RU2795545C2 - Staphylococcus aureus leukocidins, therapeutic compositions and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a composition and a method for inhibiting the onset of a Staphylococcus aureus infection in case of using such a composition. The composition specified contains a therapeutically effective amount of a S. aureus leukocidin A polypeptide analogue.

EFFECT: invention makes it possible to suppress the occurrence of S. aureus infection by administering the disclosed composition.

10 cl, 21 dwg, 5 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority date of US Provisional Application No. 61/331,550, filed May 5, 2010, the disclosure of which is hereby incorporated by reference.

SEQUENCE LISTINGS

This application contains sequence listings that have been submitted for review using the EFS-Web system and are hereby incorporated herein by reference in their entirety. The specified ASCII copy, created on May 2, 2011, is named Sequence Listing_Staphylococcus Aureus Leucocidins_ST25.txt and is 102 kilobytes in size.

PRIOR ART

The bacteria Staphylococcus aureus or staphylococcus ("staph") is commonly found on the skin or in the nasal cavity of humans and animals. Staphylococcal bacteria are usually harmless unless they enter the body through a cut or other wound. Generally, staph infections create minor skin problems for healthy people. Historically, staph infections have been treated with broad-spectrum antibiotics such as methicillin. However, certain strains of staphylococci have now emerged that are resistant to methicillin and other β-lactam antibiotics such as penicillin and cephalosporins. They are called methicillin-resistant Staphylococcus aureus (also known as multidrug-resistant Staphylococcus aureus, or "MRSA").

Staph infections, including MRSA, usually start as small red bumps that look like pimples, boils, or spider bites. Such bumps or spots can quickly develop into deep, painful abscesses that require surgical drainage. Sometimes bacteria remain within the skin. In some cases, they can penetrate far into the body, causing potentially life-threatening infections in a wide range of human tissues, including skin, soft tissues, bones, joints, surgical wounds, blood flow, heart valves, lungs, or other organs. Thus, S. aureus infections can lead to potentially fatal diseases such as necrotizing fasciitis, pneumonia, endocarditis, sepsis, toxic shock, and various forms of pneumonia. MRSA infection is particularly problematic in hospital or nursing home settings, where patients often have open wounds, undergo invasive procedures with various devices, and have weakened immune systems and thus are at greater risk of infection than the general population. Employees who do not adhere to proper sanitation procedures can carry MRSA bacteria from one patient to another.

S. aureus produces a wide range of virulence factors and toxins that allow this bacterium to neutralize and resist attacks from various types of immune cells, more specifically, subpopulations of white blood cells that form the body's primary defense system. The production of such virulence factors and toxins allows S. aureus to maintain an infectious state. See Nizet, J. Allergy Clin. Immunol. 120:13-22 (2007). Along with these virulence factors, S. aureus produces several bicomponent leukotoxins that damage the membranes of host defense cells and erythrocytes through the synergistic action of two unassociated proteins or subunits. See Supersac, et al., Infect. Immun. 61:580-7 (1993). Of these bicomponent leukotoxins, gamma-hemolysin (HlgAB and HlgCB) and Pantone-Valentine Leukocidin (PVL) are the best studied.

The toxicity of leukocidins to mammalian cells is associated with the action of two components. The first subunit is called the class S subunit (ie "slow eluting") and the second subunit is called the class F subunit (ie "fast eluting"). The S and F subunits act synergistically to form pores in white blood cells, including monocytes, macrophages, dendritic cells, and neutrophils (collectively referred to as phagocytes). See Menestrina, et al., Toxicol. 39:1661-1672 (2001). The mechanism by which bicomponent toxins form pores in target cell membranes has not been fully elucidated. The proposed mechanism of action of these toxins involves binding of the S subunit to the target cell membrane, most likely through a receptor, followed by binding of the F subunit to the S subunit, thereby forming an oligomer, which in turn forms a pre-pore. penetrating the membrane of the target cell (Jayasinghe, et al., Protein. Sci. 14:2550-2561 (2005)). Pores formed by bicomponent leukotoxins are typically cation-selective. Pore formation causes cell death due to lysis, which, in cases where white blood cells are targeted, has been reported to be caused by osmotic imbalance due to cation influx (Miles, et al., Biochemistry 40:8514-8522 (2001)).

In addition to PVL (also known as leukocidin S/F-PV or LukSF-PV) and gamma-hemolysin (HlgAB and HlgCB), the repertoire of bicomponent leukotoxins produced by S. aureus is known to include leukocidin E/D (LukED) and leukocidin M/F' (LukMF'). Thus, the S-class subunits of such bicomponent leukocidins include HlgA, HlgC, LukE, LukS-PV, and LukM, and the F-class subunits include HlgB, LukD, LukF-PV, and LukF'-PV (Menestrina, et al., supra. ). The S and F subunits of S. aureus are not leukocidin specific. This means that they are interchangeable such that other bicomponent combinations can create functional pores in the white blood cell, greatly expanding the repertoire of leukotoxins (Meyer, et al., Infect. Immun. 77:266-273 (2009)).

The development of an effective therapy for the treatment of MRSA infection is a particularly challenging problem. In addition to the aforementioned resistance to methicillin and related antibiotics, MRSA has also been found to exhibit significant levels of resistance to macrolides (eg, erythromycin), beta-lactamase inhibitor combinations (eg, unazine, augmentin), and fluoroquinolones (eg, ciprofloxacin), and also clindamycin, trimethoprim/sulfamethoxysole (Bactrim), and rifampin. In the event of a severe S. aureus infection, clinicians resort to intravenous vancomycin. However, there are reports of S. aureus resistance to vancomycin. Thus, there is a need to develop new antibiotic preparations that effectively fight S. aureus infection.

SUMMARY OF THE INVENTION

Applicants have discovered and characterized another bicomponent member of the defense system of native Staphylococcus aureus. The newly characterized polypeptide component of the native S subunit, referred to herein as "LukA", encompasses native polypeptides and their analogs having a degree of sequence similarity of at least 70% with those of the native polypeptides. Thus, one aspect of the present invention relates to isolated and/or purified LukA. Another aspect of the present invention concerns an isolated and/or purified polynucleotide encoding LukA, a transformed host (eg, cell) containing the polynucleotide, and methods for producing recombinant LukA by expressing the polynucleotide in the transformed host.

The newly characterized polypeptide component of the F-subunit, referred to herein as "LukB", encompasses native polypeptides and their analogs having a degree of sequence similarity of at least 70% with those of native polypeptides. Thus, another aspect of the present invention relates to isolated and/or purified LukB. Another aspect of the present invention relates to an isolated and/or purified polynucleotide encoding LukB, a transformed host (eg, cell) containing the polynucleotide, and methods for producing recombinant LukB by expressing the polynucleotide in the transformed host.

Yet another aspect of this application relates to therapeutic compositions useful for inhibiting the onset of or treating a Staphyloccocus aureus infection comprising a therapeutically effective amount of LukA and/or LukB in composition with a pharmaceutically acceptable carrier. Thus, in one embodiment, the therapeutic composition contains a therapeutically effective amount of LukA. In another embodiment, the therapeutic composition contains a therapeutically effective amount of LukB. In yet another embodiment, the therapeutic composition contains therapeutically effective amounts of both LukA and LukB. In yet other embodiments, the composition contains a LukA analog missing the 10 C-terminal residues, which is non-toxic (referred to here as LukAΔ10C or rLukAΔ10C). Such compositions are suitable for complex therapeutic use. In some embodiments, the compositions are referred to as anti-inflammatory compositions and may be used to treat acute inflammatory conditions or disorders, in particular localized acute inflammatory conditions.

This use takes advantage of Applicants' additional discovery that under physiological conditions (i.e., LukAB is produced directly by S. aureus), the LukAB complex has exceptional specificity for phagocytes, but not for other nucleated cells such as epithelial cells and endothelial cells. This means that the complex forms pores in the membranes of these cell types, thereby causing cell death, which is referred to herein as "LukAB-mediated cytotoxicity". On the other hand, LukAB has relatively little or negligible specificity for other mammalian nucleated cells. Thus, the anti-inflammatory compositions of the present invention exploit the specificity of LukAB for human phagocytes in order to treat acute inflammatory conditions characterized by massive infiltration of phagocytes to the site of inflammation.

In other embodiments, the therapeutic compositions may be referred to as an (active) vaccine composition. The compositions can be used to induce the production of neutralizing antibodies to LukA and to LukB in a subject at risk for S. aureus infection or a subject diagnosed with S. aureus infection, such as MRSA.

Other aspects of the present invention relate to antibodies that specifically bind LukA, antibodies that specifically bind LukB, therapeutic compositions containing LukA and/or LukB antibodies, and their use in the treatment of infectious conditions associated with S. aureus. Such therapeutic compositions may be referred to as passive vaccine compositions. Thus, in one embodiment, the therapeutic composition contains a therapeutically effective amount of anti-LukA antibodies. In another embodiment, the therapeutic composition contains a therapeutically effective amount of anti-LukB antibodies. In yet another embodiment, the therapeutic composition contains therapeutically effective amounts of both anti-LukA and anti-LukB antibodies.

The passive and active vaccine compositions of the present invention make use of another discovery made by Applicants - that infectious virulent strains of S. aureus, such as MRSA, express LukA and LukB. The persistence of LukA and LukB across a wide range of S. aureus strains allows the vaccines of the present invention to provide a full range of therapeutic efficacy. LukA, LukB, LukA antibodies and LukB antibodies are also referred to here as active agents.

Another aspect of the present invention relates to methods of using LukAB, LukA and/or LukB to identify potential inhibitors of LukAB-mediated cytotoxicity. Such methods may use the LukAB complex, per se, in combination with a phagocyte, or a phagocyte membrane-binding portion thereof. Inhibitors thus identified may be candidates for therapy to treat S. aureus infection.

Another aspect of the present invention relates to a method for predicting or assessing the severity of an S. aureus infection, comprising detecting the presence or amount of LukA and/or LukB, or detecting the corresponding LukA and/or LukB genes, in a biological sample obtained from an infected subject. This aspect of the present invention is based on yet another discovery made by the Applicants, that of the many cytotoxins produced by S. aureus, LukAB exhibits severe toxicity to human phagocytes. Thus, detection of the presence or relatively high amounts of LukA and/or LukB, or their respective genes (e.g., demonstrated by S. aureus Newman, 4645 strains and MRSA strains USA300 and USA500) compared to controls (e.g., S. aureus strains USA100 and USA400) that produces small or undetectable amounts of LukA and/or LukB indicates severe S. aureus infection.

These and other aspects of the present invention are described more fully below.

BRIEF DESCRIPTION OF THE DRAWINGS

1 is an alignment containing the amino acid sequence of the majority LukA sequence (denoted as SEQ ID NO:1) and the LukA polypeptides from thirteen (13) different strains of S. aureus to which it corresponds (denoted as SEQ ID NO:2- 14).

Figure 2 is an alignment containing the amino acid sequence of the majority LukB sequence (designated as SEQ ID NO:15) and the LukB polypeptides from the twelve (12) different strains of S. aureus to which it corresponds (designated as SEQ ID NO:16-27).

Figure 3. LukAB is a potent staphylococcal cytotoxin that targets and kills primary human phagocytes. (a) Intoxication of primary human peripheral blood mononuclear cells (PBMC) with culture filtrate (2.5% v/v) of S. aureus strain Newman (wild type, WT) and the indicated isogenic mutant strains. Cell viability is monitored using CellTiter, where cells treated with medium were taken as 100%. Results represent the mean of three replicate samples + standard deviation (S.D.). (b) Intoxication of primary human monocytes, macrophages and dendritic cells (DC) with culture filtrate (2.5% v/v) of S. aureus strain Newman (WT) and the indicated isogenic mutant strains. Cell viability is monitored as described above. Results represent the average of two donors where cells from each donor are intoxicated with three independent exoprotein preparations, + S.E.M. (standard error of the mean). (c) Intoxication of primary human PMNs (polymorphonuclear neutrophils) by various dilutions of culture filtrates of S. aureus strain Newman (WT) and the indicated isogenic mutant strains. Cell viability is monitored as described above. Results represent the mean of PMN isolated from four donors ±S.E.M. (d) Intoxication of primary human PMNs with purified rLukA, rLukB, or a combination of rLukA and rLukB at the indicated concentrations. * indicates statistical significance when compared to rLukA and rLukB combined, P<0.05. For panels (a-c), * indicates statistical significance when compared to WT, ** indicates statistical significance when compared to ΔLukAB/p, P<0.05 (Student's t-test p<0.05).

Figure 4. LukAB preferably targets human phagocytic cells. Intoxication of (a, c and d) PMN-HL60 or (b) THP1 cells with different dilutions of S. aureus WT culture filtrate Newman strain, isogenic mutant strains lacking the indicated genes/toxins, (c) S. aureus WT culture filtrate containing an empty plasmid (WT/p), a LukAB-free strain with an empty plasmid (ΔLukAB/p), and a LukAB-free strain with a LukAB-complementing plasmid (ΔLukAB/pLukAB), or (d) purified recombinant LukA (rLukA) , LukB (rLukB) or combinations of rLukA and rLukB (rLukA+rLukB) at the indicated concentrations. For intoxications with rLukA and rLukB combined, the total protein concentration is the sum of equal amounts of rLukA and rLukB (eg, 2.8 µg total protein corresponds to 1.4 µg rLukA and 1.4 µg rLukB). (e) Intoxication of these human cell lines with 10 μg/ml rLukAB. Cell viability is monitored using the CellTiter, taking the cells treated with the medium as 100%. Results represent the mean of three replicates ±S.D. An asterisk symbol (*) denotes a statistically significant difference compared to WT (one-way analysis of variance).

Figure 5. LukAB is an important toxin of various staphylococcal strains. (A) Expression of LukB by various strains of S. aureus, determined by Western blotting using anti-LukB polyclonal sera. (B) PMN-HL60 intoxication with dilutions of exoproteins from different strains of S. aureus. Cell viability was monitored using a CellTiter, taking media-treated cells as 100% viable. (C) Expression of LukB and α-toxin WT and LukAB-isogenic strains, determined by Western blotting using toxin-specific sera. (D) PMN-HL60 intoxication with exoproteins of WT-strains Newman (New.) and 4645 and LukAB-isogenic strains. Cell viability was monitored as in panel B. Results are the average of three replicates + S.D. * denotes a statistically significant difference compared to Newman (C) or WT (E) (Student's t-test p<0.05).

Figure 6. LukAB destroys the plasma membranes of target cells. (a) Light microscopy images of PMN-HL60 cells intoxicated with S. aureus culture filtrate, wild-type (WT), and isogenic LukAB-free (ΔLukAB) strains. (bc) Intoxication of PMN-HL60 cells with culture filtrates of the WT strain (WT/p), isogenic strain without LukAB (ΔLukAB/p), complemented strain (ΔLukAB/pLukAB), or pseudo-intoxication with the medium. Cells with broken membranes are stained with SYTOX Green, visualized by fluorescence microscopy (c) and the green fluorescence intensity is measured (b). (d) PMN was infected ex vivo with S. aureus, Newman strain (MSSA) or USA300 LAC strain (MRSA), and the indicated isogenic mutants at different rates of infection (MOI). Membrane damage was monitored with SYTOX green. Results represent the mean of triplicate samples ±SD. Asterisk symbols (*) denote a statistically significant difference compared to WT (Student's t-test p<0.05).

Figure 7. LukAB protects S. aureus from host-mediated death by targeting and killing phagocytes. (a) Infection of PMN-HL60 cells with S. aureus WT, IukAB-free strain and IukAB-free strain with IukAB-complementing plasmid (ΔlukAB/plukAB), at different rates of infection (MOI). Membrane-disrupted mammalian cells are monitored with SYTOX Green as described in Figure 6. Results are the mean of three replicates + SD (b) Viability of indicated S. aureus strains when ex vivo infected with human whole blood. The results represent the mean of whole blood from 12 donors + SEM (c) Viability of the indicated Newman S. aureus strains when infected with primary human neutrophils (PMN). Results represent the average of PMNs from 12 donors + SEM (d) Intoxication of primary human PMNs by various dilutions of culture filtrate of strains WT/p, ΔlukAB/p and ΔlukAB/plukAB. As an indicator of cell lysis, the release of LDH (lactate dehydrogenase) is measured. Results represent the mean of PMN obtained from 6 donors + SEM. Asterisk symbols (*) denote a statistically significant difference compared to the Newman WT strain (Student's t-test p<0.05).

Figure 8. LukAB is important for S. aureus pathogenesis in vivo , (a) Bioluminescent images of the kidneys of mice infected with a WT strain of S. aureus LAC containing pXen1 or pLukAB.Xen1. Shown are the kidneys of two typical mice in each group. (b) Bacterial load isolated from the kidneys of mice infected retroorbitally with the indicated strains of S. aureus LAC. Each data point represents the number of bacteria (CFU, colony forming units) per milliliter of tissue homogenate for an individual animal. The dotted line shows the limit of detection. For panels (AC and E), * indicates statistical significance when compared to WT, ** indicates statistical significance when compared to ΔLukAB/p, P<0.05.

Figure 9. LukAB destroys human phagocytes by pore formation in cell membranes. (a) PMN-HL60 cells are intoxicated with rLukA+rLukB and toxin binding is monitored by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and immunoblotting using antibodies specific for LukA or LukB. (b) PMN-HL60 cells are incubated with rLukAB and toxin binding is determined by FACS (fluorescence activated cell sorting) using a rabbit anti-His antibody, (c) PMN-HL60 cells are intoxicated with rLukAB and formation of LukAB oligomers in the plasma membrane is determined by SDS- PAGE and immunoblotting with anti-LukB antibody, (d) PMN-HL60 is intoxicated with rLukAB or treated with saponin in the presence or absence of PEG-400. LukAB pores are detected with ethidium bromide. (e) The viability of PMN-HL60 treated as indicated in the panel is determined using CellTiter, taking cells treated with medium as 100%. The results in panels (d) and (e) represent the mean of three replicates ±SEM. * denotes a statistically significant difference with PEG (panels d-e) (Student's t-test p<0.05).

Figure 10. Cytotoxicity of LukAB can be neutralized with α-LukA polyclonal antibody. PMN-HL60 intoxicated with 5% (v/v) S. aureus strain Newman culture filtrate was incubated with the indicated amounts of α-LukA polyclonal antibodies or non-immune sera obtained from different production bleeds from two different rabbits. Cell viability is monitored using the CellTiter, taking the cells treated with the medium as 100%. Results represent the mean of triplicate samples ± standard deviation (S.D.).

Figure 11. The C-terminal overhang of LukA is required for the cytotoxic effect of LukAB, but does not affect the recognition of α-LukA by the polyclonal antibody. (a) Alignment of the amino acid sequences of the various S-subunits of S. aureus leukotoxin (designated as SEQ ID NOS: 44-49) using the MegAlign Clustal W method of the Lasergene software application. N- and C-terminal overhangs present only in the LukA sequence are boxed. (b) Coomassie blue staining with 2 μg of recombinant LukA (rLukA), LukB (rLukB), LukA lacking C-terminal overhang (rLukAΔ10C) and LukA lacking N-terminal overhang (rΔ33NLukA) isolated from E. coli and separated by SDS-PAGE followed by PMN-HL60 intoxication with various amounts of rLukA, rLukAΔ10C and rΔ33NLukA in combination with rLukB. The final protein concentration consists of equal amounts of rLukA, rLukAΔ10C or rΔ33NLukA and rLukB. Results represent the mean of three replicates ±S.D. (c) Immunoblot showing equivalence of recognition of 6×His-labeled rLukAΔ10C by both α-LukA and α-His polyclonal antibodies.

DETAILED DESCRIPTION

The following disclosure relates, in order, to LukA polypeptides, LukB polypeptides, LukA and LukB polynucleotides, anti-LukA and anti-LukB antibodies, LukA and/or LukB-containing therapeutic compositions, or anti-LukA and/or LukB antibodies, methods of administering therapeutic compositions, methods for identifying inhibitors of LukAB-mediated cytotoxicity, and methods for predicting or assessing the severity of S. aureus infection.

LukA polypeptides

Polypeptides native to Staphylococcus aureus have been isolated and identified by Applicants as exhibiting an activity profile of the known S-subunit of leukocidins (eg LukS-PVL, LukE and HlgC). Such polypeptides, collectively referred to herein as LukA, specifically target and bind human phagocytes (but not human epithelial or human endothelial cells, or mouse cells), and after binding to the phagocyte membrane, LukA oligomerizes with the S. aureus leukocidin F subunit (e.g. , LukF-PVL, LukD and HlgB and LukB disclosed here) and after oligomerization form a transmembrane pore (which is collectively referred to as LukA activity). The alignment shown in Figure 1 contains the amino acid sequences of the majority (majority) sequence of LukA (indicated here by SEQ ID NO: 1) and LukA polypeptides from 13 different strains of S. aureus to which they correspond (indicated here by SEQ ID NO: 2-14 ).

The N-terminal 27 amino acid residues of each of SEQ ID NOs: 1-14 represent the native secretory/signal sequence. Thus, the mature secreted form of LukA represented by amino acid residues 28-351 in each of SEQ ID NOs: 1-14 may be referred to herein as "LukA(28-351)" or "mature LukA". Accordingly, the immature form of LukA may be referred to here as "LukA(1-351)".

A LukA consensus sequence determined based on SEQ ID NOs: 2-14 (which are not exhaustive of native S. aureus LukA) will thus include distinct variants at a minimum of 64 LukA positions (where successive positions of variation are denoted by X ¹ -X ⁶⁴ ), defined as follows: 8 (X ¹ =L or F), 16 (X ² =A or V), 17 (X ³ =I or L), 24 (X ⁴ =T or N), 26 (X ⁵ =Q or Ε), 31 (X ⁶ =H or N), 38 (X ⁷ =N or Τ), 46 (X ⁸ =S or A), 50 (X ⁹ =E or D), 55 (X ¹⁰ =T or N), 56 (X ¹¹ =N or D), 61 (X ¹² =S or Τ), 62 (X ¹³ =T or P), 63 (X ¹⁴ =A, G or V) , 73 (X ¹⁵ =I or V), 78 (X ¹⁶ =E or V), 77 (X ¹⁷ =T or S), 80 (X ¹⁸ =V or Ε), 83 (X ¹⁹ =E or Κ) , 84 (X ²⁰ =E or K), 105 (X ²¹ =V or I), 124 (X ²² =K or R), 125 (X ²³ =E or N), 127 (X ²⁴ =K, T or Ν), 129 (X ²⁵ =S or A), 130 (X ²⁶ =N or S), 135 (X ²⁷ =K or Q), 146 (X ²⁸ =R or S), 148 (X ²⁹ =R or Ρ), 173 (X ³⁰ =S or N), 174 (X ³¹ =S or L), 181 (X ³² =T or V), 184 (X ³³ =I or V), 195 (X ³⁴ =T or S), 202 (X ³⁵ =N or K), 208 (X ³⁶ =S or I), 214 (X ³⁷ =W or R), 221 (X ³⁸ =I or V), 229 (X ³⁹ =G or Ν), 231 (X ⁴⁰ =V or I), 237 (X ⁴¹ =E or D), 239 (X ⁴² =L or F), 243 (X ⁴³ =N or Τ), 246 (X ⁴⁴ =I or L), 247 (X ⁴⁵ =A or S), 278 (X ⁴⁶ =L or I), 283 (X ⁴⁷ =S or T), 285 (X ⁴⁸ =E or D), 288 (X ⁴⁹ =Q or R), 299 (X ⁵⁰ =I or V), 303 (X ⁵¹ =R or K), 309 (X ⁵² =A or G), 310 (X ⁵³ =P or Q), 315 (X ⁵⁴ =K or Q), 318 (X ⁵⁵ =D or Ε), 322 (X ⁵⁶ =L or F), 325 (X ⁵⁷ =T or V), 338 (X ⁵⁸ =V or I), 339 (X ⁵⁹ =D or Ε), 342 (X ⁶⁰ =S or Τ), 344 (X ⁶¹ =D, E or Q), 347 (X ⁶² =P or S), 348 (X ⁶³ =Y or F) and 349 (X ⁶⁴ = K or R).

LukB polypeptides

Polypeptides native to Staphylococcus aureus have been identified by Applicants as exhibiting an activity profile of the known F-subunit of leukocidins (eg LukF-PVL, LukD and HlgB). Such polypeptides, collectively referred to herein as LukB, specifically oligomerize with the S-subunit of S. aureus leukocidin (eg, LukS-PVL, LukE and HlgC and LukA disclosed herein) associated with a human phagocyte; and, after oligomerization, form a transmembrane pore in the phagocyte (generally referred to as LukB activity). The alignment shown in Figure 2 contains the amino acid sequences of the LukB majority sequence (herein referred to as SEQ ID NO:15) and the LukB polypeptides from the 12 different strains of S. aureus to which they correspond (herein referred to as SEQ ID NO:16-27).

The N-terminal 29 amino acid residues of each of SEQ ID NOs: 15-27 is a secretory/signal sequence. Thus, the mature secreted form of LukB represented by amino acid residues 30-339 in each of SEQ ID NOs: 16-27 may be referred to herein as "LukB(30-339)" or "mature LukB". Accordingly, the immature form of LukB may be referred to here as "LukA(1-339)".

A LukB consensus sequence determined based on SEQ ID NOs: 15-28 (which are not exhaustive of native S. aureus LukB) will thus include distinct variants at a minimum of 49 LukB positions (where successive positions of variation are denoted by X ¹ -X ⁴⁹ ), defined as follows: 5 (X ¹ =L or V), 6 (X ² =C or Υ), 13 (X ³ =S or Τ), 15 (X ⁴ =A or Τ), 16 (X ⁵ =L or I), 19 (X ⁶ =A or Τ), 20 (X ⁷ =L or F), 23 (X ⁸ =F or L), 26 (X ⁹ =S or Τ), 28 (X ¹⁰ =Y or F), 34 (X ¹¹ =E or Κ), 36 (X ¹² =K or Τ), 37 (X ¹³ =Q, T or A), 46 (X ¹⁴ =D or Ε) , 59 (X ¹⁵ =S or Τ), 60 (X ¹⁶ =Q or Ε), 62 (X ¹⁷ =N or Κ), 64 (X ¹⁸ =T or S), 75 (X ¹⁹ =P or Κ) , 95 (X ²⁰ =K or R), 98 (X ²¹ =N, d or E), 126 (X ²² =S or deletion), 159 (X ²³ =R or Q), 163 (X ²⁴ =T or Ρ), 170 (X ²⁵ =S or Κ), 187 (X ²⁶ =L or I), 190 (X ²⁷ =S or Ρ), 192 (X ²⁸ =S or Τ), 193 (X ²⁹ =S or Τ), 193 (X ²⁹ =H or N), 197 (X ³⁰ =G or A), 204 (X ³¹ =S or L), 222 (X ³² =D or N), 224 (X ³³ =T or V), 247 (X ³⁴ =N or D), 270 (X ³⁵ =N or Κ), 272 (X ³⁶ =K or Ε), 276 (X ³⁷ =R, Q or Κ), 287 (X ³⁸ = D or Ε), 290 (X ³⁹ =L or I), 294 (X ⁴⁰ =K or R), 309 (X ⁴¹ =Q or K0, 327 (X ⁴² =D or N), 329 (X ⁴³ =L or F), 330 (X ⁴⁴ =I or V), 332 (X ⁴⁵ =t or V), 333 (X ⁴⁶ =f, I or L), 336 (X ⁴⁷ =K or N) and 338 (X ⁴⁸ =K or Q).

The leukocidins LukA and LukB may differ from the native polypeptides designated SEQ ID NOs: 2-14 and 16-27, respectively, due to one or more additional amino acid insertions, substitutions, or deletions, for example, one or more amino acid residues in SEQ ID NO: 2-14 or 16-27 can be substituted with another amino acid of similar polarity that acts as a functional equivalent, resulting in a silent change. This means that the change from the native sequence will not significantly weaken the basic properties of native LukA and LukB. Examples include SEQ ID NOs: 1 and 15. Any such LukA or LukB analogue can be screened according to the protocols disclosed herein (e.g. cell toxicity assay and membrane damage assay) to determine if it retains native LukA or LukB activity. . Substitutions in such leukocidins may be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In other embodiments, non-conservative changes (eg, single or amino acid substitutions, deletions and/or additions) may be made to inactivate or detoxify LukA and LukB. In one embodiment, the non-toxic LukA analog differs from native polypeptides in that the C-terminal amino acids at positions 342-351 are deleted. With the exception of SEQ ID NO:4-6 (which contain 9 amino acids at these positions), the analog lacks 10 C-terminal amino acid residues. Collectively, such analogs are designated LukAΔ10C. Detoxified LukA and LukB can be used in the active vaccine formulations described herein. Molecular changes can be carried out by methods well known to those skilled in the art, including the method of primer extension on a plasmid template using single-stranded templates (Kunkel, Proc. Acad. Sci., USA 82:488-492 (1985)), double-stranded DNA- templates (Papworth, et al., Strategies 9(3):3-4 (1996)), and PCR cloning (Braman, J. (ed.), IN VITRO MUTAGENESIS PROTOCOLS , 2nd ed. Humana Press, Totowa, NJ (2002) Methods for determining whether a given molecular change in LukA or LukB will reduce the cytotoxicity of LukAB are described here.

Thus, given the above description and for the purposes of the present invention, LukA can be more broadly described as any of the sequences of SEQ ID NO:1-14 (e.g., SEQ ID NO:2, which is a LukA polypeptide native to strain Newman S aureus), or a (native or non-native) polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity with these sequences.

Similarly, in view of the above description and for the purposes of the present invention, LukB can be more broadly described as any of the sequences of SEQ ID NO:15-27 (for example, SEQ ID NO:27, which is a LukB polypeptide native to strain Newman S aureus), or a (native or non-native) polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity with these sequences.

Thus, unless otherwise indicated, both immature and mature forms of native LukA and LukB, and sequences having less than 100% similarity to native LukA (i.e., native sequences and analogs equally, collectively referred to herein as "LukA " and "LukB"), can be used in compositions and methods, and for the preparation of antibodies to LukA and to LukB of the present invention.

Polynucleotides encoding LukA and LukB and methods for synthesizing or isolating LukA and LukB

The leukocidins LukA and LukB can be synthesized by recombinant DNA techniques which are well known to those skilled in the art. For example, the nucleotide sequence (denoted by SEQ ID NO:28) encoding a LukA S. aureus (Newman) polypeptide (SEQ ID NO:2) is shown below. Degenerate sequences (e.g., those that may be useful given the preferred codons in hosts selected for recombinant expression) encoding this polypeptide and polynucleotides encoding other LukA polypeptides are known in the art or can be designed by those skilled in the art. the field of technology.

The nucleotide sequence (hereinafter referred to as SEQ ID NO:29) encoding the S. aureus (Newman) LukB polypeptide (SEQ ID NO:27) is shown below. Degenerate sequences (e.g., those that may be useful given the preferred codons in hosts selected for recombinant expression) encoding this polypeptide and polynucleotides encoding other LukB polypeptides are known in the art or can be designed by those skilled in the art. the field of technology.

The polynucleotides encoding LukA and LukB can be expressed in a host such as bacteria (E. coli), plants and/or yeast and then isolated and purified. Alternatively, the leukocidins LukA and LukB can be isolated from S. aureus bacteria (eg Newman strain) according to standard procedures. Thus, such leukocidins can be isolated (from native or non-native media). They may also be purified to be substantially free of other proteins and cellular components with which S. aureus LukA and LukB are associated in their native state (i.e., proteins and cellular components present in S. aureus cells) or non-native state (i.e. proteins and cellular components of the recombinant host cell). Suitable purification schemes, which typically involve a combination of at least two sequential procedures, are known to those skilled in the art. See Deutscher, Methods in Enzymology, 182 (1990); and Scopes, Protein Purification: Principles and Practice, Springer-Verlag, N.Y. (1982) using one or a combination of two or more standard techniques such as column affinity chromatography and cation exchange liquid chromatography.

Antibodies to LukA and antibodies to LukB

Aspects of the present invention relate to anti-LukA antibodies that specifically bind to LukA and anti-LukB antibodies that specifically bind to LukB, antibody-containing therapeutic compositions, and methods of using the same. For the purposes of the present invention, the term "antibody" includes monoclonal antibodies, polyclonal antibodies, antibody fragments and genetically modified forms of antibodies, and combinations thereof. More specifically, the term "antibody", which is used interchangeably with the term "immunoglobulin", includes full-length (i.e., naturally occurring or obtained by recombination processes of fragments of the normal immunoglobulin gene) immunoglobulin molecules (e.g., IgG antibody) and their immunological active fragments (ie, including the specific binding site of the full-length immunoglobulin molecule), which can also be natural or synthetic in nature. Accordingly, the term "antibody fragment" includes an antibody region such as F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, and the like. Regardless of the structure, the antibody fragment binds to the same antigen that is recognized by the full-length antibody and, in the context of the present invention, specifically binds to LukA, LukB, or the LukAB complex. Methods for obtaining and screening antibody fragments are well known to those skilled in the art.

In some embodiments, the anti-LukA antibodies of the present invention may have some degree of cross-reactivity with other Staphylococcus leukocidin S subunits such as HlgC, LukS-PVL, HlgA, LukS-I, LukE, LukEv, and LukM. Similarly, in some embodiments, the anti-LukB antibodies of the present invention may have some degree of cross-reactivity with other Staphylococcus leukocidin F subunits such as LukF'-PV, LukF-PV, LukDv, LukD, LukF-I, and HlgB. Anti-LukA and/or LukB antibodies can inhibit or decrease LukA activity and LukB activity, respectively. In some embodiments, anti-LukA and/or anti-LukB antibodies neutralize (eg, substantially abolish) LukA and LukB activity, respectively.

Natural antibodies typically have two identical heavy chains and two identical light chains, with each light chain being covalently linked to the heavy chain by an interchain disulfide bond, and a plurality of disulfide bonds further linking the two heavy chains to each other. Individual chains can fold into domains that are similar in size (110-125 amino acids) and structures, but different functions. The light chain may include one variable domain (VL) and/or one constant domain (CL). The heavy chain may also contain one variable domain (VH) and/or, depending on the class or isotype of the antibody, three or four constant domains (CHI, CH2, CH3 and CH4). IgA, IgD, IgE, IgG, and IgM isotypes occur in humans, with IgA and IgG further divided into subclasses or subtypes (IgA1-2 and IgG1-4).

In general, variable domains show significant variability in the amino acid sequences of different antibodies, especially in the region of the antigen-binding site. In each VL and VH, there are three regions, called hypervariable regions or complementarity determining regions (CDRs), which are supported by less variable regions, called framework variable regions. The antibodies of the invention include IgG monoclonal antibodies, but the antibodies of the present invention also include antibody fragments or genetically modified forms. They are, for example, Fv fragments or proteins in which the CDRs and/or variable domains of exemplary antibodies are engineered as single-stranded antigen-binding proteins.

The part of the antibody, consisting of the VL and VH domains, is designated Fv (variable fragment) and forms the antigen-binding site. A single chain Fv (scFv or SCA) is an antibody fragment containing a VL domain and a VH domain in one polypeptide chain, with the N-terminus of one domain and the C-terminus of the other domain connected by a flexible linker. Peptide linkers used to make a single chain antibody are typically flexible peptides chosen to ensure proper three-dimensional folding of the VL and VH domains. The linker typically contains 10 to 50 amino acid residues and, in some cases, is shorter, such as about 10 to 30 amino acid residues, or 12 to 30 amino acid residues, or even 15 to 25 amino acid residues. An example of such linker peptides includes repeats of four glycine residues followed by a serine residue.

Single chain antibodies do not contain some or all of the constant domains of the whole antibodies from which they are derived. Because of this, they overcome some of the problems associated with the use of whole antibodies. For example, single chain antibodies tend to lack some of the unwanted interactions between heavy chain constant regions and other biological molecules. Additionally, single chain antibodies are significantly smaller than whole antibodies and may have greater permeability than whole antibodies, allowing single chain antibodies to localize and bind target antigen-binding sites more efficiently. In addition, due to their relatively small size, single chain antibodies will be less likely to elicit an unwanted immune response in the recipient than whole antibodies.

Fab (fragment, antigen-binding) refers to antibody fragments consisting of VL, CL, VH and CH1 domains. Fragments resulting from papain hydrolysis are simply referred to as Fab and do not retain the heavy chain hinge. After hydrolysis with pepsin, various Fabs are formed that retain the hinge region of the heavy chain. Such fragments with intact interchain disulfide bonds are called F(ab')2, and in cases where disulfide bonds are not preserved, a single Fab' is formed. F(ab') ₂ fragments have higher antigen avidity than monovalent Fab fragments.

Fc (fragment, crystallizing) is used to refer to a portion or fragment of an antibody that includes paired heavy chain constant domains. In an IgG antibody, for example, Fc contains the CH2 and CH3 domains. The Fc of an IgA or IgM antibody further includes a CH4 domain. Fc is associated with Fc receptor binding, activation of complement-mediated cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC). Antibodies such as IgA and IgM, which are complexes of many IgG-like proteins, require Fc constant domains to form the complex.

Finally, the hinge region separates the Fab and Fc regions of the antibody, allowing Fab mobility with respect to each other and with respect to Fc, and also includes many disulfide bonds to covalently link the two heavy chains.

The "specificity" of an antibody refers to the selective recognition by an antibody of a particular epitope of an antigen. The term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor, or otherwise interacting with a molecule. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrates or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope may be "linear" or "conformational". In a linear epitope, all points of interaction between a protein and an interacting molecule (such as an antibody) are located linearly along the protein's primary amino acid sequence. In the conformational epitope, the interaction points are distributed over the amino acid residues of the protein located at some distance from each other, i.e. non-contiguous amino acids approaching as a result of folding the tertiary structure of the protein. Epitopes formed by contiguous amino acids typically persist after exposure to denaturing solvents, while epitopes formed by tertiary folding typically disappear after treatment with denaturing solvents. An epitope typically includes at least 3 and typically at least 5 or 8-10 amino acids with a unique spatial conformation. Antibodies recognizing the same epitope can be determined using a single immunoassay demonstrating the ability of one antibody to block the binding of another antibody to the target antigen.

The monoclonal antibodies of the present invention may be murine, human, humanized, or chimeric. A humanized antibody is a recombinant protein in which the CDRs of an antibody are of the same species; for example, rodent, rabbit, dog, goat, horse, or chicken antibodies (or any other suitable animal antibody) are transferred from the heavy and light variable chains of a rodent antibody to the heavy and light human variable domains. The constant domains of an antibody molecule are taken from a human antibody. Methods for making humanized antibodies are well known to those skilled in the art. Chimeric antibodies preferably have constant regions derived essentially or exclusively from the constant regions of a human antibody and variable regions derived essentially or exclusively from a non-human mammalian variable region sequence. The efficiency of the chimerization process can also be increased by replacing variable regions other than hypervariable regions or complementarity determining regions (CDRs) of a mouse (or other non-human mammalian) antibody with corresponding human sequences. Variable regions other than CDRs are also known as variable framework regions (FRs). Still other monoclonal antibodies of the present invention are bispecific, ie. they have specificity for both LukA and LukB. The bispecific antibodies are preferably human or humanized.

The above antibodies can be obtained in accordance with standard techniques. For example, LukA, LukB (as these terms are used herein include their non-toxic counterparts such as LukAΔ10C) or an immunologically active fragment of LukA or LukB can be administered to a subject (eg, a mammal such as a human or a mouse). Leukocidins can be used by themselves as immunogens, or they can be attached to a carrier protein or other entities such as beads, eg Sepharose beads. After the mammal has produced antibodies, a mixture of antibody-producing cells, such as splenocytes, is isolated from which polyclonal antibodies can be obtained. Monoclonal antibodies can be produced by isolating individual antibody-producing cells from a mixture and immortalizing them, for example, by fusion with tumor cells such as myeloma cells. The resulting hybridomas are maintained in culture and monoclonal antibodies are harvested from the culture medium.

Methods for preparing recombinant monoclonal antibodies are well known to those skilled in the art. Recombinant polyclonal antibodies can be produced by methods similar to those described in US Patent Application Publication 2002/0009453 using LukA, LukB or LukAB as the immunogen(s).

Therapeutic compositions

LukA and LukB may be included in a therapeutic composition for use as an anti-inflammatory agent in the treatment of acute inflammatory conditions, including localized acute inflammatory conditions. LukA and LukB may also be included in a therapeutic composition for use as an active vaccine. Anti-LukA and anti-LukB antibodies may be included in a therapeutic composition for use as a passive vaccine. Passive and active vaccines can be used prophylactically to inhibit the initial manifestations of S. aureus infection, or therapeutically to treat S. aureus infections, in particular S. aureus infections such as MRSA known to be resistant, or when a certain subject is found to be refractory to treatment with other conventional antibiotic therapies.

In embodiments where the therapeutic composition is intended for use as an active vaccine, LukA and/or LukB may be modified to exhibit reduced toxicity. Molecular changes are described above. Thus, their non-toxic counterparts such as LukAΔ10C can be used. Applicants believe that antibodies produced in response to a non-toxic immunogen will neutralize toxic native LukA or LukAB. Other modifications to reduce the toxicity of LukA and LukB include chemical processing (eg modification of certain amino acid residues) or conjugation to another moiety (eg another bacterial antigen such as a bacterial polysaccharide or bacterial glycoprotein). Other S. aureus toxins have been chemically modified to inactivate or detoxify (or reduce toxicity). Methods for determining whether a given change will reduce the toxicity of LukA or LukB are known to those skilled in the art and/or are described here.

The therapeutic compositions of the present invention are prepared by formulating LukA and LukB, or anti-LukA and anti-LukB antibodies, with a pharmaceutically acceptable carrier and, optionally, a pharmaceutically acceptable excipient. As used herein, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" (e.g., additives such as diluents, immunostimulants, adjuvants, antioxidants, preservatives, and solubilizing agents) are non-toxic to the cell or mammal that is exposed to them at the dosages used. and concentrations. Examples of pharmaceutically acceptable carriers include water, for example buffered with phosphate, citrate and other organic acids. Typical examples of pharmaceutically acceptable excipients that may be suitable for use in the present invention include antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; adjuvants (selected to avoid adjuvant-induced toxicity, such as β-glucan as described in US Pat. No. 6,355,625 or granulocyte colony stimulating factor (GCSF)); 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 such as TWEEN ^® , polyethylene glycol (PEG) and PLURONICS ^® .

As indicated elsewhere in this description, the therapeutic compositions of the present invention may additionally contain at least one additional active agent.

The therapeutic compositions of the present invention may be prepared for storage by mixing the active ingredient(s) having the desired purity with a pharmaceutically acceptable carrier and optionally an excipient and/or additional active agent, in the form of lyophilized compositions or aqueous solutions.

Use of Therapeutic Compositions - Indications

Acute inflammatory conditions

Inflammation is generally understood as a protective biological response aimed at removing harmful invading stimuli such as pathogens (eg bacteria and viruses), damaged cells and irritants and initiating healing. Inflammation is more specifically understood as the response of vascularized living tissue to injury. In essence, inflammation is a fundamental stereotypical complex of cytological and chemical reactions of diseased blood vessels and adjacent tissues in response to injury or abnormal irritation caused by a physical, chemical, or biological agent. Inflammation usually results in the accumulation of fluid and blood cells at the site of injury and is usually a healing process. Without the inflammatory process, wounds and infections would not heal and progressive tissue destruction would become life threatening. Acute inflammation refers to the body's initial response to invasive stimuli and involves the recruitment of plasma and white blood cells (leukocytes) to injured or infected tissues. Prolonged inflammation, also called chronic inflammation, is associated with a gradual change in the type of immune cells present at the site of inflammation and is characterized by the simultaneous destruction and healing of tissue from the inflammatory process.

However, inflammation sometimes causes harm, usually as a result of disruption of the normal development of inflammation. Inflammatory diseases are diseases related to, characterized by, causing, caused by, or affected by inflammation. "Acute inflammatory conditions" as used herein, in accordance with normal medical vocabulary, refers to inflammatory conditions with rapid onset and severe symptoms. The duration of the initial period, from the normal state of the patient to severe manifestations of symptoms of inflammation, usually does not exceed about 72 hours. Acute inflammatory conditions should be distinguished from chronic inflammatory conditions, which are inflammatory conditions of long duration characterized by little change or slow disease progression. The distinction between acute and chronic conditions is well known to those skilled in the art of medicine.

The major immune cells involved in the acute phase of inflammation as well as acute inflammatory disorders include mononuclear cells (eg, monocytes, which differentiate into macrophages in response to inflammation), dendritic cells, and neutrophils (which migrate to the site of inflammation). These immune cells promote an inflammatory response by releasing inflammatory mediators such as histamine, interferon-gamma, interleukin-8, leukotriene B4, nitric oxide, etc., and by engulfing bacteria, viruses, and cellular debris (a process known as phagocytosis). ). Such cells are collectively known to those skilled in the art as phagocytes.

Applicants have found that LukAB targets and kills human phagocytes and that such LukAB-mediated cytotoxicity is essentially specific to these cells, but not to other mammalian nucleated cells. Without intending to be bound by any particular theory of action, Applicants believe that the LukA/LukB complex creates pores in the plasma membrane of infiltrating phagocytes, causing cell death and thereby reducing inflammation. Thus, the anti-inflammatory compositions of the present invention may be useful in the treatment of acute inflammatory conditions in mammals, such as humans, regardless of their cause, such as any bacterial or viral infection and, in preferred embodiments, localized acute inflammatory conditions. Other examples of such conditions include allergic contact dermatitis, acute hypersensitivity, acute neurological inflammatory injury (eg, caused by acute infection), acute myocardial infarction, acute neuronal injury caused by cardiopulmonary bypass surgery, and acute localized anti-inflammatory conditions caused by bacterial or viral infection. .

In preferred embodiments, the acute inflammatory condition is an infected wound in the skin or soft tissue. Wounds treatable according to the invention may take the form of punctures, incisions, or tears in living tissue. Skin wounds can penetrate the epidermis, dermis or, in the case of deep wounds, into the subcutaneous tissues. Thus, wounds treatable with the therapeutic compositions of the present invention include deep sternal wounds, such as after open heart surgery, and postoperative wounds after abdominal and any other type of surgery. Other wounds include wounds caused by trauma such as gunshots, knives, or any other object capable of cutting or tearing the skin. Wounds that occur as side effects of medical treatment or as symptoms of various pathologies (eg, ulcers associated with Kaposi's sarcoma), as well as internal wounds (eg, anal fissures and wounds or lesions of the gastrointestinal tract, such as ulcers of the stomach or intestines) may also be treatable with the present invention.

Other acute inflammatory conditions that may be treatable with the therapeutic compositions of the present invention include conjunctivitis, iris inflammation, uveitis, central retinitis, otitis externa, otitis media suppurativa, mastoiditis, labyrinthitis, chronic rhinitis, acute rhinitis, sinusitis, pharyngitis , tonsillitis (tonsillitio), contact dermatitis, dermonecrosis, diabetic polyneuritis, polymyositis, myositis ossificans, degenerative arthritis, rheumatoid arthritis, periarthritis of the shoulder joint and osteitis deformans.

S. aureus infections

The present invention also provides a method for inhibiting the onset of or treating S. aureus infection by administering antibody compositions to a mammalian subject in need thereof. For the purposes of the present invention, the target population of subjects includes mammals, such as humans, infected with or at risk of infection with S. aureus. In some embodiments, the subject to be treated is infected with S. aureus, including MRSA, and/or has already received treatment with antibiotics or other therapeutic agents, but the treatment was unsuccessful.

Therapeutically effective amounts

In the context of the treatment of acute inflammatory conditions, amounts of LukA and LukB are therapeutically effective in the sense that the treatment achieves any one or more of the goals of reducing the number of symptoms, reducing the severity of at least one symptom, or delaying further development of at least a single symptom, or even complete relief of an acute inflammatory condition.

In the context of the use of therapeutic compositions as passive or active vaccines for S. aureus infection, therapeutically effective amounts of LukA and LukB, or anti-LukA and anti-LukB antibodies, are also prophylactically effective in that the administration of the composition achieves any one or several objectives of inhibiting or preventing S. aureus infection in subjects at risk, and in the case of mammalian subjects infected with S. aureus, reducing the number of symptoms, reducing the severity of at least one symptom, or delaying further development of at least one symptom, or even complete relief of the infection.

In general, therapeutically effective amounts of LukA, LukB, and anti-LukA and anti-LukB antibodies can be determined according to standard procedures that take into account numerous factors including, for example, the concentrations of these active agents in the formulation, the route and frequency of administration, the severity of the acute the S. aureus inflammatory condition or infection to be treated (or to be prevented) and characteristics of the subject such as age, weight and general health and immune system. General recommendations can be found, for example, in publications of the International Conference on Harmonization and REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Company 1990). The clinician may administer LukA and LukB, or anti-LukA and anti-LukB antibodies, until a dosage is reached that provides the desired or desired prophylactic or therapeutic effect. The course of such treatment can be easily monitored using routine tests.

Therapeutically effective amounts of LukA and LukB are typically 1-400 μg each of LukA and LukB, per dose or per day. Preferably, the amounts of LukA and LukB are substantially the same. Therapeutically effective amounts of antibody compositions are typically at least 50 mg of the composition per kilogram of body weight (mg/kg), including at least 100 mg/kg, at least 150 mg/kg, at least 200 mg/kg, at least 250 mg/kg, at least 500 mg/kg, at least 750 mg/kg and at least 1000 mg/kg per dose or per day. Dosages of monoclonal antibody compositions may be lower, such as about one tenth of non-monoclonal antibody compositions, such as at least about 5 mg/kg, at least about 10 mg/kg, at least about 15 mg /kg, at least about 20 mg/kg, or at least about 25 mg/kg.

Methods of administration

Before administration, the therapeutic compositions of the present invention may be sterilized, which can be easily accomplished by filtration through sterile filtration membranes, before or after lyophilization and reconstitution. Therapeutic compositions may be placed in a container having a sterile inlet, such as an intravenous solution sachet or a vial having a stopper which is pierced with a hypodermic needle.

The anti-inflammatory composition may be administered in any number of ways, in accordance with accepted medical practice. Preferred methods include intravenous, intramuscular, subcutaneous and transdermal administration, using techniques known to those skilled in the art. Other routes of administration may be contemplated. In the case of treating acute inflammatory conditions that are localized, non-systemic administration may be preferred, in which case administration of the therapeutic composition is at or around the site of the acute inflammation.

Combination Therapy

In some embodiments, the therapeutic composition is administered as part of a combination therapy in combination with another active agent, depending on the nature of the acute inflammatory condition or S. aureus infection being treated. Such additional active agents include anti-infective agents, antibiotic agents and antimicrobial agents. Typical examples of anti-infective agents that may be suitable for use in the present invention include vancomycin and lysostaphin. Representative examples of antibiotic agents and antimicrobial agents that may be suitable for use in the present invention include penicillinase-resistant penicillins, cephalosporins, and carbapenems, including vancomycin, lysostaphin, penicillin G, ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, cephalothin, cefazolin, cephalexin, cephadrin, cefamandol, cefoxitin, imipenem, meropenem, gentamicin, teicoplanin, lincomycin, and clindamycin. The dosages of these antibiotics are well known to those skilled in the art. See, for example, MERCK MANUAL OF DIAGNOSIS AND THERAPY, Section 13, Ch. 157, ^100th Ed. (Beers & Berkow, eds., 2004). Anti-inflammatory, anti-infectious, antibiotic and/or antimicrobial agents may be combined prior to administration, or may be administered simultaneously (as part of the same composition or via different compositions) or sequentially with the therapeutic compositions of the present invention.

In some embodiments, the anti-LukA and/or anti-LukB antibody composition is multivalent in that it also contains an antibody that specifically binds another bacterial antigen (and which optionally neutralizes the other bacterial antigen). Antibodies can specifically bind any of the antigens described herein in the context of vaccine compositions. Thus, for example, another antibody may specifically bind polysaccharides or glycoproteins, including S. aureus type 5, S. aureus type 8, S. aureus 336, leukocidin components such as PVL (including individual PVL subunits, LukS-PV and LukF- PV), gamma-hemolysin subunits (HlgA, HlgB, and HlgC), S. aureus LukE or LukD, S. aureus LukM or LukF'-PV, lipoteichoic acid (LTA), and protein microbial surface components recognizing adhesive matrix molecules (MSCRAMM) .

Treatment regimens

The therapeutic compositions of the present invention may be administered as a single dose or according to a multiple dose protocol. For example, a relatively small number of doses of the therapeutic composition are administered, such as one or two doses. In embodiments involving conventional antibiotic therapy, which typically involves the administration of multiple doses over several days or weeks, the antibiotics may be administered one, two, or three or more times a day over a period of time, such as at least 5 days, 10 days, or even 14 or more days, while the antibody composition is usually administered only once or twice. However, different dosages, timing of dosing, and relative amounts of therapeutic composition and antibiotics can be selected and adjusted by one of ordinary skill in the art.

Methods for identifying inhibitors of LukAB-mediated cytotoxicity and altered forms of LukA and LukB with less toxicity

Anti-LukA and anti-LukB antibodies and fragments thereof, as well as other potential therapeutic particles (eg, small organic molecules) can be used in a variety of ways (including assay formats or screens) to assess their ability to inhibit LukAB-mediated cytotoxicity. As described below, methods have been developed to identify agents that inhibit a particular aspect of the cascade of events leading to LukAB-mediated cytotoxicity and lysis of human phagocytes. Methods have also been developed to identify altered forms of LukA and LukB that have reduced toxicity compared to their native counterparts. Target events that are part of the cascade include, for example, binding of LukA to phagocyte membranes, binding of LukB to LukA (oligomerization of LukAB), and blockage of the membrane pore formed by the LukAB oligomer. Assay formats typically require the use of human phagocytes (or their LukAB membrane-binding site), suitable culture media, and purified LukA or purified LukA and LukB.

The skilled artisan will appreciate that the following protocols are illustrative only and that various operating parameters such as reaction conditions, choice of detectable label, and instrumentation (e.g., detection and quantitation tools) may vary depending on the situation.

The following methods are generally designed to identify agents that inhibit LukAB cytotoxicity without necessarily allowing the exact cascade event involved.

To identify inhibitors of LukAB cytotoxicity, human phagocytes (e.g., PMN-HL60 cells) can be plated on a 384-well black clear-bottom treated tissue culture plate (Corning) at 5 x 10 ³ cells/well in a final volume of 50 µl of medium RPMI (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells can then be contacted/mixed with/reacted/treated with test compound/molecule (about 5 µl/different concentrations) and then intoxicated with LukA and LukB, which in preferred embodiments are substantially purified (5 µl about 0.001- 2 μm solution), preferably added together, under culture conditions that allow the intoxication of phagocytes with LukA and LukB, for example, for 1 h at 37°C, 5% CO ₂ . As a control, cells can be treated with culture medium (100% viable) and 0.1% v/v. Triton X100 (100% death).

In these embodiments, the cells treated as described above can then be incubated with a cell viability monitoring dye, such as the CellTiter kit (Promega) (which allows cell viability to be determined from optical density by measuring the number of viable cells in culture by quantifying metabolic activity of the cells) and incubated for an additional period of time (eg, about 2 h at 37°C, 5% CO ₂ ). Cell viability can then be determined, for example, by measuring the colorimetric response at 492 nm using a plate reader such as the Envision 2103 Multi-label Reader (Perkin-Elmer). The percentage of viable cells can be calculated, for example, using the following equation: % Viability=100×[(Ab ₄₉₂ Sample-Ab ₄₉₂ Triton X)/(Ab ₄₉₂ Tissue Culture Medium). An increase of 100% viability suggests the presence of LukAB inhibition of cytotoxicity.

A variant of this assay is called the membrane damage assay. In such embodiments, cells treated as described above (e.g., up to and including the step of treating the cells with a test compound/molecule and then intoxicating the cells with purified LukA may then be incubated with a non-cell-penetrating fluorescent dye such as SYTOX green) (0.1 µM; Invitrogen) (according to the manufacturer's instructions) and incubated, for example, for an additional 15 minutes at room temperature in the dark Fluorescence, as an indicator of membrane damage, can then be measured using a plate reader such as as Envision 2103 Multilabel Reader (Perkin-Elmer) at 485 nm excitation wavelength, 535 nm emission wavelength Decreased fluorescence suggests inhibition of LukAB cytotoxicity.

Taken together, these assays contribute to the identification of compounds that inhibit or attenuate the cytotoxic effects of LukAB on human phagocyte cells.

Additional methods may be used independently or in combination with the methods described above; in particular, if the methods described above detect inhibitory activity, then it will allow one of skill in the art to more accurately determine which event in the biochemical cascade is affected or targeted by the agent. Such events include binding of LukA to phagocyte membranes, binding of LukB to LukA (oligomerization of LukAB), and blockage of membrane pores formed by the LukAB oligomer.

Screening for binding of LukA inhibitors to target cells

To screen for inhibitors that block or attenuate LukA binding to target cells, which is considered the first step in the intoxication process, human phagocytes (eg, PMN-HL60 cells) can be seeded in 384-well, flat-bottomed, treated tissue culture plates (Corning) at 2 .5×10 ³ cells/well in a final volume of 50 μl of RPMI medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells can then be treated with test compound/molecule (about 5 µl/various concentrations) and intoxicated with purified fluorescently labeled LukA (e.g. FITC, Cy3, Cy5, APC, ΡΕ) 5 µl about 0.01-2 µM solution for 1 hour at 37°C, 5% CO ₂ . To evaluate the potency of test compounds/molecules, cell-bound fluorescence can be measured as an indicator of LukA binding to cells, e.g. ) (excitation 485 nm, emission 535 nm)).

Screening for Inhibitors of Oligomerization/LukA-LukB Interaction

To screen for inhibitors that block or attenuate the LukA/LukB interaction, which is considered the second step in the intoxication process, human phagocytes (e.g., PMN-HL60 cells) can be seeded in 384-well flat-bottom processed tissue culture plates (Corning) at 2.5 ×10 ³ cells/well in a final volume of 50 μl of RPMI medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells can then be treated with a test compound/molecule and then intoxicated with a mixture of purified LukA and purified LukB, where LukB is a fluorescently labeled fluorescent molecule such as FITC, Cy3, Cy5, APC, and PΕ, and left until the intoxication process is complete (e.g., in for 1 h at 37°C, 5% CO ₂ ). To assess the potency of test compounds/molecules, cell-bound LukB-FITC fluorescence can be measured as an indicator of LukA/LukB-FITC interaction using, for example, an automated fluorescent microscopic imaging system designed for multi-parameter screening and multi-parameter analysis (for example, Cellomics ArrayScan HCS Reader (Thermo Scientific) (excitation 485 nm, emission 535 nm)).

Pore Inhibitor Screening LukAB

To screen for inhibitors that block or inhibit pore formation of LukAB, an effector molecule leading to cell lysis, human phagocytes (e.g., PMN-HL60 cells) can be seeded in a 384-well black clear bottom processed tissue culture plate (Corning) at 2.5×10 ³ cells/well in a final volume of 50 μl of RPMI medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells can then be treated with test compound/molecule (about 5 µl, various concentrations) and then intoxicated with purified LukAB (about 0.001-2 µM) for 10 minutes at 37° C., 5% CO ₂ . As a control, PMN-HL60 cells can be treated with culture medium (negative control) and 0.1% v/v. Triton X100 (positive control).

Ethidium bromide (EB) influx assay can be used to directly evaluate LukAB pores on the host cell surface. EB is a small cationic dye that does not penetrate healthy host cells. After the formation of LukAB cationic pores, EB enters the cells and binds to DNA, resulting in fluorescence. The cell thus treated can then be incubated with EB (5 μM) for an additional 5 minutes at room temperature in the dark. To evaluate the effectiveness of test compounds/molecules in inhibiting LukAB pore formation, fluorescence can be measured as an indicator of pore formation using a plate reader such as the Envision 2103 Multilabel Reader (Perkin-Elmer) at 530 nm excitation, 590 nm emission. This assay helps identify molecules that can block or inhibit LukAB pores, thereby reducing LukAB-mediated toxicity.

Method for Determining LukAB Production by S. aureus Clinical Isolates to Predict the Severity of Infection

Another aspect of the present invention relates to a method for predicting or assessing the severity of an S. aureus infection, comprising detecting the presence or amount of LukA and/or LukB, or their respective genes, in a biological sample obtained from an infected subject. Thus, detection of the presence or relatively high amounts of LukA and/or LukB, or detection of their respective genes (eg, observed for S. aureus strain Newman, 4645 and MRSA strains USA300 and USA500) compared to controls (eg, S. aureus strains USA100 and USA400) producing small or undetectable amounts of LukA and/or LukB is indicative of severe infection. With regard to the detection or presence of relative amounts of LukA and/or LukB, reference may be made to the illustrations shown in FIG. 4A. Typical examples of embodiments of the method are described below.

Immunoblot analysis to determine the levels of LukA and LukB

To determine LukAB levels (i.e., LukAB production), a biological sample, such as a fluid (i.e., blood) or tissue sample, is obtained from an infected subject, followed by placing the culture in suitable culture conditions to allow growth of S. aureus, a supernatant is obtained culture, isolate bacterial proteins from it, identify LukA and/or LukB, and then quantify LukA and/or LukB. More specifically, in one embodiment, clinical isolate strains can be selected and grown in a suitable culture medium, such as Royal Park Memorial Institute 1640 (RPMI; Invitrogen) culture medium supplemented with 1% casamino acids (RPMI+CAS) under suitable conditions. cultivation, for example, for 12-18 hours at 37°C with shaking at 180 rpm. The bacteria can then be pelleted by centrifugation, and culture supernatants are collected. Culture supernatants (about 30 µl) can then be mixed with 10 µl SDS-Laemmli buffer and heated to 95°C for 10 minutes. Proteins can then be separated, for example, using 15% SDS-PAGE gels and then transferred to a solid support, such as a nitrocellulose membrane. The membranes can then be incubated with antibodies directed against LukA or LukB (e.g., rabbit polyclonal antibodies) and the presence of LukA or LukB can be visualized by detecting antibody-LukA/antibody-LukB complexes with a fluorophore-conjugated secondary antibody (e.g., anti-rabbit antibody conjugated to AlexaFluor-680; Invitrogen). The membranes can then be dried and scanned with, for example, an Odyssey infrared imaging system (LI-COR Biosciences) to quantify LukA and LukB.

Polymerase chain reaction (PCR) to determine the presence of the LukA and/or LukB genes

To determine the presence of genes encoding LukAB, a biological sample is obtained from an infected subject, followed by placing the culture in suitable cultivation conditions to allow growth of S. aureus, extracting the nucleic acid from the S. aureus culture, and then performing at least one round of nucleic acid amplification using PCR or other suitable amplification protocol using LukA and/or LukB specific primers and detecting LukA and/or LukB. Thus, in one exemplary embodiment, after initial sample preparation, clinical isolate strains can be grown on a solid medium such as trypticase soy broth (TSB) hardened with 1.5% agar at 37°C. S. aureus colonies can then be selected and subjected to enzymatic digestion, for example, with 2 mg/ml lysostaphin (AMBI PRODUCTS LLC) in TSM buffer (100 mM TRIS pH 7, 500 mM sucrose, 10 mM MgCl ₂ ) for 10 minutes at 37°C. The samples can then be centrifuged, the supernatant discarded and the pellet resuspended in 100 µl of sterile water, followed by five minutes of boiling at 100° C. and centrifugation. The supernatant provides the starting material and template DNA for an amplification reaction such as PCR using LukA and/or LukB specific primers.

Working examples

The invention will be further described with reference to the following non-limiting examples. Unless otherwise stated, all shares are determined by weight.

Example 1: Expression and purification of recombinant LukA and LukB under native conditions: pMAL expression system

The LukA and LukB genes are amplified from S. aureus DNA with Taq polymerase under standard PCR conditions at an annealing temperature of 55°C using the following primers: ) and 5'-ccc-GAAGGATTCACATCATCATCATCATCACAATTCAGCTCATAAAGACTCTC-3' (SEQ ID NO:31) for LukA and 5'-CCCCGAAGGATTTCaCATCATCATCATCATCACAAGATTAATTCTGAAATCAAACAAG-3' (SEQ ID NO:32) and 5'-ccc-GTCGAC-tta-TTTCTTTTCATTATCATTAAGTACTT -3' ( SEQ ID NO:33) for LukB. The LukA and LukB gene products are digested with Nde1 and Sal1 (New England BioLabs) and ligated into the pMAL-c4X vector (New England BioLabs). The constructs are transformed into E. coli strain DH5α and the plasmid inserts are confirmed by sequencing. The transformants are grown in Terrific Broth medium with 100 μg/ml ampicillin and 0.2% glucose at 37° C. until the cultures reach an A ₆₀₀ value of approximately 0.5. Expression of 6-his-labeled MBP-LukA or 6-his-labeled MBP-LukB is induced with 0.3 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 16°C, overnight, with shaking at 180 rpm

After induction, cells are harvested by centrifugation at 4000 rpm at 4°C for 20 min and resuspended in ice-cold column buffer (20 mM Tris-HCl, 200 mM NaCl and 1 mM EDTA) supplemented with an EDTA-free protease inhibitor ( Roche). Bacterial cells are sounded on ice for 1 min (pulses 10 s). Samples are centrifuged at 10,000 rpm at 4° C. for 30 minutes and the supernatant is collected and applied to an amylose resin column. The columns are washed twice with column buffer and purified 6-his-labeled MBP-LukA or 6-his-labeled MBP-LukB is eluted in 10 fractions with column buffer supplemented with 10 mM maltose.

Example 2: Expression and purification of recombinant active toxins LukA, LukAΔ10C, Δ33NLukA and LukB: pET14b expression system

The LukA, LukAΔ10C, Δ33NLukA, and LukB genes were amplified from S. aureus DNA with Vent polymerase (New England BioLabs) under standard PCR conditions at 55°C annealing temperature using the following primers: 5'-cccc-CTCGAG-AATTCAGCTCATAAAGACTCTCAAG-3' (SEQ ID NO:34) and 5'-cccc-GGATCC-tta-TCCTTCTTTAATAAGGTTTATTGTC-3' (SEQ ID NO:35) for LukA; 5'-cccc-CTCGAG-AATTCAGCTCATAAAGACTCTCAAG (SEQ ID NO:34) and 5'-cccc-GGATCC-tta-ATATTTATCAACGACTTTAACTG (SEQ ID NO:36) for LukAΔ10C; 5'-cccc-CTCGAG-TCAACAGCACCGGATGATATTG (SEQ ID NO:37) and 5'-cccc-GGATCC-tta-TCCTTCTTATAAGGTTTATTGTC (SEQ ID NO:35) for Δ33NLukA; 5'-cccc-CTCGAG-AAGATTAATTCTGAAATCAAACAAG-3' (SEQ ID NO:38) and 5'-cccc-GGATCC-tta-TTTCTTTTCATTATCATTAAGTACTTT-3' (SEQ ID NO:39) for LukB. The gene products are digested with Xho1 and BamH1 (New England BioLabs) and ligated into the pET14b vector (Novagen) to fuse the histidine tag coding sequence to the 5' region of the genes. Expression plasmids are transformed into E. coli strain DH5α and plasmid inserts are confirmed by sequencing. Plasmids were purified and transformed into E. coli T7 lysY/lq expression strain (New England BioLabs).

For denaturing purification, transformants are grown in Terrific Broth medium with 100 μg/ml ampicillin at 37° C. until the cultures reach an A600 value of approximately 0.5. Expression of 6-his-labeled LukA or 6-hisLukB is induced with 0.4 mM IPTG at 37° C. for 3 hours with shaking at 180 rpm. After induction, cells are harvested by centrifugation at 4000 rpm at 4° C. for 15 min and then resuspended in 1x TBS (50 mM Tris, 150 mM NaCl, pH 7.5). Bacterial cells are sounded on ice for 2 min (pulses of 10 s). Sounded bacteria are subjected to ultracentrifugation for 30 min at 50,000 rpm. The pellets are resuspended in lysis buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris, 8M urea, pH 8.0) and incubated at room temperature for 30 min on a rotisserie. The samples are centrifuged at 13,000 rpm for 30 minutes and the supernatants are applied to a column containing Ni-NTA resin (Qiagen). The column was washed twice with wash buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris, 8M urea, pH 6.3) and the protein was eluted from the column with elution buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris, 8M urea ) at pH 5.9 and at pH 4.5. Fractions containing purified protein, as determined by SDS-PAGE, were pooled, diluted 1:1 with tris buffered saline (TBS; 500 mM Tris, 1.5 M NaCl, pH 7.5) and dialyzed in TBS at 4° C. overnight to remove urea and allow re-styling. Purified 6-his-labeled LukA and LukB are quantified using the Thermo Scientific Pierce BCA Protein Assay Kit.

For native purification, transformants are grown in Luria-Bertani broth with 100 μg/ml ampicillin at 37° C. until the cultures reach an A600 value of approximately 0.5. Expression of 6-his-labeled LukA, 6-his-labeled LukAΔ10C, 6-his-labeled Δ33NLukA or 6-hisLukB is induced with 0.05-0.1 mM IPTG at 25-30°C, for 3 h, with shaking at 220 rpm. After induction, cells are harvested by centrifugation at 4000 rpm at 4°C for 15 min and then resuspended in 1x TBS (50 mM Tris, 600 mM NaCl, pH 7.5) with 10 mM imidazole and a HALT cocktail of protease inhibitors, no containing EDTA (Thermo Scientific). Bacterial cells are sounded on ice. The sonicated bacteria are centrifuged for 20 minutes at 20,000 rpm. Supernatants are incubated with Ni-NTA resin (Qiagen) for 1 h at 4° C. on a rotator. Samples are applied to the column and the column is washed with 1x TBS wash buffer (50 mM Tris, 600 mM NaCl, pH 7.5) with 25 mM imidazole. The protein is eluted from the column with 50-500 mM imidazole in 1x TBS elution buffer (50 mM Tris, 600 mM NaCl, pH 7.5). Fractions containing purified protein, as determined by SDS-PAGE, were pooled, diluted 1:1 in 1× TBS (50 mM Tris, 150 mM NaCl, pH 7.5) and dialyzed in 1× TBS at 4° C. overnight. Purified 6-his-labeled LukA and LukB are quantified using the Thermo Scientific Pierce BCA Protein Assay Kit.

Example 3 Expression and Purification of Denatured Recombinant LukA and LukB

The LukA and LukB genes are amplified from S. aureus DNA with Vent polymerase (New England BioLabs) under standard PCR conditions at 55° C. annealing temperature using the following primers: 5'-ggg-CATATG-AATTCAGCTCATAAAGACTCTCAA-3' (SEQ ID NO: 40) and 5'-ccc-GTCGAC-TCCCTTCTTTATAAGGTTTATTGTC-3' (SEQ ID NO:41) for LukA, and 5'-ggg-CATATG-AAGATTAAATTCTGAAATCAAACAAG-3' (SEQ ID NO:42) and 5'-ccc-GTCGAC -TTTCTTTTCATTATCATTAAGTACTT-3' (SEQ ID NO:43) for LukB. The LukA and LukB gene products are digested with Nde1 and Sal1 (New England BioLabs) and ligated into the pET41b vector (Novagen). The constructs are first transformed into DH5α cells and then transformed into E. coli ER2566 expression strain (New England BioLabs). Transformants are grown in Terrific Broth with kanamycin, 25 μg/ml, for 2.5 h at 37°C, and LukA and LukB expression is induced with 0.3 mM IPTG at 37°C for 2 h with shaking at 180 rpm Cells are pelleted and resuspended in 1x TBS (500 mM Tris, 1.5 M NaCl, pH 7.5) and sonicated on ice for 1 min (10 s pulses). Sounded bacteria are subjected to ultracentrifugation for 30 min at 50,000 rpm.

To purify C-terminal 6-his-labeled LukA and LukB under denaturing conditions, pellets are resuspended in lysis buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris, 8 M urea, pH 8.0) and incubated at room temperature in for 30 min on a rotator. Samples are centrifuged at 13,000 rpm for 30 minutes and the supernatants are applied to a Ni-NTA column. The columns are washed twice with wash buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris, 8 M urea, pH 6.3) and LukA and LukB are eluted from the column with elution buffer (100 mM NaH ₂ PO ₄ , 10 mM Tris , 8 M urea) at pH 5.9 and at pH 4.5. Purified 6-his-labeled LukA and LukB are quantified using the BioRad DC Protein Assay.

Example 4: Production of polyclonal antibodies to LukA and to LukB

Denatured recombinant LukA (250 μg) emulsified in complete Freund's adjuvant (FCA) was injected into New Zealand white rabbits. Animals were given a booster dose of recombinant LukA (125 μg) emulsified in incomplete Freund's adjuvant (FCA) on day twenty-one (21) and day forty-nine (49).

Denatured recombinant LukB (250 μg) emulsified in complete Freund's adjuvant (FCA) was injected into New Zealand white rabbits. Animals were given a booster dose of recombinant LukB (125 μg) emulsified in incomplete Freund's adjuvant (FCA) on day twenty-one (21) and day forty-nine (49).

Example 5: Leukocidin A/B Is Primarily Responsible for Cytotoxin-Mediated Death of Human Phagocytes Due to Membrane Rupture

Used cell lines

HL-60 (ATCC CCL-240, human promyelocytic cell line) was used as a model to study how LukAB selects and kills human phagocyte cells. HL-60 cells are grown in RPMI medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37° C. with 5% CO ₂ . To differentiate HL-60 into neutrophil-like cells (PMN-HL60), 1.5% (v/v) dimethyl sulfoxide (DMSO) was added to cultures and grown for 4 days.

Methods/Assays Used

Cell toxicity analysis

To assess the viability of mammalian cells following Staphylococcus aureus leukocidin AB (LukAB) intoxication, PMN-HL60 cells were plated in 96-well flat bottom treated tissue culture plates (Corning) at 1 x 10 ⁵ cells/well in a final volume of 100 μl RPMI (Gibco ) supplemented with 10% heat-inactivated fetal bovine serum (FBS). The cells are intoxicated for 2 hours at 37° C., 5% CO ₂ with serial 2-fold dilutions of the Staphylococcus aureus strain Newman culture filtrate ranging from 20% to 0.16% v/v. with three parallel experiments. Experiments were carried out using the exoproteins of the wild-type strain and the exoproteins of the LukAB-free strain (mutant strain). Controls for 100% viability included 20% v/v. tissue culture media (RPMI+10% heat-inactivated fetal bovine serum) and 20% v/v. nutrient medium. S. aureus (RPMI + casamino acids). 0.1% v/v Triton X100 was used as a control for 100% cell death. After intoxication, 10 μl of CellTiter (Promega) is added to each well and the cells are incubated an additional 3 h at 37°C, 5% CO ₂ . CellTiter controls metabolically active cells (color change), i.e. ability lost by dead cells. Colorimetric response is measured at 492 nm using a Perkin Elmer Envision 2103 Multilabel Reader. The percentage of viable cells is calculated using the equation below: % Viability=100×[(Ab ₄₉₂ Sample-Ab ₄₉₂ Triton X)/(Ab ₄₉₂ Tissue Culture Medium)].

Membrane Damage Analysis

An alternative assay to measure LukAB-mediated cytotoxicity is to assess host cell membrane integrity. For this, the SYTOX green dye permeability assay (Invitrogen) is used. Healthy cells are impermeable to SYTOX green, but become permeable to the dye when the integrity of the cell membrane is compromised. Inside the cell, SYTOX green binds to DNA and exhibits strong fluorescence.

To assess host cell membrane integrity following LukAB intoxication or ex vivo infection with S. aureus strains, PMN-HL60 cells were plated in 96-well flat-bottom treated tissue culture plates (Corning) at 1 x 10 ⁵ cells/well in a final volume of 100 µl RPMI (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells are intoxicated with dilutions of S. aureus strain Newman culture filtrate ranging from 20% to 0.16% v/v, or infected with MOIs ranging from 1-100 in three parallel runs for 2 hours at 37°C, 5% CO ₂ . Experiments were carried out using the wild-type strain and the LukAB-free strain (mutant strain). Controls for determining the background level of fluorescence include 20% vol./about. tissue culture media (RPMI+10% heat-inactivated fetal bovine serum) and 20% v/v. S. aureus culture medium (RPMI + casamino acids). After intoxication or infection, the cells are transferred to a 96-well v-bottom plate (Corning) and centrifuged at 1500 rpm for 5 minutes. The supernatant is discarded and the pellets are resuspended in 100 μl PBS + SYTOX green (0.1 μM; Invitrogen). The cells are then transferred to a 96-well black clear bottom processed tissue culture plate (Corning) and incubated at room temperature in the dark for 10 minutes. Fluorescence is measured using a Perkin Elmer Envision 2103 Multilabel Reader (485 nm excitation, 535 nm emission).

results

LukAB targets and destroys human primary phagocytes

Intoxication of primary human peripheral mononuclear cells (PBMC) (Figure 3a), monocytes, macrophages, dendritic cells (Figure 3b) and polymorphonuclear cells (PMN) (Figure 3c) with filtered culture supernatants of S. aureus strain Newman results in severe cell death , according to the results of the determination using the analysis of cellular toxicity (Fig.3a-c). Intoxication of these cells with filtered supernatants of cultures of S. aureus strain Newman that did not contain hemolysin (hla), γ-hemolysin (hlg), leukocidin E/D (LukED), or leukocidin A/B (LukAB), demonstrated that exoproteins hla-, hlg - and LukED-negative strains are as cytotoxic as the wild-type (WT) Newman strain (as determined by cell toxicity assay), indicating that none of the above Newman-produced leukotoxins are involved in cytotoxin-mediated the death of these cells (Fig.3a-c). In contrast, very low cell death was observed when cells were intoxicated with exoproteins of the LukAB-free Newman strain (ΔlukAB). The lack of cytotoxic activity in the LukAB-free Newman strain is abolished by introducing the IukAB genes into the transformant (trans) in the plasmid (ΔlukAB/pLukAB) (FIG. 3c). It is important to note that this phenotype is completely LukAB dependent as determined by PMN intoxication with purified recombinant LukA and LukB. Individual subunits show no detectable cytotoxicity towards PMN (FIG. 3d). In contrast, the combination of both subunits results in strong cytotoxicity towards such cells in a dose-dependent manner (FIG. 3d). In summary, these results demonstrate that LukAB is responsible for the ability of S. aureus to target and destroy primary human phagocytes, key immune cells required for host defense against infectious agents.

LukAB preferentially kills human phagocytic cells

Intoxication of a neutrophil-like cell line (PMN-HL60) and a macrophage-like cell line (THP1+PMA) with filtered culture supernatants of S. aureus strain Newman resulted in severe cell death as determined by a cell toxicity assay (FIG. 3). Intoxication of these cells with filtered culture supernatants of S. aureus WT and mutant strains with isogenic cytotoxins (hla, hlgABC, LukED, and LukAB) demonstrated that LukAB is responsible for the ability of S. aureus to kill these cells, as determined by cell toxicity assay (Fig. 4a and 4b). The lack of cytotoxic activity of the LukAB-free Newman strain was abolished by transforming the LukAB-free Newman strain with a LukAB-expressing plasmid (ΔlukAB/pLukAB) (FIG. 4c). The exoproteins of this strain were extremely cytotoxic to both PMN-HL60 and THP-1+PMA cells, providing strong evidence that LukAB is a potent staphylococcal toxin that targets and kills human cells.

To further eliminate the influence of other factors present in S. aureus culture supernatants, PMN-HL60 cells are intoxicated with purified recombinant LukA or LukB. The individual subunits show no detectable cytotoxicity towards PMN-HL60 (FIG. 3d). In contrast, the combination of both subunits results in strong dose-dependent cytotoxicity towards these cells (FIG. 3d). In addition to PMN-HL60 and THP-1+PMA cells, several other human cell lines, including the myeloid progenitor from which PMN-HL60 differentiates (HL60), the monocytic progenitor from which THP-1+PMA differentiates (THP-1) , lymphocytes (HuT and Jurkat cells) and epithelial cells (293T and HepG2) are also intoxicated with recombinant LukAB (FIG. 4e). These results demonstrate that LukAB preferentially targets and kills human phagocytic cells and has no effect on human lymphocytes or epithelial cells. Taken together, these results demonstrate that LukAB plays a significant role in S. aureus-mediated phagocyte death.

LukAB is produced by clinically relevant strains of S. aureus

Immunoblot analyzes with a polyclonal antibody induced against LukB showed that LukB is produced by a number of staphylococcal strains, including MRSA strains associated with nosocomial and community-acquired infections (USA300, 400 and 500; Figure 5a). Importantly, LukB levels are associated with the cytotoxic phenotype of these strains (FIGS. 5a and 5b). Strains producing high levels of LukB (eg, Newman, 4645, USA500, and USA300) were more cytotoxic to PMN-HL60 cells than strains producing low or undetectable levels of LukB (eg, USA100 and USA400) (Fig. 5b) . To study the role of LukAB in MRSA strains, an isogenic LukAB mutant was generated in a clinical isolate of clone USA type 300 LA (FIG. 5c). As with the Newman strain, the exoproteins of the LukAB-free strain USA300 were markedly less cytotoxic than those of the parental strain (FIG. d). These data demonstrate that LukAB is an important cytotoxin produced by MRSA strains.

LukAB damages the membranes of human phagocytes

PMN-HL60 intoxication with S. aureus exoproteins results in rounding of the cell shape and swelling of the nucleus, in a LukAB dependent phenotype (FIG. 6a). This cell rounding and swelling phenotype was associated with increased membrane permeability, as determined by membrane damage assay (FIGS. 6b and 6c). Importantly, the exoproteins of the LukAB-negative strain showed little or no effect on membrane permeability, a phenotype that could be abolished by production of LukAB from the plasmid (Figure 6b), with recombinant LukAB, but not individual toxin subunits, causing dose-dependent damage. membranes (Fig. 6c). In addition, infection of primary human PMNs with methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) USA300 results in LukAB-dependent membrane damage (FIG. 6d). These results demonstrate that LukAB damages the plasma membrane of the host cell upon ex vivo infection.

LukAB protects S. aureus from host-mediated death by targeting and killing phagocytes

Infection of S. aureus WT, ΔlukAB, and ΔlukAB PMN-HL60 cells with the IukAB expression plasmid (ΔlukAB/pLukAB) showed that LukAB is necessary for S. aureus to rupture the phagocyte membrane during staphylococcus-phagocyte interaction (Fig. 7a) determined by membrane damage analysis. Importantly, S. aureus overproducing LukAB (ΔlukAB/plukAB) showed greater membrane damage than the wild-type (WT) strain (FIG. 7a), indicating that LukAB severely damages host cell membranes. Infection of human whole blood (FIG. 7b) and purified primary human neutrophils (PMN; FIG. 7c) showed that the IukAB-negative staphylococcus was killed more efficiently than the WT strain (FIGS. 7b and 7c). Importantly, the attenuated phenotype exhibited by ΔlukAB-negative staphylococci could be eliminated with the IukAB expression plasmid (FIGS. 7b and 7c). Intoxication of primary human PMNs with S. aureus WT culture filtrate, ΔlukAB, and a ΔlukAB mutant strain containing the IukAB expression plasmid showed that LukAB targets and destroys primary human PMNs (Figure 7d). These data provide strong evidence that LukAB is a potent staphylococcal cytotoxin that targets and destroys PMN by membrane rupture, thereby protecting S. aureus from PMN-mediated death.

LukAB contributes to the pathogenesis of S. aureus in vivo

Mice retroorbitally infected with S. aureus containing a luciferase reporter construct with a fused LukAB promoter (pLukAB-Xen1) showed LukAB promoter activity in renal abscesses, while the reporter lacking the promoter (pXen1) showed no activity (Figure 8a). ). These data demonstrate that LukAB is expressed in vivo in a renal abscess infection model. In addition, mice retroorbitally infected with S. aureus WT, but not with a LukAB-free S. aureus strain, exhibit extensive kidney colonization. The under-colonization seen with the LukAB-negative strain is restored to WT levels by providing LukAB in transformants (trans) (FIG. 8b). Taken together, these data demonstrate that LukAB is an important staphylococcal cytotoxin contributing to the pathogenesis of the bacterium.

LukAB forms pores in the target cell membrane that can be blocked by polyethylene glycol

Intoxication of PMN-HL60 cells with recombinant LukAB (rLukAB) showed that both rLukA and rLukB bind to target cells as determined by immunoblotting with LukA and LukB specific antibodies (FIG. 9a). Binding of 6His-labeled rLukAB to PMN-HL60 was also confirmed by fluorescence-excited cell sorting (FACS) and a His-specific antibody (FIG. 9b). rLukAB oligomers are also detected by immunoblotting on PMN-HL60 membranes after intoxication with the recombinant toxin, indicating the formation of higher order LukAB structures on the target cell membranes (FIG. 9c). Importantly, PMN-HL60 intoxication with rLukAB demonstrated that LukAB forms ethidium bromide-permeable pores on target cell membranes that can be blocked by polyethylene glycol (PEG) molecules (Figure 9d), and that blocking LukAB pores enhances viability. cells (Fig.9E). In addition, PEG specifically blocks LukAB pores because the pores formed in PMN-HL60 membranes by saponin were not blocked by PEG and, as a result, these cells were not protected from pore-mediated death. These data demonstrate that LukAB pores can be blocked by small molecules, and blocking LukAB pores protects cells from LukAB-mediated death.

Neutralization of Cytotoxicity of S. aureus α-LukA Culture Filtrate with Polyclonal Antibody

Intoxication with PMN-HL60 by a culture filtrate of S. aureus previously incubated with different amounts of α-LukA polyclonal antibodies induced in two different rabbits results in a dose-dependent decrease in the toxicity of the culture filtrate (FIG. 10). This neutralizing effect was not observed when the culture filtrate was pre-incubated with non-immune serum. Importantly, the antibodies produced by the two different rabbits behaved very similarly, and the neutralizing ability of the antibodies increased with increasing maturity, as seen by comparing the neutralizing effect of antibodies from blood samples taken at later time points with the neutralizing effect of antibodies from blood. taken earlier (Fig.10). These data indicate that the cytotoxicity observed in the culture filtrate of S. aureus can be neutralized using α-LukA polyclonal antibodies.

Identification of a non-cytotoxic LukA truncated mutant that would still be recognized by an α-LukA polyclonal antibody

LukA differs from other S-subunits of staphylococcal leukotoxin in that it has protrusions at both the N- and C-termini. These overhangs consist of 33 amino acids at the N-terminus and 10 amino acids at the C-terminus (FIG. 11a). PMN-HL60 intoxication with purified recombinant LukA lacking the N-terminal overhang (rΔ33NLukA) in combination with purified rLukB results in potent cellular cytotoxicity comparable to that of purified rLukA+rLukB (FIG. 11b). However, PMN-HL60 intoxication with purified recombinant LukA lacking the C-terminal overhang (rLukAΔ10C) in combination with rLukB did not produce a cytotoxic effect (FIG. 11b). These data demonstrate that the N-terminal overhang is not essential for the cytotoxic effect of LukA, but the C-terminal overhang is required for toxicity. Importantly, the α-LukA polyclonal antibody neutralizing the effect of LukAB (FIG. 10) continues to recognize the 6×His-labeled non-cytotoxic rLukAΔ10C mutant, as does the α-His polyclonal antibody (FIG. 11c). These data suggest that rLukAΔ10C can be used for in vivo production of α-LukA polyclonal antibodies, which are neutralizing antibodies. Thus, rLukAΔ10C can be used in an active vaccine formulation.

All patent publications and non-patent publications are indicative of the level of knowledge of those skilled in the art to which this invention pertains. All of these publications are hereby incorporated by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.

Although the invention has been described here with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It should be understood, therefore, that numerous modifications to the illustrative embodiments may be made, and that other circuits may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims (12)

1. A composition useful for inhibiting the onset of or treating a Staphylococcus aureus infection, comprising a therapeutically effective amount of a Staphylococcus aureus leukocidin A polypeptide analogue comprising an amino acid sequence of amino acid residues 28-351 of SEQ ID NO: 12 or an amino acid sequence of at least 90% identical to the amino acid sequence of amino acid residues 28-351 of SEQ ID NO: 12, wherein said leukocidin A polypeptide analog contains one or more non-conservative amino acid substitutions or deletions at amino acid residues 342-351 of SEQ ID NO: 12 that render said leukocidin A polypeptide analog non-cytotoxic in combination with Staphylococcus aureus leukocidin B polypeptide. 2. The composition of claim 1, wherein the leukocidin A polypeptide analog contains an amino acid sequence at least 95% identical to the amino acid sequence of amino acid residues 28-351 of SEQ ID NO: 12. 3. The composition of claim 1, wherein said leukocidin A polypeptide analogue does not contain the amino acid sequence of amino acid residues 342-351 of SEQ ID NO: 12. 4. The composition according to claim 1, additionally containing: a therapeutically effective amount of a Staphylococcus aureus leukocidin B polypeptide, wherein said leukocidin B polypeptide comprises an amino acid sequence of any of SEQ ID NOs: 15-27 or a sequence at least 90% identical to any of SEQ ID NOs: 15-27. 5. The composition according to claim 1, additionally containing: a therapeutically effective amount of a Staphylococcus aureus leukocidin B polypeptide comprising an amino acid sequence from amino acid residues 30-339 of SEQ ID NO: 21, or an amino acid sequence at least 90% identical to the amino acid sequence from amino acid residues 30-339 of SEQ ID NO: 21. 6. The composition of claim 5, wherein the leukocidin B polypeptide contains an amino acid sequence at least 95% identical to the amino acid sequence of amino acid residues 30-339 of SEQ ID NO: 21. 7. Composition according to claim 1, additionally containing a pharmaceutically acceptable carrier. 8. Composition according to claim 1, additionally containing an adjuvant. 9. A method for suppressing the occurrence of a Staphylococcus aureus infection, comprising administering to a mammalian subject in need thereof a composition according to claim 1. 10. The method of claim 9, wherein the Staphylococcus aureus infection is an infection caused by methicillin-resistant Staphylococcus aureus.

Images