Compositions and methods for the prevention of S. aureus infection

Abstract

The present invention relates to an immunogenic composition comprising at least one Staphylococcus aureus antigen, wherein said antigen is a polypeptide having at least 80% identity with the SdrH-like polypeptide of SEQ ID NO: 8, Nuc of SEQ ID

NO: 4, or LukG of SEQ ID NO: 12, an immunotherapeutic composition comprising a polyclonal antibody which selectively binds to at least one of said antigens, and an in vitro method of identifying an antigen conferring protection against disease caused by S. aureus in a subject.

Claims

1-14. (canceled)

15. An immunogenic composition comprising at least one Staphylococcus aureus antigen, wherein said antigen is a polypeptide having at least 80% identity with the SdrH-like polypeptide of SEQ ID NO: 8, Nuc of SEQ ID NO: 4, or LukG of SEQ ID NO: 12.

16. The immunogenic composition of claim 15, comprising an antigen having at least 80% identity with the SdrH-like polypeptide of SEQ ID NO: 8 and an antigen having at least 80% identity with LukG of SEQ ID NO: 12.

17. The immunogenic composition of claim 15, wherein said composition comprises two or more of said S. aureus antigens in the form of separate polypeptides or in the form of one or more fusion polypeptides or both in the form of separate polypeptide(s) and fusion polypeptide(s).

18. The immunogenic composition of claim 15, further comprising a pharmaceutically acceptable excipient.

19. A method for conferring protection against a disease caused by S. aureus in a subject in need thereof, said method comprising the administration to the patient of the immunogenic composition of claim 15, as a vaccine.

20. A method to promote uptake and killing of S. aureus by phagocytes in a subject in need thereof, said method comprising the administration of an immunotherapeutic composition comprising a polyclonal antibody which selectively binds to at least one antigen a polypeptide having at least 80% identity with the SdrH-like polypeptide of SEQ ID NO: 8, Nuc of SEQ ID NO: 4, or LukG of SEQ ID NO: 12 .

21. The method of claim 20, wherein said immunotherapeutic composition further comprises a pharmaceutically acceptable excipient.

22. The method of claim 20, wherein said immunotherapeutic composition is used as a passive immunotherapy conferring protection against a disease caused by S. aureus in said subject.

23. The method of claim 19, wherein said S. aureus is a methicillin-resistant S. aureus (MRSA) or a methicillin-susceptible S. aureus (MSSA).

24. The method of claim 19, wherein said subject has an osteoarticular device.

25. The method of claim 19, wherein said subject has an osteoarticular implant.

26. The method of claim 19, wherein said subject has a total joint replacement prosthesis.

27. The immunogenic composition of claim 15, further containing one or more antibiotics that are effective against a S. aureus infection.

28. An in vitro method for identifying an antigen conferring protection against disease caused by S. aureus in a subject, said method comprising: a) incubating a solution comprising S. aureus with a solution comprising antibodies raised against an S. aureus antigen, thereby obtaining a mixed suspension, b) contacting macrophages with the mixed suspension of step a), c) removing the mixed suspension from macrophages and adding fresh medium supplemented with antibiotics to kill extracellular S. aureus bacteria, and d) assessing internalization and killing of S. aureus bacteria by said macrophages, wherein said antigen is considered to confer protection against disease caused by S. aureus when said antigen induces both increased internalization and killing of S. aureus.

29. The method of claim 28, wherein step a) is performed for one hour at 35 C.

30. The method of claim 28, wherein said macrophages are an immortalized macrophage cell line.

31. The method of claim 28, wherein said macrophages are the J774.2 cell line.

32. The method of claim 28, wherein the killing of S. aureus bacteria in step d) is assessed by comparing the quantity of bacteria internalized in macrophages 3 hours after step c) with the quantity of bacteria internalized in macrophages 6 hours after step c).

33. The method of claim 20, wherein said S. aureus is a methicillin-resistant S. aureus (MRSA) or a methicillin-susceptible S. aureus (MSSA).

34. The method of claim 20, wherein said subject has an osteoarticular device.

Description

DESCRIPTION OF THE FIGURES

[0084] FIG. 1. S. aureus uptake mediated by immune sera

[0085] S. aureus uptake (3 h post-infection) at multiplicities of infections (MOIs) of 10:1 (left panels) et 25:1 (right panels), using immune sera diluted 1/1000 (upper panels) and 1/2000 (lower panels). Average fluorescence areas values (488 nm excitation, 515 nm emission) are reported, normalized by anti-GFP antibody (value of 1). Standard deviations were calculated from the values of fluorescence areas per cell, before normalization relative to anti-GFP antibody fluorescence. Statistical significance was evaluated using Graphpad Prism on the raw data. *P-value <0.05. **P-value <0.01. Proteins tested: Pbp2a (A), SspA (B), Sak (C), IsaA (D), GlpQ (E), Autolysin-like protein (F), Nuc (G), Hla (H), LukG (I), LukH (J), IsdA (K), IsdB (M), SdrD (as two partial polypeptides: N and Nb), ClfA (as two partial polypeptides: O and Ob), MntC (1), SdrH-like polypeptide (2), Lip2 (3), putative protein (4), Atl (5), and hypothetical protein (6). Grey: Nuc (G), LukG (I) and SdrH-like polypeptide (2).

[0086] As shown, only five polypeptides are associated to a significant increase of uptake in at least two different conditions: Nuc (G), Hla (H), LukG (I), IsdA (K), and SdrH-like polypeptide (2); note that the values observed for dilutions 1/1000 et 1/2000 are similar, indicating the lack of threshold effect.

[0087] FIG. 2. S. aureus killing

[0088] Killing of S. aureus (6 h post-infection) at MOls of 10:1 (left panels) and 25:1 (right panels), using immune sera diluted 1/1000 (upper panels) and 1/2000 (lower panels). The average fluorescence areas (excitation 488 nm, emission 515nnn) of the reported protein at the 6 h time point is normalized here to the value measured for the same protein at the 3 h time point (reference value of 1). Proteins tested are the same as those listed above (see legend of FIG. 1). Grey: Nuc (G), LukG (I), and SdrH-like polypeptide (2).

[0089] As shown, only three of the five antigens associated with a significant increase in

[0090] S. aureus uptake are also associated with a killing of the bacteria, in all assay conditions: Nuc (G), LukG (I), and SdrH-like polypeptide (2).

[0091] FIG. 3. Pictures of macrophages infected with S. aureus treated with anti-IsdB protein and anti-SdrH-like protein antisera at 6 h post-infection (MOI 1:10, serum dilution 1/1000).

[0092] With anti-IsdB (M) protein antiserum (panel A), myriads of bacteria (white circles) can be seen filling up cytoplasmic space; areas of cell lysis with release of extracellular bacteria can also be observed. This contrasts with the picture obtained with anti-SdrH-like polypeptide (2) antiserum (panel B): individual bacteria (white circles) can be enumerated; the cytoskeleton structure is preserved and the nuclei areas are preserved. Similar observations were made with anti-Nuc (G) protein and anti-LukG (I) protein antisera (data not shown).

EXAMPLES

[0093] The following examples are included to demonstrate preferred embodiments of the invention. All subject-matter set forth or shown in the following examples and accompanying drawings is to be interpreted as illustrative and not in a limiting sense. The following examples include any alternatives, equivalents, and modifications that may be determined by a person skilled in the art.

Example 1: Construction, Production and Purification of the S. aureus and Control Antigens

Materials and Methods

[0094] Cloning of the Genes Coding the S. aureus Antigens of the Invention and S. aureus Control Antigens into an Expression Vector

[0095] The sequenced S. aureus strain Mu50 was used as a source of genomic DNA. DNA extraction was performed using a commercial kit (DNeasy Blood and Tissue, Qiagen Hilden, Germany). S. aureus genes of interest were amplified by polymerase chain reaction (PCR) using appropriate primers, designed with AmplifX.

[0096] Nucleotide sequences of S. aureus genes are notably as provided in SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, and 79. Cloned DNA sequences are as provided in SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 75, 80, and 81. In particular, two different polypeptides were cloned for the sdrD and clfA genes (SEQ ID NOs: 74 and 75 for sdrD and SEQ ID NOs: 80 and 81 for clfA). DNA was purified prior enzymatic restriction with Sail and Stul (Thermo Scientific, Waltham, USA), as was the expression vector pET-6xHN-N (Clontech, Otsu, Japan) containing a poly-histidine tag. Restricted PCR products were then ligated into the vector. Resulting expression vectors of each gene were controlled by electrophoretic migration prior to transformation into chemocompetent DH10131 Escherichia coli (Thermo Scientific). Transformed bacteria were incubated 1 h at 35 C. in Luria-Bertani (LB) broth before being plated on LB agar with ampicillin (100 mg/L) and incubated overnight at 35 C. Isolated colonies were harvested and grown overnight in LB broth to amplify the clone. Vector DNA was then purified using a commercial kit (QlAprep Spin Miniprep, Qiagen). Sequencing was performed to validate each inserted gene sequence. The pET-6xHN-GFPuv vector (Clontech) was used for expressing the green fluorescent protein (GFP, SEQ ID NOs: 85 and 86 for cloned DNA and amino acid sequences, respectively).

Antigen Production and Purification

[0097] Verified vectors were used to transform chemocompetent BL21 (DE3) E. coli cells (Thermo Scientific) following the same protocol as used for DH1011 cells and isolated colonies similarly amplified. A 1/100 dilution of the overnight culture was incubated at 35 C. until the culture reached an optical density (OD) of 0.5. A solution of IPTG (1 mM final) was then added to the bacteria to induce antigen production at 35 C. until an OD of 1.2 was reached. Bacterial pellets obtained by centrifugation were lysed and the Histidine tagged proteins purified using a commercial kit (Proteus Metal Chelate, Generon, Slough, UK) and the recombinant His-tagged proteins eluted using a 10 mM imidazole solution. Eluted antigens were then concentrated using an Amicon Ultra-15 column (Merck, Darmstadt, Germany). Polypeptide sequences of said antigens are as provided in SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 76, and 82. Cloned polypeptide sequences, all of which further comprise an N-terminal His tag, are as provided in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 10, 44, 48, 52, 56, 60, 64, 68, 72, 77, 78, 83, and 84.

Antigen Characterization

[0098] Characterization of the purified antigens was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) colored with a Coomassie solution to evaluate the size, integrity and purity of the recombinant antigen. The concentration of the purified antigen solutions was determined using Bradford's method.

Results

[0099] A total of 20 S. aureus antigens, evaluated as 22 different polypeptides, were cloned, expressed and purified (>95% of purity), with total amounts of purified protein ranging from 1 mg to 6 mg for each recombinant protein.

[0100] Four of these proteins are included in anti-S. aureus vaccines undergoing pharmaceutical development and were used as control vaccine antigens: IsdB (described in Harro et al., 2010, Moustafa et al., 2012, Fowler et al., 2013), MntC (described in Salazar et al., 2014, Begier et al., 2017, Inoue et al., 2018), CIfA (described in Salazar et al., 2014, Begier et al., 2017, Inoue et al., 2018), and Hla alpha-toxin described in Landrum et al., 2016).

Example 2: Production of Antibodies Against S. aureus Antigens

Materials and Methods

[0101] Antibodies targeting S. aureus polypeptides and control antigens were obtained by immunization of specific pathogen free BALB/cJRj mice, specifically documented to have originated in a S. aureus-free environment. Mice were received when they were nine weeks-old and were acclimated one week prior to immunization. Groups of six mice per antigen were injected with a first dose of 20 g of purified antigen with Freund's complete adjuvant, followed by two more injections at 21 and 42 days with 20 g of antigen with Freund's incomplete adjuvant. Mice were sacrificed and the sera collected 50 days after the first injection. A first control group (n=6) was injected with GFP (non-relevant non-S. aureus antigen). A second control group (n=6) was injected with Phosphate Buffer Solution (PBS) to control for adjuvant immunogenicity.

[0102] The titer and specificity of each immune serum were verified by western-blot using purified proteins.

Results

[0103] Each serum was evaluated with serial dilutions down to a titer of 60,000. All serum sample tested showed a positive specific band at this concentration, showing the effective immunization of all animals. Sera from the six mice immunized with the same antigen were pooled to obtain a single immune serum stock for each of the 22 S. aureus polypeptides and for GFP.

Example 3: In vitro Evaluation of Immune Sera in a Macrophage-Based OPA Assay

Materials and Methods

[0104] S. aureus Strains

[0105] Experiments were performed with USA300 and its spA.sup. derivative (Frdric Laurent, Lyon).

Macrophage Culture

[0106] Cellular assays were performed on the murine BALB/c immortalized macrophage cell line J774.2 (European Collection of Authenticated Cell Lines, Porton Down, UK). Macrophages were cultured in Dulbecco's Modified Eagle Medium (DMEM) complemented with 10% fetal bovine serum at 35 C. under a 5% CO.sub.2 atmosphere. Cells were suspended in complemented DMEM, titrated, and seeded in culture plates 24 h prior to the assay.

Macrophage-Based Assay

[0107] An 18 h S. aureus culture in brain-heart infusion (BHI) broth was diluted 1/100 in fresh medium and incubated at 37 C. until the culture reached an OD of 1. Immune sera diluted 1/1000 or 1/2000 were added and allowed to bind to the bacterial surface for 1 h at 35 C. Serum-treated bacteria were then added to the titrated J774.2 cell monolayers at multiplicities of infection (MOI) of 10:1 (10 bacteria per cell) and 25:1 (25 bacteria per cell). After incubation for 1 h at 35 C. under a 5% CO.sub.2 atmosphere, wells were emptied of medium and gently washed with PBS before adding fresh DMEM with gentamicin. At appropriate times (see below: bacterial uptake, 3 h post-infection; bacterial killing, 6 h post-infection), J774.2 cells were washed with PBS, fixed with PFA 4% for 5 minutes, and then permeabilized with 0.1% Triton X100 for 5 min. Fixed cells were dyed for 30 min with Hoechst 33342 (Thermo Scientific), Phalloidin-ATTO 655 (Sigma-Aldrich, Saint-Louis, USA) and BODIPY FL Vancomycin (VMB) (Invitrogen, Carlsbad, USA), and were sealed using glass coverslips. Images were acquired using a Leica SP8 confocal microscope and analyzed using ImageJ software (National Institute of Health, Bethesda, USA).

Evaluation of Bacterial Uptake and Killing

[0108] The uptake of serum-treated bacteria was evaluated at 3 h post-infection by comparing the number of pixels with VMB fluorescence (bacterial cell wall quantification) per J774.2 cell for each antigen specific serum to the number of pixels with VMB fluorescence per J774.2 cell for the non-relevant control serum (anti-GFP). To evaluate the outcome of internalized bacteria, the area of VMB at 3 h post-infection and 6 h post-infection were compared. Bacterial growth was called when an increase of fluorescence was measured, reflecting an increase in the amount of intracellular peptidoglycan. Bacterial lysis (killing) was called when a decrease in the amount of intracellular peptidoglycan was measured, reflecting a decrease in the amount of intracellular peptidoglycan.

Results

[0109] The evaluation of bacterial uptake evaluated 3 hours after infection at MOI of 10:1 and 25:1 following S. aureus incubation with two antibody dilutions (1:1000, 1:2000) is presented here, featuring an anti-GFP control serum in each experiment for normalization. Five antigens show markedly different behaviors with a significant increase in the internalization of S. aureus bacteria in at least two conditions: proteins the SdrH-like polypeptide (2), Nuc (G), and LukG (I), Hla (H), and IsdA (K) (FIG. 1). Intracellular bacterial clearance and growth was evaluated by comparing the areas of VMB fluorescence 6 hours after infection to the areas of fluorescence observed 3 hours after infection. Among the five antigens previously shown to be associated with significant bacterial uptake, three were associated with bacterial killing in all experimental conditions: the SdrH-like polypeptide (2), Nuc (G), and LukG (I) (FIG. 2). Noticeably, a number of proteins with no significant effect on uptake were associated with bacterial growth enhancement (facilitating effect of immune sera) (see for example, proteins Pbp2a (A) and Sak (C) in FIG. 2, at a MOI of 25:1 and with a serum dilution of 1/1000). Bacterial growth was particularly intense with anti-IsdB protein sera and resulted in the destruction of the macrophage monolayer (FIG. 3), leading to underestimating the load of intracellular bacteria (compare FIGS. 1 Et 2 with FIG. 3).

Example 4: Establishment of an in vivo Model of Systemic S. aureus Infection in Mice

[0110] Previous studies have shown that BALB/c mice are highly susceptible to blood-borne S. aureus infection, due to the inability of this mouse strain to limit bacterial growth in the kidneys (von Kckritz-Bliclwede et al., 2008). However, as the course of infection may differ among S. aureus strains according to their virulence repertoire, we first determined which dose of S. aureus USA300 led to non-lethal kidney infection.

Materials and Methods

[0111] S. aureus strains

[0112] Experiments were performed with S. aureus strain USA300.

Mice

[0113] Female BALB/c mice were purchased from Janvier Labs (Le Genest Saint Isle, France). Mice were received when they were six weeks-old and were acclimatized one week prior to immunization. Animal experiments were performed according to institutional and national ethical guidelines (Agreement APAFIS #26827).

Mouse Model of Systemic S. aureus Infection

[0114] Mice were anaesthetized by intraperitoneal administration of ketamine/xylazine (50/10 mg/kg) and were inoculated with 10.sup.9, 10.sup.7 or 10.sup.5 CFU of USA300 by retro-orbital sinus injection under a volume of 1004. Mice were euthanized 3 hours and 24 hours after infection. Spleen and kidneys were harvested, homogenized, and serial dilutions were plated on Mueller Hinton 2 agar plates. CFUs were enumerated after 24 hours of incubation at 37 C. (minimal detection limit: 2.69 log.sub.10 CFU per organ).

Results

[0115] The dose of 10.sup.9 CFU caused the death of 100% of animals before the 24 h post-challenge time-point while 10.sup.5 CFU did not allow the establishment of infection in the kidneys (no bacteria detected at 3 h and 24 h post-challenge) (Table 2). Thus, 10.sup.7 CFU was the only dose to be non-lethal and to be associated with the infection of kidneys. As expected, bacterial burden was similar at 3 h and 24 h post-challenge in the spleen, suggesting infection control, while bacterial growth was dramatically increased in the kidneys (CFU differential of ca 4 log.sub.10 between 3 h and 24 h post-challenge) (Table 2).

TABLE-US-00002 TABLE 2 CFU counts at 3 h and 24 h post-challenge in non-immunized animals. Mean number of CFUs per organ, in log.sub.10 Spleen Kidneys USA300 dose 3 h 24 h 3 h 24 h 10.sup.9 CFU 6.70 .sup.b 6.68 .sup.b 10.sup.7 CFU 5.71 5.58 2.69 6.08 10.sup.5 CFU 3.66 ND ND ND Groups of four animals per organ and at each time-point. .sup.bAll animals died before the 24 h post-challenge time-point. .sup.cNot detectable (minimal detection limit, 2.69 log.sub.10 CFU).

Example 5: Evaluation of the Protective Effect of the SdrH-like Polypeptide Versus Negative Control in a Mouse Model of Systemic S. aureus Infection

[0116] BALB/c mice have been shown to be able to control S. aureus infection by developing a strong Th2 response (Nippe et al., 2011). We previously showed in the OPA assay that sera directed against the SdrH-like polypeptide, Nuc, or LukG enhanced the killing of S. aureus by phagocytes (see Example 3). We therefore studied whether the vaccination of BALB/c mice with one of these three antigens, the SdrH-like polypeptide (SdrH-like), may allow for an improved control of kidney infection in this model of systemic infection.

Materials and Methods

Production and Purification of SdrH-like, Adjuvants

[0117] SdrH-like was produced and purified as described in Example 1. Adjuvants (aluminum hydroxide gel (Alhydrogel) and aluminum phosphate gel (Adju-Phos); InVivoGen, CA, USA) were used according the manufacturer's recommendations.

Vaccination Protocol and End-Point Analysis

[0118] Mice were immunized intramuscularly once a week for 3 weeks with 10 g of purified SdrH-like (5 g with Aluminum hydroxide gel (right thigh; volume: 50 L) and 5 g with Aluminum phosphate gel (left thigh, volume: 50 L)); mice received the same quantity of adjuvants alone as a negative control.

[0119] Mice (groups of six mice per time point) were inoculated two weeks after the third immunization with a dose of 10.sup.7 CFU of USA300; the protocol was otherwise as described in Example 4.

Results

[0120] As shown in Table 3, the bacterial load at 24 h post-challenge was reduced by 0.53 log.sub.10 CFU in mice vaccinated with SdrH-like versus control mice. Although, kidney infection was not controlled in vaccinated mice, bacterial growth was substantially reduced (+1.53 log.sub.10 CFU between 3 h and 24 h post-challenge versus+2.06 log10 CFU for control mice). As expected, vaccination had a minimal impact on spleen infection.

TABLE-US-00003 TABLE 3 CFU counts at 3 h and 24 h post-challenge in animals immunized with SdrH- like versus negative control. Mean number of CFUs per organ, in log.sub.10 Time post- Negative control.sup.b SdrH-like challenge Spleen Kidneys Spleen Kidneys 3 h 5.31 3.20 5.25 3.15 24 h 4.05 5.26 4.33 4.68 3 h-24 h 1.26 +2.06 0.92 +1.53 Groups of six animals per organ and at each time-point. .sup.bAdjuvants alone.

Example 6: Evaluation of the protective effect of SdrH-like versus staphylokinase and MntC in a mouse model of systemic S. aureus infection

[0121] SdrH-like was then compared to staphylokinase and MntC. The first comparator, staphylokinase, was chosen because sera directed against this protein were paradoxically shown to favor the intracellular growth of S. aureus in the OPA assay (see Sak, C, in FIGS. 1 and 2). The second comparator, MntC, was chosen because it has been shown to be a promising vaccine candidate in various animal models (Anderson et al., 2012), while it was revealed to be inferior to SdrH-like in the OPA assay (see MntC, 1, in FIGS. 1 and 2).

[0122] Three infectious doses were tested: 10, 3x10 6 and 10 6 CFU.

Materials and Methods

[0123] SdrH-like, staphylokinase and MntC were produced and purified as described in Example 1. Vaccination protocol and end-point analysis were as described in Example 5, except that protective effect was evaluated using three doses: 10.sup.7, 310.sup.6 and 10.sup.6 CFU of USA300.

Results

[0124] The course of infection in kidneys clearly differed in the mice vaccinated with SdrH-like as compared to those vaccinated with staphylokinase (Table 4); regardless of the dose of USA300, SdrH-like reduced the bacterial load in kidneys by ca 0.80 log.sub.10 CFU as compared to staphylokinase (Table 5).

[0125] A similar CFU reduction (0.95 log.sub.10 CFU) was observed with MntC compared to staphylokinase at the lowest dose, i.e., 10.sup.6 CFU (Table 5); however, the difference was much less at 10.sup.7 and 310.sup.6 CFU (reduction of only 0.30 to 0.39 log.sub.10 CFU; Table 5).

[0126] Consistent with the above results, SdrH-like appeared to have a stronger effect than MntC on kidney infection at the two highest doses, i.e., 10.sup.7 and 310.sup.6 CFU (reduction of 0.45 and 0.47 log.sub.10 CFU, respectively; Table 5), while similar results were found at 10.sup.6 CFU. Thus, the bacterial kinetics observed in the kidneys after vaccination with SdrH-like, staphylokinase and MntC paralleled the bacterial kinetics observed with these three antigens in the OPA assay.

[0127] As expected, vaccination with each of these three antigens had a minimal impact on spleen infection.

TABLE-US-00004 TABLE 4 CFU counts at 3 h and 24 h post-challenge in animals immunized with SdrH-like versus staphylokinase and MntC. Mean number of CFUs per organ, in log.sub.10.sup.a USA300 Time post- Staphylokinase MntC SdrH-like dose challenge Spleen Kidneys Spleen Kidneys Spleen Kidneys 10.sup.7 CFU 3 h 5.28 3.00 5.09 3.20 5.24 3.55 24 h 4.41 4.77 4.55 4.58 4.27 4.48 .sup.3 h-24 h .sup.b 0.87 +1.77 0.54 +1.38 0.97 +0.93 3 10.sup.6 CFU 3 h 4.97 2.69 4.91 2.85 4.93 2.85 24 h 3.79 4.46 3.90 4.32 4.08 3.85 3 h-24 h 1.18 +1.77 1.01 +1.47 0.85 +1.00 10.sup.6 CFU 3 h 4.43 2.69 4.23 2.85 4.52 2.69 24 h 3.77 3.90 3.76 3.11 3.61 3.09 3 h-24 h 0.66 +1.21 0.47 +0.26 0.91 +0.40 .sup.aGroups of six animals per organ and at each time-point. .sup.bBacterial growth between 3 h and 24 h is indicated by +, bacterial killing by .

TABLE-US-00005 TABLE 5 Pairwise comparison of 3 h-24 h CFU differentials in the kidneys. 3 h-24 h CFU differentials, in log.sub.10 (difference).sup.a USA300 SdrH-like vs SdrH-like vs MntC vs dose staphylokinase MntC staphylokinase 10.sup.7 CFU +0.93 vs +1.77 +0.93 vs +1.38 +1.38 vs 1.77 (0.84) (-0.45) (-0.39) 3 10.sup.6 CFU +1.0 vs +1.77 +1.0 vs +1.47 +1.47 vs 1.77 (0.77) (-0.47) (-0.30) 10.sup.6 CFU +0.40 vs 1.21 +0.40 vs +0.26 +0.26 vs 1.21 (0.81) (+0.14) (0.95) In bold, differences 0.5 log.sub.10 CFU.

Conclusions

[0128] The binding of specific antibodies to S. aureus can be beneficial to the host, as they may inhibit physiological functions of extracellular antigens, increase the uptake by immune cells, facilitate phagocytosis, and/or improve bacterial targeting to phagolysosomal compartments. More particularly, antibodies against S. aureus antigens may inhibit bacterial defense mechanisms targeting the bacterium to a favorable intracellular microenvironment, enhance the immune response by increasing the processing of the bacterium for antigen presentation, and foster bacterial clearance. However, certain antibodies have deleterious effects, enhancing bacterial virulence by inhibiting the function of determinants that are adequately recognized by the immune system and which participate in the control of the infection by the host. The humoral response generated by a vaccine candidate should preferably increase bacterial uptake for optimal antigen presentation and enhance intracellular bacterial lysis.

[0129] Sera directed against the SdrH-like polypeptide, Nuc, or LukG were surprisingly shown to both promote the internalization of S. aureus by macrophages and enhance the intracellular clearance of S. aureus following phagocytosis.

[0130] It is noteworthy that none of the antisera raised against the candidate vaccine proteins Hla, MntC, and CIfA previously developed and shown to be ineffective in clinical trials combined the two properties reported here. Moreover, the IsdB vaccine candidate showed to worsen the outcome of vaccinated patients was proven to be deleterious in the macrophage assay reported here, with acute destruction of the macrophage layer following enhanced internalization. These results further confirm the pertinence of the novel macrophage based in vitro assay provided herein in identifying antigens conferring protection against disease caused by S. aureus in a subject.

[0131] In addition, the results of the macrophage based in vitro assay were confirmed in vivo in a systemic model of S. aureus infection using BALB/c mice, which are highly susceptible to S. aureus due to their inability to limit bacterial growth in the kidneys. Indeed, of the three antigens evaluated in this model, the SdrH-like polypeptide showed the strongest inhibitory effect on bacterial growth of S. aureus in the kidneys overall, followed by MntC, in-line with kinetics observed in the macrophage assay.

REFERENCES

[0132] Adlam et al., (1977) Effect immunization with highly purified alpha- and beta-toxins on staphylococcal mastitis in rabbits. Infect Immun. 17: 250-256. [0133] Anderson et al., (2012) Staphylococcus aureus manganese transport protein C is a highly conserved cell surface protein that elicits protective immunity against S. aureus and Staphylococcus epidermidis. J Infect Dis. 205(11): 1688-1696. [0134] Bubeck and Schneewind, (2008) Vaccine protection against Staphylococcus aureus pneumonia. J Exp Med. 205: 287-294. [0135] Begier, et al., (2017) SA4Ag, a 4-antigen Staphylococcus aureus vaccine, rapidly induces high levels of bacteria-killing antibodies. Vaccine. 35(8): 1132-1139. [0136] Capparelli et al., (2011) The Staphylococcus aureus Peptidoglycan Protects Mice against the Pathogen and Eradicates Experimentally Induced Infection. PLoS One. 6(12): e28377. [0137] Darouiche, (2004) Treatment of infections associated with surgical implants. N Engl J Med. 350(14):1422-9. [0138] Fitzgerald et al., (2011) A 12-year review of Staphylococcus aureus bloodstream infections in haemodialysis patients: more work to be done. J Hops Infect. 79(3):218-21. [0139] Fowler, et al., (2013) Effect of an Investigational Vaccine for Preventing Staphylococcus aureus Infections After Cardiothoracic Surgery: A Randomized Trial. JAMA. 309(13): 1368-1378. [0140] Gould, (2008) Clinical relevance of increasing glycopeptide MICs against Staphylococcus aureus. Int J Antimicrob Agents. 31 Suppl 2:1-9. [0141] Harro et al., (2010) Safety and immunogenicity of a novel Staphylococcus aureus vaccine: results from the first study of the vaccine dose range in humans. Clin. Vaccine Immunol. 17(12): 1868-1874. [0142] Inoue, et al., (2018) Safety, tolerability, and immunogenicity of a novel 4-antigen Staphylococcus aureus vaccine (SA4Ag) in healthy Japanese adults. Hum. Vaccines Immunother. 14(11): 2682-2691. [0143] Landrum, et al., (2016) Safety and immunogenicity of a recombinant Staphylococcus aureus -toxoid and a recombinant Panton-Valentine leukocidin subunit, in healthy adults. Hum. Vaccin Immunother. 13(4): 791-801. [0144] McNeely et al., (2014) Mortality among recipients of the Merck V710 Staphylococcus aureus vaccine after postoperative S. aureus infections: An analysis of possible contributing host factors. Hum Vaccin Immunother. 10(12): 3513-3516 [0145] Moustafa, et al., (2012) Phase Ila Study of the Immunogenicity and Safety of the Novel Staphylococcus aureus Vaccine V710 in Adults with End-Stage Renal Disease Receiving Hemodialysis. Clin. Vaccine Immuno!. 19(9): 1509-1516. [0146] Nanra et al., (2013) Capsular polysaccharides are an important immune evasion mechanism for Staphylococcus aureus. Hum Vaccin Immunother. 9(3): 480-487. [0147] Needleman and Wunsch (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 48(3):443-53. [0148] Nippe et al., (2011) Subcutaneous infection with S. aureus in mice reveals association of resistance with influx of neutrophils and Th2 response. J Invest Dermatol. 131: 125-132. [0149] Salazar, et al., (2014) Staphylococcus aureus Manganese Transport Protein C (MntC) Is an Extracellular Matrix- and Plasminogen-Binding Protein. PLoS ONE. 9(11): e112730. [0150] Von Kckritz-Blickwede et al., (2008) Immunological mechanisms underlying the genetic predisposition to severe Staphylococcus aureus infection in the mouse model. Am J Pathol. 173(6): 1657-1668. [0151] Zorman (2013) Naturally occurring IgG antibody levels to the Staphylococcus aureus protein IsdB in humans. Hum Vaccin Immunother. 9(9):1857-64.