID NO: 25 (Gly156Cys). In any embodiment, the polynucleotide encodes a variant LukA protein or polypeptide that includes an amino acid substitution at each of the amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO: 25. In any embodiment, the amino acid substitution at each of these amino acid residues is a cysteine ​​residue, as described above.

[0139] In any embodiment, the polynucleotide of the immunogenic composition encodes a variant LukA protein or polypeptide comprising amino acid substitutions at one or more amino acid residues corresponding to Lys83, Ser141, Val113, Val193, and Glu323 in combination with amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO: 25. In any embodiment, the polynucleotide encodes a variant LukA protein or polypeptide comprising amino acid substitutions at residues Lys83, Ser141, Val113, Val193, and Glu323 and amino acid residues corresponding to residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO: 25. In any embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a LukA variant sequence of SEQ ID NO:5 (LukA CC8 Glu320Ala, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Tyr71Cys, Asp137Cys, Gly146Cys, Gly153Cys), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:5. In any embodiment, an exemplary nucleic acid molecule of the disclosure is a nucleic acid molecule encoding a LukA variant sequence of SEQ ID NO:6 (LukA CC45 Glu321Ala, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Tyr72Cys, Asp138Cys, Gly147Cys, Gly154Cys), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:6.

[0140] In any embodiment, one or more polynucleotides of the immunogenic composition encodes a LukA variant polypeptide comprising an amino acid substitution or deletion at an amino acid residue corresponding to amino acid residue Thr249 of SEQ ID NO: 25. In any embodiment, the polynucleotide encodes a LukA variant comprising a threonine to valine substitution at this residue corresponding to position 249 of SEQ ID NO: 25. In any embodiment, the polynucleotides of the disclosure encode a LukA variant polypeptide comprising an amino acid substitution at a position corresponding to Thr249 in combination with any one or all of the amino acid substitutions at residues corresponding to Lys83, Ser141, Val113, Val193, Glu323, Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO: 25. In any embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a LukA variant sequence of SEQ ID NO: 7 (LukA CC8 Glu320Ala, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, and Thr246Val), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 7. In any embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a LukA variant sequence of SEQ ID NO: 8 (LukA CC45 Glu321Ala, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Thr247Val), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 8.

[0141] In any embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a LukA variant sequence of SEQ ID NO:9 (LukA CC8 Glu320Ala, Lys80Met, Ser138Ala, Val110Ile, Val190Ile, Thr246Val, Tyr71Cys, Asp137Cys, Gly146Cys, and Gly153Cys), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:9. In any embodiment, an exemplary nucleic acid molecule encoding this LukA CC8 variant of SEQ ID NO:9 comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to SEQ ID NO:105. In any embodiment, a nucleic acid molecule encoding this LukA CC8 variant comprises the nucleotide sequence of SEQ ID NO:105.

[0142] In any embodiment, an exemplary nucleic acid molecule is a nucleic acid molecule encoding a LukA variant sequence of SEQ ID NO: 10 (LukA CC45 Glu321Ala, Lys81Met, Ser139Ala, Val111Ile, Val191Ile, Thr247Val, Tyr72Cys, Asp138Cys, Gly147Cys, and Gly154Cys), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 10. In any embodiment, an exemplary nucleic acid molecule encoding this LukA CC45 variant of SEQ ID NO: 10 comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to SEQ ID NO: 106. In any embodiment, a nucleic acid molecule encoding this LukA CC8 variant comprises the nucleotide sequence of SEQ ID NO: 106.

[0143] In any embodiment, one or more polynucleotides of the immunogenic compositions disclosed herein further encode a LukB polypeptide disclosed herein. In any embodiment, the polynucleotide encodes a LukB polypeptide comprising the amino acid sequence of SEQ ID NO: 15. An exemplary nucleic acid molecule encoding CC8 LukB is provided herein as SEQ ID NO: 107. Thus, in any embodiment, the exemplary nucleic acid molecule is a variant of SEQ ID NO: 107, the variant comprising a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 107.

[0144] In any embodiment, the polynucleotide encodes a LukB polypeptide comprising the amino acid sequence of SEQ ID NO: 16. An exemplary nucleic acid molecule encoding CC45 LukB is provided herein as SEQ ID NO: 108. Thus, in any embodiment, the exemplary nucleic acid molecule is a variant of SEQ ID NO: 108, where the variant comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:108.

[0145] In any embodiment, the polynucleotide encodes a variant LukB polypeptide comprising an amino acid substitution or deletion at an amino acid residue corresponding to amino acid residue Val53 of SEQ ID NO: 39. In any embodiment, the amino acid substitution at Val53 comprises a valine to leucine (Val53Leu) substitution.

[0146] In any embodiment, an exemplary polynucleotide of the disclosure encodes a variant LukB protein or polypeptide of SEQ ID NO: 17 (LukB CC8 V53L), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 17. An exemplary nucleic acid molecule encoding this LukB CC8 V53L variant comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to SEQ ID NO: 109. In any embodiment, a nucleic acid molecule encoding this LukA CC8 variant comprises the nucleotide sequence of SEQ ID NO: 109.

[0147] In any embodiment, an exemplary polynucleotide of the disclosure encodes a variant LukB protein or polypeptide of SEQ ID NO: 18 (LukB CC45 V53L), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 18. An exemplary nucleic acid molecule encoding this LukB CC45 V53L variant comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to SEQ ID NO: 110. In any embodiment, a nucleic acid molecule encoding this LukA CC45 variant comprises the nucleotide sequence of SEQ ID NO: 110.

[0148] In any embodiment, the polynucleotide of the immunogenic composition encodes a variant LukB protein or polypeptide comprising an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO: 39. In any embodiment, the amino acid substitution at one or more of the foregoing residues introduces one or more cysteine ​​residues capable of forming disulfide bonds to stabilize the conformation of the LukAB heterodimeric structure. In any embodiment, the polynucleotide encodes a LukB variant protein or polypeptide comprising a glutamic acid to cysteine ​​substitution (Glu45Cys) at the amino acid residue corresponding to Glu45 of SEQ ID NO: 39, and a threonine to cysteine ​​substitution (Thr121Cys) at the amino acid residue corresponding to Thr121 of SEQ ID NO: 39. In any embodiment, the polynucleotide encodes a LukB variant protein or polypeptide that includes a glutamic acid to cysteine ​​substitution at the amino acid residue corresponding to Glu109 of SEQ ID NO:39 (Glu109Cys), and an arginine to cysteine ​​substitution at the amino acid residue corresponding to Arg154 of SEQ ID NO:39 (Arg154Cys).

[0149] In any embodiment, the polynucleotide of the immunogenic composition encodes a variant LukB protein or polypeptide comprising an amino acid substitution at each of the amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO: 39. In any embodiment, the amino acid substitution at each of these amino acid residues is accompanied by the introduction of a cysteine ​​residue as described above. In any embodiment, the polynucleotide encodes a variant LukB protein or polypeptide comprising the amino acid sequence of SEQ ID NO: 21 (LukB CC8 Glu45Cys, Glu109Cys, Thr121Cys, and Arg154Cys), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 21. In any embodiment, the polynucleotide encodes a variant LukB protein or polypeptide comprising the amino acid sequence of SEQ ID NO:22 (LukB CC45 Glu45Cys, Thr122Cys, Glu110Cys, Arg155Cys), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:22.

[0150] In any embodiment, a polynucleotide of the disclosure encodes a variant LukB protein or polypeptide that includes an amino acid substitution at an amino acid residue corresponding to Val53 of SEQ ID NO: 39 in combination with an amino acid substitution at one or more amino acid residues corresponding to Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO: 39. In any embodiment, the polynucleotide encodes a variant LukB protein or polypeptide having the amino acid sequence of SEQ ID NO: 19 (LukB CC8 Val53Leu, Glu45Cys, Glu109Cys, Thr121Cys, and Arg154Cys), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO: 19. In any embodiment, the polynucleotide encodes a variant LukB protein or polypeptide having the amino acid sequence of SEQ ID NO:20 (LukB CC45 Val53Leu, Glu45Cys, Thr122Cys, Glu110Cys, Arg155Cys), or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:20.

[0151] In any embodiment, an exemplary nucleic acid molecule of the immunogenic composition encodes the CC45 LukA variant sequence of SEQ ID NO: 4 and the CC45 LukB sequence of SEQ ID NO: 16. An exemplary nucleic acid molecule encoding this LukAB heterodimer (RARPR-15) comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 104 (CC45 LukA variant) operably linked to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 108 (CC45 LukB). An exemplary nucleic acid molecule encoding this LukAB heterodimer comprises the nucleotide sequence of SEQ ID NO: 104 operably linked to the nucleotide sequence of SEQ ID NO: 108.

[0152] In any embodiment, an exemplary nucleic acid molecule of the present disclosure encodes a CC45 LukA variant sequence of SEQ ID NO: 4 and a CC45 LukB variant sequence of SEQ ID NO: 18. An exemplary nucleic acid molecule encoding this LukAB heterodimer (RARPR-30) comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 104 (CC45 LukA variant) operably linked to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 110 (CC45 LukB variant). An exemplary nucleic acid molecule encoding this LukAB heterodimer comprises the nucleotide sequence of SEQ ID NO: 104 operably linked to the nucleotide sequence of SEQ ID NO: 110.

[0153] In any embodiment, an exemplary nucleic acid molecule of the present disclosure encodes a CC8 LukA variant sequence of SEQ ID NO: 3 and a CC8 LukB sequence of SEQ ID NO: 15. An exemplary nucleic acid molecule encoding this LukAB heterodimer (RARPR-32) comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 103 (CC8 LukA variant) operably linked to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 107 (CC8 LukB). An exemplary nucleic acid molecule encoding this LukAB heterodimer comprises the nucleotide sequence of SEQ ID NO: 103 operably linked to the nucleotide sequence of SEQ ID NO: 107.

[0154] In any embodiment, an exemplary nucleic acid molecule of the present disclosure encodes a CC8 LukA variant sequence of SEQ ID NO: 3 and a CC45 LukB variant sequence of SEQ ID NO: 18. An exemplary nucleic acid molecule encoding this LukAB heterodimer (RARPR-33) comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 103 (CC8 LukA variant) operably linked to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 110 (CC45 LukB variant). An exemplary nucleic acid molecule encoding this LukAB heterodimer comprises the nucleotide sequence of SEQ ID NO: 103 operably linked to the nucleotide sequence of SEQ ID NO: 110.

[0155] In any embodiment, an exemplary nucleic acid molecule of the present disclosure encodes a CC8 LukA variant sequence of SEQ ID NO: 3 and a CC8 LukB variant sequence of SEQ ID NO: 17. An exemplary nucleic acid molecule encoding this LukAB heterodimer (RARPR-34) comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 103 (CC8 LukA variant) operably linked to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 109 (CC8 LukB variant). An exemplary nucleic acid molecule encoding this LukAB heterodimer comprises the nucleotide sequence of SEQ ID NO: 103 operably linked to the nucleotide sequence of SEQ ID NO: 109.

[0156] Exemplary LukA and LukB nucleic acid molecule sequences of the disclosure are provided in Table 4 below. [Table 4] TIFF2024512751000012.tif189170TIFF2024512751000013.tif189170TIFF20245127510 00014.tif193170TIFF2024512751000015.tif188170TIFF2024512751000016.tif188170

[0157] In any embodiment, the immunogenic composition disclosed herein comprises a polynucleotide encoding an SpA polypeptide. In any embodiment, the polynucleotide encodes a wild-type or non-variant SpA polypeptide. In any embodiment, the polynucleotide encodes an SpA A domain comprising the amino acid sequence of SEQ ID NO: 55 or 48. In any embodiment, the polynucleotide encodes an SpA B domain comprising the amino acid sequence of SEQ ID NO: 56 or 49. In any embodiment, the polynucleotide encodes an SpA C domain comprising the amino acid sequence of SEQ ID NO: 57 or 50. In any embodiment, the polynucleotide encodes an SpA D domain comprising the amino acid sequence of SEQ ID NO: 58 or 51. In any embodiment, the polynucleotide encodes an SpA E domain comprising the amino acid sequence of SEQ ID NO: 59 or 52. In any embodiment, the polynucleotide encodes an SpA polypeptide and comprises at least two of the SpA IgG domains, at least three of the SpA IgG domains, at least four of the SpA IgG domains, or all five of the SpA IgG domains. In any embodiment, the polynucleotide encodes an SpA polypeptide comprising the amino acid sequence of SEQ ID NO:53, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:53.

[0158] In any embodiment, the polynucleotide of the immunogenic composition encodes variant E, D, A, B, and / or C domains, which comprise amino acid sequences having 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:55 or 48, SEQ ID NO:56 or 49, SEQ ID NO:57 or 50, SEQ ID NO:58 or 51, and SEQ ID NO:59 or 52, respectively. Exemplary variant E, D, A, B, and C SpA domains are described above.

[0159] In any embodiment, the polynucleotide encodes an SpA variant polypeptide having a variant E domain that includes substitutions at amino acid positions 6, 7, 33, and / or 34 of SEQ ID NO:59. In any embodiment, the polynucleotide encodes an SpA variant polypeptide having a variant D domain that includes substitutions at amino acid positions 9, 10, 36, and / or 37 of SEQ ID NO:58. In any embodiment, the polynucleotide encodes an SpA variant polypeptide having a variant A domain that includes substitutions at amino acid positions 7, 8, 34, and / or 35 of SEQ ID NO:55. In any embodiment, the polynucleotide encodes an SpA variant polypeptide having a variant B domain that includes substitutions at amino acid positions 7, 8, 34, and / or 35 of SEQ ID NO:56. In any embodiment, the polynucleotide encodes an SpA variant polypeptide having a variant C domain that includes substitutions at amino acid positions 7, 8, 34, and / or 35 of SEQ ID NO:57.

[0160] In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA variant polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90% (but not 100%) sequence identity to SEQ ID NO: 53 or 72. In any embodiment, the SpA variant polypeptide comprises an amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 53 or 72 or a fragment thereof.

[0161] In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA variant polypeptide comprising one or more amino acid substitutions in SpA domain D or at corresponding amino acid positions in other SpA IgG domains, wherein the one or more amino acid substitutions disrupt or reduce binding of the SpA variant polypeptide to IgG Fc. H 3, which disrupts or reduces binding to V of the D domain HThe present invention encodes an SpA variant polypeptide that further comprises one or more amino acid substitutions in the 3 binding subdomain or at corresponding amino acid positions in other IgG domains.

[0162] In any embodiment, the polynucleotide encodes an SpA variant polypeptide comprising a variant A domain, e.g., a variant A domain comprising the amino acid sequence of SEQ ID NO: 62, 67, 88, or 93. In any embodiment, the polynucleotide encodes an SpA variant polypeptide comprising a variant B domain, e.g., a variant B domain comprising the amino acid sequence of SEQ ID NO: 63, 68, 89, or 94. In any embodiment, the polynucleotide encodes an SpA variant polypeptide comprising a variant C domain, e.g., a variant C domain comprising the amino acid sequence of SEQ ID NO: 64, 69, 90, or 95. In any embodiment, the polynucleotide encodes an SpA variant polypeptide comprising a variant D domain, e.g., a variant D domain comprising the amino acid sequence of SEQ ID NO: 66, 71, 91, or 96. In any embodiment, the polynucleotide encodes an SpA variant polypeptide comprising a variant E domain, e.g., a variant E domain comprising the amino acid sequence of SEQ ID NO: 65, 70, 92, or 97.

[0163] In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA variant polypeptide comprising variant A, B, C, D, and E domains comprising amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:62 or 67, SEQ ID NO:63 or 68, SEQ ID NO:64 or 69, SEQ ID NO:66 or 71, and SEQ ID NO:65 or 70, respectively.

[0164] In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA variant polypeptide comprising variant A, B, C, D, and E domains that comprise amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical identity to SEQ ID NO:88 or 93, SEQ ID NO:89 or 94, SEQ ID NO:90 or 95, SEQ ID NO:91 or 96, and SEQ ID NO:92 or 97, respectively.

[0165] In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA variant polypeptide comprising a variant D domain, wherein the variant D domain comprises substitutions at amino acid positions corresponding to 9, 10, and / or 33 of SEQ ID NO:58.

[0166] In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA variant polypeptide comprising (i) a lysine substitution of a glutamine amino acid residue in each of the SpA A-E domains at amino acid positions corresponding to positions 9 and 10 of the SpA D domain (SEQ ID NO:58), and (ii) a glutamic acid substitution of a serine amino acid residue in each of the SpA A-E domains at amino acid position corresponding to position 33 of the SpA D domain (SEQ ID NO:58). In any embodiment, the polynucleotide encodes an SpA E domain having an amino acid sequence of SEQ ID NO:65. In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA D domain having an amino acid sequence of SEQ ID NO:66. In any embodiment, the polynucleotide encodes an SpA A domain having an amino acid sequence of SEQ ID NO:62. In any embodiment, the polynucleotide encodes an SpA B domain having an amino acid sequence of SEQ ID NO:63. In any embodiment, the polynucleotide encodes an SpA C domain having an amino acid sequence of SEQ ID NO:64. In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA variant polypeptide having the amino acid sequence of SEQ ID NO:60.

[0167] In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA variant polypeptide comprising (i) a lysine substitution for a glutamine amino acid residue in each of the SpA A-E domains at amino acid positions corresponding to positions 9 and 10 of the SpA D domain (SEQ ID NO:58), and (ii) a threonine substitution for a serine amino acid residue in each of the SpA A-E domains at amino acid position corresponding to position 33 of the SpA D domain (SEQ ID NO:58). In any embodiment, the polynucleotide encodes an SpA E domain having an amino acid sequence of SEQ ID NO:70. In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA D domain having an amino acid sequence of SEQ ID NO:71. In any embodiment, the polynucleotide encodes an SpA A domain having an amino acid sequence of SEQ ID NO:67. In any embodiment, the polynucleotide encodes an SpA B domain having an amino acid sequence of SEQ ID NO:68. In any embodiment, the polynucleotide encodes an SpA C domain having an amino acid sequence of SEQ ID NO:69. In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA variant polypeptide having the amino acid sequence of SEQ ID NO:61.

[0168] In any embodiment, the polynucleotide of the immunogenic composition encodes an SpA variant polypeptide comprising variant A, B, C, D, and / or E domains. In any embodiment, the polynucleotide encodes an SpA variant polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:60 or 61. In any embodiment, the polynucleotide encodes an SpA variant polypeptide comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:54.

[0169] In some embodiments, the nucleic acid molecules encoding the S. aureus polypeptides described herein are codon-optimized for expression in mammalian cells, preferably human cells. Methods of codon optimization are known and have been previously described (e.g., International Patent Application Publication No. 1996 / 09378 to Seed, incorporated herein by reference in its entirety). A sequence is considered to be codon-optimized if, compared to the wild-type sequence, at least one non-preferred codon is replaced with a more preferred codon. As used herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon that codes for the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a particular organism can be found in codon frequency tables that are well known and available in the art. Preferably, more than one non-preferred codon, e.g., more than 10%, 40%, 60%>, 80%> non-preferred codons, preferably most (e.g., at least 90%) or all non-preferred codons are replaced with more preferred codons. Preferably, the codons most frequently used in the organism are used in the codon-optimized sequence. Substitution with preferred codons generally results in higher expression.

[0170] The polynucleotides of the present disclosure may be cloned using conventional molecular biology techniques or may be produced de novo by DNA synthesis, which can be performed using routine procedures by service companies operating in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Invitrogen, Eurofins).

[0171] In some embodiments, the nucleic acid molecules described above are inserted into a vector, such as an expression vector for use in the immunogenic compositions described herein. Alternatively, these nucleic acid molecules may be inserted into expression vectors that are transformed or transfected into a suitable host cell for expression and isolation of the encoded SpA polypeptide, LukA variant polypeptide, LukB protein, or LukAB complex (as a stable heterodimer) disclosed herein.

[0172] According to this aspect of the disclosure, the nucleic acid molecules encoding the S. aureus polypeptides described herein may be incorporated into any expression vector capable of expressing the polypeptide encoded by the nucleic acid sequence construct. Suitable expression vectors contain nucleic acid sequence elements that control, regulate, cause, or enable expression of the polypeptide encoded by such vector. Such elements may include transcription enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate expression of the encoded polypeptide in a given expression system. Suitable vectors include, but are not limited to, DNA vectors, plasmid vectors, linear nucleic acids, and viral vectors, such as adenoviral vectors.

[0173] In one embodiment, the expression vector is a circular plasmid (see, e.g., Muthumani et al., "Optimized and Enhanced DNA Plasmid Vector Based In vivo Construction of a Neutralizing anti-HIV-1 Envelope Glycoprotein Fab," Hum. Vaccin. Immunother. 9:2253-2262 (2013), which is incorporated by reference in its entirety). The plasmid can transform the target cell by integration into the cell genome or can exist extrachromosomally (e.g., an autonomously replicating plasmid with an origin of replication). Exemplary plasmid vectors include, but are not limited to, pCEP4, pREP4, pVAX, pcDNA3.0, provax, or any other plasmid expression vector capable of expressing a variant LukA and / or variant LukB protein or polypeptide encoded by a recombinant nucleic acid sequence construct.

[0174] In another embodiment, the expression vector is a linear expression cassette ("LEC"). The LEC can be efficiently delivered to a subject via electroporation to express the SpA, LukA, and / or LukB polypeptides encoded by the recombinant nucleic acid molecules described herein. The LEC can be any linear DNA lacking a phosphate backbone. In one embodiment, the LEC does not contain any antibiotic resistance genes and / or a phosphate backbone. In another embodiment, the LEC does not contain other nucleic acid sequences unrelated to the desired gene expression.

[0175] LEC may be introduced into any plasmid that can be linearized. The plasmid may be capable of expressing the polypeptide encoded by the recombinant nucleic acid molecule described herein. Exemplary plasmids include, but are not limited to, pNP (Puerto Rico / 34), pM2 (New Caledonia / 99), WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector that can express the polypeptide encoded by the recombinant nucleic acid sequence construct.

[0176] In another embodiment, the expression vector is a viral vector. Suitable viral vectors capable of expressing a polypeptide include, for example, adeno-associated viral (AAV) vectors (see, e.g., Krause et al., "Delivery of Antigens by Viral Vectors for Vaccination," Ther. Deliv. 2(1):51-70 (2011); Ura et al., "Developments in Viral Vector-Based Vaccines," Vaccines 2:624-641 (2014); Buning et al., "Recent Developments in Adeno- associated Virus Vector Technology," J. Gene Med. 10:717-733 (2008) each of which is incorporated by reference in its entirety), lentiviral vectors (see, e.g., Ura et al., "Developments in Viral Vector-Based Vaccines," Vaccines 2:624-641 (2014); and Hu et al., "Immunization Vectors that can be used include those derived from viruses such as vaccinia virus, replication-deficient adenovirus vectors, and gutless adenovirus vectors (see, e.g., Ura et al., "Developments in Viral Vector-Based Vaccines," Vaccines 2:624-641 (2014) which is incorporated by reference in its entirety), retroviral vectors (see, e.g., Ura et al., "Developments in Viral Vector-Based Vaccines," Vaccines 2:624-641 (2014) which is incorporated by reference in its entirety), vaccinia virus, replication-deficient adenovirus vectors, and gutless adenovirus vectors (see, e.g., U.S. Pat. No. 5,872,005 which is incorporated by reference in its entirety).Methods for generating and isolating adeno-associated viruses (AAV) suitable for use as vectors are known in the art (see, e.g., Grieger & Samulski, "Adeno-associated Virus as a Gene Therapy Vector: Vector Development, Production and Clinical Applications," Adv. Biochem. Engin / Biotechnol. 99:119-145 (2005); Buning et al., "Recent Developments in Adeno-associated Virus Vector Technology," J. Gene Med. 10:717-733 (2008), each of which is incorporated herein by reference in its entirety).

[0177] Polynucleotides encoding SpA, LukA, and / or LukB polypeptides described herein are typically combined with promoter, translation initiation, 3' untranslated region, polyadenylation, and transcription termination sequences in an expression vector construct to achieve maximum expression. Suitable promoter sequences for driving expression of the polypeptides described herein include, but are not limited to, elongation factor 1-alpha (EF1a) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus immediate early gene promoter (CMV), chimeric liver specific promoter (LSP), cytomegalovirus enhancer / chicken beta actin promoter (CAG), tetracycline responsive promoter (TRE), transthyretin promoter (TTR), simian virus 40 promoter (SV40), and CK6 promoter. Other promoters suitable for driving gene expression in host cells known in the art are also suitable for incorporation into the expression constructs disclosed herein.

[0178] Another aspect of the present disclosure is directed to a host cell comprising a nucleic acid molecule encoding an S. aureus polypeptide described herein, or a vector containing these polynucleotides. Expression constructs encoding the SpA, LukA, and LukB proteins or polypeptides described herein may be co-transfected, sequentially transfected, or separately transfected into a host cell. Suitable host cells may include, but are not limited to, primary cells, cell lines, mixed cell lines, immortalized cell populations, or cells of a clonal population of immortalized cells, as known in the art (see, for example, Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2002; and others, incorporated herein by reference in their entireties). nd (See, for example, Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001). Such host cells may be eukaryotic, bacterial, plant, or archaeal cells.

[0179] In some embodiments, a suitable host cell for the S. aureus polynucleotides described herein is a bacterial cell. Suitable bacterial host cells include, but are not limited to, Escherichia host cells, Pseudomonas host cells, Staphylococcus host cells, Streptomyces host cells, Mycobacterium host cells, and Bacillus host cells. In some embodiments, the host cell is an Escherichia coli host cell. In some embodiments, the host cell is a S. aureus host cell.

[0180] In some embodiments, suitable host cells for the S. aureus polynucleotides described herein are eukaryotic cells. Exemplary eukaryotic cells may be of mammalian, insect, avian, or other animal origin. Mammalian eukaryotic cells include immortalized cell lines, such as hybridoma or myeloma cell lines, such as SP2 / 0 (American Type Culture Collection (ATCC), Manassas, Va., CRL-1581), NSO (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) mouse cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHO-K1SV (Lonza Biologics, Walkersville, Md.), CHO-K1 (ATCC CRL-61), or DG44.

[0181] The SpA, LukA, and LukB polypeptides described herein can be prepared by any of a variety of techniques using the isolated polynucleotides, vectors, and host cells described above. In general, proteins are produced by standard cloning and cell culture techniques commonly used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover proteins or polypeptides from culture media. Transfection into host cells can be performed using a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as by electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

[0182] The polypeptides described herein may be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation, or non-natural covalent modifications, such as the addition of polyethylene glycol (PEG) moieties (pegylation) and lipidation. Such modifications may occur in vivo or in vitro.

[0183] In some embodiments, the SpA, LukA, and LukB polynucleotides and / or polypeptides described herein are preferably "isolated" polynucleotides and / or polypeptides. When used to describe the polynucleotides and polypeptides disclosed herein, "isolated" means that the polynucleotide or polypeptide has been identified, separated, and / or recovered from components of its production environment. Preferably, an isolated polynucleotide or polypeptide is free of association with other components from its production environment. Contaminating components of the production environment, such as those resulting from recombinant transduced cells, are typically substances that may interfere with the use of the pharmaceutical product and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Polynucleotides or polypeptides are recovered and purified from recombinant cell cultures by known methods, including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") may also be used for purification.

[0184] Adjuvants for Immunogenic Compositions As used herein, the term "adjuvant" refers to a compound that enhances, strengthens, and / or promotes the immune response to SpA polypolypeptides, LukA polypeptides, LukB polypeptides, and / or polynucleotides encoding them when administered in conjunction with or as part of an immunogenic composition described herein. However, when administered alone, the adjuvant compound does not generate an immune response to the aforementioned polypeptides or polynucleotides. Adjuvants can enhance the immune response by several mechanisms, including, for example, lymphocyte recruitment, stimulation of B cells and / or T cells, and stimulation of antigen-presenting cells.

[0185] The immunogenic compositions described herein comprising SpA, LukA, and LukB polypeptides and / or polynucleotides encoding them may comprise or be administered in combination with an adjuvant. Adjuvants for administration in combination with the immunogenic compositions of the invention may be administered prior to, concomitantly with, or after administration of the immunogenic composition.

[0186] Specific examples of adjuvants include aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and aluminum oxide, including nanoparticles containing alum or nanoalum formulations), calcium phosphate (e.g., Masson JD et al, Expert Rev Vaccines 16:289-299 (2017), which are incorporated by reference in their entireties), monophosphoryl lipid A (MPL) or 3-De-O-acylated monophosphoryl lipid A (3D-MPL) (see, e.g., GB 2220211, EP 0971739, EP 1194166, US 6491919, which are incorporated by reference in their entireties), AS01, AS02, AS03, and AS04 (see, e.g., EP 1126876, which are incorporated by reference in their entireties, and US 7357936 for AS04). EP0671948, EP0761231, US5750110 for AS02), imidazopyridine compounds (see WO2007 / 109812, which is incorporated herein by reference in its entirety), imidazoquinoxaline compounds (see WO2007 / 109813, which is incorporated herein by reference in its entirety), delta-inulin (e.g., Petrovsky N and PD Cooper, Vaccine 33:5920-5926 (2015), which is incorporated herein by reference in its entirety), STING-activating synthetic cyclic dinucleotides (e.g., US2015 / 0056224, which is incorporated herein by reference in its entirety), combinations of lecithin and carbomer homopolymers (e.g., US6676958), and saponins such as Quil A and QS21 (see, e.g., Zhu D and W Tuo, 2016, Nat Prod Chem Res 3:e113 (doi:10.4172 / 2329-6836.1000e113)), optionally in combination with QS7 (Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell &Adjuvants include, but are not limited to, Freund's adjuvant (complete or incomplete). In any embodiment, the adjuvant includes Quil-A, commercially available, for example, from Brenntag (now Croda) or Invivogen. Quil-A contains the water-extractable fraction of saponins from the Quillaja saponaria Molina tree. These saponins belong to a group of triterpenoid saponins that share a common triterpenoid backbone structure. Saponins are known to induce strong adjuvant responses to T-dependent and T-independent antigens, as well as strong cytotoxic CD8+ lymphocyte responses, and enhance responses to mucosal antigens. Saponins can be combined with cholesterol and phospholipids to form immune stimulating complexes (ISCOMs), and the QuilA adjuvant can activate both antibody- and cell-mediated immune responses to a wide range of antigens of different origins. In a particular embodiment, the adjuvant is AS01, e.g., AS01B. AS01 is a saponin-containing adjuvant that is a component of the saponin family of adjuvants, including MPL (3-O-desacyl-4'-monophosphoryl lipid A), QS21 (Quillaja saponaria), and glyceryl esters of ...Molina, fraction 21), and liposomes. In certain embodiments, AS01 is commercially available or can be made as described in WO96 / 33739, which is incorporated herein by reference in its entirety. Certain adjuvants include emulsions, which are mixtures of two immiscible fluids, such as oil and water, one of which is suspended as small droplets inside the other, which are stabilized by a surfactant. An oil-in-water emulsion has water forming a continuous phase surrounding small droplets of oil, and a water-in-oil emulsion has oil forming a continuous phase. Certain oil-in-water emulsions include squalene (a metabolizable oil). Certain adjuvants include block copolymers, which are copolymers formed when two monomers cluster together to form a block of repeating units. An example of a water-in-oil emulsion that includes a block copolymer, squalene, and a particulate stabilizer is TiterMax®, which can be obtained commercially from Sigma-Aldrich.

[0187] Optionally, the emulsion may be combined with or contain additional immune stimulating components, such as TLR4 agonists. Suitable but non-limiting examples of adjuvant combinations for use in the compositions disclosed herein include oil-in-water emulsions (such as squalene or peanut oil), optionally combined with immune stimulants, such as monophosphoryl lipid A and / or QS21, such as AS02 (see Stoute et al., 1997, N. Engl. J. Med. 336, 86-91, which is incorporated herein by reference in its entirety), also used in MF59 and AS03 (see, for example, EP0399843, US6299884, US6451325). Further examples of adjuvants are liposomes containing immune stimulants, such as MPL and QS21, such as AS01E and AS01B (see, for example, US2011 / 0206758, which is incorporated herein by reference in its entirety). Other examples of adjuvants are CpG and imidazoquinolines (such as imiquimod and R848) (see, e.g., Reed G, et al., 2013, Nature Med, 19:1597-1608, which is incorporated by reference in its entirety). In certain preferred embodiments according to the invention, the adjuvant is a Th1 adjuvant.

[0188] In any embodiment, the adjuvant comprises a water-extractable fraction of a saponin, preferably a saponin obtained from Quillaja saponaria. In certain embodiments, the adjuvant comprises QS-21.

[0189] In any embodiment, the adjuvant of the immunogenic composition disclosed herein comprises a Toll-like receptor 4 (TLR4) agonist, alone or in combination with another adjuvant. TLR4 agonists are well known in the art, see, for example, Ireton GC and SG Reed, 2013, Expert Rev Vaccines 12:793-807, which is incorporated herein by reference in its entirety. In any embodiment, the adjuvant is a TLR4 agonist, including lipid A, or an analog or derivative thereof.

[0190] As used herein, the term "lipid A" refers to the hydrophobic lipid portion of the LPS molecule that contains glucosamine and is linked to keto-deoxyoctulosonate in the inner core of the LPS molecule via a ketosidic bond that anchors the LPS molecule to the outer leaflet of the outer membrane of gram-negative bacteria. As used herein, lipid A includes naturally occurring lipid A, mixtures, analogs, derivatives, and precursors thereof. The term includes monosaccharides, such as the precursor of lipid A, called lipid X, disaccharide lipid A, heptaacyl lipid A, hexaacyl lipid A, pentaacyl lipid A, tetraacyl lipid A, such as the tetraacyl precursor of lipid A, called lipid IVA, dephosphorylated lipid A, monophosphoryl lipid A, diphosphoryl lipid A, such as lipid A from Escherichia coli and Rhodobacter sphaeroides. Some immunostimulatory lipid A structures contain six acyl chains. The four primary acyl chains that are directly attached to the glucosamine sugar are 3-hydroxyacyl chains that are usually 10-16 carbons long. Two additional acyl chains are often attached to the 3-hydroxy group of the primary acyl chains. E. coli lipid A, as an example, typically has four C14 3-hydroxyacyl chains attached to the sugar, and one C12 and one C14 attached to the 3-hydroxy group of the primary acyl chain at the 2' and 3' positions, respectively.

[0191] As used herein, the term "lipid A analog or derivative" refers to a molecule that resembles the structure and immunological activity of lipid A, but does not necessarily occur naturally in nature. Lipid A analogs or derivatives may be modified to be shortened or condensed, and / or to replace their glucosamine residues with another amine sugar residue, such as a galactosamine residue, to contain 2-deoxy-2-aminogluconic acid in place of glucosamine-1-phosphate at the reducing end, or to have a galacturonic acid moiety in place of the phosphate at the 4' position. Lipid A analogs or derivatives may be prepared from lipid A isolated from bacteria, e.g., by chemical derivatization, or by chemical synthesis, e.g., by first determining the structure of a preferred lipid A and synthesizing the analog or derivative thereof. Lipid A analogs or derivatives are also useful as TLR4 agonist adjuvants (see, e.g., Gregg KA et al, 2017, MBio 8, eDD492-17, doi:10.1128 / mBio.00492-17, incorporated by reference in its entirety).

[0192] For example, lipid A analogs or derivatives can be obtained by deacylation of wild-type lipid A molecules, e.g., by alkaline treatment. Lipid A analogs or derivatives can be prepared, for example, from lipid A isolated from bacteria. Such molecules can also be chemically synthesized. Another example of a lipid A analog or derivative is a lipid A molecule isolated from a bacterial cell carrying a mutation, or a deletion or insertion, of an enzyme involved in lipid A biosynthesis and / or lipid A modification.

[0193] MPL and 3D-MPL are lipid A analogs or derivatives modified to attenuate lipid A toxicity. Lipid A, MPL, and 3D-MPL have a sugar backbone with long fatty acid chains attached, the backbone containing two 6-carbon sugars in glycosidic linkages and a phosphoryl moiety at the 4-position. Typically, 5-8 long chain fatty acids (usually 12-14 carbon atoms) are attached to the sugar backbone. Being derived from natural sources, MPL or 3D-MPL can exist as a complex or mixture of many fatty acid substitution patterns with various fatty acid lengths, e.g., heptaacyls, hexacyls, pentaacyls, etc. This is also true for some of the other lipid A analogs or derivatives described herein, but synthetic lipid A variants can also be defined and homogenous. MPL and its production are described, for example, in US 4,436,727, which is incorporated herein by reference in its entirety. 3D-MPL is described, for example, in US 4,912,094 B1 (hereby incorporated by reference in its entirety) and differs from MPL by the selective removal of the 3-hydroxymyristate acyl residue, an ester attached at the 3-position to the reducing-end glucosamine.Examples of lipid A (analogs, derivatives) suitable for inclusion in the immunogenic compositions described herein include MPL, 3D-MPL, RC529 (e.g., EP1385541, which is incorporated herein by reference in its entirety), PET-Lipid A, GLA (glycopyranosyl lipid adjuvant, a synthetic disaccharide glycolipid, see, e.g., US2010 / 0310602 and US8722064, which are incorporated herein by reference in their entireties), SLA (see, e.g., Carter D et al., which are incorporated herein by reference in their entireties, describing a structure-function approach to optimize TLR4 ligands for human vaccines). al, 2016, Clin. Transl. Immunology. 5:e108 (doi:10.1038 / cti.2016.63)), PHAD (phosphorylated hexaacyl disaccharide, the structure of which is the same as that of GLA), 3D-PHAD, 3D-(6-acyl)-PHAD (3D(6A)-PHAD) (PHAD, 3D-PHAD, and 3D(6A)PHAD are synthetic lipid A variants, which also provide the structures of these molecules), E6020 (CAS number 287180-63-6), ONO4007, OM-174, and the like. In certain preferred embodiments, the TLR4 agonist adjuvant comprises a lipid A analog or derivative selected from 3D-MPL, GLA, or SLA. In certain embodiments, the lipid A analog or derivative is formulated in a liposome.

[0194] Preferably, the adjuvants comprising a TLR4 agonist may be formulated in different ways, e.g., in emulsions, e.g., water-in-oil (w / o) emulsions or oil-in-water (o / w) emulsions (examples are MF59, AS03), stable (nano-)emulsions (SE), lipid suspensions, liposomes, (polymeric) nanoparticles, virosomes, alum adsorbed, aqueous formulations (AF), etc., which represent different delivery systems for the immunomodulatory molecules and / or immunogens in the adjuvant (see, e.g., Reed et al, 2013, supra, and Alving CR et al, 2012, Curr Opin Immunol 24:310-315, which are incorporated herein by reference in their entirety).

[0195] In any embodiment, the immunostimulatory TLR4 agonist can optionally be combined with other immunomodulatory components, such as squalene oil-in-water emulsions (e.g., MF59, AS03), saponins (e.g., QuilA, QS7, QS21, Matrix M, Iscom, Iscomatrix, etc.), aluminum salts, activators for other TLRs (e.g., imidazoquinolines, flagellins, dsRNA analogs, TLR9 agonists such as CpG, etc.), etc. (see, e.g., Reed et al., 2013, supra).

[0196] In any embodiment, the adjuvant of the immunogenic composition disclosed herein is a combination of a TLR4 agonist, such as GLA, formulated as a stable emulsion (i.e., GLA-SE). The stable emulsion used in GLA-SE is an oil-in-water emulsion, and the oil is squalene (see, e.g., WO2013 / 119856). In any embodiment, the adjuvant of the immunogenic composition disclosed herein is combined with a TLR4 agonist, such as GLA combined with a saponin (e.g., GLA-QS21). In any embodiment, the aforementioned adjuvant may be formulated as a liposome. Thus, an exemplary adjuvant includes a synthetic TLR4 agonist (e.g., MPL[GLA]) and a saponin (e.g., QS21), and also includes GLA-LSQ formulated as a liposome.

[0197] Additional exemplary adjuvants for use in the immunogenic compositions described herein that comprise a lipid A analog or derivative include SLA-SE (synthetic MPL [SLA], squalene oil / water emulsion), SLA-Nanoalum (synthetic MPL [SLA], aluminum salt), GLA-Nanoalum (synthetic MPL [GLA], aluminum salt), SLA-AF (synthetic MPL [SLA], aqueous suspension), GLA-AF (synthetic MPL [GLA], aqueous suspension), SLA-alum (synthetic MPL [SLA], aluminum salt), GLA-alum (synthetic MPL [GLA], aluminum salt), AS01 (MPL, QS21, liposomes), AS02 (MPL, QS21, oil / water emulsion), AS25 (MPL, oil / water emulsion), AS04 (MPL, aluminum salt), and AS15 (MPL, QS21, CpG, liposomes). See, for example, WO2008 / 153541, WO2010 / 141861, WO2013 / 119856, WO2019 / 051149, WO2013 / 119856, WO2006 / 116423, U.S. Pat. No. 4,987,237, U.S. Pat. No. 4,436,727, U.S. Pat. No. 4,877,611, U.S. Pat. No. 4,866,034, U.S. Pat. See U.S. Pat. Nos. 4,912,094, 4,987,237, 5,191,072, 5,593,969, 6,759,241, 9,017,698, 9,149,521, 9,149,522, 9,415,097, 9,415,101, and 9,504,743, supra; Reed G. et al., 2013; Johnson et al., 1999, J Med Chem, 42:4640-4649; and Ulrich and Myers, 1995, Vaccine Design: The Subunit and Adjuvant Approach; Powell and Newman, Eds.; Plenum: New York, 495-524.

[0198] S. aureus immunogenic composition and method of use In one aspect, the immunogenic composition disclosed herein comprises any one or more of the SpA polypeptides and LukA variant polypeptides described herein, or one or more nucleic acid molecules encoding the polypeptides described herein. In another aspect, the immunogenic composition disclosed herein comprises any one or more of the SpA polypeptides and LukB variant polypeptides described herein, or one or more nucleic acid molecules encoding the polypeptides described herein. In another aspect, the immunogenic composition disclosed herein comprises any one or more of the SpA polypeptides, LukA variant polypeptides, and LukB variant polypeptides described herein, or one or more nucleic acid molecules encoding the polypeptides described herein.

[0199] In any embodiment, the immunogenic composition comprises one or more SpA polypeptides (variant or non-variant), CC45 LukA variant polypeptides, CC45 LukB polypeptides (variant or non-variant), or polynucleotides encoding them. For example, the immunogenic composition of the present disclosure may comprise an SpA variant polypeptide, a CC45 LukA variant polypeptide, a CC45 LukB non-variant polypeptide, or polynucleotides encoding them, as described herein. An exemplary immunogenic composition according to this embodiment comprises an SpA variant polypeptide comprising at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58, and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58. An exemplary SpA variant polypeptide comprises an amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:60. The composition further comprises a CC45 LukA variant polypeptide comprising a lysine to methionine substitution at amino acid position corresponding to position 81 of SEQ ID NO:2, a serine to alanine substitution at amino acid position corresponding to position 139 of SEQ ID NO:2, a valine to isoleucine substitution at amino acid positions corresponding to positions 111 and 191 of SEQ ID NO:2, and a glutamic acid to alanine substitution at amino acid position corresponding to position 321 of SEQ ID NO:2. In any embodiment, the LukA variant polypeptide has an amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:4. The composition further comprises a CC45 LukB polypeptide, such as a polypeptide of SEQ ID NO:16, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:16.

[0200] Another exemplary immunogenic composition according to the foregoing embodiment comprises an SpA variant polypeptide, a CC45 LukA variant polypeptide, a CC45 LukB variant polypeptide, or a polynucleotide encoding the same, as described herein. An exemplary immunogenic composition comprises an SpA variant polypeptide comprising at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58, and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58. An exemplary SpA variant polypeptide comprises an amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:60. The composition further comprises a CC45 LukA variant polypeptide comprising a lysine to methionine substitution at amino acid position corresponding to position 81 of SEQ ID NO:2, a serine to alanine substitution at amino acid position corresponding to position 139 of SEQ ID NO:2, a valine to isoleucine substitution at amino acid positions corresponding to positions 111 and 191 of SEQ ID NO:2, and a glutamic acid to alanine substitution at amino acid position corresponding to position 321 of SEQ ID NO:2. In some embodiments, the LukA variant polypeptide has an amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:4. The composition further comprises a CC45 LukB variant polypeptide comprising an amino acid substitution corresponding to Val53Leu of SEQ ID NO:16. In some embodiments, the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:18, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:18.

[0201] Other immunogenic compositions according to this embodiment comprise an SpA variant polypeptide comprising a sequence of SEQ ID NO:60, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:60, a CC45 LukA variant of SEQ ID NO:2, in combination with a CC45 LukB variant of SEQ ID NO:16, or a variant sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:16. In some embodiments, the CC45 LukB variant sequence comprises an amino acid sequence selected from SEQ ID NOs:18, 20, and 22. For example, in some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 4, in combination with a CC45 LukB variant sequence of SEQ ID NO: 16, or a variant thereof having greater than 85% sequence identity to CC45 LukB of SEQ ID NO: 16, such as a CC45 LukB variant sequence selected from SEQ ID NOs: 18, 20, and 22. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 6, in combination with a CC45 LukB variant sequence of SEQ ID NO: 16, or a variant thereof having greater than 85% sequence identity to CC45 LukB of SEQ ID NO: 16, such as a CC45 LukB variant sequence selected from SEQ ID NOs: 18, 20, and 22. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 8, in combination with a CC45 LukB sequence of SEQ ID NO: 16, or a variant thereof having greater than 85% sequence identity to CC45 LukB of SEQ ID NO: 16, e.g., a CC45 LukB variant selected from SEQ ID NOs: 18, 20, and 22.In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 10, in combination with a CC45 LukB sequence of SEQ ID NO: 16, or a variant thereof having greater than 85% sequence identity to CC45 LukB of SEQ ID NO: 16, such as a CC45 LukB variant selected from SEQ ID NOs: 18, 20, and 22. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 11, in combination with a CC45 LukB sequence of SEQ ID NO: 16, or a variant thereof having greater than 85% sequence identity to CC45 LukB of SEQ ID NO: 16, such as a CC45 LukB variant selected from SEQ ID NOs: 18, 20, and 22. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 12, in combination with a CC45 LukB sequence of SEQ ID NO: 16, or a variant thereof having greater than 85% sequence identity to CC45 LukB of SEQ ID NO: 16, e.g., a CC45 LukB variant selected from SEQ ID NOs: 18, 20, and 22. In one embodiment, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant having an amino acid sequence of SEQ ID NO: 4, in combination with a CC45 LukB having an amino acid sequence of SEQ ID NO: 16. In one embodiment, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant having an amino acid sequence of SEQ ID NO: 11, in combination with a CC45 LukB having an amino acid sequence of SEQ ID NO: 16. In one embodiment, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant having an amino acid sequence of SEQ ID NO: 12, in combination with a CC45 LukB having an amino acid sequence of SEQ ID NO: 16. In one embodiment, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO:60, a CC45 LukA variant having the amino acid sequence of SEQ ID NO:8, in combination with CC45 LukB having the amino acid sequence of SEQ ID NO:16.In one embodiment, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO:60, a CC45 LukA variant having the amino acid sequence of SEQ ID NO:4, in combination with a CC45 LukB variant having the amino acid sequence of SEQ ID NO:18.

[0202] In another embodiment, the immunogenic composition comprises an SpA polypeptide, a CC8 LukA variant polypeptide, and a CC8 LukB polypeptide (variant or non-variant) disclosed herein, or a polynucleotide encoding the polypeptide. For example, the immunogenic composition comprises an SpA variant polypeptide, a CC8 LukA variant polypeptide, and a CC8 LukB polypeptide described herein, or a polynucleotide encoding the same. An exemplary composition comprises an SpA variant polypeptide comprising at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58, and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58. An exemplary SpA variant polypeptide comprises an amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:60. The composition further comprises a CC8 LukA variant polypeptide comprising a lysine to methionine substitution at amino acid position corresponding to position 80 of SEQ ID NO:1, a serine to alanine substitution at amino acid position corresponding to position 138 of SEQ ID NO:1, a valine to isoleucine substitution at amino acid positions corresponding to positions 110 and 190 of SEQ ID NO:1, and a glutamic acid to alanine substitution at amino acid position corresponding to position 320 of SEQ ID NO:1. In any embodiment, the LukA variant polypeptide comprises an amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:3. The composition further comprises a CC8 LukB polypeptide, such as a polypeptide of SEQ ID NO:15, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:15.

[0203] Another immunogenic composition according to this embodiment comprises an SpA variant polypeptide, a CC8 LukA variant polypeptide, a CC8 LukB variant polypeptide, or a polynucleotide encoding them, as disclosed herein. An exemplary immunogenic composition comprises an SpA variant polypeptide comprising at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58, and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58. An exemplary SpA variant polypeptide comprises an amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:60. The composition further comprises a CC8 LukA variant polypeptide comprising a lysine to methionine substitution at amino acid position corresponding to position 80 of SEQ ID NO:1, a serine to alanine substitution at amino acid position corresponding to position 138 of SEQ ID NO:1, a valine to isoleucine substitution at amino acid positions corresponding to positions 110 and 190 of SEQ ID NO:1, and a glutamic acid to alanine substitution at amino acid position corresponding to position 320 of SEQ ID NO:1. In any embodiment, the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:3. The composition further comprises a CC8 LukB variant polypeptide comprising a valine to leucine amino acid substitution at amino acid position corresponding to position 53 of SEQ ID NO:15. In any embodiment, the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:17, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:17.

[0204] Other immunogenic compositions according to this embodiment comprise an SpA variant polypeptide comprising a sequence of SEQ ID NO:60, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:60, a CC8 LukA variant of SEQ ID NO: 1, in combination with a CC8 LukB variant of SEQ ID NO: 15, or a variant sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15. In some embodiments, the CC8 LukB sequence variant sequence comprises an amino acid sequence selected from SEQ ID NOs: 17, 19, and 21. For example, in some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC8 LukA variant of SEQ ID NO: 3, and a CC8 LukB sequence of SEQ ID NO: 15, or a variant thereof having 85% or more sequence identity to CC8 LukB of SEQ ID NO: 15, such as a CC8 LukB variant sequence selected from SEQ ID NOs: 17, 19, and 21. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC8 LukA variant of SEQ ID NO: 5 in combination with a CC8 LukB sequence of SEQ ID NO: 15, or a variant thereof having more than 85% sequence identity to CC8 LukB of SEQ ID NO: 15, such as a CC8 LukB variant sequence selected from SEQ ID NOs: 17, 19, and 21. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC8 LukA variant of SEQ ID NO: 7, and a CC8 LukB sequence of SEQ ID NO: 15, or a variant thereof having greater than 85% sequence identity to CC8 LukB of SEQ ID NO: 15, e.g., a CC8 LukB variant sequence selected from SEQ ID NOs: 17, 19, and 21.In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC8 LukA variant of SEQ ID NO: 9, and a CC8 LukB sequence of SEQ ID NO: 15, or a variant thereof having greater than 85% sequence identity to CC8 LukB of SEQ ID NO: 15, e.g., a CC8 LukB variant sequence selected from SEQ ID NOs: 17, 19, and 21.

[0205] In another embodiment, the immunogenic composition comprises an SpA polypeptide (variant or non-variant), a CC8 LukA variant polypeptide, and a CC45 LukB polypeptide (variant or non-variant) described herein, or a polynucleotide encoding them. For example, the composition comprises an SpA variant polypeptide, a CC8 LukA variant polypeptide, and a CC45 LukB polypeptide, or a polynucleotide encoding them. An exemplary immunogenic composition according to this embodiment comprises an SpA variant polypeptide comprising at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58, and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58. An exemplary SpA variant polypeptide comprises an amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:60. The composition further comprises a CC8 LukA variant polypeptide comprising a lysine to methionine substitution at amino acid position corresponding to position 80 of SEQ ID NO:1, a serine to alanine substitution at amino acid position corresponding to position 138 of SEQ ID NO:1, a valine to isoleucine substitution at amino acid positions corresponding to positions 110 and 190 of SEQ ID NO:1, and a glutamic acid to alanine substitution at amino acid position corresponding to position 320 of SEQ ID NO:1. In any embodiment, the LukA variant polypeptide comprises an amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:3. The composition further comprises a CC45 LukB polypeptide, such as a polypeptide of SEQ ID NO:16, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:16.

[0206] Another immunogenic composition according to this embodiment comprises an SpA variant polypeptide, a CC8 LukA variant polypeptide, and a CC45 LukB variant polypeptide disclosed herein, or a polynucleotide encoding them. An exemplary immunogenic composition according to this embodiment comprises an SpA variant polypeptide comprising at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58, and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58. An exemplary SpA variant polypeptide comprises an amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:60. The composition further comprises a CC8 LukA variant polypeptide comprising a lysine to methionine substitution at amino acid position corresponding to position 80 of SEQ ID NO:1, a serine to alanine substitution at amino acid position corresponding to position 138 of SEQ ID NO:1, a valine to isoleucine substitution at amino acid positions corresponding to positions 110 and 190 of SEQ ID NO:1, and a glutamic acid to alanine substitution at amino acid position corresponding to position 320 of SEQ ID NO:1. In any embodiment, the LukA variant polypeptide comprises an amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:3. The composition further comprises a CC45 LukB variant polypeptide comprising an amino acid substitution corresponding to Val53Leu of SEQ ID NO:16. In some embodiments, the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO:18, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:18.

[0207] Other immunogenic compositions according to this embodiment include an SpA variant polypeptide comprising a sequence of SEQ ID NO: 60, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 60, a CC8 LukA variant of SEQ ID NO: 1, and a CC45 LukB variant of SEQ ID NO: 16, or a variant sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16. In some embodiments, the CC45 LukB variant sequence comprises an amino acid sequence selected from SEQ ID NOs: 18, 20, and 22. For example, in some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC8 LukA variant of SEQ ID NO: 3, and a CC45 LukB sequence of SEQ ID NO: 16, or a variant thereof having greater than 85% sequence identity to CC45 LukB of SEQ ID NO: 16, such as a CC45 LukB variant sequence selected from SEQ ID NOs: 18, 20, and 22. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC8 LukA variant of SEQ ID NO: 5, and a CC45 LukB sequence of SEQ ID NO: 16, or a variant thereof having greater than 85% sequence identity to CC45 LukB of SEQ ID NO: 16, such as a CC45 LukB variant sequence selected from SEQ ID NOs: 18, 20, and 22. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC8 LukA variant of SEQ ID NO: 7, and a CC45 LukB sequence of SEQ ID NO: 16, or a variant thereof having greater than 85% sequence identity to CC45 LukB of SEQ ID NO: 16, e.g., a CC45 LukB variant sequence selected from SEQ ID NOs: 18, 20, and 22.In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC8 LukA variant of SEQ ID NO: 9, and a CC45 LukB sequence of SEQ ID NO: 16, or a variant thereof having greater than 85% sequence identity to CC45 LukB of SEQ ID NO: 16, e.g., a CC45 LukB variant sequence selected from SEQ ID NOs: 18, 20, and 22. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC8 LukA variant having an amino acid sequence of SEQ ID NO: 5, and a CC45 LukB variant having an amino acid sequence of SEQ ID NO: 16. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC8 LukA variant having an amino acid sequence of SEQ ID NO: 5, and a CC45 LukB variant having an amino acid sequence of SEQ ID NO: 22. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC8 LukA variant having an amino acid sequence of SEQ ID NO: 5, and a CC45 LukB variant having an amino acid sequence of SEQ ID NO: 18. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO:60, a CC8 LukA variant having the amino acid sequence of SEQ ID NO:5, and a CC45 LukB variant having the amino acid sequence of SEQ ID NO:20.

[0208] In another embodiment, the immunogenic composition comprises an SpA polypeptide (variant or non-variant), a CC45 LukA variant polypeptide, and a CC8 LukB polypeptide (variant or non-variant) described herein, or a polynucleotide encoding them. For example, an immunogenic composition of the present disclosure may comprise an SpA variant polypeptide, a CC45 LukA variant polypeptide, and a CC8 LukB polypeptide. An exemplary immunogenic composition comprises an SpA variant polypeptide comprising at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58, and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58. An exemplary SpA variant polypeptide comprises an amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:60. The composition further comprises a CC45 LukA variant polypeptide comprising a lysine to methionine substitution at amino acid position corresponding to position 81 of SEQ ID NO:2, a serine to alanine substitution at amino acid position corresponding to position 139 of SEQ ID NO:2, a valine to isoleucine substitution at amino acid positions corresponding to positions 111 and 191 of SEQ ID NO:2, and a glutamic acid to alanine substitution at amino acid position corresponding to position 321 of SEQ ID NO:2. In some embodiments, the LukA variant polypeptide has an amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:4. The composition further comprises a CC8 LukB polypeptide, e.g., a LukB polypeptide of SEQ ID NO:15, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:15.Alternatively, the composition further comprises a CC8 LukB variant polypeptide comprising a valine to leucine amino acid substitution at an amino acid position corresponding to position 53 of SEQ ID NO: 15. In any embodiment, the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the amino acid sequence of SEQ ID NO:17.

[0209] Other immunogenic compositions according to this embodiment comprise an SpA variant polypeptide comprising a sequence of SEQ ID NO:60, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:60, a CC45 LukA variant of SEQ ID NO:2, in combination with a CC8 LukB sequence of SEQ ID NO:15, or a variant sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:15. In some embodiments, the CC8 LukB variant sequence comprises an amino acid sequence selected from SEQ ID NOs:17, 19, and 21. For example, in some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 4, and a CC8 LukB sequence of SEQ ID NO: 15, or a variant thereof having greater than 85% sequence identity to the CC8 LukB sequence of SEQ ID NO: 15, such as a variant sequence selected from SEQ ID NOs: 17, 19, and 21. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 6, and a CC8 LukB sequence of SEQ ID NO: 15, or a variant thereof having greater than 85% sequence identity to the CC8 LukB sequence of SEQ ID NO: 15, such as a variant sequence selected from SEQ ID NOs: 17, 19, and 21. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 8, and a CC8 LukB sequence of SEQ ID NO: 15, or a variant thereof having greater than 85% sequence identity to the CC8 LukB sequence of SEQ ID NO: 15, e.g., a variant sequence selected from SEQ ID NOs: 17, 19, and 21.In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 9, and a CC8 LukB sequence of SEQ ID NO: 15, or a variant thereof having greater than 85% sequence identity to the CC8 LukB sequence of SEQ ID NO: 15, such as a variant sequence selected from SEQ ID NOs: 17, 19, and 21. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 10, and a CC8 LukB sequence of SEQ ID NO: 15, or a variant thereof having greater than 85% sequence identity to the CC8 LukB sequence of SEQ ID NO: 15, such as a variant sequence selected from SEQ ID NOs: 17, 19, and 21. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 11, and a CC8 LukB sequence of SEQ ID NO: 15, or a variant thereof having greater than 85% sequence identity to the CC8 LukB sequence of SEQ ID NO: 15, such as a variant sequence selected from SEQ ID NOs: 17, 19, and 21. In some embodiments, the immunogenic composition comprises an SpA variant polypeptide of SEQ ID NO: 60, a CC45 LukA variant of SEQ ID NO: 12, and a CC8 LukB sequence of SEQ ID NO: 15, or a variant thereof having greater than 85% sequence identity to the CC8 LukB sequence of SEQ ID NO: 15, such as a variant sequence selected from SEQ ID NOs: 17, 19, and 21.

[0210] Another aspect of the present disclosure is directed to an immunogenic composition comprising an SpA polypeptide as described herein, and any of the variant LukB proteins or polypeptides as described herein, or a nucleic acid molecule encoding the SpA and LukB variants as described herein. Specifically, the variant LukB polypeptide of the immunogenic composition comprises one or more amino acid residue insertions, substitutions, and / or deletions as described herein. In some embodiments, the composition comprises an SpA variant polypeptide comprising a sequence of SEQ ID NO:60, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:60, and a LukB variant of SEQ ID NO:15 (CC8). Exemplary CC8 LukB variants include, but are not limited to, LukB variants of SEQ ID NOs:17, 19, and 21. In some embodiments, the composition comprises an SpA variant polypeptide comprising a sequence of SEQ ID NO:60, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:60, and a LukB variant of SEQ ID NO:16 (CC45). Exemplary CC45 LukB variants include, but are not limited to, LukB variants of SEQ ID NOs:18, 20, and 22.

[0211] The immunogenic composition according to this embodiment may further comprise a LukA protein or polypeptide. For example, a composition comprising an SpA variant and a LukB variant as described in the previous paragraph further comprises a CC8 LukA sequence of SEQ ID NO: 1, or a variant sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. Alternatively, an immunogenic composition comprising an SpA variant and a LukB variant as described in the previous paragraph further comprises a CC45 LukA sequence of SEQ ID NO: 2, or a variant sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2.

[0212] The immunogenic compositions described herein may further comprise one or more additional S. aureus antigens. Suitable S. aureus antigens include, but are not limited to, serotype 336 polysaccharide antigen, clumping factor A, clumping factor B, fibrinogen binding protein, collagen binding protein, elastin binding protein, MHC-like protein, polysaccharide intracellular adhesion, beta-hemolysin, delta-hemolysin, Panton-Valentine leukocidin, leukocidin M, exfoliative toxin A, exfoliative toxin B, V8 protease, hyaluronate lyase, lipase, staphylokinase, enterotoxin, enterotoxin superantigen SEA, enterotoxin superantigen SAB, toxic shock syndrome toxin-1, poly-N-succinyl beta-1→6 glucosamine, catalase, beta-lactamase, teichoic acid, peptidoglycan, penicillin binding protein, chemotaxis inhibitory protein, complement inhibitor, Sbi, type 5 antigen, type 8 antigen, and lipoteichoic acid.Other S. aureus antigens suitable for inclusion in the immunogenic composition include, but are not limited to, CP5, CP8, Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, EsxAB (fusion), IsdA, IsdB, IsdC, MntC, rTSST-1, rTSST-1v, TSST-1, SasF, vWbp, vWh vitronectin binding protein, Aaa, Aap, Ant, autolysin glucosaminidase, O -tricin amidase, Can, collagen binding protein, Csa1A, EFB, elastin binding protein, EPB, FbpA, fibrinogen binding protein, fibronectin binding protein, FhuD, FhuD2, FnbA, FnbB, GehD, HarA, HBP, immunodominant ABC transporter, IsaA / PisA, laminin receptor, lipase GehD, MAP, Mg2+ transporter, MHC II analogue, MRPII, NPase, RNA III-activating protein (RAP), SasA, SasB, SasC, SasD, SasK, SBI, SEA exotoxin, SEB exotoxin, mSEB, SitC, Ni ABC transporter, SitC / MntC / saliva binding protein, SsaA, SSP-1, SSP-2, Spa5, SpAKKAA, SpAkR, Sta006, and Sta011.

[0213] The immunogenic compositions of the present disclosure are prepared by formulating the SpA, LukA polypeptide, and / or LukB polypeptide described herein with a pharma- ceutically acceptable carrier and, optionally, a pharma- ceutically acceptable excipient. As used herein, the terms "pharma-ceutically acceptable carrier" and "pharma-ceutically acceptable excipient" (e.g., additives such as diluents, immunostimulants, adjuvants, antioxidants, preservatives, and solubilizers) are non-toxic to subjects receiving the composition at the dosages and concentrations used. Examples of pharma-ceutically acceptable carriers include water, e.g., water buffered with phosphate, citrate, and other organic acids. Representative examples of pharma- ceutically acceptable excipients that may be useful in the present disclosure include antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants.

[0214] Formulation of pharma- ceutically active ingredients with pharma- ceutically acceptable carriers is known in the art, e.g., Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005), and later editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tonicity regulating agents, preservatives, stabilizers, and chelating agents. One or more pharma-ceutically acceptable carriers can be used in formulating the pharmaceutical compositions of the present invention.

[0215] In any embodiment, the immunogenic compositions described herein are liquid formulations. A preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation that includes water. Liquid formulations may include solutions, suspensions, emulsions, microemulsions, gels, and the like. Aqueous formulations typically include at least 50% w / w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w / w water.

[0216] In any embodiment, the immunogenic composition can be formulated as an injectable that can be injected, for example, via an injection device (e.g., a syringe or an infusion pump). The injection can be delivered, for example, intramuscularly, intraperitoneally, intravitreally, or intravenously.

[0217] The immunogenic composition of the present disclosure may be formulated for parenteral administration. Solutions, suspensions, or emulsions of the composition may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Exemplary oils are those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0218] Pharmaceutical immunogenic compositions suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0219] In any embodiment, the immunogenic compositions described herein are solid formulations, e.g., freeze-dried or spray-dried compositions, which can be used as is or to which solvents and / or diluents are added prior to use by the physician or patient. Solid dosage forms can include tablets, e.g., compressed tablets, and / or coated tablets, as well as capsules (e.g., hard or soft gelatin capsules). The immunogenic compositions can also be in the form of, for example, sachets, dragees, powders, granules, lozenges, or powders for reconstitution.

[0220] The dosage form of the immunogenic composition may be immediate release, which may include a water soluble or dispersible carrier, or may be delayed, sustained, or modified release, which may include a water insoluble polymer that modulates the dissolution rate of the dosage form in the gastrointestinal tract or under the skin.

[0221] In other embodiments, the immunogenic composition may be delivered intranasally, bucally, or sublingually.

[0222] The pH of the aqueous formulation of the immunogenic composition may be from pH 3 to pH 10. In one embodiment, the pH of the immunogenic composition is from about 7.0 to about 9.5. In another embodiment, the pH of the immunogenic composition is from about 3.0 to about 7.0.

[0223] Another aspect of the present disclosure relates to a method of using the immunogenic composition described herein. Thus, one aspect is directed to a method for treating or preventing Staphylococcus infection in a subject in need thereof, comprising administering an effective amount of the immunogenic composition disclosed herein. Another aspect is directed to a method for inducing an immune response against Staphylococcus bacteria in a subject in need thereof, comprising administering an effective amount of the immunogenic composition disclosed herein. Another aspect is directed to a method for decolonizing or preventing colonization or recolonization of Staphylococcus bacteria in a subject in need thereof, comprising administering an effective amount of the immunogenic composition disclosed herein. According to this aspect, the method described herein is suitable for treating or preventing short-term and persistent colonization or recolonization of Staphylococcus bacteria in a subject in need thereof.

[0224] The methods of the disclosure include administering any one of the immunogenic compositions described above. Subjects suitable for treatment according to these aspects of the disclosure are those at risk of developing a S. aureus infection, at risk of exposure to S. aureus bacteria, and / or who have been exposed to S. aureus bacteria.

[0225] According to this aspect of the disclosure, a prophylactically effective amount of the immunogenic composition is administered to a subject to generate an immune response against S. aureus infection. A prophylactically effective amount is an amount necessary to generate or induce a humoral (i.e., antibody-mediated) and cellular (T cell) immune response. The induced humoral response is sufficient to prevent or at least reduce the extent of a S. aureus infection that would otherwise develop in the absence of such a response. Preferably, administration of a prophylactically effective amount of the immunogenic composition described herein induces a neutralizing immune response against S. aureus in the subject. To effect an effective immune response in the subject, the composition may further contain one or more additional S. aureus antigens or adjuvants as described above. In an alternative embodiment, an adjuvant is administered to the subject separately from the composition, either before, after, or simultaneously with administration of the composition of the present disclosure.

[0226] For the purposes of this aspect of the disclosure, the target "subject" includes any animal, preferably a mammal, and more preferably a human. In the context of administering an immunogenic composition to prevent, inhibit, or reduce the severity of S. aureus infection and S. aureus colonization in a subject, the target subject includes any subject at risk of being infected with S. aureus. Particularly susceptible subjects include immunocompromised infants, juveniles, adults, and the elderly. However, any infant, juvenile, adult, or elderly at risk of S. aureus infection may be treated according to the methods and immunogenic compositions described herein. Particularly suitable subjects include subjects at risk of infection with methicillin-resistant S. aureus (MRSA) or methicillin-susceptible S. aureus (MSSA). Other suitable subjects include those who may have or may be at risk of developing a condition resulting from S. aureus infection, i.e., an S. aureus-associated condition, such as, for example, skin wounds and infections, tissue abscesses, folliculitis, osteomyelitis, pneumonia, scalded skin syndrome, sepsis, septicemia, septic arthritis, myocarditis, endocarditis, and toxic shock syndrome.

[0227] In some embodiments, the subject is at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, or 90 years of age (or any range derivable therein). In certain embodiments, the subject or patient described herein, such as a human subject, is a pediatric subject. A pediatric subject is a subject defined as being under 18 years of age. In some embodiments, a pediatric subject is 2 years of age or younger. In some embodiments, a pediatric subject is under 1 year of age. In some embodiments, a pediatric subject is under 6 months of age. In some embodiments, the pediatric subject is 2 months of age or younger. In some embodiments, the human patient is 65 years of age or older. In some embodiments, the human patient is a healthcare worker. In some embodiments, the patient is a patient undergoing a surgical procedure.

[0228] A number of other factors may also be considered when administering the immunogenic composition under conditions effective to induce a robust immune response. These factors include, for example, but are not limited to, the concentration of the active agent in the composition, the mode and frequency of administration, and subject details such as age, weight, and overall health and immune status. General guidance can be found, for example, in publications of the International Conference on Harmonization and REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Company 1990), which are incorporated herein by reference in their entirety. The clinician may administer the immunogenic composition described herein until a dosage is reached that provides the desired or required prophylactic effect, e.g., the desired antibody titer. The progress of the prophylactic response can be easily monitored by conventional assays.

[0229] In one embodiment of the disclosure, the immunogenic compositions described herein are administered prophylactically to prevent, delay, or inhibit the onset of S. aureus infection in a subject at risk of infection with S. aureus or at risk of developing an associated condition. In some embodiments of the disclosure, prophylactic administration of the immunogenic composition is effective to completely prevent S. aureus infection in the individual. In other embodiments, prophylactic administration is effective to prevent the full extent of infection that would otherwise develop in the absence of such administration, i.e., to substantially prevent or inhibit S. aureus infection in the individual.

[0230] In connection with the use of prophylactic compositions to prevent S. aureus infection, the dosage of the composition is sufficient to generate antibody titers capable of neutralizing S. aureus LukAB-mediated cytotoxicity and SpA-mediated toxic activity, and can achieve a reduction in many symptoms, a decrease in the severity of at least one symptom, or a delay in further progression of at least one symptom, or even a complete alleviation of the infection.

[0231] The prophylactically effective amount of the immunogenic compositions described herein depends on whether an adjuvant is co-administered, with higher doses being required in the absence of an adjuvant. The amount of SpA, LukA, and LukB polypeptides and / or polynucleotides encoding them can vary from 1 μg to 500 μg per patient. In some embodiments, 5, 10, 20, 25, 50, or 100 μg are used for each human injection. In some cases, higher doses of 1 to 50 mg per injection are used. In some embodiments, about 10, 20, 30, 40, or 50 mg are used for each human injection. The timing of injections can vary widely, from once a year to once every 10 years. In general, effective dosages can be monitored by obtaining a body fluid sample, typically a serum sample, from the subject and determining the titer of antibodies developed against SpA, LukA, LukB, or LukAB, using methods that are well known in the art and can be readily adapted to the particular antigen being measured. Ideally, a sample is taken before the first dose and subsequent samples are taken after each immunization to determine the titer. Generally, a dose or dosing schedule that provides a detectable titer at least 4-fold higher than the control or "background" level at a serum dilution of 1:100 is desirable, where background is defined relative to control serum or relative to the plate background of an ELISA assay.

[0232] The immunogenic compositions of the disclosure may be administered by parenteral, topical, intravenous, oral, intraperitoneal, intranasal, or intramuscular means for prophylactic treatment.

[0233] Embodiment The present invention also provides the following non-limiting embodiments. In embodiment 1, (i) a Staphylococcus aureus protein A (SpA) polypeptide; (ii) an S. aureus LukA variant polypeptide, comprising an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Lys83, Ser141, Val113, and Val193 of SEQ ID NO: 25; and In embodiment 2, (i) a Staphylococcus aureus protein A (SpA) polypeptide; (ii) a combination of two or more immunogenic compositions comprising, together with an S. aureus LukA variant polypeptide, the S. aureus LukA variant polypeptide comprising amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Lys83, Ser141, Val113, and Val193 of SEQ ID NO:25. Embodiment 3 is a combination of the immunogenic composition described in embodiment 1 or the immunogenic composition described in embodiment 2, wherein the LukA variant polypeptide comprises an amino acid substitution at an amino acid residue corresponding to Glu323 of SEQ ID NO:25. Embodiment 4 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 1 to 3, wherein the LukA variant polypeptide comprises amino acid substitutions at amino acid residues corresponding to Lys83, Ser141, Val113, Val193, and Glu323 of SEQ ID NO:25. Embodiment 5 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 4, wherein the amino acid substitutions include Lys83Met, Ser141Ala, Val113Ile, Val193Ile, and Glu323Ala. Embodiment 6 is an immunogenic composition or a combination of immunogenic compositions described in any one of embodiments 1 to 5, wherein the LukA variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4. Embodiment 7 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 1 to 6, wherein the LukA variant polypeptide further comprises amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149 and Gly156 of SEQ ID NO:25. Embodiment 8 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 7, wherein the amino acid substitutions include Tyr74Cys, Asp140Cys, Gly149Cys, and Gly156Cys. Embodiment 9 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 7 or 8, wherein the LukA variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6. Embodiment 10 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 1 to 9, wherein the variant LukA protein or polypeptide further comprises an amino acid substitution at the amino acid residue corresponding to amino acid residue Thr249 of SEQ ID NO:25. Embodiment 11 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 10, wherein the LukA variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 8, an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10. Embodiment 12 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 1 to 11, wherein the SpA polypeptide is an SpA variant polypeptide. Embodiment 13 is an immunogenic composition or combination of immunogenic compositions according to embodiment 12, wherein the SpA variant polypeptide has at least one amino acid substitution that disrupts Fc binding and at least a second amino acid substitution that disrupts VH3 binding. Embodiment 14 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 12 or 13, wherein the SpA variant polypeptide comprises an SpA D domain, the SpA D domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:58. Embodiment 15 is an immunogenic composition or combination of immunogenic compositions according to embodiment 14, wherein the SpA variant polypeptide further comprises an SpA E, A, B and / or C domain. Embodiment 16 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 15, wherein the SpA E domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 59, the SpA A domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 55, the SpA B domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 56, and the SpA C domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 57. Embodiment 17 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 12 or 13, wherein the SpA variant polypeptide comprises an SpA E, D, A, B or C domain, wherein the SpA E domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 52, the SpA D domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 51, the SpA A domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 48, the SpA B domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 49, and the SpA C domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 50. Embodiment 18 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 12 to 17, wherein the SpA variant polypeptide comprises the SpA E, D, A, B and C domains consecutively. Embodiment 19 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 18, wherein the SpA variant polypeptide comprises the SpA E, D, A, B, and C domains and has an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:53. Embodiment 20 is an immunogenic composition or combination of immunogenic compositions according to any one of embodiments 12 to 17, wherein each SpA E, D, A, B, and C domain has an amino acid substitution at one or both of the amino acid positions corresponding to amino acid positions 9 and 10 of SEQ ID NO:58. Embodiment 21 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 20, wherein the amino acid substitution at one or both amino acid positions corresponding to positions 9 and 10 of SEQ ID NO: 58 is a lysine residue for a glutamine residue. Embodiment 22 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 21, wherein the SpA variant polypeptide comprises the SpA E, D, A, B, and C domains and has an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:54. Embodiment 23 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 12 to 22, wherein the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, and at least one domain has (i) a lysine substitution at glutamine residues corresponding to positions 9 and 10 of SEQ ID NO: 58, and (ii) a glutamic acid substitution at the amino acid position corresponding to position 33 of SEQ ID NO: 58. Embodiment 24 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 23, wherein the SpA E domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 65, the SpA D domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 66, the SpA A domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 62, the SpA B domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 63, and the SpA C domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 64. Embodiment 25 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 23, wherein the SpA E domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 92, the SpA D domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 91, the SpA A domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 88, the SpA B domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 89, and the SpA C domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 90. Embodiment 26 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 23, wherein the SpA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 60. Embodiment 27 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 12 to 22, wherein the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, and at least one domain has (i) a lysine substitution at the glutamine residues corresponding to positions 9 and 10 of SEQ ID NO: 58, and (ii) a threonine substitution at the amino acid position corresponding to position 33 of SEQ ID NO: 58. Embodiment 28 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 27, wherein the SpA E domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 70, the SpA D domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 71, the SpA A domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 67, the SpA B domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 68, and the SpA C domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 69. Embodiment 29 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 27, wherein the SpA E domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 97, the SpA D domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 96, the SpA A domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 93, the SpA B domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 94, and the SpA C domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 95. Embodiment 30 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 27, wherein the SpA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 61. Embodiment 31 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 12 to 22, wherein the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, and at least one domain has (i) a lysine substitution at glutamine residues corresponding to positions 9 and 10 of SEQ ID NO: 58, and (ii) an amino acid substitution at the amino acid position corresponding to position 29 of SEQ ID NO: 58. Embodiment 32 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 1 to 31, wherein the composition further comprises a S. aureus Leukocidin B (LukB) polypeptide or a variant thereof. Embodiment 33 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 32, wherein the LukB polypeptide is a LukB polypeptide of SEQ ID NO: 15 or a LukB polypeptide of SEQ ID NO: 16. Embodiment 34 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 32, wherein the LukB polypeptide is a LukB variant polypeptide. Embodiment 35 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 34, wherein the LukB variant polypeptide comprises an amino acid sequence having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 16. Embodiment 36 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 35, wherein the LukB variant polypeptide comprises an amino acid substitution at the amino acid position corresponding to SEQ ID NO:15 and position 53 of SEQ ID NO:16. Embodiment 37 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 36, wherein the amino acid substitution is a valine to leucine substitution. Embodiment 38 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 34 to 37, wherein the LukB variant polypeptide comprises amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO: 15. Embodiment 38 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 34 to 37, wherein the LukB variant polypeptide comprises amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Glu45, Glu110, Thr122, and Arg155 of SEQ ID NO: 16. Embodiment 40 is an immunogenic composition or a combination of immunogenic compositions described in any one of embodiments 34 to 39, wherein the LukB variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 17 to 22. Embodiment 41 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 32, wherein the composition comprises a LukA variant polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4, and a LukB polypeptide comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16. Embodiment 42 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 32, wherein the composition comprises a LukA variant polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, and a LukB polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15. Embodiment 43 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 32, wherein the composition comprises a LukA variant polypeptide comprising the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, and a LukB polypeptide comprising the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18. Embodiment 44 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 41 to 43, wherein the SpA polypeptide is an SpA variant polypeptide. Embodiment 45 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 44, wherein the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, and at least one domain has (i) a lysine substitution with glutamine residues corresponding to positions 9 and 10 of SEQ ID NO:58, and (ii) a glutamic acid substitution at the amino acid position corresponding to position 33 of SEQ ID NO:58. Embodiment 46 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 32, wherein (i) the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58 and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58, (ii) the LukA variant polypeptide comprises a CC8 LukA variant polypeptide comprising a methionine substitution at amino acid position corresponding to position 80 of SEQ ID NO:1, an alanine substitution at amino acid position corresponding to position 138 of SEQ ID NO:1, an isoleucine substitution at amino acid positions corresponding to positions 110 and 190 of SEQ ID NO:1, and an alanine substitution at amino acid position corresponding to position 320 of SEQ ID NO:1, and (iii) the LukB polypeptide is a CC45 LukB variant polypeptide comprising a leucine substitution at amino acid position corresponding to position 53 of SEQ ID NO:16. Embodiment 47 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 46, wherein the SpA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 60, the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 3, and the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 18. Embodiment 48 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 32, wherein (i) the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58 and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58, (ii) the LukA variant polypeptide comprises a CC8 LukA variant polypeptide comprising a methionine substitution at amino acid position corresponding to position 80 of SEQ ID NO:1, an alanine substitution at amino acid position corresponding to position 138 of SEQ ID NO:1, an isoleucine substitution at amino acid positions corresponding to positions 110 and 190 of SEQ ID NO:1, and an alanine substitution at amino acid position corresponding to position 320 of SEQ ID NO:1, and (iii) the LukB polypeptide is a CC8 LukB variant polypeptide comprising a leucine substitution at amino acid position corresponding to position 53 of SEQ ID NO:15. Embodiment 49 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 48, wherein the SpA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 60, the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 3, and the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 17. Embodiment 50 is an immunogenic composition or combination of immunogenic compositions according to embodiment 32, wherein (i) the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58 and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58, (ii) the LukA variant polypeptide comprises a CC8 LukA variant polypeptide comprising a methionine substitution at amino acid position corresponding to position 80 of SEQ ID NO:1, an alanine substitution at amino acid position corresponding to position 138 of SEQ ID NO:1, an isoleucine substitution at amino acid positions corresponding to positions 110 and 190 of SEQ ID NO:1, and an alanine substitution at amino acid position corresponding to position 320 of SEQ ID NO:1, and (iii) the LukB polypeptide is a CC8 LukB polypeptide comprising the amino acid sequence of SEQ ID NO:15. Embodiment 51 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 50, wherein the SpA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 60, the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 3, and the LukB polypeptide comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 15. Embodiment 52 is an immunogenic composition or combination of immunogenic compositions according to embodiment 32, wherein (i) the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58 and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58; (ii) the LukA variant polypeptide comprises a CC45 LukA variant polypeptide comprising a methionine substitution at amino acid position corresponding to position 81 of SEQ ID NO:2, an alanine substitution at amino acid position corresponding to position 139 of SEQ ID NO:2, an isoleucine substitution at amino acid positions corresponding to positions 111 and 191 of SEQ ID NO:2, and an alanine substitution at amino acid position corresponding to position 321 of SEQ ID NO:2; and (iii) the LukB polypeptide is a CC45 LukB variant polypeptide comprising a leucine substitution at amino acid position corresponding to position 53 of SEQ ID NO:16. Embodiment 53 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 52, wherein the SpA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 60, the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 4, and the LukB variant polypeptide comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 18. Embodiment 54 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 32, wherein (i) the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, at least one domain having a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58 and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58, (ii) the LukA variant polypeptide comprises a CC45 LukA variant polypeptide comprising a methionine substitution at amino acid position corresponding to position 81 of SEQ ID NO:2, an alanine substitution at amino acid position corresponding to position 139 of SEQ ID NO:2, an isoleucine substitution at amino acid positions corresponding to positions 111 and 191 of SEQ ID NO:2, and an alanine substitution at amino acid position corresponding to position 321 of SEQ ID NO:2, and (iii) the LukB polypeptide is a CC45 LukB polypeptide comprising the amino acid sequence of SEQ ID NO:16. Embodiment 55 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 54, wherein the SpA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 60, the LukA variant polypeptide comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 4, and the LukB polypeptide comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 90% sequence similarity to the amino acid sequence of SEQ ID NO: 16. Embodiment 56 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 1 to 55, further comprising an adjuvant. Embodiment 57 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 56, wherein the adjuvant comprises an aluminum salt, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and aluminum oxide. Embodiment 58 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 56, wherein the adjuvant comprises aluminium hydroxide or aluminium phosphate. Embodiment 59 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 56, wherein the adjuvant comprises a stable oil-in-water emulsion. Embodiment 60 is an immunogenic composition or combination of immunogenic compositions according to embodiment 56, wherein the adjuvant comprises a saponin. Embodiment 61 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 60, wherein the saponin is QS21. Embodiment 62 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 56, wherein the adjuvant comprises a TLR4 agonist. Embodiment 63 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 62, wherein the TLR4 agonist is lipid A or an analogue or derivative thereof. Embodiment 64 is an immunogenic composition or combination of immunogenic compositions according to embodiment 62, wherein the TLR4 agonist comprises MPL, 3D-MPL, RC529, GLA, SLA, E6020, PET-Lipid A, PHAD, 3D-PHAD, 3D-(6-acyl)-PHAD, ONO4007, or OM-174. Embodiment 65 is an immunogenic composition or combination of immunogenic compositions according to embodiment 62, wherein the TLR4 agonist is a glycopyranosyl lipid adjuvant (GLA). Embodiment 66 is an immunogenic composition or combination of immunogenic compositions according to embodiment 62, wherein the adjuvant comprises a TLR4 agonist in combination with a stable oil-in-water emulsion. Embodiment 67 is an immunogenic composition or combination of immunogenic compositions according to embodiment 62, wherein the adjuvant comprises a TLR4 agonist formulated in a stable oil-in-water emulsion. Embodiment 68 is an immunogenic composition or combination of immunogenic compositions according to embodiment 65, wherein the adjuvant comprises GLA-SE. Embodiment 69 is an immunogenic composition or combination of immunogenic compositions according to embodiment 62, wherein the adjuvant comprises a TLR-4 agonist in combination with a saponin. Embodiment 70 is an immunogenic composition or combination of immunogenic compositions according to embodiment 65, wherein the adjuvant comprises GLA-LSQ. Embodiment 71 is an immunogenic composition or a combination of immunogenic compositions, the composition comprising one or more isolated nucleic acid molecules encoding a Staphylococcus aureus protein A (SpA) polypeptide or a variant thereof, a LukA variant polypeptide, and a LukB polypeptide or a variant thereof of the immunogenic composition of any one of embodiments 1 to 55. Embodiment 72 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 71, wherein the composition comprises one or more nucleic acid molecules encoding a Staphylococcus aureus protein A (SpA) polypeptide or a variant thereof, and a nucleic acid molecule encoding a LukAB heterodimer (RARPR-15), wherein the nucleic acid molecule encoding the LukAB heterodimer comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 104 operably linked to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 108. Embodiment 73 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 71, wherein the composition comprises one or more nucleic acid molecules encoding a Staphylococcus aureus protein A (SpA) polypeptide or a variant thereof, and a nucleic acid molecule encoding a LukAB heterodimer (RARPR-30), wherein the nucleic acid molecule encoding the LukAB heterodimer comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 104 operably linked to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 110. Embodiment 74 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 71, wherein the composition comprises one or more nucleic acid molecules encoding a Staphylococcus aureus protein A (SpA) polypeptide or a variant thereof, and a nucleic acid molecule encoding a LukAB heterodimer (RARPR-32), wherein the nucleic acid molecule encoding the LukAB heterodimer comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 103 operably linked to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 107. Embodiment 75 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 71, wherein the composition comprises one or more nucleic acid molecules encoding a Staphylococcus aureus protein A (SpA) polypeptide or a variant thereof, and a nucleic acid molecule encoding a LukAB heterodimer (RARPR-33), wherein the nucleic acid molecule encoding the LukAB heterodimer comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 103 operably linked to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 110. Embodiment 76 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 71, wherein the composition comprises one or more nucleic acid molecules encoding a Staphylococcus aureus protein A (SpA) polypeptide or a variant thereof, and a nucleic acid molecule encoding a LukAB heterodimer (RARPR-34), wherein the nucleic acid molecule encoding the LukAB heterodimer comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 103 operably linked to a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 109. Embodiment 77 is an immunogenic composition or a combination of immunogenic compositions according to embodiment 71, wherein the one or more nucleic acid molecules are contained in one or more vectors. Embodiment 78 is an immunogenic composition according to embodiment 71 or 77, wherein the composition comprises a host cell, the host cell comprising the one or more nucleic acid molecules or the one or more vectors. Embodiment 79 is a method for treating or preventing a Staphylococcus infection in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of an immunogenic composition or combination of immunogenic compositions described in any one of embodiments 1 to 78. Embodiment 80 is a method for inducing an immune response against Staphylococcus bacteria in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of an immunogenic composition or combination of immunogenic compositions described in any one of embodiments 1 to 78. Embodiment 81 is a method for decolonizing or preventing colonization or recolonization of Staphylococcus bacteria in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of an immunogenic composition or combination of immunogenic compositions described in any one of embodiments 1 to 78. Embodiment 82 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 1 to 78 for use in a method for generating an immune response against S. aureus in a subject. Embodiment 83 is an immunogenic composition or a combination of immunogenic compositions according to any one of embodiments 1 to 78 for use as a medicament. EXAMPLES

[0234] The following examples are provided to illustrate embodiments of the present disclosure, but are not intended to limit its scope in any way.

[0235] Example 1: Exemplary LukA variant polypeptides, LukB variant polypeptides, and stable LukAB heterodimeric complexes For expression of the LukAB heterodimeric proteins, E. coli BL21(DE3) cells were co-transformed with the lukA construct cloned in pCDFDuet-1 and the lukB construct cloned in pETDuet-1. Transformants were grown overnight at 37°C with shaking at 190 rpm in Luria-Bertani broth for selection of pETDuet-1 and pCDFDuet-1 with 50 μg / mL ampicillin and 50 μg / mL spectinomycin, respectively. Cultures were grown at OD 600Fresh Terrific Broth medium was inoculated with a 1:50 dilution of the culture grown overnight at 37°C with shaking at 190 rpm until a NA of 0.05 was reached. Expression was then induced via addition of isopropyl β-d-1-thiogalactopyranoside to a final concentration of 1 mM, and induction was continued for an additional 5 h at 37°C. Expression of LukAB heterodimers containing pairs of cysteine ​​substitutions in LukA and / or LukB to support disulfide bond formation was expressed in the cytoplasm of E. coli Origami 2(DE3) cells. Expression of LukA monomers in the periplasm of E. coli BL21(DE3) was achieved via transformation of the lukA construct in pD861-CH and induced with a final concentration of 4 mM rhamnose in Terrific Broth (supplemented with 30 μg / mL kanamycin) for 4 h at 37°C. After induction of both cytoplasmic and periplasmic expression constructs, cells were harvested by centrifugation at 4000 rpm for 15 min at 4°C and then resuspended in lysis buffer (94% Bugbuster [EMD Millipore] + 6% 5M NaCl + 0.4% 4M imidazole + protease inhibitor cocktail [ProteaseArrest, G-Biosciences]). After lysis for 20 min at room temperature, the lysate was incubated on ice for 45 min and then centrifuged at 16100 x g for 35 min at 4°C. Proteins were purified via the 6xHis-tag at the N-terminus of LukA using an AKTA Pure 25M FPLC and a HisTrap column and eluted using an imidazole gradient (50-500 mM imidazole in 50 mM sodium phosphate buffer, pH 7.4, 200 mM NaCl). Fractions containing purified protein as determined by SDS-PAGE were pooled and dialyzed overnight in 50 mM sodium phosphate buffer, pH 7.4, 200 mM NaCl, 10% glycerol at 4° C. Purified protein was quantified via the bicinchoninic acid (BCA) protein assay (Pierce). [Table 5] TIFF2024512751000018.tif205170

[0236] Example 2: Cytotoxicity of Wild-type, LukA, and LukAB Toxoids The cytotoxicity of the LukAB toxoid protein (defined in Table 5) was evaluated in comparison to wild-type LukAB toxin using either the promonocytic cell line THP-1 or freshly isolated primary human polymorphonuclear leukocytes (hPMN).

[0237] THP-1 cells were differentiated in the presence of phorbol 12-myristate 13-acetate before testing for cytotoxicity. For the THP-1 cytotoxicity assay, a total of 1 × 10 5 Cells were added to each well of a 96-well plate. LukAB toxin and toxoid proteins were adjusted to standard protein concentrations, serially diluted in ice-cold RPMI medium, and a volume of 50 μL of each was added to the appropriate wells. Triton X-100 was added to a final concentration of 0.1% as a positive control, in addition to a negative control of RPMI only. Plates were incubated at 37°C, 5% CO2 for 2 hours, after which the release of the cytosolic enzyme lactate dehydrogenase, which serves as a marker of membrane integrity, was assessed using the CytoTox-ONE assay (Promega).

[0238] The cytotoxicity of LukA and LukAB toxoids against differentiated THP-1 cells is provided in Table 6 below. Differentiated THP-1 cells were sensitive to wild-type toxins, as both CC8 and CC45 LukAB wild-type toxins killed more than 30% of the cell population at toxin concentrations as low as 0.313 μg / mL. Deletion of the last 10 amino acid residues at the C-terminus of LukA (Delta10) reduced the cytotoxicity of CC8 Delta10 toxin to less than 5% cell death at 40 μg / mL, but did not reduce the cytotoxicity of CC45 Delta10 toxin against differentiated THP-1 cells. None of the LukA monomers showed cytotoxicity against differentiated THP-1 cells. This result was expected, as LukA should not form an active pore complex in the absence of LukB. Each of the LukAB dimeric toxoids containing RARPR-33, RARPR-34, and RARPR-15 exhibited significantly reduced cytotoxicity against differentiated THP-1 cells, with less than 1% cell death for each of the toxoids tested at the highest test concentration of 40 μg / mL. [Table 6]

[0239] For hPMNs, all toxins were normalized to 2.5 μg / mL (per subunit) prior to intoxication, then 20 μL of toxin was pipetted into the top wells of a 96-well plate and serially diluted 2-fold in 10 μL of 1X PBS. PMNs were isolated and normalized to 200,000 cells per 90 μL of RPMI (10 mM HEPES + 0.1% HSA). 90 μL of PMNs were then pipetted into each well, and the toxin-PMN mixture was incubated for 1 h in a 37°C + 5% CO2 incubator. To assess toxicity, 10 μL of CellTiter 96 Aqueous One Solution (CellTiter, Promega) was added to the 96-well plate, and the mixture was incubated for 1.5 h at 37°C, 5% CO2. PMN viability was assessed using a PerkinElmer EnVision 2103 Multilabel Reader at absorbance of 492 nm.

[0240] The cytotoxicity of LukA monomer and LukAB dimer toxoids against human primary PMN cells is provided in Table 7 below. Wild-type CC8 and CC45 toxins exhibited greater than 90% killing of primary human PMNs at toxin concentrations of 0.313 μg / mL and 1.25 μg / mL, respectively. In comparison, the LukAB toxoids and LukA monomers each exhibited greatly reduced cytotoxicity against these cells. Deletion of 10 C-terminal residues in CC8 LukA essentially eliminated cytotoxicity against differentiated THP-1 cells, yet the toxin retained cytotoxicity against hPMNs, with greater than 20% killing observed at concentrations of 5 μg / mL or higher. CC8 and CC45 LukA monomers exhibited little cytotoxicity against hPMNs, as would be expected for a toxoid lacking the LukB component critical for the formation of the active pore complex. Each of the LukAB dimeric toxoids had significantly reduced cytotoxicity against hPMN cells compared with the CC8 and CC45 wild-type LukAB toxins. The RARPR-33 LukAB toxoid, as well as the related toxoids RARPR-32 and -34, were less cytotoxic than CC8delta10, with RARPR-33 killing only 15% of the cell population at the highest concentration tested, 20 μg / mL. [Table 7]

[0241] Example 3: Cytotoxicity of RARPR-33 LukAB toxin and other variants of WT LukAB toxin Additional experiments were performed to evaluate the cytotoxicity and immunogenicity of RARPR-33 and different variants of WT LukAB toxin and LukA monomer. Mice were used for immunogenicity studies.

[0242] The cytotoxicity of LukAB toxins, toxoids, and monomers was evaluated in human PMNs. Prior to intoxication, all toxins were normalized to 100 μg / mL (per subunit), then 20 μL of toxin was pipetted into the top wells of a 96-well plate and serially diluted 2-fold in 10 μL of 1X PBS. PMNs were isolated from different donors and normalized to 200,000 cells per 90 μL of RPMI (10 mM HEPES + 0.1% HSA). 90 μL of PMNs were pipetted into each well, and the toxin-PMN mixture was incubated for 1 h in a 37°C + 5% CO2 incubator. To evaluate toxicity, 10 μL of CellTiter 96 Aqueous One Solution (CellTiter, Promega) was added to the 96-well plate, and the mixture was incubated for 1.5 h at 37°C, 5% CO2. PMN viability was assessed using a PerkinElmer EnVision 2103 Multilabel Reader at absorbance of 492 nm. The percentage of dead cells was calculated by subtracting the background (healthy cells + PBS) and normalizing to Triton X100-treated cells, which were set to 100% dead.

[0243] The cytotoxicity of LukA monomer and LukAB toxins against human primary PMN cells is provided in FIG. 3. Wild-type LukAB CC8 and CC45 toxins showed greater than 90% killing of primary human PMNs at toxin concentrations of 2.5 μg / mL and 5 μg / mL, respectively. Maximal killing was also observed for the LukAB hybrid toxins CC8 / CC45 and CC45 / CC8 at 2.5 μg / mL. In comparison, LukAB toxoids and LukA monomers showed significantly reduced cytotoxicity against these cells. Deletion of the 10 C-terminal residues in CC8 LukA retained cytotoxicity against hPMNs, with greater than 20% killing observed at concentrations of 5 μg / mL and above. CC8 and CC45 LukA monomers, as well as combinations of these monomers, showed little cytotoxicity against hPMNs. The LukAB toxoid of RARPR-33 was less cytotoxic than CC8Δ10C, with RARPR-33 killing only 15% of the cell population at the highest tested concentration of 20 μg / mL.

[0244] Example 4: Immunogenicity of LukAB variants in mice To determine the immunogenicity of the different LukAB variants, Envigo Hsd:ND4 (4 week old) mice (n=5 / antigen) were subcutaneously administered 20 μg of LukAB in 50 μl of 10% glycerol 1× TBS mixed with 50 μl of the adjuvant TiterMax® Gold. Cohorts of 5 mice were also mock immunized with the same volume of 10% glycerol 1× TBS and TiterMax® Gold. After two boosts with the same antigen-adjuvant cocktail at 2-week intervals, mice were bled via cardiac puncture and serum was obtained.

[0245] To determine anti-LukAB antibody titers, an ELISA was performed. WT LukAB CC8 or CC45 was diluted to 2 μg / ml in 1× PBS and coated at 100 μl in 96-well Immulon 2HB plates (Thermo Fisher, Cat. No. 3455) and incubated overnight at 4° C. The plates were then washed three times with wash buffer (1× PBS+0.05% Tween) and then blocked with 200 μl of blocking buffer (2.5% emulsion in 1× PBS) for 1 hour. Five-fold serial dilutions of serum starting at 1:500 into blocking buffer were made and allowed to incubate for 1 hour on a rocker. The plates were then washed again three times and mouse IgG-HRP (Biorad) antibody diluted 1:5,000 in blocking buffer was added and allowed to incubate for 1 hour at room temperature. Unbound secondary antibody was washed away with three successive washes with wash buffer. Room temperature TMB (100 μl) was added to each well, covered and incubated for 25 minutes. After completion of the reaction, an equal volume of 2N sulfuric acid was added to each reaction well to stop the reaction. The plate was then read on an Envision plate reader for absorbance at 450 nm. The heat maps depicted in Figures 4A and 4B show the average absorbance values ​​of duplicate measurements, with black representing high absorbance and antibody binding to the coating antigen and white representing low absorbance and no antibody binding.

[0246] RARPR-33 induced robust anti-CC8 and anti-CC45 LukAB IgG antibody titers (Figure 4A and Figure 4B). RARPR-33 immunization induced anti-CC8 IgG responses comparable to immunization with CC8 WT toxin, CC8 / CC45 hybrid toxin, and CC8Δ10C toxoid. Anti-CC8 LukAB IgG titers induced by individual CC8 LukA monomers were not as high as those induced by CC8 LukAB toxin or CC8 / CC45 hybrid toxin, CC45 / CC8 hybrid toxin, and RARPR 33 hybrid antigen (Figure 4A).

[0247] Anti-CC45 LukAB titers in RARPR-33 immunized mice were higher than those induced by CC8 / CC45 WT hybrid antigen and comparable to those induced by CC45 WT antigen. Combining CC8 and CC45 LukA monomers induced antibody titers against both CC8 and CC45 LukAB (Figure 4B). However, these anti-CC8 and anti-CC45 LukAB titers induced by combining CC8 and CC45 LukA monomers were not as high as those induced by RARPR33. Individual CC45 LukA monomers induced very high anti-CC45 LukAB titers, which were similar to the levels induced by CC45 / CC8 hybrids and only slightly lower than those induced by RARPR-33 or CC45 WT toxins. These results indicate that RARPR-33 immunization induces high magnitude antibody responses against both LukAB CC8 and CC45.

[0248] Example 5: Antibody-Mediated Neutralization of Toxin Cytotoxicity Antibody-mediated neutralization of toxin cytotoxicity was assessed with sera obtained from mice immunized as described above in Example 4. Heat-inactivated pooled sera were normalized to 40% serum in PBS, and then 20 μL of serum was pipetted into the top wells of a 96-well plate and serially diluted 2-fold with 10 μL of 1× PBS. LD 90Each of the LukAB toxin clone complex sequence variants was added to the plate (10 μL / well) for 15 min at room temperature. Freshly isolated human primary polymorphonuclear leukocytes (hPMNs) normalized to 200,000 cells per 80 μL of RPMI (10 mM HEPES + 0.1% HSA) were then added to the serum-toxin mixture and incubated for 1 h at 37 °C + 5% CO2. To assess toxicity, 10 μL of CellTiter 96 Aqueous One Solution (CellTiter, Promega) was added to the 96-well plate and the mixture was incubated for 1.5 h at 37 °C, 5% CO2. PMN viability was assessed using a PerkinElmer EnVision 2103 Multilabel Reader at absorbance at 492 nm. Antibody neutralization data are presented in Figure 5.

[0249] Sera from RARPR-33 immunized mice showed the most potent and broadest LukAB neutralization capacity of all antigens (Figure 5). Sera from RARPR33 immunized mice strongly neutralized the cytotoxic effect of all 11 LukAB variants tested at as low as 0.25% serum, and most LukAB variants also provided protection at as low as 0.063-0.125% serum (Figure 5). Immunization with individual CC8 and CC45 LukA monomers resulted in sera with LukAB neutralization capacity highly biased towards the antigen scaffold (Figure 5). A combination of CC8 LukA monomer and CC45 LukA administered at 20 μg of each monomer (40 μg total protein) for each immunization yielded sera with broad and potent LukAB neutralizing capacity, neutralizing all 11 LukAB variants tested with as little as 0.5% of the sera (Figure 5), which was lower than that observed with sera from RARPR-33-immunized mice.

[0250] Combined, the data presented in Examples 3-5 show that attenuating and stabilizing mutations incorporated into the CC8 / CC45 LukAB backbone of RARPR-33 improve the broad immunogenic effect of the CC8 / CC45 WT LukAB hybrid (Figures 4 and 5), while simultaneously highly attenuating RARPR-33 compared to the CC8 / CC45 WT LukAB toxin (Figure 3).

[0251] Example 6: Antiserum Toxin Neutralization Antibody-mediated neutralization of toxin cytotoxicity was assessed with sera obtained from mice immunized with wild-type LukAB, wild-type LukAB hybrids (i.e., CC8 LukA / CC45 LukB and CC45 LukA / CC8 LukB), LukA monomer, or LukAB toxoid. Heat-inactivated pooled sera were normalized to 40% serum in PBS, and then 20 μL of serum was pipetted into the top wells of a 96-well plate and serially diluted 2-fold with 10 μL of 1X PBS. LD 90 Each of the LukAB toxin clone complex sequence variants was added to wells of plates (10 μL / well) containing either 2%, 1%, or 0.5% serum for 15 min at room temperature. Freshly isolated human primary polymorphonuclear leukocytes (hPMN) from different donors, normalized to 200,000 cells per 80 μL of RPMI (10 mM HEPES + 0.1% HSA), were then added to the serum-toxin mixture and incubated for 1 h at 37 °C + 5% CO2. To assess toxicity, 10 μL of CellTiter 96 Aqueous One Solution (CellTiter, Promega) was added to the 96-well plate and the mixture was incubated for 1.5 h at 37 °C, 5% CO2. PMN viability was assessed using a PerkinElmer EnVision 2103 Multilabel Reader at absorbance of 492 nm. Antibody neutralization data are presented in the tables of Figure 6A (2% antibody serum), Figure 6B (1% antibody serum), and Figure 6C (0.5% antibody serum).

[0252] Immunization with wild-type CC8 and CC45 LukAB induced antibodies that neutralized naturally occurring sequence variants of LukAB toxins in a pattern that reflected the sequence composition of the immunization antigens. Antibodies induced by CC8 LukAB toxin potently neutralized toxins from CC8, CC1, CC5, and other S. aureus strains, but did not provide complete neutralization of toxins from CC30, CC45, or ST22A S. aureus. Similarly, immunization with CC45 LukAB toxin induced antibodies that potently neutralized toxins from CC30, CC45, or ST22A S. aureus strains, but not from other strains.

[0253] Immunization of mice with the non-natural hybrid LukAB, either CC8 LukA combined with CC45 LukB or CC45 LukA combined with CC8 LukB, elicited antibodies that showed broader neutralization of LukAB sequence variants compared to the naturally occurring dimer combinations. Of the non-natural hybrid dimers, CC8 LukA and CC45 LukB showed slightly better neutralization profiles than the opposite combination, and this pattern was retained in proteins carrying a Glu to Ala substitution at the penultimate residue of LukA (E323A). As observed for antibodies elicited against wild-type toxin, LukA monomers elicited antibodies that showed a neutralization pattern indicative of their sequence composition. The combination of CC8 LukA and CC45 LukA monomers (RARPR-31+CC45 LukA W97) elicited antibodies that displayed a broad neutralizing pattern, but with reduced neutralizing potency compared to the dimeric antigen, as evidenced by reduced neutralization levels at 1% or 0.5% serum.

[0254] Of the dimeric toxoids, RARPR-15, RARPR-33, and RARPR-34 demonstrated broadly neutralizing antibody responses against all LukAB sequence variants tested. The unnatural wild-type dimer combination also demonstrated a broad neutralizing profile, although the potency of the neutralizing response was less than that observed for some toxoids. Both the hybrid wild-type and toxoid antigens demonstrated broad neutralizing profiles when tested with 2% (Figure 6A) and 1% (Figure 6B) serum, but improved potency of the response to the toxoid was evident when tested with 0.5% serum (Figure 6C). At this lowest tested concentration, RARPR-15, RARPR-32, RARPR-33, and RARPR-34 each demonstrated a broadly neutralizing response. RARPR-33 in particular elicited sera that retained broadly neutralizing responses, whereas the hybrid wild-type antigen and E323A toxoid failed to elicit broadly protective responses at 0.5% serum. Also, the neutralization pattern elicited by the CC45 toxoid RARPR-15 at the lowest tested concentration reflected its sequence composition, as high levels of neutralization were observed only with CC30, CC45, and ST22A LukAB toxins. The hybrid dimeric toxoid RARPR-33 elicited potent and broadly neutralizing immune responses.

[0255] Example 7: Cytotoxicity of RARPR-33 at high concentrations. method: Cytotoxicity assay: To evaluate the cytotoxicity of each respective LukAB protein complex, freshly isolated primary human polymorphonuclear leukocytes (PMN) were intoxicated with S. aureus toxin. PMN were isolated from different donors and normalized to 200,000 cells per 50 μl of RPMI (10 mM HEPES + 0.1% HSA). 50 μl of toxin in PBS was added to the cells and the toxin-PMN mixture was incubated for 1 h in an incubator at 37 °C + 5% CO2. To evaluate toxicity, 10 μl of CellTiter 96 Aqueous One Solution (CellTiter, Promega) was added to a 96-well plate and the mixture was incubated for 1.5 h at 37 °C, 5% CO2. PMN viability was evaluated using a PerkinElmer EnVision 2103 Multilabel Reader at absorbance of 492 nm. The % dead cells are calculated by subtracting the background (healthy cells + PBS) and normalizing to TritonX treated cells, which are set to 100% dead.

[0256] LDH assay: To assess whether each respective LukAB protein complex could cause cell lysis, freshly isolated primary human polymorphonuclear leukocytes (PMN) from different donors were intoxicated with S. aureus LukAB toxin and LDH release was measured. WT toxin was serially diluted 2-fold in PBS and tested at concentrations ranging from 5 to 0.0024 μg / ml. LukAB toxoid was diluted in PBS and tested at 2.5, 2, 1, 1.5, and 0.5 mg / ml. PMN were isolated and normalized to 200,000 cells per 50 μl of RPMI (10 mM HEPES + 0.1% HSA). Then, 50 μl of PMN were pipetted into each well and 50 μl of diluted toxin was added per well. The toxin-PMN mixture was incubated for 2 h in a 37°C + 5% CO2 incubator. To assess LDH release, plates were centrifuged at 1500 rpm for 5 min, then 25 μl of supernatant was removed from each well and transferred to a 96-well black clear bottom plate. 25 μl of CytoTox-ONE homogeneous membrane integrity reagent (Promega) was added to the black clear bottom 96-well plate, and the mixture was incubated in the dark for 10 min at room temperature. Cell lysis was assessed by recording fluorescence at an excitation wavelength of 560 nm and an emission wavelength of 590 nm using a PerkinElmer EnVision 2103 Multilabel Reader. The % dead cells were calculated by subtracting the background (healthy cells + PBS) and normalizing to TritonX-treated cells, which were set to 100% dead.

[0257] result: In the previous examples, the cytotoxicity of the LukAB toxoid RARPR-33 against hPMNs was determined up to concentrations of 20 μg / ml. Next, the cytotoxicity of human PMNs was monitored in the presence of high concentrations (up to 2.5 mg / ml) of RARPR-33. Maximal cytotoxicity of human PMNs (4-6 donors) based on CellTiter measurements was observed for WT LukAB CC8, CC45 and CC8 / CC45 toxins at 1 h of intoxication with approximately 0.156 μg / ml toxin (Figure 7A). For RARPR-33 and CC8 LukA monomer, the percentage of dead cells measured by CellTiter was approximately 10% at a concentration of 0.5 mg / ml (Figure 7B). Incubation of PMNs with concentrations of RARPR-33 or CC8 LukA monomer up to 2.5 mg / ml did not further increase the percentage of dead cells determined by CellTiter measurements.

[0258] LD 15 Values ​​indicate the concentration of antigen that induces 15% cell death. D15 was determined using linear regression. For CC8 WT LukAB, LD 15 The LD for CC45 WT LukAB was 0.013 μg / ml. 15 The LD for the CC8 / CC45 LukAB hybrid was 0.004 μg / ml. 15 The LD of LukAB RARPR-33 was 0.002 μg / ml. 15 The LD of RARPR-33 was 2.5 mg / ml. 15 The concentration is the LD 15 Divide by the concentration to find the LD 15 Based on these observations, LukAB RARPR-33 toxicity is >192,308-fold lower than LukAB CC8 WT, >625,000-fold lower than LukAB CC45 WT, and >1,250,000-fold lower than the LukAB CC8 / CC45 hybrid.

[0259] Furthermore, LDH assays were performed to evaluate plasma membrane damage after 2 h of incubation with the different WT toxins, CC8 LukA monomer, or RARPR-33. Human PMN cytotoxicity was induced after 2 h of exposure to WT toxin, CC8 WT, CC45 WT, or CC8 / CC45 toxin hybrid (Figure 7C). Maximal cell death, as determined by LDH, was observed at a concentration of 0.625 μg / ml toxin. In contrast, no plasma membrane damage of human PMN was observed after 2 h of exposure to RARPR-33 or CC8 LukA monomer at concentrations up to 2.5 mg / ml (Figure 7D). These data indicate that RARPR-33 is detoxified and cannot induce cell death of human PMN at concentrations up to 2.5 mg / ml. Mutations engineered into the CC8 / CC45 LukAB backbone of RARPR-33 significantly attenuated cytotoxicity compared with the CC8 / CC45 WT LukAB toxin.

[0260] Example 8: Comparison of RARPR-33 and D39A / R23E toxoid A LukAB toxoid based on the CC8 backbone was generated in which LukA carries a D39A mutation and LukB carries an R23E point mutation. This "D39A / R23E toxoid" is described in Kailasan, S. et al, "Rational Design of Toxoid Vaccine Candidates for Staphylococcus aureus Leukocidin AB (LukAB)," Toxins 11(6): (2019), which is incorporated herein by reference in its entirety. This toxoid was generated on the LukAB CC8 backbone and was described as >36,000-fold attenuated in toxicity compared to the WT CC8 LukAB toxin. Cytotoxicity was determined using an HL-60 cell line differentiated to be PMN-like. In this experiment, a comparison was made between the D39A / R23E toxoid and RARPR-33. Cytotoxicity against human polymorphonuclear leukocytes (PMN) was determined and the ability to induce broadly toxin-neutralizing antibodies upon immunization was assessed.

[0261] method: Cytotoxicity assay: To evaluate the cytotoxicity of each respective LukAB protein complex, freshly isolated primary human polymorphonuclear leukocytes (PMN) from different donors were intoxicated with S. aureus toxin. PMN were isolated and normalized to 200,000 cells per 50 μl RPMI (10 mM HEPES + 0.1% HSA). To the cells, 50 μl of toxin in PBS was added and the toxin-PMN mixture was incubated for 2 h in an incubator at 37 °C + 5% CO2. To evaluate toxicity, 10 μl of CellTiter 96 Aqueous One Solution (CellTiter, Promega) was added to a 96-well plate and the mixture was incubated for 1.5 h at 37 °C, 5% CO2. PMN viability was evaluated using a PerkinElmer EnVision 2103 Multilabel Reader at absorbance of 492 nm. The percentage of dead cells is calculated by subtracting the background (healthy cells + PBS) and normalizing to TritonX-treated cells, which are set to 100% dead.

[0262] LDH assay: To assess whether each respective LukAB protein complex causes cell lysis, freshly isolated primary human polymorphonuclear leukocytes (PMN) from different donors were intoxicated with S. aureus LukAB toxin and LDH release was measured. WT toxin was serially diluted 2-fold in PBS and tested at concentrations ranging from 0.5 μg / ml to 0.00024 μg / ml. LukAB toxoid was diluted in PBS and tested at concentrations ranging from 1 mg / ml to 0....

Claims

1. (i) a Staphylococcus aureus protein A (SpA) polypeptide; and (ii) an S. aureus LukA variant polypeptide, the S. aureus LukA variant polypeptide comprising an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Lys83, Ser141, Val113, and Val193 of SEQ ID NO:

25.

2. (i) a Staphylococcus aureus protein A (SpA) polypeptide; and (ii) a combination of two or more immunogenic compositions comprising, together with an S. aureus LukA variant polypeptide, the S. aureus LukA variant polypeptide comprising an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Lys83, Ser141, Val113, and Val193 of SEQ ID NO:

25. (a) the LukA variant polypeptide comprises an amino acid substitution at an amino acid residue corresponding to Glu323 of SEQ ID NO:25; and / or (b) the LukA variant polypeptide comprises amino acid substitutions at amino acid residues corresponding to Lys83, Ser141, Val113, Val193, and Glu323 of SEQ ID NO:25, optionally wherein the amino acid substitutions comprise Lys83Met, Ser141Ala, Val113Ile, Val193Ile, and Glu323Ala; and / or (c) the LukA variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, and / or (d) the LukA variant polypeptide further comprises amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Tyr74, Asp140, Gly149, and Gly156 of SEQ ID NO:25, optionally wherein the amino acid substitutions comprise Tyr74Cys, Asp140Cys, Gly149Cys, and Gly156Cys, and further optionally, the LukA variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6; and / or (e) the variant LukA protein or polypeptide further comprises an amino acid substitution at an amino acid residue corresponding to amino acid residue Thr249 of SEQ ID NO: 25, and optionally The LukA variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

8. A combination of the immunogenic composition of claim 1 or the immunogenic composition of claim 2.

4. The SpA polypeptide is an SpA variant polypeptide, and optionally (a) the SpA variant polypeptide has at least one amino acid substitution that disrupts Fc binding and at least a second amino acid substitution that disrupts VH3 binding; and / or (b) the SpA variant polypeptide comprises an SpA D domain, the SpA D domain comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:58, and optionally (i) the SpA variant polypeptide has an amino acid substitution at one or both of the amino acid positions corresponding to positions 9 and 10 of SEQ ID NO: 58; and / or (ii) the SpA variant polypeptide further comprises an SpA E, A, B, or C domain, and further optionally, the SpA variant polypeptide comprises the SpA E, A, B, and C domains and has an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 54; and / or each of the SpA E, A, B, and C domains has an amino acid substitution at one or both amino acid positions corresponding to amino acid positions 9 and 10 of SEQ ID NO:58; and / or (iii) the amino acid substitutions at one or both amino acid positions corresponding to positions 9 and 10 of SEQ ID NO: 58 are lysine residues for glutamine residues; and / or (c) the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, wherein said at least one domain has (i) a lysine substitution at glutamine residues corresponding to positions 9 and 10 of SEQ ID NO:58, and (ii) a glutamic acid substitution at the amino acid position corresponding to position 33 of SEQ ID NO:58; An immunogenic composition or a combination of immunogenic compositions according to any one of claims 1 to 3.

5. The composition further comprises a S. aureus leukocidin B (LukB) polypeptide or a variant thereof, and optionally (a) the LukB polypeptide is a LukB polypeptide of SEQ ID NO: 15 or a LukB polypeptide of SEQ ID NO: 16; or (b) the LukB polypeptide is a LukB variant polypeptide, optionally wherein the LukB variant polypeptide comprises an amino acid sequence having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 16, and further optionally wherein the LukB variant polypeptide comprises an amino acid substitution at an amino acid position corresponding to position 53 of SEQ ID NO: 15 and SEQ ID NO: 16, and further optionally wherein the amino acid substitution is a valine to leucine substitution. An immunogenic composition or a combination of immunogenic compositions according to any one of claims 1 to 4. (a) the LukB variant polypeptide comprises amino acid substitutions at one or more amino acid residues corresponding to amino acid residues Glu45, Glu109, Thr121, and Arg154 of SEQ ID NO:15; or (b) the LukB variant polypeptide comprises an amino acid substitution at one or more amino acid residues corresponding to amino acid residues Glu45, Glu110, Thr122, and Arg155 of SEQ ID NO: 16; or (c) the LukB variant polypeptide comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 17-22; An immunogenic composition or combination of immunogenic compositions according to claim 5(b). (a) the composition comprises a LukA variant polypeptide comprising an amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4, and a LukB polypeptide comprising an amino acid sequence of SEQ ID NO:16, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:16; or (b) the composition comprises a LukA variant polypeptide comprising an amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3, and a LukB polypeptide comprising an amino acid sequence of SEQ ID NO:15, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:15; or (c) the composition comprises a LukA variant polypeptide comprising an amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, and a LukB polypeptide comprising an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18; and / or (d) the SpA polypeptide is an SpA variant polypeptide, optionally wherein the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, wherein the at least one domain has (i) a lysine substitution at glutamine residues corresponding to positions 9 and 10 of SEQ ID NO:58, and (ii) a glutamic acid substitution at an amino acid position corresponding to position 33 of SEQ ID NO:

58. An immunogenic composition or a combination of immunogenic compositions according to claim 5.

8. (i) the SpA variant polypeptide comprises at least one SpA A, B, C, D, or E domain, wherein the at least one domain has a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58, and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:58; (ii) the LukA variant polypeptide comprises a CC8 LukA variant polypeptide comprising a methionine substitution at amino acid position corresponding to position 80 of SEQ ID NO:1, an alanine substitution at amino acid position corresponding to position 138 of SEQ ID NO:1, an isoleucine substitution at amino acid positions corresponding to positions 110 and 190 of SEQ ID NO:1, and an alanine substitution at amino acid position corresponding to position 320 of SEQ ID NO:1; and (iii) the LukB polypeptide is a CC45 LukB variant polypeptide comprising a leucine substitution at amino acid position corresponding to position 53 of SEQ ID NO:16; The immunogenic composition or combination of immunogenic compositions of claim 5, comprising consecutive E, D, A, B and C domains, each of which has a lysine substitution at amino acid positions corresponding to positions 9 and 10 of SEQ ID NO:58, and a glutamic acid substitution at amino acid position corresponding to position 33 of SEQ ID NO:

58.

9. Further comprising an adjuvant, optionally (a) the adjuvant is a stable oil-in-water emulsion, or (b) the adjuvant comprises a saponin, optionally wherein the saponin is QS21; or (c) the adjuvant comprises a TLR4 agonist, and optionally (i) the TLR4 agonist is lipid A or an analogue or derivative thereof; (ii) the TLR4 agonist is a glycopyranosyl lipid adjuvant (GLA), optionally wherein the adjuvant comprises GLA-SE or GLA-LSQ; or (iii) the adjuvant comprises a TLR-4 agonist in combination with a stable oil-in-water emulsion; or (iv) the adjuvant comprises a TLR-4 agonist in combination with a saponin; An immunogenic composition or a combination of immunogenic compositions according to any one of claims 1 to 7.

10. An immunogenic composition or combination of immunogenic compositions, said composition comprising one or more nucleic acid molecules encoding a Staphylococcus aureus protein A (SpA) polypeptide or variant thereof, said LukA variant polypeptide, and said LukB polypeptide or variant thereof of the immunogenic composition of any one of claims 1 to 8, and optionally (a) the one or more nucleic acid molecules are contained in one or more vectors; and / or (b) the composition comprises a host cell, the host cell comprising the one or more nucleic acid molecules or the one or more vectors; Immunogenic composition or combination of immunogenic compositions.

11. An immunogenic composition or combination of immunogenic compositions according to any one of claims 1 to 10 for use in a method for the treatment or prevention of a Staphylococcal infection in a subject in need thereof, said method comprising: An immunogenic composition or combination of immunogenic compositions comprising administering to a subject in need thereof an effective amount of the immunogenic composition or combination of immunogenic compositions according to any one of claims 1 to 10.

12. An immunogenic composition or combination of immunogenic compositions according to any one of claims 1 to 10 for use in a method for inducing an immune response against Staphylococcus bacteria in a subject in need thereof, said method comprising: An immunogenic composition or combination of immunogenic compositions comprising administering to a subject in need thereof an effective amount of the immunogenic composition or combination of immunogenic compositions according to any one of claims 1 to 10.

13. 11. An immunogenic composition or combination of immunogenic compositions according to any one of claims 1 to 10 for use in a method for decolonising or preventing colonisation or recolonisation of Staphylococcus bacteria in a subject in need thereof, said method comprising: An immunogenic composition or combination of immunogenic compositions comprising administering to a subject in need thereof an effective amount of the immunogenic composition or combination of immunogenic compositions according to any one of claims 1 to 10.

14. An immunogenic composition or combination of immunogenic compositions according to any one of claims 1 to 10 for use in a method for generating an immune response against S. aureus in a subject.

15. An immunogenic composition or a combination of immunogenic compositions according to any one of claims 1 to 10 for use as a medicament.

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