IMMUNIZATION AGAINST STAPHYLOCOCCAL INFECTIONS OF BONES AND JOINTS

专利摘要:
Immunization with EsxA, EsxB, FhuD2, StaOll, and / or Hla leads to a significantly lower bacterial load in the joints of animals that are exposed to infections with S. aureus. Thus, the invention provides an effective means of preventing and / or treating S. aureus infections of bones and joints, thereby avoiding disorders such as osteomyelitis, septic arthritis, and prosthetic infection. articulation.
公开号:BE1022359B1
申请号:E2015/5154
申请日:2015-03-16
公开日:2016-03-25
发明作者:Giuliano Bensi；Emiliano Chiarot；Alessia Corrado
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

IMMUNIZATION AGAINST STAPHYLOCOCCAL INFECTIONS OF BONE AND
JOINTS
This application claims the priority of European Patent Application 14160390.2 (filed March 17, 2014), the entire contents of which are hereby incorporated by reference for all purposes.
Technical area
This invention relates to immunization against infection of bones and joints by S. aureus.
State of the art context
Staphylococcus aureus is a gram-positive spherical bacterium. Annual mortality in the United States exceeds that of any other infectious disease, including HIV / AIDS, and S. aureus is the leading cause of infections in the bloodstream, lower respiratory tract, skin and soft tissues . It is also the leading cause of bone infections worldwide, and these infections are painful, debilitating and difficult to treat. S. aureus infections can occur in the bone marrow and / or in the joints, and can lead to osteomyelitis, septic arthritis, and joint prosthesis infection [1].
An object of the invention is to provide vaccines which are useful for the prevention and / or treatment of S. aureus infections of bones and joints.
Description of the invention The invention provides the use of one or more antigens for immunizing a mammal to prevent or treat a S. aureus infection of its bones and joints, where the antigen / antigens is / are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOll; and Hla. The inventors have shown that immunization with these antigens can lead to a significantly lower bacterial burden in the joints of animals that are exposed to S. aureus infections. Compared with control animals, knee joint washings show anti-S antibodies. aureus, a reduction in immune cell death, and inferior inflammatory responses. Thus, the invention provides an effective means for preventing and / or treating S. aureus infections of bones and joints.
S. aureus antigens The invention uses 1, 2, 3, 4, or all 5 of the following antigens: EsxA; EsxB; FhuD2; StaOll; and Hla. These five antigens are already known from the state of the art (for example, see references 2 to 8) and further details are given below. A particularly useful composition comprises the five of these antigens (preferably with a nontoxic mutant form of Hla). The "EsxA" antigen in strain NCTC 8325 has the amino acid sequence SEQ ID NO: 1 (GI: 88194063). The EsxA antigens used in the present invention can elicit an antibody (e.g., when administered to a human) that recognizes SEQ ID NO: 1 and / or may comprise an amino acid sequence (a) having 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%, 99.5% or more) with SEQ ID NO: 1; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 1, where "n" is 7 or greater (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more). These EsxA polypeptides comprise variants of SEQ ID NO: 1. The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 1. To other preferred fragments, one or more amino acids are missing (for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and / or one or more amino acids (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 1 while retaining at least one epitope of SEQ ID NO: 1. The "EsxB" antigen in strain NCTC 8325 has the amino acid sequence SEQ ID NO: 2 (GI: 88194070). The EsxB antigen used in the present invention can elicit an antibody (e.g., when administered to a human) that recognizes SEQ ID NO: 2 and / or may comprise an amino acid sequence: (a) presenting 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 2; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 2, where "n" is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more). These EsxB polypeptides comprise variants of SEQ ID NO: 2. The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 2. To other preferred fragments, one or more fragmented amino acids are missing (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and / or one or more amino acids (e.g. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 2 while retaining at least one epitope of SEQ ID NO: 2. To a useful EsxB antigen, the internal cysteine residue of SEQ ID NO: 2 is missing, for example, it comprises SEQ ID NO: 35, where the residue X at position 30 is either absent or is a residue. of amino acid without free thiol group (under reducing conditions) for example, it is any natural amino acid with the exception of a cysteine. The "FhuD2" antigen is annotated as "ferrichrome binding protein", and has also been studied in the literature [9]. It is also known as "Sta006" (for example, in references 2 to 8). In strain NCTC 8325, the FhuD2 antigen has the amino acid sequence SEQ ID NO: 3 (GI: 88196199). The FhuD2 antigen used in the present invention can elicit an antibody (e.g., when administered to a human) that recognizes SEQ ID NO: 3 and / or may comprise an amino acid sequence: (a) presenting 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 3; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 3, where "n" is 7 or greater (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These polypeptides. FhuD2 include variants of SEQ ID NO: 3. The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 3. To other preferred fragments, one or more amino acids are missing (for example, 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and / or one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 3 while retaining at least one epitope of SEQ ID NO The first 17 N-terminal amino acids of SEQ ID NO: 3 may be conveniently omitted (to provide SEQ ID NO: 6). Mutant forms of FhuD2 are reported in reference 10. To a useful FhuD2 antigen, the cysteine residue of SEQ ID NO: 3 is missing, for example, it comprises SEQ ID NO: 34 and does not include any amino acid residue with a free thiol group (under reducing conditions) for example, it is devoid of cysteine. An FhuD2 antigen may be lipidated, for example, with an N-terminal acylated cysteine. A useful sequence of FhuD2 is SEQ ID NO: 7, which has a Met-Ala-Ser- sequence at the N-terminus; SEQ ID NO: 37 is another such sequence, but it lacks the cysteine present in SEQ ID NO: 7. The "StaOll" antigen has the amino acid sequence SEQ ID NO: 4 (GI: 88193872) in the strain NCTC 8325. The StaOll antigens used in the invention can elicit an antibody (e.g., when administered to a human being) that recognizes SEQ ID NO: 4 and / or may comprise an amino acid sequence (a) with 50% or more identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 4; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 4, where "n" is 7 or greater (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These StaOll polypeptides include variants of SEQ ID NO: 4. The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 4. To other preferred fragments, one or more amino acids are missing (for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and / or one or more amino acids (e.g. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO: 4 while retaining at least one epitope of SEQ ID NO: 4. The first 23 N-terminal amino acids of SEQ ID NO: 4 can be conveniently omitted (to provide SEQ ID NO: 33). To a useful StaOll antigen, the cysteine residue of SEQ ID NO: 4 is missing, for example, it comprises SEQ ID NO: 36 and does not include any amino acid residue with a free thiol group (under reducing conditions) by for example, it is devoid of cysteine. StaOll antigen can be lipidated, for example, with N-terminal acylated cysteine. A useful sequence of StaOll is SEQ ID NO: 8, which has an N-terminal methionine; SEQ ID NO: 39 is another such sequence, but it lacks the cysteine present in SEQ ID NO: 8. Alternative forms of SEQ ID NO: 4 that can be used as such or to prepare StaOll antigens include, but are not limited thereto, SEQ ID Nos. 9, 10 and 11 with various Ile / Val / Leu substitutions (and Cys-free variants of these sequences may also be used in the invention). StaOll may exist as a monomer or oligomer, with Ca ++ ions promoting oligomerization. The invention can use StaOll monomers and / or oligomers. The "Hla" antigen is the "precursor of alpha-hemolysin" also known as "alpha toxin" or simply "hemolysin". In strain NCTC 8325, Hla has the amino acid sequence SEQ ID NO: 5 (GI: 88194865). Hla is an important virulence determinant produced by most strains of S. aureus, possessing pore and hemolytic activity. Anti-Hla antibodies can neutralize the deleterious effects of the toxin in animal models, and Hla is particularly useful for protection against pneumonia.
Useful Hla antigens can elicit an antibody (e.g., when administered to a human) that recognizes SEQ ID NO: 5 and / or may comprise an amino acid sequence: (a) having 50% or more of identity (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.5% or more) with SEQ ID NO: 5; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 5, where "n" is 7 or greater (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). These Hla antigens include variants of SEQ ID NO: 5. The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 5. To other preferred fragments, one or more amino acids are missing (for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and / or one or more amino acids (e.g. 2,3,4,5,6,7,8,9,10,15,20,25 or more) from the N-terminus of ID NO: 5. The first 26 N-terminal amino acids of SEQ ID NO: 5 can be conveniently omitted (e.g., to give SEQ ID NO: 12). Truncation at the C-terminus may also be used, for example, leaving only 50 amino acids (residues 27 to 76 of SEQ ID NO: 5) [11].
The toxicity of Hla can be avoided by chemical inactivation (for example, using formaldehyde, glutaraldehyde or other cross-linking reagents). However, instead, it is preferable to use mutant forms of Hla that eliminate its toxic activity while maintaining its immunogenicity. Such detoxified mutants are already known from the state of the art. A preferred Hla antigen is a S. aureus mutant hemolysin having a mutation at residue 61 of SEQ ID NO: 5, which is the residue of the mature antigen (i.e. first N-terminal amino acids = residue of SEQ ID NO: 12). Thus, residue 61 may not be histidine, and may instead be, for example, Ile, Val or preferably Leu. His-Arg mutation at this position can also be used. For example, SEQ ID NO: 13 is the mature mutant sequence of Hla-H35L (i.e., SEQ ID NO: 12 with an H35L mutation) and a useful Hla antigen comprises SEQ ID NO: 13. Useful muta-tion replaces a long loop with a short sequence, for example, to replace 39-mer at residues 136 to 174 of SEQ ID NO: 5 with a tetramer such as PSGS (SEQ ID NO: 14), as in SEQ ID NO: 15 (which also includes the H35L mutation) and SEQ ID NO: 16 (which does not include the H35L mutation). Another useful mutation replaces the Y101 residue, for example, with leucine (SEQ ID NO: 17). Another useful mutation replaces D152, for example, with leucine (SEQ ID NO: 18). Another useful mutant replaces residues H35 and Y101, for example, with leucine (SEQ ID NO: 19). Another useful mutant replaces residues H35 and D152, for example, with leucine (SEQ ID NO: 20). Other useful Hla antigens are disclosed in references 12 and 13. SEQ ID NO: 21, 22 and 23 are three useful fragments of SEQ ID NO: 5 ("Hla2 -76", "Hla27-89" and "Hla27 -79 ", respectively). SEQ ID NO: 24, 25 and 26 are the corresponding fragments from SEQ ID NO: 13.
A useful sequence of Hla is SEQ ID NO: 27. It comprises an N-terminal Met, then an Ala-Ser dipeptide from the expression vector, then SEQ ID NO: 13 (from strain NCTC8325).
When a composition comprises both EsxA and EsxB antigens, these may be present as a single polypeptide (i.e., as a fusion polypeptide). Thus, a single polypeptide can elicit antibodies (e.g., when administered to a human) that recognize both SEQ ID NO: 1 and SEQ ID NO: 2. The only polypeptide can include: (i) a first polypeptide sequence having 50% or more identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 1 and / or comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 1, such as it has been defined above for EsxA; and (ii) a second polypeptide sequence having 50% or more identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 2 and / or comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 2, as defined above for EsxB. The first and second polypeptide sequences may be in one order or the other, from the N-terminus to the C-terminus. SEQ ID NO: 28 ("EsxAB") and 29 ("EsxBA") are examples of such polypeptides, both of which include ASGGGS hexapeptide linkers (SEQ ID NO: 30). Another hybrid of "EsxAB" includes SEQ ID NO: 31, which may be provided with an N-terminal methionine (e.g., SEQ ID NO: 32). To a useful variant of EsxAB, the internal cysteine residue of EsxB is missing, for example it comprises SEQ ID NO: 40 where the residue X at position 132 is either absent or is an amino acid residue without a free thiol group (in reducing conditions) for example, it is any natural amino acid with the exception of a cysteine. Thus, a preferred EsxAB antigen for use according to the invention has the amino acid sequence SEQ ID NO: 38.
Thus, a useful polypeptide comprises an amino acid sequence (a) having 80% or more identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, 99.5% or more) with SEQ ID NO: 31; and / or (b) comprising both a fragment of at least "n" consecutive amino acids of amino acids 1 to 96 of SEQ ID NO: 31 and a fragment of at least "n" consecutive amino acids of amino acids 103 to 205 of SEQ ID NO: 31, where "n" is 7 or greater (e.g., 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Such polypeptides (e.g., SEQ ID NO: 32) can elicit antibodies (e.g., when administered to a human) that recognize both the wild-type staphylococcal protein comprising SEQ ID NO: 1 and the protein Staphylococcal wild-type comprising SEQ ID NO: 2. Thus, the immune response will recognize both EsxA and EsxB antigens. The preferred fragments of (b) provide an epitope derived from SEQ ID NO: 1 and an epitope derived from SEQ ID NO: 2.
A preferred composition thus comprises all four of: (i) a single polypeptide comprising both an EsxA antigen and an EsxB antigen, for example, comprising SEQ ID NO: 31; (ii) an FhuD2 antigen, for example, comprising SEQ ID NO: 6; (iii) a StaOll antigen, for example, comprising SEQ ID NO: 33; and (iv) an H35L mutant form of Hla, for example, comprising SEQ ID NO: 13. In some embodiments, a composition may comprise one or more other polypeptides; in other embodiments, the only polypeptides in a composition are these four specified polypeptides, and these polypeptides may even be the only immunogenic components in a composition.
Although SEQ ID NO: 31, 6, 33 and 13 are amino acid sequences useful in combination, the invention is not limited to these precise sequences. Thus, 1, 2, 3 or the 4 of these sequences can be independently modified by up to 5 single amino acid changes (i.e., 1, 2, 3, 4 or 5 substitutions, deletions and or single amino acid insertions) provided that the modified sequence can elicit antibodies that will always bind to a polypeptide consisting of the unmodified sequence.
Another useful composition comprises the four of: (i) a first polypeptide having the amino acid sequence SEQ ID NO: 32; (ii) a second polypeptide having the amino acid sequence SEQ ID NO: 7; (iii) a third polypeptide having the amino acid sequence SEQ ID NO: 8; and (iv) a fourth polypeptide having the amino acid sequence SEQ ID NO: 27. In some embodiments, a composition may comprise one or more other polypeptides; in other embodiments, the only polypeptides in the composition are these four specified polypeptides, and these polypeptides may even be the only immunogenic components in a composition.
Although SEQ ID NO: 32, 7, 8 and 27 are amino acid sequences useful in combination, the invention is not limited to these precise sequences. Thus, 1, 2, 3 or all 4 of these four sequences can be independently modified by 1, 2, 3, 4 or 5 single amino acid changes (i.e., 1, 2, 3, 4 or single amino acid substitutions, deletions and / or insertions) provided that the modified sequence can elicit antibodies that will always bind to a polypeptide consisting of the unmodified sequence. In a preferred embodiment, a composition thus comprises these four polypeptides specified with 1, 2, 3 or 4 of SEQ ID NO: 32, 7, 8 and 27 independently modified by substitution, deletion and / or simple insertion of amino acid.
For example, wild-type polypeptide sequences of FhuD2, StaOll and EsxAB (e.g., SEQ ID NOS: 6, 31 and 33) each comprise a single cysteine residue that can lead to inter-polypeptide disulfide bridges, forming both homodimers and heterodimers. Such inter-linked polypeptides are undesirable and thus the Sta006, StaOll and EsxB sequences can be modified to remove their natural cysteine residues, so that they do not contain free thiol groups (under reducing conditions). . The wild type cysteine can be deleted or it can be substituted with a different amino acid.
Thus: an FhuD2 antigen may comprise SEQ ID NO: 34; a StaOll antigen may comprise SEQ ID NO: 36; and an EsxB antigen may comprise SEQ ID NO: 35 (e.g., as an EsxAB hybrid comprising SEQ ID NO: 40). Examples of such sequences include, but are not limited to, SEQ ID NO: 37, 39, and 38. These sequences can be used separately as substitutes for the corresponding wild-type sequences, or in combination. Thus, a particularly useful composition includes all four of: (i) a first polypeptide having the amino acid sequence SEQ ID NO: 38; (ii) a second polypeptide having the amino acid sequence SEQ ID NO: 37; (iii) a third polypeptide having the amino acid sequence SEQ ID NO: 39; and (iv) a fourth polypeptide having the amino acid sequence SEQ ID NO: 27. In some embodiments, a composition may comprise one or more other polypeptides; in other embodiments, the only polypeptides in a composition are these four specified polypeptides.
When more than one polypeptide is present, they can be present at substantially equal masses, that is to say that the mass of each of them is + 5% of the average mass of all the polypeptides. Thus, when four polypeptides are present, they may be present in a mass ratio of a / b / c / d, where each of a to d is between 0.95 and 1.05. Apart from EsxA, EsxB, Hla, FhuD2 and StaOll, there are other S. aureus antigens, and a composition may optionally include one or more other S. aureus antigens. For example, both saccharide and polypeptide antigens are known for S. aureus. Thus, a composition may comprise a S. aureus saccharide antigen, for example known saccharide antigens include S. aureus exopolysaccharide, which is a poly-N-acetylglucosamine (PNAG), and capsular saccharides of S. aureus, which may be, for example, type 5, type 8 or type 336. A composition may also comprise a ClfA antigen, an IsdA antigen, an IsdB antigen, an IsdC antigen, and / or an IsdH antigen (each as defined on pages 15 to 17 of reference 3).
In some embodiments, a composition comprises a S. aureus antigen as defined above, and also an antigen from a different organism (e.g., a virus or other bacterium).
S. aureus infections of mammalian bones and joints The invention consists of immunizing a mammal in such a way that it has an increased immune response which is then useful for the prevention of future S. aureus infection of its bones. and joints, or may help treat an existing S. aureus infection of this type. S. aureus infects various mammals (including cows, dogs, horses, and pigs), but the preferred mammal for use of which the invention is the human being.
By preventing or treating S. aureus infection of bones and joints, the invention is thus suitable for the prevention or treatment of particular disorders including, but not limited to, osteomyelitis, septic arthritis, and infection. articular prosthesis (IPA). In many cases, these disorders may be associated with the formation of a S. aureus biofilm. Osteomyelitis is an infection and inflammation of a bone or bone marrow. Symptoms include pain and tenderness in the area of the affected bone. S. aureus can reach one bone in two main ways: from an infection in another part of the body through the bloodstream, that is, hematogenous osteomyelitis, which is often seen in children; or as a result of an injury, particularly a deep cut that reaches or exposes a bone (with or without fracture). It can affect any bone, but the most commonly affected are the femur, tibia, fibula, humerus, vertebrae, maxillary, and mandibular bodies. Risk factors include bone fracture, bone prosthesis, bone surgery, immunosuppression, immunodeficiency, drug use, kidney dialysis, steroid use, and (less commonly for S. aureus) sickle cell disease . Osteomyelitis may be suppurative (including acute and chronic suppurative osteomyelitis) or nonsuppurative (usually sclerosing, including diffuse sclerosing osteomyelitis, focal sclerosing osteomyelitis, and Garré sclerosing osteomyelitis).
A bone infection can spread to a joint (especially in children) and infect the synovial membrane of a joint, leading to arthrosynovitis and possibly septic arthritis (often called suppurative arthritis). In children, large subperiosteal abscesses can form because the periosteum is loosely attached to the surface of the bone.
In patients who receive a bone or joint prosthesis, S. aureus can cause an IPA, whose diagnosis is detailed in references 14 to 16. Thus, a mammal that is immunized according to the invention can have a bone or joint prosthesis, or it may be a recipient intended for such a prosthesis (for example, a patient of orthopedic surgery in preoperative). In this way, the invention may be useful for the prevention or treatment of nosocomial S. aureus infection, which is often associated with prostheses. The invention is useful for the prevention or treatment of any of these disorders that arise in bones or joints. A patient may have a bone disorder, joint disorder, or both.
Immunogenic compositions and drugs
Immunogenic compositions according to the invention may be useful as vaccines. The vaccines of the invention may be either prophylactic (i.e., to prevent infection) or therapeutic (i.e., to treat an infection) but will generally be prophylactic.
The compositions can thus be pharmaceutically acceptable. They will usually include components in addition to antigens, for example, they will generally comprise one or more pharmaceutical carrier (s) and / or excipient (s). A detailed description of these components is available in reference 121.
The compositions will generally be administered to a mammal in aqueous form. However, prior to administration, the composition may have been in a non-aqueous form. For example, although some vaccines are manufactured in aqueous form, then packaged and dispensed and administered in aqueous form, other vaccines are lyophilized during manufacture and are reconstituted in aqueous form at the time of use. Thus, a composition can be dried, such as a lyophilized formulation. Reference 4 discloses the use of lyophilization with immunogenic compositions of S. aureus.
A composition may include preservatives such as thiomersal or 2-phenoxyethanol. However, it is preferred that the vaccine be substantially free of (i.e., less than 5 μg / ml) mercurial material, e.g., free of thiomersal. Vaccines containing no mercury are more preferred. Vaccines lacking a preservative are particularly preferred.
To improve thermal stability, a composition may include a temperature-protective agent (see below).
To control tonicity, it is best to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at a concentration between 1 and 20 mg / ml, for example about 10 + 2 mg / ml NaCl. Other salts that may be present include potassium chloride, potassium dihydrogenphosphate, dehydrated disodium phosphate, magnesium chloride, calcium chloride, and the like.
The compositions will generally have an osmolality of between 200 mOsm / kg and 400 mOsm / kg, preferably between 240 and 360 mOsm / kg, and will more preferably be in the range of 290 to 310 mOsm / kg.
The compositions may comprise one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (in particular with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will generally be included in the range of 5 to 20 mM.
The compositions may include a metal ion chelator, particularly a divalent metal ion chelator such as EDTA. Reference 4 discloses that the inclusion of EDTA can improve the stability of the compositions described therein. The final concentration of EDTA in an immunogenic composition may be from about 1 to 50 mM, about 1 to 10 mM or about 1 to 5 mM, preferably about 2.5 mM.
The pH of a composition will generally be between 5.0 and 8.1, and more generally between 6.0 and 8.0, for example 6.5 and 7.5, or between 7.0 and 7.8.
The composition is preferably sterile. The composition is preferably non-pyrogenic, for example containing <1 EU (endotoxin unit, a standard measurement) per dose, and preferably <0.1 EU per dose. The composition is preferably free of gluten.
The composition may comprise the material for a single immunization, or it may comprise the material for multiple immunizations (i.e., a "multi-dose" kit). The inclusion of a preservative is preferred in multi-dose arrangements. Alternatively (or additionally) to the inclusion of a preservative in the multidose compositions, the compositions may be contained in a container provided with an aseptic adapter for the removal of the material.
S. aureus infections can affect various areas of the body and thus a composition can be prepared in various forms. For example, a composition can be prepared as injectables, either as solutions or as liquid suspensions. Solid forms suitable for solution, or suspension, in liquid vehicles prior to injection may also be prepared (for example, a freeze-dried composition or a spray-dried composition). A composition may be prepared for topical administration, for example as an ointment, cream or powder. A composition may be prepared for oral administration, for example in the form of a tablet or capsule, in the form of a spray, or in the form of a syrup (optionally flavored). A composition may be prepared for pulmonary administration, for example in the form of an inhaler, using a fine powder or a spray. A composition can be prepared in the form of a suppository or an egg. A composition may be prepared for nasal, atrial or ocular administration, for example in the form of drops. A composition may be in kit form, so designed that a combined composition is reconstituted just prior to administration to a patient. Such kits may comprise one or more antigens in liquid form and one or more lyophilized antigens.
When a composition is to be prepared extemporaneously before use (for example, when a component is in freeze-dried form) and is in the form of a kit, the kit may comprise two vials, or it may comprise a pre-filled syringe and a vial, with the contents of the syringe used to reactivate the contents of the vial before injection.
Human vaccines are generally administered in a dose volume of about 0.5 ml, although half a volume (i.e., about 0.25 ml) may also be useful, for example, for children.
The immunogenic compositions administered according to the invention may also comprise one or more immunoregulatory agents. Preferably, one or more of the immunoregulatory agents comprises one or more adjuvants (see below).
The compositions can elicit both a cell-mediated immune response as well as a humoral immune response. This immune response will preferably induce long-lasting (e.g., neutralizing) antibodies and cell-mediated immunity that can respond rapidly upon exposure to S. aureus.
The immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen (s), as well as any other component, as necessary. By "immunologically effective amount" it is meant that administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This quantity varies according to the health and the physical condition of the individual to be treated, the age, the taxonomic group of the individual to be treated (for example, non-human primate, primate, etc.), the capacity of the system immune system of the individual to synthesize antibodies, the degree of protection desired, the vaccine formulation, the physician's estimate of the medical situation, and other relevant factors. The quantity is expected to be in a relatively wide range that can be determined by routine testing. When more than one antigen is included in a composition, then two antigens may be present at the same dose with respect to each other or in different doses.
As mentioned above, a composition may comprise a temperature-protecting agent, and this component may be particularly useful in adjuvanted compositions (particularly those containing a mineral adjuvant, such as an aluminum salt). As described in reference 17, a liquid temperature protective agent may be added to an aqueous vaccine composition to lower its freezing point, for example to reduce the freezing point below 0 ° C. Thus, the composition can be stored below 0 ° C, but above its freezing point, to inhibit thermal degradation. The temperature-protecting agent also allows the composition to be frozen while protecting the mineral-based adjuvants against agglomeration or sedimentation after freezing and thawing, and it can also protect the composition at elevated temperatures, for example above 40 ° C. A starting aqueous vaccine and the liquid temperature protective agent may be mixed such that the liquid temperature protective agent forms from 1 to 80% by volume of the final mixture. Suitable temperature-protecting agents should be safe for administration to humans, easily miscible / water-soluble, and they should not damage other components (eg, antigen and adjuvant) in the composition. Examples include glycerine, propylene glycol, and / or polyethylene glycol (PEG). Suitable PEG's may have an average molecular weight in the range of 200 to 20,000 Da. In a preferred embodiment, the polyethylene glycol may have an average molecular weight of about 300 Da ("PEG-300").
Methods of treatment and administration of an immunogenic composition The invention provides a method of preventing or treating a S. aureus infection of bones and joints of a mammal by administering to the mammal at least one antigen selected from the group consisting of: EsxA; EsxB; FhuD2; StaOll; and Hla. The invention also provides the use of at least one antigen in the manufacture of a medicament for the prevention or treatment of S. aureus infection of bones and joints of a mammal where the antigen is present. or the antigens are / are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOll; and Hla. The invention also provides at least one antigen for use in immunizing a mammal to prevent or treat a S. aureus infection of its bones and joints, where the antigen or antigens is / are selected ( s) in the group consisting of: EsxA; EsxB; FhuD2; StaOll; and Hla. The invention also provides at least one antigen for use in a method of preventing or treating a S. aureus infection of bones and joints of a mammal by administering the antigen or antigens to the mammal. wherein the antigen or antigens is / are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOll; and Hla.
These methods, uses, and antigens elicit an immune response that is effective in preventing or treating S. aureus infections of bones and joints. The immune response may involve antibodies and / or cell-mediated immunity.
As mentioned above, the mammal is preferably a human. The human being can be a child (for example, a young child or an infant), a teenager, or an adult. In some embodiments, the human may have a bone or joint prosthesis, or it may be a recipient provided for such a prosthesis (for example, a preoperative orthopedic patient). A vaccine intended for children can also be administered to adults, for example to estimate the absence of risk, the dosage, the immunogenicity, etc. However, vaccines are not only appropriate for these groups, and they can be used more generally in a human population. Other mammals that can be usefully immunized according to the invention are cows, dogs, horses, and pigs.
One means for verifying the effectiveness of a therapeutic treatment involves monitoring S. aureus infection of a joint or bone after administration of the compositions of the invention. One way to verify the efficacy of prophylactic treatment involves monitoring immune responses, at the systemic level (such as monitoring IgG1 and IgG2a production rate) and / or at the mucosal level (such as rate monitoring). IgA production), against the antigens in the composition administered after its administration. Another means of estimating immunogenicity from the compositions is to express the antigens in a recombinant manner for screening sera and mucosal secretions of patients by immunoblot and / or microchips. A positive reaction between the protein and the patient's sample indicates that the patient has developed an immune response against the protein in question. The efficacy of the vaccine compositions can also be determined in vivo using animal models of S. aureus infection, for example, guinea pigs or mice, with the vaccine compositions. Generally, there are three animal models useful for the study of infectious disease due to S. aureus, namely: (i) the model of murine abscess [18], (ii) the model of murine lethal infection [ 18], and (iii) the model of murine pneumonia [19]. However, in relation to the evaluation of the efficacy for the prevention / treatment of S. aureus infection of the bones and joints of a mammal, different models are needed, for example such as those disclosed in the examples below.
The compositions will generally be administered directly to a patient. Direct administration may be accomplished by parenteral injection (eg, subcutaneously, intraperitoneally, intravenously, intramuscularly, or into the interstitial space of a tissue), or mucosally, as by rectal, oral administration ( for example, a tablet, a spray), vaginal, topical, transdermal or transcutaneous, intranasal, ocular, auricular, pulmonary or other mucosal administration. Intramuscular injection is the most general route for administering compositions according to the invention. The invention can be used to elicit systemic and / or mucosal immunity, preferably to trigger enhanced systemic and / or mucosal immunity. Preferably, the enhanced systemic and / or mucosal immunity is reflected by an amplified TH1 and / or TH2 immune response. Preferably, the amplified immune response comprises an increase in the production of IgG1 and / or IgG2a and / or IgA.
The dosage can be obtained by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a sensitization immunization schedule and / or in a booster immunization schedule. In a multiple dose schedule, the various doses may be given by the same or different routes, for example parenteral sensitization and mucosal booster, mucosal sensitization and parenteral booster, etc. Multiple doses will usually be given at least 1 week apart (eg, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks , etc.)
The immunogenic compositions may be administered to the patient at substantially the same time as (for example, during the same medical consultation or visit to a health professional or a vaccination center) other vaccines.
The immunogenic compositions may be administered to the patient in combination with an antibiotic. For example, they may be administered at substantially the same time as an antibiotic. Similarly, they may be administered to a subject receiving antibiotic treatment. Similarly, they may be administered as part of a co-treatment which involves the administration of both a composition as described herein and an antibiotic. The antibiotic will be effective against S. aureus bacteria, for example a beta-lactam.
Strains and variants
The antigens are described above with reference to the existing nomenclature (eg "EsxA") and sequence examples given in GI numbers and also in the sequence listing. The invention is not limited to these precise sequences. The genomic sequences of several S. aureus strains are available, including those of MRSA strains N315 and Mu50 [20], MW2, N315, COL, MRSA252, MSSA476, RF122, USA300 (very virulent), JH1, JH9, NCTC 8325, and Newman. Conventional search and alignment techniques can be used to identify in any of these genomic sequences or other genomic sequences the homologue of any particular sequence mentioned herein. In addition, the specific sequences disclosed herein can be used to design primers for amplification of homologous sequences from other strains. Thus, the invention encompasses such variants and homologues from any S. aureus strain as well as unnatural variants. In general, suitable variants of a particular SEQ ID NO include its allelic variants, polymorphic forms, homologues, orthologs, paralogs, mutants, and the like.
Thus, for example, the polypeptides used in the invention may, as compared to SEQ ID NO herein, include one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.). amino acid substitutions, such as conservative substitutions (i.e., substitutions of one amino acid with another that has a related side chain). Genetically encoded amino acids are generally divided into four families: (1) acids, i.e., aspartate, glutamate; (2) basic, i.e. lysine, arginine, histidine; (3) non-polar, that is, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes jointly classified as aromatic amino acids. In general, simple amino acid substitution within these families has no major effect on biological activity. The polypeptides may also include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) single amino acid deletions with respect to SEQ ID NO. The polypeptides may also include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) insertions (e.g., each of 1, 2, 3, 4 or 5 acids amino) with respect to the sequences SEQ ID NO.
Similarly, a polypeptide used in the invention may comprise an amino acid sequence that: (a) is identical (i.e., 100% identical) to a sequence disclosed in the sequence listing ( b) shares sequence identity (eg, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5 % or more) with a sequence disclosed in the sequence listing; (c) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or more) single amino acid modifications (deletions, insertions, substitutions), which may be at the level of distinct locations or which may be contiguous, compared to the sequences of (a) or (b); or (d) when aligned with a particular Sequence Listing Sequence using a pairwise alignment algorithm, each x-amino acid moving window from N-terminus to C-terminus ( as for an alignment extending to p amino acids, where p> x, there are p-x + 1 of these windows) has at least xy identical aligned amino acids, where: x is selected from 20, 25, 30 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200; y is selected from 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0, 91, 0.92, 0, 93, 0.94, 0, 95, 0, 96, 0, 97, 0.98, 0.99; and if x-y is not an integer, then it is rounded to the nearest integer. The preferred pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm [21], using default parameters (for example, with a gap opening penalty = 10.0, and with a gap extension penalty = 0.5, using score matrix EBLOSUM62). This algorithm is conveniently implemented in the needle tool of EMBOSS [22],
When hybrid polypeptides are used, the individual antigens within the hybrid (i.e., the individual -X- radicals) may be from one or more strains. When n = 2, for example, X2 can come from the same strain as Xi or from a different strain. When n = 3, the strains can be (i) Xi = X2 = X3 (ii) Xi = X2 / X3 (iii) Xi X2 = X3 (iv) Xi X2 = / = X3 or (v) Xi = X3 / X2, etc.
Within group (c), deletions or substitutions may be at the N-terminus and / or at the C-terminus, or may be between the two ends. Thus, truncation is an example of a deletion. Truncations may involve a deletion of up to 40 (or more) amino acids at the N-terminus and / or at the C-terminus. Truncation at the N-terminus can eliminate the leader peptides, for example to facilitate recombinant expression in a heterologous host. Truncation at the C-terminus may eliminate anchor sequences, for example to facilitate recombinant expression in a heterologous host.
In general, when an antigen comprises a sequence that is not identical to a complete S. aureus sequence from the sequence listing (for example, when it comprises a sequence listing sequence with <100% d sequence identity therewith, or when it comprises a fragment thereof), it is preferable in each individual case that the antigen can elicit an antibody that recognizes the respective complete sequence of S. aureus.
Polypeptides used in the invention
The polypeptides used in the invention may take various forms (for example, native, fusions, glycosylated, non-glycosylated, lipidated, non-lipidated, phosphorylated, non-phosphorylated, myristoylated, non-myristoylated, monomeric, multimeric, etc.).
The polypeptides used in the invention can be prepared by various means (e.g., recombinant expression, purification from cell culture, chemical synthesis, etc.). Proteins expressed recombinantly are preferred, particularly for hybrid polypeptides.
The polypeptides used in the invention are preferably provided in a purified or substantially purified form, i.e., substantially free of other polypeptides (e.g., lacking naturally occurring polypeptides), particularly other staphylococcal polypeptides or the host cell, and are generally at least about 50% (by weight), and usually at least about 90% pure, that is, less than about 50% pure. and more preferably less than about 10% (eg 5%) of a composition is other expressed polypeptides. Thus, the antigens in the compositions are separated from the whole organism with which the molecule is expressed.
The term "polypeptide" refers to amino acid polymers of any length. The polymer may be linear or branched, may include modified amino acids, and may be interrupted by non-amino acids. The term also encompasses an amino acid polymer that has been modified naturally or by intervention; for example, formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included are, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids, etc.), as well as other known modifications of the state of the amino acid. art. The polypeptides may appear as simple chains or associated chains.
Although expression of the polypeptides of the invention may occur in a Staphylococcus, the invention will usually employ a heterologous host for expression (recombinant expression). The heterologous host may be prokaryotic (e.g., a bacterium) or eukaryotic. This may be E. coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria (eg, M. tuberculosis), yeast, etc. Compared to the wild-type S. aureus genes encoding the polypeptides of the invention, it is useful to change codons for optimization. The efficiency of expression in such hosts without affecting the encoded amino acids.
admixtures
As mentioned above, the immunogenic compositions according to the invention may comprise one or more adjuvants. Adjuvants which may be used in the invention include, but are not limited to: A. Compositions containing minerals
Mineral-containing compositions suitable for use as adjuvants in the invention include inorganic salts, such as aluminum salts and calcium salts (or mixtures thereof). Calcium salts include calcium phosphate (e.g., "CAP" particles disclosed in reference 23). Aluminum salts include hydroxides and phosphates etc., with salts from any suitable form (e.g., gel, crystalline, amorphous, etc.). Adsorption on its salts is preferred (e.g., all antigens can be adsorbed). Compositions containing minerals may also be formulated as a metal salt particle [24].
Adjuvants known as aluminum hydroxide and aluminum phosphate may be used. These names are conventional, but are used only for practical reasons, since neither is an accurate description of the actual chemical compound that is present (for example, see Chapter 9 of Reference 25). The invention may use any of the "hydroxide" or "phosphate" adjuvants which are generally used as adjuvants. Adjuvants known as "aluminum hydroxide" are generally aluminum oxyhydroxide salts, which are usually at least partially crystalline. Adjuvants known as "aluminum phosphate" are generally aluminum hydroxyphosphates, often also containing a small amount of sulfate (i.e., aluminum sulfate hydroxyphosphate). They can be obtained by precipitation, and the reaction conditions and concentrations during the precipitation influence the degree of substitution of the phosphate for the hydroxyl in the salt.
Fibrous morphology (e.g. as seen on transmission electron micrographs) is typical of aluminum hydroxide adjuvants. The amount of aluminum hydroxide adjuvants is generally about 11, i.e., the adjuvant itself has a positive surface charge at physiological pH. Adsorption capacities between 1.8 and 2.6 mg protein per mg Al +++ at pH 7.4 have been reported for aluminum hydroxide adjuvants.
The aluminum phosphate builders generally have a molar ratio of PO4 / Al of between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably 0.95 + 0.1. Aluminum phosphate will generally be amorphous, particularly for the hydroxyphosphate salts. A typical adjuvant is amorphous aluminum hydroxyphosphate with a molar ratio of PO4 / Al of between 0.84 and 0.92, including 0.6 mg of Al3 + / ml. The aluminum phosphate will generally be particulate (for example, a plate-like morphology as observed on transmission electron micrographs). Typical particle diameters are in the range of 0.5 to 20 μm (eg, about 5 to 10 μm) after any antigen adsorption. Adsorption capacities of between 0.7 and 1.5 mg protein per mg Al +++ at pH 7.4 have been reported for aluminum phosphate adjuvants.
The zero charge point (PCN) of aluminum phosphate is inversely related to the degree of substitution of phosphate with hydroxyl, and this degree of substitution can vary depending on the reaction conditions and the concentration of the reagents used to prepare the salt. by precipitation. PCN is also modified by changing the concentration of free phosphate ions in solution (more phosphate = more acidic PCN) or by adding a buffer like histidine buffer (makes the NCP more basic). The aluminum phosphates used according to the invention will generally have a PCN between 4.0 and 7.0, more preferably between 5.0 and 6.5, for example about 5.7.
The aluminum salt suspensions used to prepare the compositions of the invention may contain a buffer (for example, a phosphate buffer or a histidine buffer or a Tris buffer), but this is not always necessary. The suspensions are preferably sterile and pyrogen-free. A suspension may comprise free aqueous phosphate ions, for example present at a concentration of between 1.0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM. The suspensions may also include sodium chloride. The invention can use a mixture of both aluminum hydroxide and aluminum phosphate. In this case, there may be more aluminum phosphate than hydroxide, for example a weight ratio of at least 2/1 for example> 5/1,> 6/1,> 7/1,> 8 / 1,> 9/1, etc.
The concentration of Al +++ in a composition for administration to a patient is preferably less than 10 mg / ml, for example <5 mg / ml, <4 mg / ml, <3 mg / ml, <2 mg / ml, < 1 mg / ml, etc. A preferred range is between 0.3 and 1 mg / ml. A maximum of 0.85 mg / dose is preferred. B. Emulsions oil in water
Oil-in-water emulsion compositions suitable for use as adjuvants in the invention include squalene emulsions in water, such as MF59 (see Chapter 10 of Reference 25, see also Reference 26). ) and AS03 [27].
Various adjuvants of oil-in-water emulsions are known, and they generally comprise at least one oil and at least one surfactant, the oil / oils and the surfactant (s) being biodegradable (metabolizable) and biocompatible. The emulsion will include submicron droplets of oil, and emulsions with droplets having a diameter of less than 220 nm are preferred because they can be sterilized by filtration. The emulsion comprises one or more oils. Suitable oil / oils include those from, for example, an animal source (such as a fish) or a plant source. The oil is ideally biodegradable (metabolizable) and biocompatible. Sources of vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify nut oils. Jojoba oil can be used, for example obtained from jojoba seeds. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like can be also used. The 6- to 10-carbon fatty acid esters of glycerol and 1,2-propanediol, while not naturally occurring in seed oils, can be prepared by hydrolysis, separation and esterification of appropriate materials. starting with nut oils and seeds. Fats and oils from mammalian milk are metabolizable and so can be used. Procedures for separation, purification, saponification and other means necessary to obtain pure oils from animal sources are well known in the art.
Most fish contain metabolizable oils that can be easily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify many of the fish oils that can be used here. A number of branched chain oils are synthesized by biochemistry in 5-carbon isoprene units and are generally referred to as terpenoids. Preferred emulsions include squalene, a shark liver oil which is a branched unsaturated terpenoid. Squalane, the saturated analogue of squalene, can also be used. Fish oils, including squalene and squalane, are readily available from commercial sources or can be obtained by methods known in the art. Other useful oils are tocopherols, especially in combination with squalene. When the oily phase of an emulsion comprises a tocopherol, any of α, β, γ, δ, ε or □ tocopherols may be used, but α-tocopherols are preferred. Both D-α-tocopherol and DL-α-tocopherol can be used. A preferred α-tocopherol is DL-α-tocopherol. A combination of oils comprising squalene and a tocopherol (e.g., DL-α-tocopherol) can be used. The oil in the emulsion may comprise a combination of oils, for example squalene and at least one other oil.
The aqueous component of the emulsion may be pure water (e.g., water for injection) or it may comprise other components, for example solutes. For example, it may include salts to form a buffer, for example citrate or phosphate salts, such as sodium salts. Typical buffers include: a phosphate buffer; a tampon
Tris; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. A buffered aqueous phase is preferred, and the buffers will generally be in the range of 5 to 20 mM.
In addition to the oil and cationic lipid, an emulsion may comprise a nonionic surfactant and / or a zwitterionic surfactant. Such surfactants include, but are not limited to: polyoxyethylene (commonly called Tween) sorbitan ester surfactants, especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the trade name DOWFAX ™, such as EO / PO linear block copolymers ; octoxynols, which may vary in the number of repeating ethoxy groups (oxy-1,2-ethanediyl), with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; 1 '(octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), triethylene glycol monolauryl ether (Brij 30); polyoxyethylene-9-lauryl ether; and sorbitan esters (commonly known as Span), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Preferred surfactants for inclusion in the emulsion are polysorbate 80 (Tween 80, polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.
Surfactant mixtures may be used, for example mixtures of Tween 80 / Span 85, or mixtures of Tween 80 / Triton-X100. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene (Tween 80) sorbitan monooleate and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Another useful combination includes laureth 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol. Useful mixtures may include a surfactant with a BHL value in the range of 10 to 20 (for example, polysorbate 80, with a BHL of 15.0) and a surfactant with a BHL value in the range of 1 at 10 (for example, sorbitan trioleate, with a BHL of 1.8).
Preferred amounts of oil (% by volume) in the final emulsion are between 2 and 20%, for example 5 to 15%, 6 to 14%, 7 to 13%, 8 to 12%. A squalene content of about 4 to 6% or about 9 to 11% is particularly useful.
The preferred amounts of the surfactants (% by weight) in the final emulsion are between 0.001% and 8%. For example: the polyoxyethylene sorbitan esters (such as polysorbate 80) 0.2 to 4%, in particular between 0.4 and 0.6%, between 0.45 and 0.55%, about 0.5% or between 1.5 and 2%, 1.8 to 2.2%, 1.9 to 2.1%, about 2%, or 0.85 to 0.95%, or about 1%; sorbitan esters (such as sorbitan trioleate) 0.02 to 2%, in particular about 0.5% or about 1%; octyl- or nonyl-phenoxy-polyoxyethanols (such as Triton X-100) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 8%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.
The absolute amounts of oil and surfactant, and their ratio, can vary within wide limits while still forming an emulsion. Those skilled in the art can easily vary the relative proportions of the components to obtain a desired emulsion, but a weight ratio of between 4/1 and 5/1 for the oil and the surfactant is typical (excess oil).
An important parameter for ensuring the immunostimulatory activity of an emulsion, especially in large animals, is the size of the oil droplets (diameter). The most efficient emulsions have a droplet size in the submicron range. Suitably, the droplet sizes will be in the range of 50 to 750 nm. Most desirably, the average size of the droplets is less than 250 nm, for example less than 200 nm, less than 150 nm. The average size of the droplets is conveniently in the range of 80 to 180 nm. Ideally, at least 80% (by number) of the oil droplets of the emulsion are less than 250 nm in diameter, and preferably at least 90%. These droplet sizes can be conveniently obtained by techniques such as microfluidization. Apparatus for determining the average droplet size in an emulsion, and the size distribution, are commercially available. These typically utilize dynamic light scattering techniques and / or single particle optical detection, for example the Accusizer ™ and Nicomp ™ series of instruments available from Partile Sizing Systems (Santa Barbara, USA), or Zetasizer ™ instruments from Malvern Instruments (UK), or Partiele Size Distribution instruments
Horiba Analyzer (Kyoto, Japan).
Ideally, the droplet size distribution (in number) has only one maximum, ie there is only one population of droplets distributed around an average (mode), rather than having two maxima. Preferred emulsions have a polydispersity <0.4, for example 0.3, 0.2, or less.
The specific adjuvants of the oil-in-water emulsions useful in the invention include, but are not limited to: □ A submicron emulsion of squalene, Tween 80, and Span 85. The composition of the emulsion in volume may be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. In terms of weight, these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85. This adjuvant is known as "MF5 9" [28 to 30], as further described in Chapter 10 of Reference 31 and Chapter 12 of Reference 32. The MF59 emulsion comprises advantageously citrate ions, for example 10 mM sodium citrate buffer. An emulsion comprising squalene, a tocopherol, and polysorbate 80. The emulsion may comprise a phosphate buffer solution. These emulsions may have a volume of 2 to 10% of squalene, 2 to 10% of tocopherol and 0.3 to 3% of polysorbate 80, and the weight ratio of squalene / tocopherol is preferably <1 (for example, 0.90) as this can provide a more stable emulsion. Squalene and polysorbate 80 may be present in a volume ratio of about 5/2 or in a weight ratio of about 11/5. Thus, the three components (squalene, tocopherol, polysorbate 80) may be present in a weight ratio of 1068/1186/485 or about 55/61/25. Such an emulsion ("AS03") can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g of DL-α-tocopherol and ml of squalene), then microfluidization of the mixture. The resulting emulsion may have submicron oil droplets, for example with a mean diameter of between 100 and 250 nm, preferably about 180 nm. The emulsion may also comprise a 3-de-O-acylated monophosphoryl lipid A (3d MPL). Another useful emulsion of this type may comprise, per human dose, 0.5 to 10 mg of squalene, 0.5 to 11 mg of tocopherol, and 0.1 to 4 mg of polysorbate 80 [33] for example, in reports discussed above. □ An emulsion of squalene, a tocopherol, and a Triton detergent (eg, Triton X-100). The emulsion may also include a 3d-MPL (see below). The emulsion may contain a phosphate buffer. An emulsion comprising a polysorbate (e.g., polysorbate 80), a Triton detergent (e.g., Triton X-100) and a tocopherol (e.g., α-tocopherol succinate). The emulsion can comprise these three components in a weight ratio of about 75/11/10 (for example, 750 μg / ml of polysorbate 80, 110 μg / ml of Triton X-100 and 100 μg / ml of succinate of α-tocopherol), and these concentrations should include any contribution of these components from the antigens. The emulsion may also include squalene. The emulsion may also include a 3d-MPL (see below). The aqueous phase may contain a phosphate buffer. □ An emulsion of squalane, polysorbate 80 and poloxamer 401 ("Pluronic ™ L121"). The emulsion can be formulated in phosphate buffer solution, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and it has been used with threonyl-MDP in the adjuvant "SAF-1" [34] (0.05 to 1% Thr-MDP, % squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without Thr-MDP, as in the adjuvant "AF" [35] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidization is preferred. An emulsion comprising squalene, an aqueous solvent, a hydrophilic nonionic surfactant of polyoxyethylene alkyl ether (e.g., polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic surfactant (e.g., a sorbitan ester or a mannide ester, such as sorbitan monooleate or "Span 80"). The emulsion is preferably thermoreversible and / or at least 90% of the oil droplets (by volume) with a size less than 200 nm [36]. The emulsion may also include one or more of: alditol; a cryoprotectant (e.g., a sugar, such as dodecylmaltoside and / or sucrose); and / or an alkylpolyglycoside. The emulsion may include a TLR4 agonist [37]. Such emulsions can be lyophilized. □ An emulsion of sgualene, poloxamer 105 and Abil-Care [38]. The final concentration (weight) of these components in adjuvanted vaccines are 5% squalene, 4% poloxamer 105 (pluronic polyols) and 2% Abil-Care 85 (Bis-PEG / PPG-16/16 PEG / PPG -16/16 dimethicone, caprylic / capric triglyceride). An emulsion having from 0.5 to 50% of an oil, 0.1 to 10% of a phospholipid, and 0.05 to 5% of a nonionic surfactant. As described in reference 39, the preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. The submicron sizes of the droplets are advantageous. □ An oil emulsion in submicron water of a non-metabolizable oil (such as a light mineral oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80). Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophilic conjugate (such as GPI-0100, described in reference 40, produced by addition of an aliphatic amine to a deacyl-saponin via the group carboxyl of glucuronic acid), dimethyldioctadecylammonium bromide and / or N, N-dioctadecyl-N, N-bis (2-hydroxyethyl) propanediamine. □ An emulsion in which a saponin (eg, QuilA or QS21) and a sterol (eg, cholesterol) are combined in the form of helicoidal micelles [41]. An emulsion comprising a mineral oil, a nonionic lipophilic ethoxylated fatty alcohol, and a nonionic hydrophilic surfactant (for example, an ethoxylated fatty alcohol and / or a polyoxyethylene-polyoxypropylene block copolymer) [42]. An emulsion comprising a mineral oil, a nonionic hydrophilic ethoxylated fatty alcohol, and a nonionic lipophilic surfactant (for example, an ethoxylated fatty alcohol and / or a polyoxyethylene-polyoxypropylene block copolymer) [42].
In some embodiments, an emulsion may be mixed with one or more antigens extemporaneously at the time of administration, and thus the adjuvant and antigen or antigens may be kept separately in a packaged or dispensed vaccine, ready for use. the final formulation at the time of use. In other embodiments, an emulsion is mixed with an antigen during manufacture, and thus the composition is packaged in a liquid adjuvant form. The antigen will generally be in an aqueous form, so that the vaccine is finally prepared by mixing two liquids. The ratio of the volumes of the two liquids for mixing can vary (for example, between 5/1 and 1/5) but is generally about 1/1. When the concentrations of the components are given in the above descriptions of specific emulsions, these concentrations are generally for an undiluted composition, and thus the concentration after mixing with a solution of the antigen will decrease. C. Saponin Formulations [Chapter 22 of Reference 25]
Saponin formulations may also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponins from the bark of the Quillaia saponaria Molina tree have been widely used as adjuvants. Saponins can also be obtained commercially from Smilax ornata (sarsaprilla), Gypsophilla paniculata (bridal veil), and Saponaria officianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs. QS21 is marketed under the name of Stimulon ™.
The saponin compositions were purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of producing QS21 is disclosed in reference 43. The saponin formulations may also include a sterol, such as cholesterol [44].
Combinations of saponin and cholesterol can be used to form particles called ISCOM (chapter 23 of reference 25). ISCOMs generally comprise a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, ISCOM comprises one or more of QuilA, QHA and QHC. The ISCOMs are further described in references [44 to 46].
Optionally, ISCOMs may lack additional detergent [47].
A description of the development of saponin adjuvant can be found in references 48 and 49. D. Bacterial or Microbial Derivatives
Suitable adjuvants for use in the invention include bacterial or microbial derivatives such as non-toxic enterobacterial lipopolysaccharide (LPS) derivatives, lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives of those -this.
Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-0-deacylated MPL (3dMPL). 3dMPL is a mixture of monophosphoryl lipid A 3-de-O-acylated with 4, 5 or 6 acylated chains. A preferred form of "small particles" of 3-de-O-acylated monophosphoryl lipid A is disclosed in reference 50. Such "small particles" of 3dMPL are small enough to be sterilized by filtration through a membrane of 0. 22 μπι [50]. Other non-toxic LPS derivatives include monophosphoryl lipid A mimetics, such as aminoalkylglucosaminide phosphate derivatives, e.g. RC-529 (see below).
The lipid A derivatives include Escherichia coli lipid A derivatives such as OM-174. OM-174 is described, for example in references 51 and 52.
Immunostimulatory oligonucleotides suitable for use as an adjuvant in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly (dG) sequences have also been shown to be immunostimulatory.
CpGs may include nucleotide modifications / analogs such as phosphorothioate modifications and may be double stranded or single stranded. References -53, 54 and 55 disclose possible analogous substitutions, for example, replacement of guanosine with 2'-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is further described in references 56 to 61.
The CpG sequence may be directed against TLR9, such as GTCGTT or TTCGTT [62]. The CpG sequence may be specific to induce a Th1 immune response, such as a CpG-A ODN, or it may be more specific for the induction of a B cell response, such as a CpG-B ODN. The CpG-A and CpG-B ODNs are described in references 63-65. Preferably, the CpG is a CpG-A ODN.
Preferably, the CpG oligonucleotide is constructed such that the 5 'end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3 'ends to form "immunomers". See, for example, references 62 and 66 to 68.
A useful CpG adjuvant is CpG7909, also known as ProMune ™ (Coley Pharmaceutical Group, Inc.). Another is CpG1826. As an alternative, or in addition, to the use of CpG sequences, TpG sequences may be used [69], and these oligonucleotides may be free of unmethylated CpG motifs. The immunostimulatory oligonucleotide may be rich in pyrimidine. For example, it may comprise more than one consecutive thymidine nucleotide (e.g., TTTT, as described in reference 69), and / or may have a nucleotide composition with> 25% thymidine (e.g.,> 35% ,> 40%,> 50%,> 60%,> 80%, etc.). For example, it may comprise more than one consecutive cytosine nucleotide (e.g. CCCC, as described in reference 69), and / or may have a nucleotide composition with> 25% cytosine (e.g.,> 35% ,> 40%,> 50%,> 60%,> 80%, etc.). These oligonucleotides may be devoid of unmethylated CpG motifs. The immunostimulatory oligonucleotides will generally comprise at least 20 nucleotides. They may comprise less than 100 nucleotides.
A particularly useful adjuvant based around immunostimulatory oligonucleotides is known as IC-31 ™ [70]. Thus, an adjuvant used in the invention may comprise a mixture of (i) an oligonucleotide (e.g., between 15 and 40 nucleotides) including at least one (and preferably multiple) Cpl motifs (i.e. ie, an inosin-linked cytosine to form a dinucleotide), and (ii) a polycationic polymer, such as an oligopeptide (for example, between 5 and 20 amino acids) including at least one (and preferably multiple) tripeptide sequences Lys-Arg-Lys. The oligonucleotide may be a deoxynucleotide comprising a 26-mer 5 '- (IC) 13-3' sequence (SEQ ID NO: 41). The polycationic polymer may be a peptide comprising an 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 42). The oligonucleotide and the polymer may form complexes, for example as described in references 71 and 72.
ADP-ribosylating bacterial toxins and their detoxified derivatives can be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (heat labile enterotoxin "LT" from E. coli), cholera ("CT"), or pertussis ("PT"). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in reference 73 and as parenteral adjuvants in reference 74. The toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits. Preferably, subunit A contains a detoxifying mutation; preferably, the subunit B is not mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins and their detoxified derivatives, particularly LT-K63 and LT-R72, as adjuvants can be found in references 75 to 82. A useful CT mutant is CT-E29H [83]. ]. The reference numeral for amino acid substitutions is preferably based on the A and B subunit alignments of the ADP-ribosylating toxins presented in reference 84, specifically incorporated herein by reference in its entirety.
E. TLR agonists
The compositions may comprise a TLR agonist, i.e. a compound that can exert an agonist effect on a Toll type receptor. Most preferably, a TLR agonist is an agonist of a human TLR. The TLR agonist can activate any of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 or TLR11; preferably it can activate human TLR4 or human TLR7. The agonist activity of a compound against any particular Toll type receptor may be determined by standard assays. Companies such as Imgenex and Invivogen provide cell lines that are stably cotransfected with human TLR genes and NFkB, plus appropriate reporter genes, to measure TLR activation pathways. They are designed for sensitivity, wide range dynamics, and can be used for high throughput screening. The constitutive expression of one or two specific TLRs is typical in such cell lines. See also reference 85.
Many TLR agonists are known in the state of the art, for example, reference 86 describes certain lipopeptide molecules which are TLR2 agonists, references 87 to 90 each describe small molecule agonist classes of the TLR2 agonists. TLR7, and references 91 and 92 describe TLR7 and TLR8 agonists for the treatment of diseases.
A TLR agonist used in the invention ideally comprises at least one adsorption entity. The inclusion of such entities in TLR agonists allows them to be adsorbed on insoluble aluminum salts (for example, by ligand exchange or any other suitable mechanism) and improve their immunological behaviors [93]. Phosphorus-containing adsorption entities are particularly useful, and thus an adsorption entity can include a phosphate, a phosphonate, a phosphinate, a phosphonite, a phosphinite, and the like. Preferably, the TLR agonist comprises at least one phosphonate group.
Thus, in preferred embodiments, a composition comprises a TLR agonist (more preferably, a TLR7 agonist) which comprises a phosphonate group. This phosphonate group allows adsorption of the agonist onto an insoluble aluminum salt [93].
The TLR agonists useful in the invention may comprise a single adsorption entity, or they may comprise more than one, for example between 2 and 15 adsorption entities. Generally, a compound will comprise 1, 2 or 3 adsorption entities.
Useful phosphorus-containing TLR agonists can be represented by the formula (A1):
(Al) wherein:
Rx and RY are independently selected from H and C1-C6 alkyl; X is selected from a covalent bond, O and NH; Y is selected from a covalent bond, O, C (O), S and NH; L represents a linker, for example selected from C 1 to C 6 alkylene, C 1 to C 6 alkenylene, arylene, heteroarylene, C 1 to C 6 alkyleneoxy and - ((CH 2) pO) q (CH 2) p each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C1-C4 alkyl, -0P (O) (OH) 2 and -P (O) (OH) 2; each p is independently selected from 1, 2, 3, 4, 5 and 6; q is selected from 1, 2, 3 and 4; n is selected from 1, 2 and 3; and A represents a TLR agonist entity.
In one embodiment, the TLR agonist according to the formula (A1) is as follows: Rx and RY are H; X represents O; L is selected from C1 to C6 alkylene and - ((CH2) p0) q (CH2) p- each optionally substituted with 1 to 2 halogen atoms; p is selected from 1, 2 and 3; q is selected from 1 and 2; and n is 1. Thus, in these embodiments, the adsorption entity comprises a phosphate group. Other useful TLR agonists of formula (A1) are disclosed on pages 6 to 13 of reference 94.
The compositions may include an imidazoquinolone compound, such as Imiquimod ("R-837") [95,96], Resiquimod ("R-848") [97], and the like; and their salts (for example, hydrochloride salts). Further details regarding immunostimulatory imidazoquinolines can be found in references 98-102.
The compositions may comprise a TLR4 agonist, and most preferably a human TLR4 agonist. TLR4 is expressed by cells of the innate immune system, including dendritic cells and traditional macrophages [103]. Triggering via TLR4 induces a signaling cascade that utilizes both the MyD88- and TRIF-dependent pathways, leading to the activation of NF-κΒ and IRF3 / 7, respectively. Activation of TLR4 generally induces robust IL-12p70 production and strongly enhances Th1-like cellular and humoral immune responses.
Various useful TLR4 agonists are known in the art, many of which are endotoxin or lipopolysaccharide (LPS) analogs. For example, the TLR4 agonist may be: 3d-MPL (i.e., 3-O-deacylated monophosphoryl lipid A present in GSK adjuvant "AS04" along with other details in references 104 to 107, glucopyranosyl lipid A (GLA) [108] or its ammonium salt, aminoalkylglucosaminide phosphate, such as RC-529 or CRX-524 [109 to 111]; E5564 [112,113] or a compound of formula I, II or III as defined in reference 114, or one of its salts, such as compounds "ER 803058", "ER 803732", "ER 804053", "ER 804058", ER 804059, ER 804442, ER 804680, ER 803022, ER 804764 or ER 804057 (also known as E6020) .The invention is particularly useful when using agonists human TLR7, such as a compound of formula (K) These agonists are discussed in detail in reference 115:
(K) wherein: R1 is H, C1-C6 alkyl, -C (R5) 20H, -L1R5, -L1R6, -L2R5, -L2R6, -OL2R5, or -OL2R6; L1 represents-C (O) - or -O-; L 2 represents a C 1 -C 6 alkylene, C 2 -C 6 alkenylene, arylene, heteroarylene or - ((CR 4 R 4) pO) q (CH 2) p - group, where the C 1 -C 6 alkylene and C 2 -C 6 alkenylene groups of L 2 are optionally substituted with 1 to 4 fluoro groups; each L3 is independently selected from C1 to C6 alkylene and - ((CR4R4) p0) q (CH2) P-, wherein the C1 to C6 alkylene group of L3 is optionally substituted with 1 to 4 fluoro groups; L4 represents an arylene or heteroarylene group; R2 is C1-C6alkyl; R3 is selected from C1-4alkyl, -L3R5, -L1, -L3R7, -L3L4L3R7, -L3L4R5, -L3L4L3R5, -OL3R5, -OL3R7, -OL3L4R7, -OL3L4L3R7, -OR8, -OL3L4R5, - OL3L4L3R5 and -C (R5) 20H; each R4 is independently selected from H and fluoro; R5 is -P (O) (OR9) 2; R6 is -CF2P (O) (OR9) 2 or -C (O) OR10; R7 is -CF2P (O) (OR9) 2 or -C (O) OR10; R8 is H or C1-C4alkyl; each R9 is independently selected from H and C1-C6 alkyl; R10 is H or C1-C4alkyl; each p is independently selected from 1, 2, 3, 4, 5 and 6, and q is 1, 2, 3 or 4.
The compound of formula (K) is preferably of formula (K '):
(K1) wherein: P1 is selected from H, C1-C6 alkyl optionally substituted with COOH and -Y-L-X-P (O) (ORX) (OR4); P2 is selected from H, C1-C6 alkyl, C1-C5 alkoxy and -Y-L-X-P (O) (ORX) (OR4); with the proviso that at least one of P1 and P2 is -Y-L-X-P (O) (ORX) (ORY); RB is selected from H and C1-C6 alkyl;
Rx and RY are independently selected from H and C1-C6 alkyl; X is selected from a covalent bond, O and NH; Y is selected from a covalent bond, O, C (O), S and NH; L is selected from covalent C 1 -C 6 alkylene, C 1 -C 6 alkenylene, arylene, heteroarylene, C 1 -C 6 alkylenoxy and - ((CH 2) PO) q (CH 2) p- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C1-C4 alkyl, -OP (O) (OH) 2 and -P (O) (OH) 2; each p is independently selected from 1, 2, 3, 4, 5 and 6; and q is selected from 1, 2, 3 and 4.
In certain embodiments of formula (K '): P1 is selected from C1-6alkyl optionally substituted with COOH and -Y-L-X-P (O) (ORX) (OR4); P2 is selected from C1-C6 alkoxy and -Y-L-X-P (O) (ORX) (OR4); RB represents a C1-C6 alkyl group; X represents a covalent bond; L is selected from C1 to C6 alkylene and - ((CH2) PO) q (CH2) p- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C1-C4 alkyl, -OP (O) (OH) 2 and -P (O) (OH) 2; each p is independently selected from 1, 2 and 3; q is selected from 1 and 2.
A preferred compound of formula (K) for use in which the invention is 3- (5-amino-2- (2-methyl-4- (2- (2 - (2-phosphonoethoxy) ethoxy) ethoxy ) phenethyl) benzo [f] [1,7] -naphthyridin-8-yl) propanoic acid, or "Kl" compound:
(Kl)
This compound can be used in free base form or in the form of a pharmaceutically acceptable salt, for example an arginine salt [116], F. Microparticles
Microparticles may also be used as adjuvants in the invention. The microparticles (i.e., a particle of -100 nm to ~ 150 μm in diameter, more preferably ~ 200 nm to -30 μm in diameter, and most preferably -500 nm to -10 pm of diameter) formed from materials that are biodegradable and non-toxic (eg, a poly (α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with a poly (lactide -co-glycolide) are preferred, optionally treated to have a negatively charged surface (e.g., with SDS) or a positively charged surface (e.g., with a cationic detergent, such as CTAB).
Combinations of adjuvants
The individual adjuvants listed above may also be included in combinations. For example, a combination of aluminum hydroxide and aluminum phosphate adjuvant may be used. Similarly, a combination of aluminum phosphate and 3dMPL can be used.
A particularly preferred combination of adjuvants is an insoluble metal salt (e.g., an aluminum salt, such as an aluminum hydroxide) and a TLR agonist (e.g., a human TLR7 agonist, such as the compound "K2 Identified above), as disclosed in references 3 and 93. The TLR agonist is preferably adsorbed on the metal salt, and the antigen / antigens of S. aureus can also be adsorbed on the metal salt.
A composition comprising a TLR agonist of the invention adsorbed on a metal salt may also comprise a buffer (e.g., a phosphate buffer or a histidine buffer or a Tris buffer). However, when such a composition comprises a phosphate buffer, it is preferable that the concentration of phosphate ions in the buffer is less than 50 mM, for example <40 mM, <30 mM, <20 mM, <10 mM, or <5 mM mM, or between 1 and 15 mM. A histidine buffer is preferred, for example, between 1 and 50 mM, between 5 and 25 mM, or about 10 mM.
A composition may comprise a mixture of both aluminum oxyhydroxide and aluminum hydroxyphosphate, and a TLR agonist may be adsorbed on one or both of these salts.
As mentioned above, a maximum of 0.85 mg / dose of Al +++ is preferred. Because the inclusion of a TLR agonist can enhance the adjuvant effect of the aluminum salts, then the invention advantageously allows lower amounts of Al +++ per dose, and thus a composition can conveniently include 10 and 250 μg Al +++ per unit dose. Current pediatric vaccines generally include at least 300 μg Al +++. In terms of concentration, a composition may have a concentration of Al +++ between 10 and 500 μg / ml, for example between 10 and 300 μg / ml, between 10 and 200 μg / ml, or between 10 and 100 μg / ml.
In general, when a composition comprises both a TLR agonist and an aluminum salt, the weight ratio of the agonist to Al +++ will be less than 5/1, for example less than 4/1, less than 3 / 1, less than 2/1, or less than 1/1. Thus, for example, with a concentration of Al +++ of 0.5 mg / ml, the maximum concentration of TLR agonist will be 1.5 mg / ml. But higher or lower rates can be used.
When a composition comprises a TLR agonist and an insoluble metal salt, it is preferable that at least 50% (by weight) of the agonist in the composition is adsorbed on the metal salt, for example> 60%, > 70%,> 80%,> 85%,> 90%,> 92%,> 94%,> 95%,> 96%,> 97%,> 98%,> 99%, or even 100%.
Thus, in one embodiment, the invention uses an immunogenic composition comprising: an aluminum hydroxide adjuvant; A TLR7 agonist of formula (K), such as compound K2; A first polypeptide comprising SEQ ID NO: 6, or a modified amino acid sequence which differs from SEQ ID NO: 6 by up to 5 single amino acid changes provided that the modified sequence can elicit antibodies that bind to a polypeptide consisting of SEQ ID NO: 6; A second polypeptide comprising SEQ ID NO: 13, or a modified amino acid sequence which differs from SEQ ID NO: 13 by up to 5 single amino acid changes provided that the modified sequence can elicit antibodies which bind to a polypeptide consisting of SEQ ID NO: 13; A third polypeptide comprising SEQ ID NO: 31, or a modified amino acid sequence which differs from SEQ ID NO: 31 by up to 5 single amino acid changes provided that the modified sequence can elicit antibodies that bind to a polypeptide consisting of SEQ ID NO: 31; A fourth polypeptide comprising SEQ ID NO: 33, or a modified amino acid sequence which differs from SEQ ID NO: 33 by up to 5 single amino acid changes provided that the modified sequence can elicit antibodies that bind to a polypeptide consisting of SEQ ID NO: 33, wherein the TLR7 agonist and / or at least one of the polypeptides are adsorbed on the aluminum hydroxide adjuvant.
For example, as is explained in more detail elsewhere in this document: the first polypeptide may comprise SEQ ID NO: 34; the second polypeptide may comprise SEQ ID NO: 13; the third polypeptide may comprise SEQ ID NO: 40; and the fourth polypeptide may comprise SEQ ID NO: 36. Thus, the composition may use a mixture of four polypeptides having SEQ ID NOs: 37, 27, 38 and 39. General
The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the abilities of those skilled in the art. These techniques are fully explained in the literature. See, for example, references 117 to 124, etc.
The numbering "GI" is used above. A GI number, or "Genlnfo Identifier", is a series of digits assigned consecutively to each sequence record processed by the NCBI when sequences are added to its databases. The GI number has no relation to the accession number of the record of the sequence. When a sequence is updated (for example, for a correction, or to add other annotations or information) then it receives a new GI number. Thus, the sequence associated with a given GI number is never changed.
When the invention relates to an "epitope", this epitope may be a B cell epitope and / or a T cell epitope. Such epitopes may be empirically identified (e.g., using PEPSCAN [125,126] or methods similar), or they can be predicted (for example, using the Jameson-Wolf antigenic index [127], matrix-based approaches [128], ΜΆΡΙΤΟΡΕ [129], TEPITOPE [130,131], neural networks [ 132], OptiMer and EpiMer [133, 134], ADEPT [135], Tsites [136], hydrophilicity [137], antigenic index [138] or methods disclosed in references 139 to 143, etc.) . Epitopes are the parts of an antigen that are recognized by and bind to antigen binding sites of antibodies or T cell receptors, and they may also be called "antigenic determinants".
When an "area" of antigen is omitted, this may involve the omission of a signal peptide, a cytoplasmic domain, a transmembrane domain, an extracellular domain, etc.
The term "comprising" includes "including" as well as "consisting of", for example a composition "comprising" X may consist exclusively of X or it may comprise something else, for example X + Y.
The term "about" in relation to a numerical value x is optional and means, for example, x + 10%.
References to percent sequence identity between two amino acid sequences mean that, when aligned, these amino acid percentages are identical by comparing the two sequences. This alignment and the percentage of homology or sequence identity can be determined using known state-of-the-art software programs, for example those described in section 7.7.18 of reference 144. An alignment preferred is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap opening penalty of 12 and a gap extension penalty of 2, the BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in reference 145.
The phosphorus-containing adjuvants used in the invention can exist in a number of protonated and deprotonated forms depending on the pH of the immediate environment, for example the pH of the solvent in which they are dissolved. Therefore, although a particular form can be illustrated, it is intended that these illustrations are merely representative and not limiting to a specific protonated or deprotonated form. For example, in the case of a phosphate group, it has been illustrated as -0P (O) (OH) 2 but the definition includes the protonated forms [O (O) (OH) (OH)] + and - [OP (O) (OH) 2] 2+ that can exist under acidic conditions and the deprotonated forms ~ [OP (0) (OH) (O)] "and [0P (0) (0) 2] 2_ which can exist under basic conditions.
The compounds may exist in the form of pharmaceutically acceptable salts. Thus, the compounds (e.g., adjuvants) can be used in the form of their pharmaceutically acceptable salts, i.e., physiologically or toxicologically tolerable salts (which includes, where appropriate, salts thereof). addition of pharmaceutically acceptable bases and pharmaceutically acceptable acid addition salts).
The term "substantially" does not exclude "completely", for example, a composition that is "substantially devoid" of Y may be completely devoid of Y. When necessary, the term "substantially" may be omitted from the definition. of the invention.
Embodiments of the invention
Template system
Bioluminescent strains of S. aureus were used to infect mice in the lateral caudal vein (iv) and the progression of infection was monitored for at least 1 week after injection using an IVIS 100 ™ machine (Perkin Eimer). The i.v. route of infection was chosen by trying to mimic a haematogenous source of infection. One day after the injection, S. aureus reached the knee joints and was able to establish a local infection that persisted for at least 7 days. The bioluminescence values showed a very good correlation when compared to colony forming units (CFU) counted after knee joint washes on the last day of the experiment. Various different strains had the ability to reach the knee joint. Finally, the presence of bacteria in the knee joints after i.v. inoculation was confirmed using confocal microscopy. The in vivo imaging approach was used to better understand whether bacteria that had reached the joint could invade bone tissue (thus potentially causing osteomyelitis). The mice were again injected i.v. with a bioluminescent strain and followed for 1 week after infection. The IVIS spectrum-CT ™ system, which combines a camera capable of acquiring bioluminescent signals and a tomography system that can scan sections of the animal body, revealed that bioluminescent bacteria were not only present in the area of the body. knee joint but also in the tibia.
These findings suggested that S. aureus-mediated hematogenous infection could cause arthrosynovitis and osteomyelitis. Histopathological analysis of tissues obtained from infected mice showed that the normal architecture of the bones and joints was cleared by the presence of mixed inflammatory cells (with a prevalence of neutrophils and macrophages) compared to control animals. In particular, the tibia was destroyed and granulomas / abscesses were observed. Periosteal inflammation was severe, causing thickening of the periosteal tissue. Inflammation was evident in the synovium, which appeared severely thickened. It was possible to observe elongated cells that formed a wall around foci of inflammation, composed of neutrophils and macrophages. In the inflammatory focus, aggregates of pinkish eosinophilic slightly hyaline material consistent with the presence of bacteria have been observed. Indeed, immunohistochemical staining demonstrated that S. aureus was present in bone abscesses and that a neutrophil wall was built around these structures.
To develop an infection-specific arthrosynovitis mouse model for long-term in vivo studies, the Newman strain was chosen because it has been widely used in research around the world as being well-suited to animal studies. The first objective was to find a dose high enough to homogeneously infect one group of mice at both the systemic level and in the knee joints, but without inducing severe disease and / or death. The mice were infected intravenously with doses in the range of 1 x 107 to 1 x 104 CFU / mouse and followed for 3 weeks after infection. The kidneys of the infected animals were removed and washings of the knee joints were performed. The mice treated with the dose of 1 x 107 showed obvious signs of disease and, as early as four days after infection, the mice began to die, while low doses of 1 x 105 to 1 x 104 produced a low or even absent infection. For these reasons, a dose of 1 x 106 was chosen for our purposes since no animal died during the time. and all of them were significantly infected at both the systemic and the local levels.
The animals were inoculated with 106 CFU and the progression of S. aureus spread was followed for 90 days after infection. CFU numbers in the kidneys have been used as a marker of systemic infection and CFU numbers in joint lavage fluids as a marker of local infection in the knee joints. CFUs in the blood were used as a second possible marker of systemic infection since it was reported that during the chronization of the pathology, S. aureus could escape organs and exploit the bloodstream to spread further .
The peak of infection appeared to be reached between 1 and 2 weeks after inoculum when CFUs recovered in the joints and kidneys were maximal. Starting at 1 month after infection, CFUs began to decrease in both the kidneys and the knee joints when the infection was controlled by the host to some extent. On the other hand, CFU in the blood appeared to increase over time reaching the highest rate at the latest time point, 90 days after infection.
To better understand if the infection has been controlled or has become chronic, it has been used to color H & E cuts of the knee joints. These analyzes demonstrated that after intravenous infection with S. aureus, the mice developed arthrosynovitis and osteomyelitis and that both an acute phase (7-14 days) and a subacute / chronic phase (90 days) could be clearly detected.
The humoral response over time both in situ and systemically was also analyzed. IgM and IgG against Hla were titrated both in the sera and in the lavage fluids of the mice infected at each time point as a marker of progression of the infection. Anti-Hla IgM peaked seven days after bacterial injection in both the knee joints and serum, while IgG levels peaked at 14 and 30 days post-inoculum. joint and serum, respectively, subsequently decreasing in the knee washings, this accompanied by a decrease in CFU, while remaining almost stable over time in the sera.
Because cytokine secretion can be considered a reliable indicator of immune activation, cytokine levels at different time points after infection have been estimated. In order to understand whether the local inflammatory response was specific and whether there were differences with the systemic response, cytokine levels were measured both in the serum and directly in situ. In the knee joints, all cytokines that increased (IL-Ια, IL-1β, IL-6, IL-10, IL-12 (p40), IL-17, eotaxin, G-CSF, GM-CSF, IFNγ, KC, MCP-1, MIP-1α, MIP-1β, RANTES) compared to time 0 showed an expression pattern that correlated somewhat with the observed trend for variation in the number of CFUs during infection. Some of the cytokines found in the knee joint washings correlated well with the CFUs recovered at this site at different time points. In particular, for most of them, this correlation could be detected 14 days after infection (IL-6, IL-12 (p40), IL-13, IL-17, G-CSF, KC, MCP -1, MIP-1ß, RANTES), whereas for IL-12 (p40) and ΜΙΡ-Ια, a good correlation was also found at 30 and 90 days post-infection, respectively.
Immune cell populations recruited from the knee joint space and present in the blood of infected animals were also monitored. As early as 1 week after infection, the immune cells counted in the knee joints of infected animals were 100 times higher than those recovered in uninfected mice. The next step was staining of these cells with specific antibodies directed against different cell markers to better characterize multiple cell populations. Neutrophils, macrophages (only in knee joint lavage fluids), monocytes, dendritic cells, eosinophils (only in the blood), B cells, CD4 + and CD8 + T cells, NK cells have all summers detected.
Locally, in the knee joints, cells belonging to the myeloid lineage increased starting from three days after infection with an obvious peak 1-2 weeks after inoculum. Neutrophils were the most abundant population increasing up to 1000-fold compared to baseline, but also monocytes and macrophages showed a pronounced increase (10 to 50-fold). A similar situation, although less obvious, was observed when myeloid cells were stained and enumerated in the blood. In this case, a decrease in eosinophils, especially immediately after infection, was observed. While lymphoid cells decreased in the blood beginning 3 days after injection with S. aureus, they increased in the joints. This was true for all lineages but not for B lymphocytes which significantly decreased in numbers also in the joints. During the chronic phase, the cell composition in both the blood and the knee joint returned to baseline.
Antibiotic treatment (for comparison)
In humans, antibiotics are the first and most commonly used treatment for septic arthritis and osteomyelitis due to S. aureus. However, sometimes they are not able to eradicate the bacteria from these sites because of low vascularization of bones and joints and increased antibiotic-resistant forms of S. aureus. In order to investigate whether conventional antibiotic therapy will be able to reduce bacterial burden in the mouse model, i.v. infected mice with about 2 x 106 Newman strain bacteria were treated with i.p. with 100 μg / g of ampicillin weight 24 and 48 hours after infection [146]. Five days later, they were sacrificed, kidneys were removed and knee joints were washed.
A significant reduction in CFU numbers was observed in the kidneys, indicating that antibiotic treatment had some positive effect on the containment of systemic infection. On the contrary, there was no reduction in CFU in the knee joints, which could be expected on the basis of reported difficulties in successfully treating such localized infections with antibiotics.
When the cell composition in the knee joint washings was analyzed, no substantial differences were observed except for B cells, which appeared to be more abundant in animals treated with ampicillin. .
Vaccination
A tetravalent vaccine with the antigens consisting of SEQ ID NO: 7, 8, 27 and 32 was used to immunize mice. Different groups of mice received two immunizations at an interval of two weeks, either with the tetravalent vaccine (10 μg per antigen per dose) adjuvanted with aluminum hydroxide, or with the adjuvant alone as a negative control. Ten days after the second dose, the animals were infected with the Newman strain, approximately 2 x 106 CFU / mouse intravenously. They were sacrificed 7 days later and then the kidneys, blood, serum were taken together with knee joint washings for microbiological and immunological analysis. Immunization produced a significantly lower bacterial load in both the knee joints (approximately 2 log lower, p <0.05, Mann-Whitney t test) and in the kidneys (approximately 1.5 log lower; <0.05), suggesting that immunization is more effective than a conventional therapeutic approach (ie, antibiotic therapy) by reducing S. aureus-mediated infection in the joints.
A similar experiment was performed using S. aureus strain Xen36. Immunization with the tetravalent vaccine substantially reduced the bacterial burden in the knee joints (again about 2 log lower). The results were confirmed by bioluminescence tests. Further analyzes were then performed in order to understand the possible mechanism (s) of action of the vaccine. For this purpose, the humoral and cellular immune components were analyzed. Antibody titers against each single vaccine antigen in the knee joint washings were measured, and IgG titers against the four antigens were observed (p <0.0001, Mann-Whitney U test). ), demonstrating that all antigens induced seroconversion in immunized animals and that measurable levels of antibody could be detected in the knee joints.
The production of functional antibodies in response to the tetravalent vaccine has been confirmed by the use of rabbit sera in passive mouse immunization. The mice were passively immunized with sera from rabbits vaccinated with either the tetravalent / adjuvant vaccine or the adjuvant alone as a negative control, and infected intravenously with S. aureus. The log numbers of CFUs from the knee joint washings showed that the bacterial load in mice immunized with rabbit sera vaccinated with the tetravalent vaccine was lower than the mice immunized with the control sera (p <0.05). Mann-Whitney U test).
In addition, the immune cells were analyzed by comparing the results obtained for the actively vaccinated animals with those obtained for the negative controls. Fewer dead immune cells were found in the knee joint lavage fluids of mice immunized with the combination and, although the number of total cells recovered was more or less the same in both samples, the number of neutrophils recruited was lower in the immunized group indicating the lower state of general inflammation. B cells were preserved in the knee joints after immunization, implying that vaccination could increase the number of cells recruited and / or protect them from S. aureus-mediated toxicity.
Cytokine content in knee joint washings from vaccinated mice was also analyzed. IL-1alpha, IL-1beta, IL-17, G-CSF and MIP-lalpha levels were all reduced in mice vaccinated with the tetravalent / adjuvant vaccine compared to mice vaccinated with the alum adjuvant. alone.
It should be understood that the invention has been described by way of example only and that modifications may be made while remaining within the scope and spirit of the invention.

权利要求:
Claims (15)
[1]
A method of preventing or treating S. aureus infection of bones and joints of a mammal by administering to the mammal at least one antigen selected from the group consisting of: EsxA; EsxB; FhuD2; StaOll; and Hla.
[2]
2. Use of one or more antigens to immunize a mammal to prevent or treat a S. aureus infection of its bones and joints, wherein the antigen / antigens is / are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOll; and Hla.
[3]
3. Use of at least one antigen in the manufacture of a medicament for the prevention or treatment of S. aureus infection of bones and joints of a mammal, where the antigen / antigens is / are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOll; and Hla.
[4]
4. One or more antigen (s) for use in a method of preventing or treating S. aureus infection of bones and joints of a mammal by administering the antigen or antigens to the mammal, wherein the antigen / antigens is / are selected from the group consisting of: EsxA; EsxB; FhuD2; StaOll; and Hla.
[5]
A method, use, or antigen (s) according to any one of the preceding claims, wherein the mammal receives a composition comprising 2, 3, 4 or 5 of EsxA, EsxB, FhuD2, StaOll, and / or Hla; for example, including the five of EsxA, EsxB, FhuD2, StaOll, and Hla.
[6]
The method, use, or antigen (s) according to any one of the preceding claims, wherein: the EsxA antigen elicits antibodies in the mammal that recognize SEQ ID NO: 1; the EsxB antigen elicits antibodies in the mammal that recognize SEQ ID NO: 2; the FhuD2 antigen elicits antibodies in the mammal that recognize SEQ ID NO: 3; StaOll antigen elicits antibodies in the mammal that recognize SEQ ID NO: 4; and / or the Hla antigen elicits antibodies in the mammal that recognize SEQ ID NO: 5.
[7]
A method, use, or antigen (s) according to any one of the preceding claims, wherein the mammal receives a composition comprising (i) a detoxified Hla and / or a fusion polypeptide of EsxA and EsxB.
[8]
A method, use, or antigen (s) according to any one of the preceding claims, wherein the mammal receives a composition comprising all four of: (i) a single polypeptide comprising both an EsxA antigen and an EsxB antigen, for example, comprising SEQ ID NO: 31; (ii) an FhuD2 antigen, for example, comprising SEQ ID NO: 6; (iii) a StaOll antigen, for example, comprising SEQ ID NO: 33; and (iv) an H35L mutant form of Hla, for example comprising SEQ ID NO: 13; The four of: (i) a first polypeptide having the amino acid sequence SEQ ID NO: 32; (ii) a second polypeptide having the amino acid sequence SEQ ID NO: 7; (iii) a third polypeptide having the amino acid sequence SEQ ID NO: 8; and (iv) a fourth polypeptide having the amino acid sequence SEQ ID NO: 27; or all four of: (i) a first polypeptide having the amino acid sequence SEQ ID NO: 38; (ii) a second polypeptide having the amino acid sequence SEQ ID NO: 37; (iii) a third polypeptide having the amino acid sequence SEQ ID NO: 39; and (iv) a fourth polypeptide having the amino acid sequence SEQ ID NO: 27.
[9]
The method, use, or antigen (s) according to any one of the preceding claims, wherein the mammal receives a composition comprising: (i) EsxA, EsxB, FhuD2, StaOll, and / or Hla; and (ii) an adjuvant, such as an aluminum salt, for example, aluminum hydroxide and / or aluminum phosphate. ✓
[10]
The method, use, or antigen (s) according to claim 9, wherein the adjuvant comprises a human TLR agonist, such as a TLR7 agonist, for example a compound of formula (K) such as 3- ( 5-amino-2- (2-methyl-4- (2- (2- (2-phosphonoethoxy) ethoxy) ethoxy) phenethyl) benzo [f] [1,7] naphthyridin-8-yl) propanoic acid.
[11]
The method, use, or antigen (s) according to any one of claims 9 and 10, wherein the adjuvant comprises a human TLR agonist adsorbed on an aluminum salt.
[12]
The method, use, or antigen (s) according to any one of the preceding claims, wherein the mammal is a human.
[13]
13. A method, use, or antigen (s) according to any one of the preceding claims, for the treatment or prevention of (i) osteomyelitis; (ii) septic arthritis; or (iii) a prosthetic joint infection.
[14]
The method, use, or antigen (s) according to any one of the preceding claims, wherein the mammal has a bone or joint prosthesis, or is a recipient provided for a bone or joint prosthesis.
[15]
The method, use, or antigen (s) according to any one of the preceding claims, wherein the mammal receives the antigen (s) by injection and / or the mammal receives the antigen (s) together with an antibiotic.

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同族专利:

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公开号 | 公开日

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WO2015140108A1|2015-09-24|

引用文献:

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CS277676B6|1991-06-13|1993-03-17|Lekarska Fakulta Up|Vaccine for the prevention, prophylaxis and therapy of infectious diseases of bones|

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法律状态:
2018-03-07| FG| Patent granted|Effective date: 20160325 |

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2018-03-07| MM| Lapsed because of non-payment of the annual fee|Effective date: 20170331 |

优先权:

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申请号 | 申请日 | 专利标题

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EP14160390|2014-03-17|

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EP14160390.2|2014-03-17|

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