MUTANT STAPHYLOCOCCAL ANTIGENS

专利摘要:
A mutant SpA with decreased affinity for the Fc □ part of human IgG is provided. Immunization with EsxA, EsxB, FhuD2, Sta011, Hla and said mutant SpA provides surprising results in a model of renal abscess of S. aureus infection.
公开号:BE1022744B1
申请号:E2015/5176
申请日:2015-03-24
公开日:2016-08-29
发明作者:Fabio Bagnoli；Luigi Fiaschi；Maria Scarselli
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

MUTANT STAPHYLOCOCCAL ANTIGENS
This application claims the benefit of European Patent Applications 14161861.1 (filed March 26, 2014) and 14192913.3 (filed November 12, 2014), all of which is incorporated herein by reference for all purposes.
Technical area
This invention relates to immunization against S. aureus infection.
11 art background
Staphylococcus aureus is a gram-positive spherical bacterium. Annual mortality in the United States exceeds that of all other infectious diseases, including HIV / AIDS, and S. aureus is the leading cause of infections in the bloodstream, lower respiratory tract, skin and tissues soft. It is also the predominant cause of bone infections worldwide, and these conditions are painful, debilitating and difficult to treat.
The treatment of S. aureus is increasingly difficult because of the development of antibiotic resistance by many strains of S. aureus. S. aureus methicillin-resistant (MRSA) is found in more than half of all community-acquired and nosocomial infections. Recent years have seen the emergence of MRSA strains that are also resistant to vancomycin, the antibiotic of last resort, and are essentially intractable.
There is currently no licensed vaccine. A vaccine based on a mixture of surface polysaccharides from bacterial types 5 and 8, StaphVAX ™, failed to reduce infections compared to the placebo group in a phase III clinical trial. Similarly, the V710 [1] vaccine, based on IsdB antigen [2], failed to reduce the rate of postoperative S. aureus infections [3].
The need for a vaccine is particularly acute because of the problem of antibiotic resistance and the fact that S. aureus infection does not provide immunity against future infection because of its well-developed immune evasion capabilities. . The immune evasion properties of S. aureus in turn make the development of effective vaccines more difficult. The mechanisms of immune evasion are not fully understood, but they are at least partly due to the staphylococcal protein A (SpA), a surface molecule of S. aureus that binds to immunoglobulin (Ig) Fc. and the Fab part of VH3 B cell receptors. The interaction of SpA with B-cell receptors leads to clonal expansion and subsequent cell death of B-cell populations, leading to the removal of the adaptive immune response. SpA binding to Ig Fc interferes with opsonophagocytic clearance of staphylococci by polynuclear leukocytes.
Mutant forms of SpA with reduced affinity for immunoglobulins have been developed. WO 2011/005341 discloses SpA with point mutations in each of the five Ig binding domains that reduce the ability of the protein to bind to IgGs.
Reference 4 discloses various approaches to provide improved vaccines against S. aureus, including two combinations of preferred immunogens called "Combo-1" and "Combo-2". "Combo-1" included five antigens EsxA, EsxB, a mutant Hla, FhuD2, and StaOll, while "Combo-2" used an IsdA fragment in place of the mutant Hla. Both of these combinations were tested with aluminum hydroxide adjuvants and increased the median survival time in an infection mouse model compared to the single buffer or IsdB antigen. "Combo-1" was also tested with aluminum hydroxide adjuvant and TLR7 agonist, and the addition of the TLR7 agonist improved the responses [5].
Despite positive results with "Combo-1" and "Combo-2", there remains a need for other and improved compositions for immunization against S. aureus.
Disclosure of the invention
Protein A (SpA) (SEQ ID NO: 43), a cell surface anchored surface protein of Staphylococcus aureus, is responsible for the bacterial escape of innate and adaptive immune responses. Protein A binds immunoglobulins at their Fc portion, interacts with the B-cell receptor VH3 domain that inappropriately stimulates B-cell proliferation and apoptosis, binds to Al domains of von Willebrand factor to activate the intracellular coagulation, and also binds to TNF receptor 1 to contribute to the pathogenesis of staphylococcal pneumonia. Because protein A captures immunoglobulins and displays toxic attributes, the possibility that this surface molecule can function as a vaccine in humans has not been rigorously pursued. Here, the inventors demonstrate that variants of protein A that are no longer able to bind to immunoglobulins, which are thereby rid of their toxigenic potential, i.e., are non-toxigenic, stimulate immune responses humoral that protect against staphylococcal disease.
Therefore, the invention provides mutant SpA antigens as described below. Said mutants preferably have decreased affinity for the FcD portion of human IgGs relative to unmodified SpA. The mutants may also have decreased affinity, relative to the unmodified SpA, for the VH3-containing human B cell receptor Fab part.
The inventors have discovered that the known "Combo-1" vaccine can be improved by adding a mutant Staphylococcal A (SpA) protein that has been modified to decrease its affinity for the FcDI part of human IgGs and for the Fab part of lymphocyte receptors. B containing VH3. This additional antigen increases the protective efficacy of the combination and provides surprising results in a model of renal abscess. None of the combinations of antigens tested in reference 4 included SpA antigen.
Therefore, the invention further provides an immunogenic composition comprising the antigens EsxA, EsxB, FhuD2, StaOll, and Hla, characterized in that the composition further comprises a mutant SpA antigen, wherein the mutant has a decreased affinity, relative to with unmodified SpA, for the FcD portion of human IgGs and for the Fab portion of human B-cell receptors containing VH3. The invention also provides an immunogenic composition comprising: (i) at least one antigen selected from the group consisting of EsxA, EsxB, FhuD2, StaOll, and Hla antigens; and (ii) a mutant SpA antigen that exhibits decreased affinity, relative to the unmodified SpA, for the FcD portion of human IgGs and for the Fab portion of the VH3-containing human B-cell receptors. Thus, 1, 2, 3, 4 or preferably EsxA, EsxB, FhuD2, StaOll, and Hla can be used in combination with the mutant SpA.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.
For convenience, the meaning of certain terms and phrases used in the specification, examples and claims is provided.
Antigens of S. aureus The invention relates inter alia to a mutant SpA antigen. Wild-type SpA (Staphylococcal Protein A) is a cell-wall-anchored surface protein that is a crucial virulence factor for pulmonary infections, sepsis, and abscess development and is expressed by most clinical isolates of S. aureus. Wild-type SpA binds to the Fc part of human IgG, to BH-cell receptors containing VH3, to von Willebrand factor at its Al domain, and to TNF-D receptor 1. The interaction of SpA with B-cell receptors leads to clonal expansion and subsequent cell death in B-cell populations with effects on adaptive and innate immune responses, whereas its binding to Fc □ of IgG interferes with opsonophagocytic clearance of staphylococci by polymorphonuclear leukocytes. The N-terminal part of mature SpA is composed of four or five Ig binding domains of 56 to 61 residues, which fold into triple helical bundles linked by short linkers, and are designated in order by E , D, A, B, and C [6]. These domains display ~ 80% identity at the amino acid level, 56 to 61 residues in length, and are organized as tandem repeats [7]. The C-terminal region is composed of "Xr", a highly repetitive yet variable octapeptide, and "Xc", a domain that adjoins the cell wall anchor structure of SpA.
In strain NCTC 8325, spa is SAOUHSC_00069 and has the amino acid sequence SEQ ID NO: 43 (GI: 88193885). In the Newman strain, it is nwmn_0055 (GI: 151220267). The SpA antigens used with the invention may elicit an antibody (for example, when administered to a human being) that recognizes SEQ ID NO: 43 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 greater) with SEQ ID NO: 43; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 43, 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 SpA antigens include variants of SEQ ID NO: 43. The preferred fragments of (b) comprise an epitope from SEQ ID NO: 43. 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: 43. The final 35 N-terminal amino acids of SEQ ID NO: 43 can be conveniently omitted. The first 36 N-terminal amino acids of SEQ ID NO: 43 may be omitted in a useful manner. Reference 8 suggests that individual IgG binding domains could be useful immunogens, alone or in combination.
A useful fragment of SEQ ID NO: 43 corresponds to amino acids 37 to 325. This fragment contains the five SpA Ig binding domains (which are naturally arranged from the N-terminus to the C-terminus in the order E, D, A, B, C) and includes the most exposed domain of SpA. It also reduces the similarity of the antigen with human proteins. Other useful fragments may omit 1, 2, 3 or 4 domains A, B, C, D and / or E to prevent excessive expansion of B cells and then apoptosis that could occur if spa functions as a superantigen As mentioned in reference 18, other useful fragments may comprise only 1, 2, 3 or 4 of the natural domains A, B, C, D and / or E, for example comprising only the SpA domain. (A) but not B to E, or include only the SpA domain (D) but not A, B, C or E, etc. Thus, a spa antigen useful with the invention may comprise 1, 2, 3, 4 or 5 IgG binding domains, but ideally comprises 4 or less.
Thus, another useful fragment of SpA comprises or consists of the amino acid sequence of SEQ ID NO: 50 in which the amino acid pair at positions 61 and 61 is not Gln-Gln. Said doublet can be mutated as described below, for example in Lys-Arg. Thus, said fragment may comprise or consist of SEQ ID NO: 51 or 52.
If an antigen comprises only one type of spa domain (for example, only the SpA (A), SpA (D) or SpA (E) domain), it may comprise more than one copy of this domain, for example multiple SpA (E) domains in a single polypeptide chain. It may also include one type of SpA domain and another protein or polypeptide. Thus, an antigen of the invention may be a fusion protein comprising only one type of SpA domain, such as the SpA (E) domain, and another protein antigen, such as EsxA; EsxB; FhuD2; StaOll; and Hla.
The SpA antigens of the invention are mutated with respect to SEQ ID NO: 43, such that they exhibit decreased affinity for the FcD part of human IgGs. For example, QQ dipeptide at residues 60-61 of SEQ ID NO: 43 can be mutated to reduce affinity for immunoglobulins. Dipeptide substitutions useful for a QQ dipeptide are discussed below, and a preferred substitution is a KR peptide. Thus, a useful SpA antigen may comprise SEQ ID NO: 49, wherein one or more (preferably all) of the XX dipeptides differ from the corresponding dipeptides within SEQ ID NO: 43. For example, the SpA antigen may include SEQ ID NO: 46, and a preferred example of SEQ ID NO: 46 is SEQ ID NO: 47. When expressed with an N-terminal methionine, the SpA antigen comprising SEQ ID NO: 47 may consist of SEQ ID NO: 48.
The SpA antigens used with the invention may be further mutated with respect to SEQ ID NO: 43, such that they exhibit decreased affinity for the FcD portion of human IgGs and for the Fab portion of human B-cell receptors. containing VH3. This can be achieved and estimated, for example, by following the indications in reference 9. Thus, at least one Gln-Gln dipeptide in wild-type SpA can be mutated (for example, in Lys-Lys, other mutations). Arg-Arg, Arg-Lys, Lys-Arg, Ala-Ala, Ser-Ser, Ser-Thr, Thr-Thr, etc.) and / or at least one Asp-Asp dipeptide in wild type SpA can to be mutated (for example, in Ala-Ala, other possible mutations include Lys-Lys, Arg-Arg, Lys-Arg, Arg-Lys, His-His, Val-Val, etc.). These target sequences for mutation are highlighted below, where separate indents Ig binding five areas: MKKKNIYSIRKLGVGIASVTLGTLLISGGVTP-AANAAQHDEAQQNAFYQVLNMPNLNADQRNGF IQSLKDDPSQSANVLGEAQKLNDSQAPK-ADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQS LKDDPSQSTNVLGEAKKLNESQAPK-ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPS QSANLLSEAKKLNESQAPK-ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLL AEAKKLNDAQAPK-ADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKL NDAQAPK-EEDNNKPGKEDNNKPGKEDNNKPGKEDNNKPGKEDNNKPGKEDGNKPGKEDNKKPGK EDGNKPGKEDNKKPGKEDGNKPGKEDGNKPGKEDGNGVHWKPGDTVNDIAKANGTTADKIAADN KLADKNMIKPGQELWDKKQPANHADANKAQALPETGEENPFIGTTVFGGLSLALGAALLAGRRR EL (SEQ ID NO : 43)
An individual domain within the antigen may be mutated at the level of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids relative to SEQ ID NO: 43 (e.g. see above in connection with the Gln-Gln and Asp-Asp sequences, but see also reference [22] which discloses mutations at residues 3 and / or 24 of domain D at residue 46 and / or 53 of domain A, etc.). Such mutations should not eliminate the ability of the antigen to elicit an antibody that recognizes SEQ ID NO: 43, but will remove antigen binding to IgG and / or other human proteins (such as blood proteins). human) as noted above. In particular, the mutant SpA antigen is of sequence comprising or consisting of SEQ ID NO: 43 mutated in at least 1, more particularly at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and even more particularly 20 amino acids at position 43, 44, 70, 71, 104, 105, 131, 132, 162, 163, 190 , 191, 220, 221, 247, 248, 278, 279, 305 and / or 306 of SEQ ID NO: 43. Substitutions useful for these positions are mentioned above.
In addition, the native N-terminus may be removed, and the first 36 amino acids of SEQ ID NO: 43 may be omitted in a useful manner. Similarly, the native C-terminus can be removed, and the sequence downstream of the fifth Ig binding domain can be conveniently omitted (i.e., downstream of Lys-327 in SEQ ID NO: 43). Thus, a useful SpA antigen comprises SEQ ID NO: 44:
AQHDEAXXNAFYQVLNMPNLNADQRNGFIQSLKXXPSQSANVLGEAQKLNDSQAPKADAQQNNFN
KDXXSAFYEILNMPNLNEAQRNGFIQSLKXXPSQSTNVLGEAKKLNESQAPKADNNFNKEXXNAF
YEILNMPNLNEEQRNGFIQSLKXXPSQSANLLSEAKKLNESQAPKADNKFNKEXXNAFYEILHLP
NLNEEQRNGFIQSLKXXPSQSANLLAEAKKLNDAQAPKADNKFNKEXXNAFYEILHLPNLTEEQR
NGFIQSLKXXPSVSKEILAEAKKLNDAQAPK in which the underlined XX dipeptides differ from the corresponding dipeptides within SEQ ID NO: 43. Thus a QQ at these positions in SEQ ID NO: 43 will not be QQ in SEQ ID NO: 44, and ideally not. does not include glutamine residue, for example, it is KK. Similarly, a DD at these positions in SEQ ID NO: 43 will not be DD in SEQ ID NO: 44, and ideally does not include aspartate residue, for example, it is AA. The preferred form of SpA for use with the invention thus comprises SEQ ID NO: 45.
In addition to XX-dipeptide substitutions in SEQ ID NO: 44, it is possible to modify the amino acid sequence with up to 5 single amino acid changes provided that the modified sequence can trigger antibodies that still bind to a polypeptide consisting of SEQ ID NO: 44. Thus, SEQ ID NO: 45 can be modified by 1, 2 or 3 substitutions at positions outside the XX dipeptides within SEQ ID NO: 44. the QQ dipeptide at residues 60-61 of SEQ ID NO: 44 can be mutated to further reduce the affinity for immunoglobulins. The dipeptide substitutions useful for a QQ dipeptide are discussed above, and a preferred substitution is the KR dipeptide. Thus, a useful SpA antigen may comprise SEQ ID NO: 49, wherein one or more (preferably all) of the XX dipeptides differ from the corresponding dipeptides within SEQ ID NO: 43. For example, the SpA antigen may include SEQ ID NO: 46, and a preferred example of SEQ ID NO: 46 is SEQ ID NO: 47. When expressed with an N-terminal methionine, the SpA antigen comprising SEQ ID NO: 47 may consist of SEQ ID NO: 48.
As discussed above, a useful fragment of SpA can comprise only 1, 2, 3 or 4 of the A, B, C, D and / or E natural domains, for example, to include only the SpA (E) domain. but not D, A, B or C. Thus, a SpA antigen useful with the invention may comprise only the mutated SpA (E) domain as described above, i.e. amino acid comprising or consisting of SEQ ID NO: 54 mutated in at least 1 amino acid at positions 60 and 61 of SEQ ID NO: 54, amino acids 1 to 67 of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 49, or amino acids 1-68 of SEQ ID NO: 48. For example, said antigen may comprise SEQ ID NO: 50, SEQ ID NO : 51, SEQ ID NO: 52 or SEQ ID NO: 53. Said antigen preferably will not comprise a sequence other than SpA. It may comprise more than one copy of the SpA domain (E), and / or further comprise another protein antigen such as EsxA, EsxB, FhuD2, StaOll, or Hla antigen as described herein.
The antigen combinations of the invention use 1, 2, 3, 4 or the following 5 antigens: EsxA; EsxB; FhuD2; StaOll; and Hla. These five antigens are already known in the art (for example, see references 4-514) and further details are given below. A particularly useful composition comprises the five of these antigens (preferably where the Hla is a nontoxic (i.e., detoxified) mutant form). The "EsxA" antigen in strain NCTC 8325 has the amino acid sequence SEQ ID NO: 1 (GI: 88194063). The EsxA antigens used with the present invention can elicit an antibody (e.g., when administered to a human being) 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 include 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 antigens used with the present invention can elicit an antibody (e.g., when administered to a human being) that recognizes SEQ ID NO: 2 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: 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 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: 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, in which residue X at position 30 is absent or is an amino acid residue without a 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 [15]. It has also been known as "Sta006" (for example, in references 4-14). In strain NCTC 8325, FhuD2 has the amino acid sequence SEQ ID NO: 3 (GI: 88196199). The FhuD2 antigens used with the present invention can elicit an antibody (e.g., when administered to a human being) 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 FhuD2 polypeptides comprise 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. 2,3,4,5,6,7,8,9,10,15,20,25 or more) from the N-terminus of ID NO: 3. The first 17 N-terminal amino acids of SEQ ID NO: 3 can be conveniently omitted (to provide SEQ ID NO: 6). Mutant forms of FhuD2 are reported in reference 16. 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 residues. with a free thiol group (under reducing conditions), for example, it is devoid of cysteine. An FhuD2 antigen can be lipidated, for example with an acylated N-terminal cysteine. A useful FhuD2 sequence 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 with the present invention can elicit an antibody (for example, 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. 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., 1, 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), for example, it is devoid of cysteine. StaOll antigen can be lipidated, for example, with acylated N-terminal cysteine. A useful StaOll sequence 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 or to prepare StaOll antigens include but are not limited thereto, SEQ ID NO: 9, 10 and 11 with various Ile / Val / Leu substitutions (and Cys-free variants of these sequences may also be used with 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 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) [17].
In particular, the Hla antigen is a detoxified Hla antigen, i.e., a mutant form of Hla, where the natural toxicity of Hla has been eliminated. The toxicity of Hla can be avoided by chemical inactivation (for example, using formaldehyde, glutaraldehyde or other cross-linking reagents). However, instead, it is preferred to use mutant forms of Hla that eliminate its toxic activity while maintaining its immunogenicity. More particularly, the detoxified Hla antigen is a mutant form of Hla, where the toxicity of Hla has been eliminated while the immunogenicity of Hla has been retained. Such detoxified mutants are already known in 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 can not be a 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 sequence of the mature H35L mutant form of Hla (e.g., SEQ ID NO: 12 with an H35L mutation) and a useful detoxified Hla antigen is of sequence comprising or consisting of SEQ ID NO: 13. Another useful mutation 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 the D152 residue, 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 18 and 19. SEQ ID NOs: 21, 22 and 23 are three useful fragments of SEQ ID NO: 5 (Hla27-76 ',' Hla27-89 'and' Hla27- 79 ', respectively). SEQ ID NO: 24, 25 and 26 are the corresponding fragments from SEQ ID NO: 13.
A useful Hla sequence is SEQ ID NO: 27. It has an N-terminal Met, then an Ala-Ser dipeptide from the expression vector, then SEQ ID NO: 13 (from strain NCTC8325) including the mutation. H35L.
When a composition comprises both EsxA and EsxB antigens, these may be present as a single polypeptide (i.e., as a fusion polypeptide comprising or consisting of both EsxA and EsxB). 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 single polypeptide may 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 any order 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 "EsxAB" includes SEQ ID NO: 31, which may be provided with an N-terminal methionine (e.g., SEQ ID NO: 32). A useful variant of EsxAB lacks the internal cysteine residue of EsxB, for example, it comprises SEQ ID NO: 40 in which the residue X at position 132 is either absent or is an amino acid residue without a free thiol group. (under reducing conditions), for example, it is any natural amino acid with the exception of a cysteine. Thus, a preferred EsxAB antigen for use with 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 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 more (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 type 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 from SEQ ID NO: 2. The invention uses 1, 2, 3, 4 or 5 of EsxA, EsxB, FhuD2, StaOll , and Hla (preferably a nontoxic mutant Hla). As mentioned above, a particularly useful composition includes the five of these antigens, but in some embodiments the invention comprises only 1, 2, 3 or 4 of these five antigens, i.e. say, 1, 2, 3 or 4 of EsxA, EsxB, FhuD2, StaOll, and Hla is absent from the composition.
A preferred composition 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.
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 still 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.
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 the 4 of these 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 still 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 NOs: 32, 7, 8 and 27 independently modified by substitution, deletion and / or insertion of acid. simple amine.
For example, the wild-type FhuD2, StaOll, and EsxAB polypeptide sequences (e.g., SEQ ID NOS: 6, 31, and 33) each comprise a single cysteine residue that can lead to interpolypeptide disulfide bridges, forming both homodimers and heterodimers. Such interleaved polypeptides are undesirable and thus the Sta006, StaOll and EsxAB sequences can be modified to remove their natural cysteine residues, so that they do not contain a free thiol group (under reducing conditions). Wild type cysteine may be deleted or may 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; an EsxB antigen may comprise SEQ ID NO: 35 (for example, in the form of 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 may be used singly 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.
Thus, a preferred composition of the invention comprises the five 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; (iv) an H35L mutant form of Hla, for example, comprising SEQ ID NO: 13; and (v) a mutant SpA, for example, comprising SEQ ID NO: 45 or 47, or an antigen comprising a single domain thereof, for example, SEQ ID NO: 50, 51, 52 or 53. Thus, In particular, the composition according to the invention comprises: (i) a single polypeptide comprising both an EsxA antigen and an EsxB antigen, particularly of sequence comprising or consisting of SEQ ID NO: 31; (ii) an FhuD2 antigen, particularly of sequence comprising or consisting of SEQ ID NO: 6; (iii) a StaOll antigen, particularly of sequence comprising or consisting of SEQ ID NO: 33; (iv) an H35L mutant form of HLA, particularly a sequence comprising or consisting of SEQ ID NO: 13; and (v) a mutant SpA antigen, particularly of sequence comprising or consisting of SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 52.
Another preferred composition according to the invention comprises: (i) a first sequence polypeptide comprising or consisting of SEQ ID NO: 32, more particularly consisting of SEQ ID NO: 32; (ii) a second sequence polypeptide comprising or consisting of SEQ ID NO: 7, more particularly consisting of SEQ ID NO: 7; (iii) a third sequence polypeptide comprising or consisting of SEQ ID NO: 8, more particularly consisting of SEQ ID NO: 8; (iv) a fourth sequence polypeptide comprising or consisting of SEQ ID NO: 27, more particularly consisting of SEQ ID NO:. 27; and (v) a fifth sequence polypeptide comprising or consisting of SEQ ID NO: 52, more particularly comprising or consisting of SEQ ID NO: 45 modified with up to 3 amino acid substitutions (eg, comprising SEQ ID NO: 47 or consisting of SEQ ID NO: 48).
Another preferred composition according to the invention comprises: (i) a first sequence polypeptide comprising or consisting of SEQ ID NO: 38, more particularly consisting of SEQ ID NO: 38; (ii) a second sequence polypeptide comprising or consisting of SEQ ID NO: 37, more particularly consisting of SEQ ID NO: 37; (iii) a third sequence polypeptide comprising or consisting of SEQ ID NO: 39, more particularly consisting of SEQ ID NO: 39; (iv) a fourth sequence polypeptide comprising or consisting of SEQ ID NO: 27, more particularly consisting of SEQ ID NO: 27; and (v) a fifth sequence polypeptide comprising or consisting of SEQ ID NO: 52, more particularly comprising or consisting of SEQ ID NO: 45 modified with up to 3 amino acid substitutions (eg, comprising SEQ ID NO: 47 or consisting of SEQ ID NO: 48).
The proteins (i) to (v) in these combinations can, as explained above, be independently modified by up to 5 single amino acid changes provided that the modified sequence can elicit antibodies that bind always to a polypeptide consisting of the unmodified sequence. In some embodiments, a composition may comprise one or more other polypeptides; in other embodiments, the only peptides in a composition are these five specified polypeptides, and these polypeptides may even be the only immunogenic components in a composition.
When more than one polypeptide is present, they may be present at substantially equal masses, i.e. the mass of each of them is within ± 5% of the average mass of all the polypeptides. Thus, when five polypeptides are present, they can be present in a mass ratio of a / b / c / d / e, where each of a to e is between 0.95 and 1.05.
Apart from EsxA, EsxB, Hla, FhuD2, StaOll, and SpA, 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 include a S. aureus saccharide antigen, for example, the 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 such as defined on pages 15 to 17 of reference 5).
In some embodiments, a composition comprises a S. aureus antigen as defined above, and also an antigen from a different organism (e.g. from a virus or other bacterium).
Immunogenic compositions and medications
The immunogenic compositions of 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 comprise components in addition to the antigens, for example, they will generally comprise one or more pharmaceutically acceptable carriers and / or excipients. A full discussion of such 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 poured and distributed and also 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 10 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 (i.e., less than 5 μg / ml) of 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 the tone, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at a concentration of between 1 and 20 mg / ml, for example, about 10 ± 2 mg / ml NaCl. Other salts which 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 (particularly with 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 10 discloses that the inclusion of EDTA can improve the stability of the compositions disclosed herein. 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 material for a single immunization, or it may comprise material for multiple immunizations (i.e., a "multi-dose" kit). The inclusion of a preservative is preferred in multi-dose arrangements. As an alternative (or in addition) to the inclusion of a preservative in the multidose compositions, the compositions may be contained in a container having an aseptic adapter for taking the material.
S. aureus infections can affect various areas of the body and so a composition can be prepared in various forms. For example, a composition can be prepared in the form of injectables, either in the form of liquid solutions or in the form of 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 just before use (for example, when a component is presented in freeze-dried form) and is presented 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 being used to reactivate the contents of the vial prior to injection.
Human vaccines are generally administered in a dosage volume of about 0.5 ml, although half a volume (i.e., about 0.25 ml) may 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 an immune response to cell mediation 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 to exposure to S. aureus.
The immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen (s), as well as any other optional components, as needed. 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 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 ability of the immune system to the individual to synthesize antibodies, the physician's estimate of the medical situation, and other relevant factors. The quantity is expected to be within 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 20, 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-protective 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 a human, easily miscible / soluble in water, 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 relates to the immunogenic composition according to the invention for use as a medicament. The invention also relates to the immunogenic composition according to the invention for use as a medicament for the prevention and / or treatment of S. aureus infection. The invention also provides a method for the prevention and / or treatment of S. aureus infection in a mammal comprising the step of administering to a mammal in need of an immunologically effective amount of an immunogenic composition according to the invention as defined above. Advantageous embodiments are as defined above. The invention also provides the use of (i) at least one antigen selected from the group consisting of EsxA, EsxB, FhuD2, StaOll, and Hla antigens, and (ii) a mutant SpA antigen that has a decreased affinity, relative to to unmodified SpA, for the FcD part of human IgGs and for the Fab part of human B-lymphocyte receptors containing Vh3, in the manufacture of a medicament for the prevention or treatment of S. aureus infection in a mammal. Advantageous embodiments are as defined above. The invention also provides (i) at least one antigen selected from the group consisting of EsxA, EsxB, FhuD2, StaOll, and Hla antigens, and (ii) a mutant SpA antigen that has decreased affinity, compared to a non-SpA. modified, for the FcD portion of human IgGs and for the Fab portion of human V-lymphocyte receptors containing Vh3, for use in immunizing a mammal to prevent or treat S. aureus infection. Advantageous embodiments are as defined above. The invention also provides (i) at least one antigen selected from the group consisting of EsxA, EsxB, FhuD2, StaOll, and Hla antigens, and (ii) a mutant SpA antigen that has decreased affinity, compared to a non-SpA. modified, for the FcD part of human IgGs and for the VH3-containing human B cell receptor Fab part, for use in a method of immunizing a mammal to prevent or treat S. aureus infection by the administration a therapeutically effective amount of the antigens to the mammal. Advantageous embodiments are as defined above.
As noted above, 1, 2, 3, 4 or preferably EsxA, EsxB, FhuD2, StaOll, and Hla can be used in combination with the mutant SpA. In this manner, the methods, uses, compositions and combinations of antigens of the invention elicit an immune response that is effective for the prevention or treatment of S. aureus infections. The immune response may involve antibodies and / or cell-mediated immunity. By raising an immune response in the mammal by these uses and methods, the mammal can be protected against S. aureus infection, including nosocomial infection. More particularly, the mammal may be protected against skin infection, pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, and / or sepsis. The invention is also useful for protecting against S. aureus infection of bones and joints of a mammal (and thus for preventing disorders including, but not limited to, osteomyelitis, septic arthritis, and infection of joint prostheses). In many cases, these disorders may be associated with the formation of a S. aureus biofilm. S. aureus infects various mammals (including cows, dogs, horses, and pigs), but the preferred mammal for use with the invention is 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 an intended recipient of such prostheses (for example, a preoperative orthopedic patient). A vaccine intended for children may also be administered to adults, for example, to estimate safety, dosage, immunogenicity, etc. However, vaccines are not appropriate only for these groups and they can be used more generally in a human population.
One way to verify the effectiveness of a therapeutic treatment involves monitoring S. aureus infection after administration of the compositions or antigens according to the invention. One way to verify the effectiveness of prophylactic treatment involves monitoring immune responses, at the systemic level (such as monitoring the production rate of IgG1 and IgG2a) and / or at the mucosal level (such as monitoring rate of production). IgA) against antigens in the administered composition after administration. Another way to estimate the immunogenicity of the compositions is to express the antigens in a recombinant manner to screen the sera or 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 mounted an immune response against the protein in question. The effectiveness of the vaccine compositions can also be determined in vivo by provoking animal models of S. aureus infection, for example, guinea pigs or mice, with the vaccine compositions. There are three animal models that are generally useful for the study of an S. aureus infectious disease, namely: (i) the mouse abscess model [21], (ii) the mouse lethal infection model [21] , and (iii) the murine pneumonia model [22]. The abscess model examines the number of mice that survive after being infected with a normally lethal dose of S. aureus intravenously or intraperitoneally. The pneumonia model also examines the survival rate, but uses an intranasal infection. Other useful models are disclosed in reference 23 for the study of both S. aureus disease in connection with biofilm-mediated implant infection, skin and soft tissue infection. (IPTM), and sepsis. A useful vaccine can be effective in one or more of these models. For example, for certain clinical situations, it may be desirable to protect against pneumonia, without the need to prevent haematic dissemination or to promote opsonization; in other situations, the main wish may be to prevent haematic dissemination or sepsis. Different antigens, and different combinations of antigens, can contribute to different aspects of an effective vaccine.
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, rectally, orally (eg, tablet, spray), vaginal, topical, transdermal or transcutaneous, intranasal, ocular, atrial, pulmonary or other mucosal administration. Intramuscular injection is the most typical route for the administration of the 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, 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 may be administered 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 route or by 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 patients substantially simultaneously (for example, during the same medical consultation or visit to a health professional or a vaccination center) as other vaccines.
The immunogenic compositions may be administered to patients in combination with an antibiotic. For example, they can be administered substantially simultaneously with an antibiotic. Similarly, they may be administered to a subject receiving antibiotic treatment. Similarly, they can be administered as part of a co-treatment which involves the administration of both a composition as discussed herein and an antibiotic. The antibiotic will be effective against S. aureus bacteria, for example, a beta-lactam.
Strains and variants
The antigens are discussed above with reference to an existing nomenclature (eg, "EsxA") and examples of sequences 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 [24], 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 among any of these other genomic sequences the homologue of any particular sequence mentioned herein. In addition, the specific sequences disclosed herein may 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 allelic variants, polymorphic forms, homologues, orthologues, paralogs, mutants, and the like.
Thus, for example, the polypeptides used with the invention may, as compared to SEQ ID NO herein, include one or more (e.g., 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 having a related side chain). The 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, i.e., 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 classified together as aromatic amino acids. In general, substitution of single amino acids 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 with the invention may comprise an amino acid sequence that: (a) is identical (i.e., 100% identical) with 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 (ideally over the entire length of said sequence); (c) has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or more) single amino acid changes (deletions, insertions, substitutions), which may be at separate locations or which may be contiguous, compared to the sequences of (a) or (b); or (d) when aligned with a particular sequence from the sequence listing using a pair alignment algorithm, each x-amino acid moving window from the N-terminus to the C-terminus (so that for an alignment that extends to p amino acids, where p> x, yap-x + l 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 [25] using the 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 in EMBOSS [26].
When hybrid polypeptides are used, the individual antigens within the hybrid (i.e., the -X- individual entities) 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) Χι = Χ2 = / = Χ3 (iii) Xi ^ X2 = X3 (iv) Xi ^ X2 / X3 or (v) Xi = X3 ^ X2, etc.
Within group (c), the deletions or substitutions may be at the N-terminus and / or the C-terminus, or they may be between the two ends. Thus, truncation is an example of a deletion. Truncations may involve the deletion of up to 40 (or more) amino acids at the N-terminus and / or at the C-terminus. N-terminal truncation can eliminate leader peptides, for example, to facilitate recombinant expression in a heterologous host. C-terminal truncation can eliminate anchor sequences, for example, to facilitate recombinant expression in a heterologous host.
In general, when an antigen comprises a sequence which is not identical to a complete sequence of S. aureus from the sequence listing (for example, when it comprises a sequence listing with <100% identity sequence with it, or when it comprises a fragment thereof), it is preferred in each individual case that the antigen can elicit an antibody that recognizes the respective complete sequence of S. aureus.
Polypeptides used with the invention
The polypeptides used with the invention may take various forms (e.g., native, fusion, glycosylated, non-glycosylated, lipidated, non-lipidated, phosphorylated, unphosphorylated, myristoylated, non-myristoylated, monomeric, multimeric, etc.).
The polypeptides used with the invention may be prepared by various means (e.g., recombinant expression, purification from cell cultures, chemical synthesis, etc.). Proteins expressed recombinantly are preferred, particularly for hybrid polypeptides.
The polypeptides used with 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 of other staphylococcal polypeptides or host cell, and are generally at least about 50% (by weight) pure, and usually at least about 90% pure, i.e., less than about 50% and more preferably less than about 10% (e.g., 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 naturally modified 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 modifications known in the art. The polypeptides may appear as simple chains or associated chains.
Although the expression of the polypeptides of the invention may take place 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 (for example, M. tuberculosis), yeast, etc. Compared to the S. aureus wild-type genes encoding the polypeptides of the invention, it is useful to change the codons to optimize the expression efficiency in such hosts without affecting the encoded amino acids.
admixtures
As mentioned above, the immunogenic compositions used according to the invention may comprise one or more adjuvants. Adjuvants that may be used with 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 27). The aluminum salts include hydroxides and phosphates, etc., with salts of any suitable form (e.g., gel, crystalline, amorphous, etc.). Adsorption to these salts is preferred (e.g., all antigens can be adsorbed). The mineral-containing compositions can also be formulated as a metal salt particle [28].
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 29). 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 hydroxyphosphate sulfate). 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 observed 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 adjuvants generally have a PO4 / Al molar ratio 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 PO 4 / Al between 0.84 and 0.92, including 0.6 mg of Al 3+ / ml. Aluminum phosphate will generally be particulate (eg, plate-like morphology as observed on transmission electron micrographs). Typical particle diameters are in the range of about 0.5 to about 20 microns (eg, about 5 to 10 microns) after adsorption of any antigen. Adsorption capacities between 0.7 and 1.5 mg protein per mg Al +++ at pH 7.4 have been reported for aluminum phosphate adjuvants.
The zero point of charge (PZC) of the aluminum phosphate is inversely proportional to the degree of substitution of the phosphate with the hydroxyl, and this degree of substitution can vary according to the reaction conditions and the concentration of the reagents used to prepare the salt by precipitation. . PZC is also modified by changing the concentration of free phosphate ions in solution (more phosphate = more acidic PZC) or by adding a buffer like a histidine buffer (makes the PZC more basic). The aluminum phosphates used according to the invention will generally have a PZC 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 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 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. Oily emulsions
Oily emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 [Chapter 10 of Ref. 29; see also 30] and AS03 [31].
Various adjuvants based on oil-in-water emulsions are known, and they generally comprise at least one oil and at least one surfactant, with the oil / oils and the surfactant (s) being biodegradable (metabolizable) and biocompatible. The emulsion will include submicron oil droplets, 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 animal (such as fish) or vegetable sources. The oil is ideally biodegradable (metabolizable) and biocompatible. Sources for 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 the jojoba bean. 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. 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 the appropriate materials starting from nuts and seeds oils. Mammalian milk fats and oils are metabolizable and can therefore be used in the practice of this invention. 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 herein. 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 squalene-saturated analogue, 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, particularly in combination with squalene. When the oily phase of an emulsion comprises a tocopherol, any of the α, β, γ, δ, ε or to tocopherols may be used, but D-tocopherols are preferred. D-O-tocopherol and DL-D-tocopherol may both be used. A preferred D-tocopherol is DL-D-tocopherol. A combination of oils comprising squalene and a tocopherol (e.g., DL-D-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 Tris buffer; 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, particularly 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 Malvern Instruments Zetasizer ™ instruments (United Kingdom), or Horiba Partiele Size Distribution Analyzer instruments (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 with the invention include, but are not limited to: □ A squalene submicron emulsion, Tween 80, and
Span 85. The composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. In terms of weight, these ratios become 4.3% of squalene, 0.5% polysorbate 80 and 0.48% Span 85. This adjuvant is known as "MF59" [32 to 34], as further described in Chapter 10 of Reference 35 and Chapter 12 of reference 36. The MF59 emulsion advantageously comprises citrate ions, for example 10 mM sodium citrate buffer. An emulsion comprising squalene, a tocopherol, and polysorbate 80. The emulsion may comprise 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 [37] 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., o-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 oi-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" [38] (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" [39] (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 has at least 90% of the oil droplets (by volume) with a size less than 200 nm [40]. 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 comprise a TLR4 agonist [41]. Such emulsions can be lyophilized. □ An emulsion of squalene, poloxamer 105 and Abil-Care [42]. 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 43, the preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipid. 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 saponin QuilA, cholesterol, a saponin-lipophilic conjugate (such as GPI-0100, described in reference 44, produced by adding 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 [45]. 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) [46]. 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) [46].
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 distributed 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 Ref. 29] 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 (sarsaparilla), Gypsophilla paniculata (bridal veil), and Saponaria officinalis (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 47. The saponin formulations may also include a sterol, such as cholesterol [48].
Combinations of saponins and cholesterols can be used to form particles called ISCOM (chapter 23 of reference 29). 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. ISCOMs are further described in references 48 to 50. Optionally, the ISCOMs may be devoid of additional detergent [51].
A description of the development of saponin adjuvant can be found in references 52 and 53. D. Bacterial or Microbial Derivatives
Adjuvants suitable 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 thereof. 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-des-O-acylated monophosphoryl lipid A is disclosed in reference 54. Such "small particles" of 3dMPL are small enough to be sterilized by filtration through a membrane of 0. 22 pm [54]. 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 55 and 56.
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 57, 58 and 59 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 60 to 65.
The CpG sequence may be directed against TLR9, such as the GTCGTT or TTCGTT motif [66]. 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 67 to 69. 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 66 and 70 to 72.
A useful CpG adjuvant is CpG7909, also known as ProMune ™ (Coley Pharmaceutical Group, Inc.). Another is CpGl826. As an alternative, or in addition, to the use of CpG sequences, TpG sequences can be used [73], 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 73), and / or it 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 73), 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 ™ [74]. Thus, an adjuvant used with 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 75 and 76.
ADP-ribosylating bacterial toxins and their detoxified derivatives can be used as adjuvants in the invention. Preferably, the protein is derived from S. coli (heat labile enterotoxin "LT" of E. coli), cholera ("CT"), or pertussis ("PT"). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in reference 77 and as parenteral adjuvants in reference 78. 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 79 to 86. A useful CT mutant is CT-E29H [87]. ]. 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 88, 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 89. Many TLR agonists are known in the art, for example, reference 90 discloses certain lipopeptide molecules that are TLR2 agonists, references 91 to 94 each describe small agonist classes. TLR7 molecule, and references 95 and 96 describe TLR7 and TLR8 agonists for the treatment of diseases.
A TLR agonist used with 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 (eg, by ligand exchange or other suitable mechanism) and improve their immunological behavior [97]. 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 the adsorption of the agonist on an insoluble aluminum salt [97].
The TLR agonists useful with 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 by 1-4 substituents independently selected from halogeno, 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 is 0; L is selected from C1 to C6 alkylene and - ((CH2) pO) 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 98.
The compositions may comprise an imidazoquinolone compound, such as Imiquimod ("R-837") [99,100], Resiquimod ("R-848") [101], and their analogs; and their salts (for example, hydrochloride salts). Further details regarding immunostimulatory imidazoquinolines can be found in references 102-106.
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 [107]. 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., 3-0-deacylated monophosphoryl lipid A present in GSK adjuvant "AS04" along with other details in references 108 to 111; glucopyranosyl lipid A (GLA) [112] or its ammonium salt; an aminoalkylglucosaminide phosphate, such as RC-529 or CRX-524 [113 to 115]; E5564 [116,117] or a compound of formula I, II or III as defined in reference 118, 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 ref.
(K) wherein: R1 is H, C1-C6 alkyl, -C (R5) 2OH, -L1R5, -L1R6, -L2R5, -L2R6, -OL2Rs, or -OL2R6; L1 represents-C (O) - or -O-; L 2 is C 1 -C 6 alkylene, C 2 -C 6 alkenylene, arylene, heteroarylene or - ((CR 4 R 4) pO) q (CH 2) p-, where C 1 -C 6 alkylene and C 2 -C 6 alkenylene 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-C4 alkyl, -L3R5, -L1R5, -L3R7, -L3L4L3R7, -L3L4R5, -L3L4L3R5, -OL3R5, -OL3R7, -OL3L4R7, -OL3L4L3R7, -OR8, -OL3L4R5, -OL3L4L3R5 and -C (R5) 2OH; each R4 is independently selected from H and fluoro; R5 is -P (O) (OR9) 2; R6 is -CF2P (O) (OR9) 2 or -C (O) OR1; 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 *):
(K ') 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-C6alkyl, C1-C6alkoxy 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 alkyleneoxy and - ((CH 2) p0) q (CH 2) p- each optionally substituted with 1 to 4 substituents independently selected from halo, OH, C1-C4alkyl, -0P (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 the 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 with 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 [120]. F. Microparticles
Microparticles may also be used as adjuvants in the invention. 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 K1). Identified above), as is disclosed in references 5 and 97. Thus, in particular, said adjuvant is selected from the group consisting of: aluminum salts, particularly aluminum hydroxides and aluminum phosphates ; human TLR agonists, particularly TLR7 agonists; and - a mixture of these. The TLR agonist is preferably adsorbed on the metal salt, and the S. aureus antigen / antigens can also be adsorbed onto 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 preferred 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 0.8b 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 preferred that at least 50% (by weight) of the agonist in the composition is adsorbed on the metal salt, e.g. 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 K1; 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; and □ a fifth polypeptide comprising SEQ ID NO: 45, or a modified amino acid sequence which differs from SEQ ID NO: 45 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: 43; and wherein the TLR7 agonist and / or at least one of the polypeptides is adsorbed on the aluminum hydroxide adjuvant.
For example, as is explained in more detail elsewhere herein: the first polypeptide may comprise SEQ ID NO: 34; the second polypeptide may comprise SEQ ID NO: 13; the third polypeptide may comprise bbQ 1D tiu: 4L); and the fourth polypeptide may comprise SEQ ID NO: 36; and the fifth polypeptide may comprise SEQ ID NO: 45, optionally modified with up to 3 amino acid substitutions (other than at the positions that are X in SEQ ID NO: 44). Thus, the composition may use a mixture of five polypeptides having SEQ ID NO: 37, 27, 38, 39, and 45 (except that SEQ ID NO: 45 may be modified by up to 3 amino acid substitutions as discussed above).
Chemical groups
Unless otherwise specifically defined elsewhere, the chemical groups discussed herein have the following meaning when used in this specification:
The term "alkyl" includes saturated hydrocarbon residues comprising: - linear groups up to 10 atoms (Ci to Cio), or up to 6 atoms (Ci to Ce), or up to 4 atoms ( Ci to C4). Examples of such alkyl groups include, but are not limited to, C1-methyl, C2-ethyl, C3-propyl, and C4 n-butyl. branched groups of 3 to 10 atoms (C3 to C10), or of up to 7 atoms (C3 to C7), or of up to 4 atoms (C3 to C4). Examples of such alkyl groups include, but are not limited to, C3 iso-propyl, C4 sec-butyl, C4 iso-butyl, C4 tert-butyl and C5 neo-pentyl.
The term "alkylene" refers to the divalent hydrocarbon radical derived from an alkyl group, and should be interpreted in accordance with the above definition.
The term "alkenyl" includes monounsaturated hydrocarbon residues comprising: - linear groups of 2 to 6 atoms (C2 to Ce). Examples of such alkenyl groups include, but are not limited to, C2-vinyl, 1-propenyl, C3-allyl, C3, 2-butenyl, C4-groups. branched groups of 3 to 8 atoms (C3 to C8). Examples of such alkenyl groups include, but are not limited to, C 2 -C 4 and C 2 -methyl-2-butenyl 2-methyl-propenyl.
The term alkenylene refers to the divalent hydrocarbon radical derived from an alkenyl group, and should be interpreted in accordance with the above definition.
The term "alkoxy" includes O-linked hydrocarbon residues comprising: - linear groups of 1 to 6 atoms (Ci to Ce), or 1 to 4 atoms (Ci to C4). Examples of such alkoxy groups include, but are not limited to, C1-methoxy, C2-ethoxy, C3-n-propoxy, and C4 n-butoxy. branched groups of 3 to 6 atoms (C3 to Ce) or of 3 to 4 atoms (C3 to C4). Examples of such alkoxy groups include, but are not limited to, C3 iso-propoxy, and C4 sec-butoxy and tert-butoxy.
Halogeno is selected from Cl, F, Br and I. Halogeno is preferably F.
The term "aryl" includes a single or fused aromatic ring system containing 6 to 10 carbon atoms; wherein, unless otherwise indicated, at each occurrence the aryl group may be optionally substituted with up to 5 substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, OH, halo, CN, COOR14, CF3 and NR14R15 ; as defined above. Generally, the aryl group will be optionally substituted with 1, 2 or 3 substituents. The optional substituents are chosen from those indicated above. Examples of suitable aryl groups include phenyl and naphthyl groups (each optionally substituted as indicated above). Arylene refers to the divalent radical derived from an aryl group, and should be interpreted in accordance with the definition above.
The term "heteroaryl" includes a monocyclic or bicyclic aromatic ring of 5, 6, 9 or 10 members, containing 1 or 2 N atoms and, optionally, an NR 14 atom, or NR 14 atom and an S or O atom, or an atom S, or an O atom; wherein, unless otherwise indicated, said heteroaryl group may be optionally substituted with 1, 2 or 3 substituents independently selected from C 1 -C 6 alkyl, C 1 -C 6 alkoxy, OH, halo, CN, COOR 14, CF 3 and NR 14 R 15; as defined below. Examples of suitable heteroaryl groups include thienyl, furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, benzimidazolyl, benzotriazolyl, quinolinyl. and isoquinolinyl (optionally substituted as indicated above). Heteroarylene refers to the bivalent radical derived from a heteroaryl group, and should be interpreted in accordance with the definition above.
The term "heterocyclyl" is a C-linked or N-linked monocyclic or bicyclic non-aromatic ring of 3 to 10 members, wherein said heterocycloalkyl ring contains, where possible, 1, 2 or 3 heteroatoms independently selected from N, NR 14 , S (O) q and O; and said heterocycloalkyl ring optionally contains, where possible, 1 or 2 double bonds, and is optionally substituted on the carbon by 1 or 2 substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, OH, CN, CF3, halo, COOR14, NR14R15 and aryl.
In the above definitions, R14 and R15 are independently selected from H and C1-C6alkyl.
When a structural formula is defined with a substituent attached to the core of the molecule by an unspecified or "floating" bond, for example, as for the P3 group in the case of formula (C), this definition includes cases where the Unspecified substituent is attached to any of the atoms on the ring in which the floating bond is located, while observing the permissible valence for that atom.
In the case of the compounds of the invention which may exist in tautomeric forms (i.e., in keto or enol forms), for example the compounds of formula (C) or (H), the reference to a particular compound optionally comprises all these tautomeric forms. Overview
The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology within the skill of the art. Such techniques are fully explained in the literature. See, for example, references 121-128, 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 can be a B cell epitope and / or a T cell epitope. Such epitopes can be identified empirically (for example, using PEPSCAN [129,130] or methods similar), or they can be predicted (for example, using the Jameson-Wolf antigenic index [131], matrix-based approaches [132], MAPITOPE [133], TEPITOPE [134,135], neural networks [ 136], OptiMer and EpiMer [137,138], ADEPT [139], Tsites [140], hydrophilicity [141], antigenic index [142] or methods disclosed in references 143 to 147, 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, and so on.
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 software programs known in the art, for example those described in section 7.7.18 of reference 148. A preferred alignment 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. Smith-Waterman's homology search is disclosed in reference 149. The percentage identity with any particular sequence (for example, at a particular SEQ ID) is ideally calculated over the entire length of that sequence. The binding affinity can be determined by any method known in the art, including surface plasmon resonance, isothermal titration calorimetry, competitive binding assays, thermal shift assay, and the like. Although the absolute numbers obtained using the different methods may vary, it is contemplated that the relative binding affinity to the determination of one protein over another does not depend on the method used.
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 (0) (OH2) (OH)] + and - [0P (O) (OH) 2] 2+ which can exist under acidic conditions and the deprotonated forms - [O (0) (OH) (O)] and [O (0) (O) 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 Mutant SpAkR
SpA is a crucial virulence factor in S. aureus and acts by interfering with the opsonophagocytic clearance of the bacterium and by ablation of adaptive immune responses. The wild type sequence SEQ ID NO: 43 comprises five Ig binding domains (IgBD), as shown above, and it is known to mutate amino acid residues within these domains to abolish the Fc / Fab binding activity while maintaining immunogenicity, for example, see reference [19], which replaces Gln-Gln dipeptides with Lys-Lys and / or replaces Asp-Asp dipeptides with Ala-Ala.
A new mutant SpA has been produced in which another Gln-Gln dipeptide is mutated to Lys-Arg. This dipeptide was identified based on bioinformatic analysis and predictions of an additional site involved in Ig binding. In particular, residues 96 and 97 of SEQ ID NO: 43 were thought to be involved in Ig binding. This site has been neglected in previous work because it lies outside the conserved IgBD and is not part of the resolved structures. The new mutant is named "SpAkR" and it has the following amino acid sequence, wherein the substitution QQ / additional KR relative to mutant "SpAkkAA" prior maqhdeakknafyqvlnmpnlnadqrngfiqslkaapsqsanvlgeaqklndsqapkadä is framed | kr [nnf NKDKKSAFYEILNMPNLNEAQRNGFIQSLKAAPSQSTNVLGEAKKLNESQAPKADNNFNKEKKNA FYEILNMPNLNEEQRNGFIQS LKAAPSQSANLLSEAKKLNE SQAPKADNKFNKEKKNAFYEILHL PNLNEEQRNGFIQSLKAAPSQSANLLAEAKKLNDAQAPKADNKFNKEKKNAFYEILHLPNLTEEQ RNGFIQSLKAAPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 48)
The SpAkR mutant was confirmed to have reduced affinity for immunoglobulins compared to known mutants, while maintaining immunogenicity.
CD and DSC analyzes showed that the introduction of the KR mutant did not materially affect the SpA structure. The CD spectra of SpAkkAA and SpAkR were identical. The Tml and Tm2 from the DSC analysis were as follows. Tml: SpAkkAA 48.9 ° C; SpAKR 51.1 ° C. Tm2: SpAkkAA 68.1 ° C; SpAKR 68.0 ° C. The affinity of wild-type SpA, and SpAkkAA and SpAkR mutants for human IgG, IgA and IgM was tested using surface plasmon resonance. The proteins were immobilized on a sensor chip and IgG, IgA and human IgM were used as the analytes. Both mutants displayed greatly reduced binding capacity compared to the wild-type, but while SpAkkAA showed residual binding to IgG and IgM, no detectable interaction with any immunoglobulin was observed with SpAkR. The residual binding activities of SpAkkAA to IgG and IgM were very low (11 to 12 times lower than the wild type), but reproducible and concentration dependent.
The survival of S. aureus in the presence of SpA (wild type, SpAkkAA and SpAkR) was estimated in a whole blood survival test (WBA). 1 ml of whole blood from healthy donors, supplemented with 50 mg / l of lepirudin anticoagulant (10 μl / ml of blood), was incubated with 0.15 cm SpA or with PBS, for 15 min. 37 ° C, before adding approximately 2.5 x 105 CFU of S. aureus USA300 LAC (OD600 / 0.4) diluted in BHI. Aliquots of this culture were plated on BHI agar to determine CFU initially. After incubation at 37 ° C for 2 h with shaking (180 rpm), the neutrophils were lysed at 0.5% saponin-PBS for 3 min on ice. The number of viable bacteria was determined by tenth serial dilutions in BHI and deposition on nutrient agar plates. Colonies were counted after incubation of the plates at 37 ° C for 18 h. The control was the blood sample preincubated with PBS. Relative survival was calculated based on initial CFUs as compared to CFUs after incubation. The experiments were performed in triplicate; there was little variation between the experiments.
In the WBA test, the relative survival of S. aureus in human whole blood was significantly lower when incubated with SpAkR compared to wild-type (p <0.001, Mann-Whitney) or SpAkkAA ( p <0.05). Incubation with wild type SpA resulted in increased survival of S. aureus compared to the control (p <0.001), whereas survival in the WBA test incubated with SpAkkAA was comparable to the control. Incubation with SpAkR reduced survival by about half that of the control.
It has been found that SpAkkAA and SpAkR formulated with aluminum hydroxide adjuvant are poorly immunogenic in the mice per se, but the inclusion of the adsorbed TLR7 "Kl" agonist significantly increased the antibody titres. Because the mechanism of action associated with SpA immunization appears to be primarily directed by antibodies, this represents a significant improvement.
Vaccines against S. aureus
SpAkR, SpAkkAA, SpAkR E domain and SpAkR domain E fused to HlaH35L were tested in the renal abscess model, adjuvated with 2 mg / ml aluminum hydroxide (Al-H). ml (total salt). The antigens were each present at 10 μg in a dose of 100 μg for intramuscular injection.
Mice (CDI) four or five weeks old were immunized intramuscularly (IM) with reminder sensitization injections with an interval of 14 days. Control mice received equal amounts of adjuvants alone. Serum was taken from mice both before and after vaccination to document serum antibody titers against each protein component in the combined vaccine. These titers were measured by Luminex technology using recombinant vaccine antigens conjugated to microspheres.
Renal abscess model: Immunized animals were challenged at day 24 by intravenous injection of a sub-lethal dose of the Newman strain of S. aureus (~ 2 to 6 x 107 CFU). At day 28, the mice were euthanized and the kidneys were removed and homogenized in 2 ml of PBS and plated on duplicate agar medium for determination of colony forming units (CFU).
A comparable log reduction of CFU / ml (reduction around 1 log) was obtained with vaccination with SpAkR, SpAkkAA, domain E of SpAkR alone and domain E of SpAkR fused to HlaH35L, compared to adjuvant alone.
Combined vaccines against S. aureus
A pentavalent vaccine and a hexavalent vaccine were prepared. The pentavalent vaccine included antigens consisting of SEQ ID NOs: 7, 8, 27 and 32 (FhuD2, StaOll, Hla-H35L, and EsxAB); the hexavalent vaccine also included the SpAkR mutant. The vaccines were adjuvated with: (i) aluminum hydroxide, Al-H; (ii) Al-H + TLR7 Kl agonist adsorbed; or (iii) the MF59 oil-in-water emulsion. Al-H was used at 2 mg / ml (total salt), KI was present at 50 μg per dose, and MF59 was mixed with the antigens in a volume ratio of 1/1. The antigens were each present at 10 μg in a 100 μl dose for intramuscular injection.
Mice (CDI) four or five weeks old were immunized with reminder sensitization injections with an interval of 14 days. Control mice received equal amounts of adjuvants alone. Serum was taken from mice both before and after vaccination to document serum antibody titers against each protein component in the combined vaccine. These titers were measured by Luminex technology using recombinant vaccine antigens conjugated to microspheres.
Renal abscess model: Immunized animals were challenged at day 24 by intravenous injection of a sub-lethal dose of S. aureus (~ 2 to 6 x 107 CFU, where the specific inoculum varied according to the strain of provocation). At day 28, the mice were euthanized and the kidneys were removed and homogenized in 2 ml of PBS and plated on duplicate agar medium for determination of colony forming units (CFU). The kidneys were also treated for histopathology.
Peritonitis model: Separately, immunized animals were challenged at day 24 by intraperitoneal injection of a lethal dose of S. aureus (~ 2 to 5 x 108 CFU) and then monitored daily for 14 days.
Model of infection of the skin: Immunized mice were inoculated by subcutaneous injection into the right flank shaved with 2 x 107 CFU of the S. aureus strain LAC (clone übAJUu, which is one of the clones of the most important in the world and is highly associated with skin infections acquired in community). Mass and abscess formation (size and dermonecrosis) were monitored at 24-hour intervals over a 7-day period. The size of an abscess and the underlying underlying dermonecrotic lesion was determined using image analysis software. The skin of the mice and the abscesses were harvested at day 7 after inoculation for counting CFUs. This model was only used with Al-H / Kl adjuvant.
The results were as follows:
These results demonstrate that the SpAkR mutant enhances the pentavalent Combo-1 product in both models. In addition, the best results were observed using Al-H / Kl adjuvant. Surprisingly, with the results in the renal abscess model, the hexavalent vaccine with Al-H / K approached infertility.
In the peritonitis model, the hexavalent vaccine with Al-H / K1 was statistically superior compared to the negative control (see table above), and also compared to the pentavalent vaccine with Al-H. In the abscess model, the hexavalent vaccine with Al-H / K1 was statistically superior compared to the negative control (see table above), pentavalent vaccine Al-H, and pentavalent vaccine Ά1-Η / Κ1 , thus showing that the contribution of the SpA mutant goes beyond the amplification that was only due to the Kl agonist.
In the skin infection model, pentavalent and hexavalent vaccines significantly reduced abscess formation and the number of CFUs (see table above). Dermonecrosis was absent in the vaccinated mice while it was observed in all mice that received the adjuvant alone ("N / A" in the table). In addition, the reduction in CFU was significantly improved by inclusion of SpAkR in the vaccine, and fewer mice were observed with macroscopically distinguishable abscesses (71% vs. 88%).
Anti-SpA antibody titers were also compared for the three adjuvants. Median titres using Al-H or MF59 were not significantly different, but the titre using Al-H / Kl was significantly higher compared to using Al-H alone (p = 0, 0047, Mann-Whitney test) and MF59 (p = 0.01). Thus the formulation that worked best in functional tests was the one that triggered the highest titres of anti-SpA antibodies, in line with the hypothesis that the activity of SpA as a vaccine antigen depends on antibodies. which can bind it and inhibit its immune evasion activity.
It should be understood that the invention has been described by way of example only and that modifications can be made while remaining within the scope and spirit of the invention. References [1] Sheridan (2009) Nature Biotechnology 27: 499-501.
[2] Kuklin et al. (2006) Infect Immun. 74 (4): 2215-23.
[3] Fowler et al. (2013) JAMA 309 (13): 1368-78.
[4] WO 2010/119343.
[5] WO 2013/030378.
[6] Sjodahl (1977) J. Biochem. 73: 343-351.
[7] Uhlen et al. (1984) J. Biol. Chem. 259: 1695-1702 &amp; 13628 (Corr.).
[8] WO 2007/071692.
[9] Kim et al. (2010) J Exp Med 207 (9): 1863-70.
[10] WO 2013/092985.
[11] WO 2014/033190.
[12] WO 2014/033191.
[13] WO 2014/033192.
[14] WO 2014/033193.
[15] Sebulsky &amp; Heinrichs (2001) J. Bacteriol 183: 4994-5000.
[16] Sebulsky et al. (2003) J Biol Chem 278: 49890-900.
[17] Rable &amp; Wardenburg (2009) Infect Immun 77: 2712-8.
[18] WO 2007/145689.
[19] WO 2009/029831.
[20] WO 2006/110603.
[21] Stranger-Jones et al. (2006) PNAS USA 103: 16942-7.
[22] Wardenburg et al. (2007) Infect Immun 75: 1040-4.
[23] Prabhakara et al. (2011) J Immunol 186: 155.29 [24] Kuroda et al. (2001) Lancet 357: 1225-1240.
[25] Needleman &amp; Wunsch (1970) J. Mol. Biol. 48, 443-453.
[26] Rice et al. (2000) Trends Genet 16: 276-277.
[27] US Patent 6355271.
[28] WO 00/23105.
[29] Vaccine Design ... (1995) eds. Powell &amp; Newman. ISBN: 030644867X. Plenum.
[30] WO 90/14837.
[31] Boy et al. (2012) Expert Rev Vaccines 11: 349-66.
[32] WO 90/14837.
[33] Podda &amp; Del Giudice (2003) Expert Rev Vaccines 2: 197-203.
[34] Podda (2001) Vaccine 19: 2673-2680.
[35] Vaccine Design: The Subunit and Adjuvant Approach (eds Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X).
[36] Adjuvant Vaccine: Preparation Methods and Research
Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed O'Hagan.
[37] WO 2008/043774.
[38] Allison &amp; Byars (1992) Res Immunol 143: 519-25.
[39] Hariharan et al. (1995) Cancer Res 55: 3486-9.
[40] US-2007/014805.
[41] US-2007/0191314.
[42] Suli et al. (2004) Vaccine 22 (25-26): 3464-9.
[43] WO 95/11700.
[44] US Patent 6,080,725.
[45] WO 2005/097181.
[46] WO 2006/113373.
[47] US 5,057,540.
[48] WO 96/33739.
[49] EP-A-0109942.
[50] WO 96/11711.
[51] WO 00/07621.
[52] Barr et al. (1998) Advanced Drug Delivery Reviews 32: 247-271.
[53] Sjolanderet et al. (1998) Advanced Drug Delivery Reviews 32: 321-338.
[54] EP-A-0689454.
[55] Meraldi et al. (2003) Vaccine 21: 2485-2491.
[56] Pajak et al. (2003) Vaccine 21: 836-842.
[57] Kandimalla et al. (2003) Nucleic Acids Research 31: 2393-2400.
[58] WO 02/26757.
[59] WO 99/62923.
[60] Krieg (2003) Nature Medicine 9: 831-835.
[61] McCluskie et al. (2002) FEMS Immunology and Medical Microbiology 32: 179-185.
[62] WO 98/40100.
[63] US 6,207,646.
[64] US 6,239,116.
[65] US 6,429,199.
[66] Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3): 654-658.
[67] Blackwell et al. (2003) J Immunol 170: 4061-4068.
[68] Krieg (2002) Trends Immunol 23: 64-65.
[69] WO 01/95935.
[70] Kandimalla et al. (2003) BBRC 306: 948-953.
[71] Bhagat et al. (2003) BBRC 300: 853-861.
[72] WO 03/035836.
[73] WO 01/22972.
[74] Schellack et al. (2006) Vaccine 24: 5461-72.
[75] Kamath et al. (2008) Eur J Immunol 38: 1247-56.
[76] Riedl et al. (2008) Vaccine 26: 3461-8.
[77] WO 95/17211.
[78] WO 98/42375.
[79] Beignon et al. (2002) Infect Immun 70: 3012-3019.
[80] Pizza et al. (2001) Vaccine 19: 2534-2541.
[81] Pizza et al. (2000) Int J Med Microbiol 290: 455-461.
[82] Scharton-Kersten et al. (2000) Infect Immun 68: 5306-5313.
[83] Ryan et al. (1999) Infect Immun 67: 6270-6280.
[84] Partidos et al. (1999) Immunol Lett 67: 209-216.
[85] Peppoloni et al. (2003) Expert Rev Vaccines 2: 285-293.
[86] Pine et al. (2002) J Control Release 85: 263-270.
[87] Tebbey et al. (2000) Vaccine 18: 2723-34.
[88] Domenighini et al. (1995) Mol Microbiol 15: 1165-1167.
[89] Rosenberg et al. (2010) J. Immunol 184: 136.20.
[90] US-4,666,886.
[91] WO 2009/118296.
[92] WO 2008/005555.
[93] WO 2009/111337.
[94] WO 2009/067081.
[95] WO 2007/040840.
[96] WO 2010/014913.
[97] WO 2012/031140.
[98] WO 2013/132041.
[99] US 4,680,338.
[100] US 4,988,815.
[101] WO 92/15582.
[102] Stanley (2002) Clin Exp Dermatol 27: 571-577.
[103] Wu et al. (2004) Antiviral Res. 64 (2): 79-83.
[104] Vasilakos et al. (2000) Cell Immunol. 204 (1): 64-74.
[105] US Patents 4689338, 4929624, 5238944, 5266575, 5268376, 5346905, 5352784, 5389640, 5395937, 5482936, 5494916, 5525612, 6083505, 6440992, 6627640, 6656938, 6660735, 6660747, 6664260, 6664264, 6664265, 6667312, 6670372. , 6677347, 6677348, 6677349, 6683088, 6703402, 6743920, 6800624, 6809203, 6888000 and 6924293.
[106] Jones (2003) Curr Opin Investig Drugs 4: 214-218.
[107] Steinhagen et al. (2011) Vaccine 29: 3341-55.
[108] Myers et al. (1990) pp 145-156 of Cellular and Molecular Aspects of Endotoxin Reactions.
[109] Ulrich (2000) Chapter 16 (pages 273-282) of reference 40.
[110] Johnson et al. (1999) J Med Chem 42: 4640-9.
[111] Baldrick et al. (2002) Regulatory Toxicol Pharmacol 35: 398-413.
[112] Coler et al. (2011) PLoS ONE 6 (l): el6333.
[113] Johnson et al. (1999) Bioorg Med Chem Lett 9: 2273-2278.
[114] Evans et al. (2003) Expert Rev Vaccines 2: 219-229.
[115] Bazin et al. (2006) Tetrahedron Lett 47: 2087-92.
[116] Wong et al. (2003) J Clin Pharmacol 43 (7): 735-42.
[117] US 2005/0215517.
[118] WO 03/011223.
[119] WO 2011/027222.
[120] WO 2013/131985 [121] Gennaro (2000) Remington: The Science and Practice of
Pharmacy. 20th edition, ISBN: 0683306472.
[122] Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.) [123] Handbook of Experimental Immunology, Vols. I-IV (DM Weir and C. C. Blackwell, eds, 1986, Blackwell Scientific Publications) [124] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition (Cold Spring Harbor Laboratory Press).
[125] Handbook of Surface and Colloidal Chemistry (Birdi, K. S. Ed., CRC Press, 1997) [126] Ausubel et al. (eds) (2002) Short Protocols in Molecular Biology, 5th Edition (Current Protocols).
[127] Molecular Biology Techniques: An Intensive Laboratory
Course, (Ream et al., Eds., 1998, Academic Press) [128] PCR (Introduction to Biotechnical Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag) [129] Geysen et al. (1984) PNAS USA 81: 3998-4002.
[130] Carter (1994) Methods Mol Biol 36: 207-23.
[131] Jameson, BA et al. 1988, CABIOS 4 (1): 181-186.
[132] Raddrizzani &amp; Hammer (2000) Brief Bioinform 1 (2): 179-89.
[133] Bublil et al. (2007) Proteins 68 (1): 294-304.
[134] De Lalla et al. (1999) J. Immunol. 163: 1725-1729.
[135] Kwok et al. (2001) Trends Immunol 22: 583-88.
[136] Brusic et al. (1998) Bioinformatics 14 (2): 121-30 [137] Meister et al. (1995) Vaccine 13 (6): 581-91.
[138] Roberts et al. (1996) AIDS Res Hum Retroviruses 12 (7): 593-610.
[139] Maksyutov &amp; Zagrebelnaya (1993) Comput Appl Biosci 9 (3): 291-7.
[140] Feiler &amp; de la Cruz (1991) Nature 349 (6311): 720-1.
[141] Hopp (1993) Peptide Research 6: 183-190.
[142] Welling et al. (1985) FEBS Lett. 188: 215-218.
[143] Davenport et al. (1995) Immunogenetics 42: 392-297.
[144] Tsurui &amp; Takahashi (2007) J Pharmacol Sci. 105 (4): 299-316.
[145] Tong et al. (2007) Brief Bioinform. 8 (2): 96-108.
[146] Schirle et al. (2001) J Immunol Methods. 257 (1-2): 1-16.
[147] Chen et al. (2007) Amino Acids 33 (3): 423-8.
[148] Current Protocols in Molecular Biology (F.M. Ausubel et al., Eds., 1987) Supplement 30 [149] Smith &amp; Waterman (1981) Adv. Appl. Math. 2: 482-489.
Sequence list SEQIDNO: 1 (EsxA)
MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLE
EIKQQLNSTADAVQEQDQQLSNNFGLQ SEQID NO: 2 (EsxB)
MGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELV
QPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP SEQ ID NO: 3 (FhuD2)
MKKLLLPLIIMLLVLAACGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAWAPTYAGGLKKLGANIVA VNQQVDQSKVLKDKFKGVTKIGDGDVEKVAKEKPDLIIVYSTDKDIKKYQKVAPTVWDYNKHKYLEQQE MLGKIVGKEDKVKAWKKDWEETTAKDGKEIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGL KMQPEQQKLTAKAGWAEVKQEEIEKYAGDYIVSTSEGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWY NDPYTLDFMRKDLKEKLIKAAK SEQ ID NO: 4 (Sta011)
MMKRLNKLVLGIIFLFLVISITAGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGT WIINSEMVIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKE IKDEKIKKEIENFKFFVQYGDFKNLKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAP KLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 5 (Hla)
MKTRIVSSVTTTLLLGSILMNPVANAADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFID
DKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMS
TLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDR
DSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRD
## EQU1 ##
CGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAWAPTYAGGLKKLGANIVAVNQQVDQSKVLKDKFKG
VTKIGDGDVEKVAKEKPDLIIVYSTDKDIKKYQKVAPTVWDYNKHKYLEQQEMLGKIVGKEDKVKAWKK
DWEETTAKDGKEIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAGWAE
VKQEEIEKYAGDYIVSTSEGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLKEKL
IKAAK SEQ ID NO: 7 (FhuD2 sequence example)
MASCGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAWAPTYAGGLKKLGANIVAVNQQVDQSKVLKDK FKGVTKIGDGDVEKVAKEKPDLIIVYSTDKDIKKYQKVAPTWVDYNKHKYLEQQEMLGKIVGKEDKVKA WKKDWEETTAKDGKEIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAG WAEVKQEEIEKYAGDYIVSTSEGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLK EKLIKAAK SEQ ID NO: 8 (e exempl sequence Sta011)
MGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWIINSEMVIQPNNEDMVAKGMV
LYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKEIKDEKIKKEIENFKFFVQYGDF
KNLKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEF
TFVEKKEENIYFSDSLDYKKSGDV SEQID NO: 9 (example sequence of Sta011)
MMKRLNKLVLGIIFLFLVISITAGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGT
WIINSEMVIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKE
IKDEKLKKEIENFKFFVQYGDFKNIKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAP
KLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKSGDV SEQID NO: 10 (example sequence of Sta011)
MMKRLNKLVLGIIFLFLVISITAGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGT
WIINSEMVIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKE
IKDEKVKKEIENFKFFVQYGDFKNIKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAP
KLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 11 (free from Sta011 sequence)
MMKRLNKLVLGIIFLFLVISITAGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGT
WIINSEMVIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKE
IKDEKLKKEIENFKFFVQYGDFKNVKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAP
KLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 12 (HLatronquered at the endN -ministeredSEQ ID NO: 5)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV SIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE RYKIDWEKEEMTN SEQ ID NO: 13 (mature-Hla H35L)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV SIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE RYKIDWEKEEMTN SEQ ID NO: 14 (tetramer)
PSGS SEQ ID NO: 15 (Hla-H35L with substitution of PSGS)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTPSGSVQPDFKTILESPTDKKVGWKVIFNNMV NQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNI DVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRS SERYKIDWEKEEMTN SEQ ID NO: 16 (Hla with substitution PSGS)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTPSGSVQPDFKTILESPTDKKVGWKVIFNNMV NQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNI DVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN SEQ ID NO: 17 (Hla with Y101 mutation)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE EGANKSGLAWPSAFKVQLQLPDNEVAQISDLYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV SIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE RYKIDWEKEEMTN SEQID NO: 18 (Hla mutation with D152)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV SIGHTLKYVQPLFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE RYKIDWEKEEMTN SEQID NO: 19 (Hla with H35 and Y101 mutations)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE EGANKSGLAWPSAFKVQLQLPDNEVAQISDLYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV SIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE RYKIDWEKEEMTN SEQID NO: 20 (Hla with H53 and D152 mutations)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV SIGHTLKYVQPLFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE RYKIDWEKEEMTN SEQ ID NO: 21 (fragment IIIa)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNK SEQ ID NO: 22 (fragment of Hla)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAG SEQ ID NO: 23 (fragment of Hla)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLL SEQ ID NO: 24 (fragment of Hla-H35L)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNK SEQ ID NO: 25 (fragment of Hla-H35L)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAG SEQ ID NO: 26 (fragment of Hla-H35L)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLL SEQID NO: 27 (HLA sequence useful with H35L mutation)
MASADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRV YSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIG ANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAA DNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDR SSERYKIDWEKEEMTN SEQ ID NO: 28 (example of EsxAB)
MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLE
EIKQQLNSTADAVQEQDQQLSNNFGLQASGGGSMGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQ
LAEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP SEQID NO: 29 (example of EsxBA)
MGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELV
QPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNPASGGGSMAMIKMSPEEIRAKSQSYGQGSDQIRQILS
DLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEEIKQQLNSTADAVQEQDQQLSNNFGLQ SEQ ID NO: 30 (Meur)
ASGGGS SEQ ID NO: 31 (example of EsxAB)
AMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEE
IKQQLNSTADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLA
EYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP SEQ ID NO: 32 (example of EsxAB)
MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLE
EIKQQLNSTADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQL
## STR1 ##
GCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWIINSEMVIQPNNEDMVAKGMVL
YMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKEIKDEKIKKEIENFKFFVQYGDFK
NLKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEFT
FVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 34 (FhuD2)
GNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAWAPTYAGGLKKLGANIVAVNQQVDQSKVLKDKFKGV
TKIGDGDVEKVAKEKPDLIIVYSTDKDIKKYQKVAPTVWDYNKHKYLEQQEMLGKIVGKEDKVKAWKKD
WEETTAKDGKEIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAGWAEV
KQEEIEKYAGDYIVSTSEGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLKEKLI
KAAK SEQ ID NO: 35 (sequence of EsxB devoid of Cys)
GGYKGIKADGGKVDQAKQLAAKTAKDIEAXQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQ
PMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP SEQ ID NO: 36 (non-Cys version of Sta011 SEQ ID NO: 4)
GIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWIINSEMVIQPNNEDMVAKGMVLYM
NRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKEIKDEKIKKEIENFKFFVQYGDFKNL
KNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEFTFV
EKKEENIYFSDSLDYKKSGDV SEQ ID NO: 37 (to which there is no Cys of FhuD2 SEQ ID NO: 7)
MASGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAWAPTYAGGLKKLGANIVAVNQQVDQSKVLKDKF
KGVTKIGDGDVEKVAKEKPDLIIVYSTDKDIKKYQKVAPTVWDYNKHKYLEQQEMLGKIVGKEDKVKAW
KKDWEETTAKDGKEIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAGW
AEVKQEEIEKYAGDYIVSTSEGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLKE
KLIKAAK SEQ ID NO: 38 (Cys-Ala mutant from EsxAB)
MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLE EIKQQLNSTADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEAAQKQTQQL AEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP SEQID NO: 39 (séquencede useful Sta011)
MGSGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWIINSEMVIQPNNEDMVAKGMV
LYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKEIKDEKIKKEIENFKFFVQYGDF
KNLKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEF
TFVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 40 (example of EsxAB without Cys)
AMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEE
IKQQLNSTADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEAXQKQTQQLA
EYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP SEQ ID NO: 41 icicicicicicicicicicicicic SEQID NO: 42
KLKLLLLLKLK SEQ ID NO: 43 (SpA)
MKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANAAQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKD
DPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVL
GEAKKLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAP
KADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQN
AFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPKEEDNNKPGKEDNNKPGKEDNNKP
GKEDNNKPGKEDNNKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDGNKPGKEDGN
GVHWKPGDTVNDIAKANGTTADKIAADNKLADKNMIKPGQELWDKKQPANHADANKAQALPETGEENP
FIGTTVFGGLSLALGAALLAGRRREL SEQ ID NO: 44 (SpAkkAA)
AQHDEAXXNAFYQVLNMPNLNADQRNGFIQSLKXXPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDXXS
AFYEILNMPNLNEAQRNGFIQSLKXXPSQSTNVLGEAKKLNESQAPKADNNFNKEXXNAFYEILNMPNLN
EEQRNGFIQSLKXXPSQSANLLSEAKKLNESQAPKADNKFNKEXXNAFYEILHLPNLNEEQRNGFIQSLK
XXPSQSANLLAEAKKLNDAQAPKADNKFNKEXXNAFYEILHLPNLTEEQRNGFIQSLKXXPSVSKEILAE
AKKLNDAQAPK SEQID NO: 45 (SpAkkAA)
AQHDEAKKNAFYQVLNMPNLNADQRNGFIQ SLKAAPSQ SANVLGEAQKLNDSQAPKADAQQNNFNKDKKS AFYEILNMPNLNEAQRNGFIQSLKAAPSQSTNVLGEAKKLNESQAPKADNNFNKEKKNAFYEILNMPNLN EEQRNGFIQSLKAAPSQSANLLSEAKKLNESQAPKADNKFNKEKKNAFYEILHLPNLNEEQRNGFIQSLK AAPSQSANLLAEAKKLNDAQAPKADNKFNKEKKNAFYEILHLPNLTEEQRNGFIQSLKAAPSVSKEILAE AKKLNDAQAPK SEQ ID NO: 46 (SpAkR)
AQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPKADAXXNNFNKDKKS AFYEILNMPNLNEAQRNGFIQSLKAAPSQSTNVLGEAKKLNESQAPKADNNFNKEKKNAFYEILNMPNLN EEQRNGFIQSLKAAPSQSANLLSEAKKLNESQAPKADNKFNKEKKNAFYEILHLPNLNEEQRNGFIQSLK AAPSQSANLLAEAKKLNDAQAPKADNKFNKEKKNAFYEILHLPNLTEEQRNGFIQSLKAAPSVSKEILAE AKKLNDAQAPK SEQ ID NO: 47 (SpAkR)
AQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPKADAKRNNFNKDKKS AFYEILNMPNLNEAQRNGFIQSLKAAPSQS TNVLGEAKKLNE SQAPKADNNFNKEKKNAFYEILNMPNLN EEQRNGFIQSLKAAPSQSANLLSEAKKLNESQAPKADNKFNKEKKNAFYEILHLPNLNEEQRNGFIQSLK AAPSQSANLLAEAKKLNDAQAPKADNKFNKEKKNAFYEILHLPNLTEEQRNGFIQSLKAAPSVSKEILAE AKKLNDAQAPK SEQID NO: 48 (used in Example SpAkR)
MAQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPKADAKRNNFNKDKK SAFYEILNMPNLNEAQRNGFIQSLKAAPSQS TNVLGEAKKLNE SQAPKADNNFNKEKKNAFYEILNMPNL NEEQRNGFIQSLKAAPSQSANLLSEAKKLNESQAPKADNKFNKEKKNAFYEILHLPNLNEEQRNGFIQSL KAAPSQSANLLAEAKKLNDAQAPKADNKFNKEKKNAFYEILHLPNLTEEQRNGFIQSLKAAPSVSKEILA EAKKLNDAQAPK SEQID NO: 49 (SpAkR)
AQHDEAXXNAFYQVLNMPNLNADQRNGFIQSLKXXPSQSANVLGEAQKLNDSQAPKADAXXNNFNKDXXS AFYEILNMPNLNEAQRNGFIQ S LKXX P SQS TNVLGEAKKLNE SQAPKADNNFNKEXXNAFYEILNMPNLN EEQRNGFIQSLKXXPSQSANLLSEAKKLNESQAPKADNKFNKEXXNAFYEILHLPNLNEEQRNGFIQSLK XXPSQSANLLAEAKKLNDAQAPKADNKFNKEXXNAFYEILHLPNLTEEQRNGFIQSLKXXPSVSKEILAE AKKLNDAQAPK SEQ ID NO: 50 (not domai E SpAkR)
## EQU2 ##
AQHDEAXXNAFYQVLNMPNLNADQRNGFIQSLKXXPSQSANVLGEAQKLNDSQAPKADAKRNNFNKD SEQ ID NO: 52 (Domain E of SpAkR)
AQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPKADAKRNNFNKD SEQ ID NO: 53 (SpAkR E domain used in the example)
MAQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPKADAKRNNFNKD SEQ ID NO: 54 (Domain E of SpA)
AQH DEAQQNAFYQVLNMPNLNADQRNGFIQS LKDDP S Q SANVLGEAQKLNDSQAPKADAQQNNFNKD

权利要求:
Claims (16)
[1]
A mutant SpA antigen, wherein the antigen comprises an amino acid sequence comprising or consisting of SEQ ID NO: 50 in which the dipeptide at positions 60 and 61 is not QQ; wherein said dipeptide is optionally KR; wherein said sequence is optionally SEQ ID NO: 51 or SEQ ID NO: 52.
[2]
The mutant SpA antigen of claim 1, wherein the antigen comprises an amino acid sequence comprising or consisting of SEQ ID NO: 49 in which the dipeptide at positions 60 and 61 is not QQ; wherein said dipeptide is optionally KR; wherein said sequence is optionally SEQ ID NO: 47.
[3]
The mutant SpA antigen of claim 1 which comprises more than one copy of said amino acid sequence comprising or consisting of SEQ ID NO: 50 in which the dipeptide at positions 60 and 61 is not QQ.
[4]
A mutant SpA antigen according to any one of claims 1 to 3 wherein the antigen elicits antibodies in a mammal that recognize SEQ ID NO: 43 and / or SEQ ID NO: 54.
[5]
A mutant SpA antigen according to any one of claims 1 to 4, which has decreased affinity, relative to the unmodified SpA, for the Fcγ portion of human IgGs.
[6]
The mutant SpA antigen of claim 5 which has decreased affinity, relative to the unmodified SpA, for the VH3-containing human B cell receptor Fab portion.
[7]
A mutant SpA antigen according to any one of claims 1 to 6, wherein the mutant SpA antigen is of sequence comprising or consisting of SEQ ID NO: 43 mutated in at least 1, particularly at least 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and more particularly 20 amino acids at position 43, 44, 70, 71, 96, 97 , 104, 105, 131, 132, 162, 163, 190, 191, 220, 221, 247, 248, 278, 279, 305 and / or 306 of SEQ ID NO: 43; even more preferably is a sequence comprising or consisting of SEQ ID NO: 47 or SEQ ID NO: 49.
[8]
8. SpA mutant antigen according to any one of claims 1 to 7, particularly of sequence comprising SEQ ID NO: 52, SEQ ID NO: 47 or SEQ ID NO: 48.
[9]
A fusion protein comprising a mutant SpA antigen according to any one of claims 1 to 8.
[10]
An immunogenic composition comprising a mutant SpA antigen according to any of claims 1 to 8 or a fusion protein according to claim 9.
[11]
11. Composition according to claim 10, wherein the composition also comprises an adjuvant.
[12]
The composition of claim 11, wherein said adjuvant is selected from the group consisting of: - aluminum salts, particularly aluminum hydroxides and aluminum phosphates; human TLR agonists, particularly TLR7 agonists; and - a mixture of these.
[13]
The composition of claim 12, wherein said TLR7 agonist is a compound of the following formula (K):

wherein: R1 is H, C1-C6 alkyl, -C (R5) 2OH, -L1R5, -L1R6, -L2R5, -L2R6, -OL2R5, or -OL2R6; L1 represents-C (O) - or -O-; L 2 represents C 1 -C 6 alkylene, C 2 -C 6 alkenylene, arylene, heteroarylene or - ((CR 4 R 4) p0) q (CH 2) p-, where C 1 -C 6 alkylene and C 2 -C 6 alkenylene 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-C4 alkyl, -L3R5, -L1R5, -L3R7, -L3L4L3R7, -L3L4R5, -L3L4L3R5, -0L3R5, -OL3R7, -OL3L4R7, -OL3L4L3R7, -OR8, -OL3L4R5, -OL3L4L3R5 and -C (R5) 2OH; 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; especially 3- (5-amino-2- (2-methyl-4- (2- (2- (2-phosphonoethoxy) ethoxy) ethoxy) phenethyl) benzo [f] [1,7] naphthyridine-8-acid; -yl) propanoic (K1).
[14]
The composition of claim 12 or 13, wherein the adjuvant comprises a human TLR agonist adsorbed on an aluminum salt.
[15]
15. An immunogenic composition according to any one of claims 1 to 14 for use as a medicament.
[16]
An immunogenic composition according to any one of claims 1 to 15 for use as a medicament for the prevention and / or treatment of S. aureus infection, optionally in a human being.

类似技术:

---

公开号 | 公开日 | 专利标题

---

BE1022744B1|2016-08-29|MUTANT STAPHYLOCOCCAL ANTIGENS

---

BE1022875B1|2016-09-30|COMPOSITIONS FOR IMMUNIZATION AGAINST STAPHYLOCOCCUS AUREUS

---

JP2015500864A|2015-01-08|Stable composition for immunizing against Staphylococcus aureus

---

US20180256701A1|2018-09-13|Pseudomonas antigens and antigen combinations

---

EP2890394B1|2019-05-01|Stabilised proteins for immunising against staphylococcus aureus

---

US20150202277A1|2015-07-23|Stabilised proteins for immunising against staphylococcus aureus

---

BE1022857B1|2016-09-27|IMMUNIZATION AGAINST STAPHYLOCOCCUS AUREUS

---

BE1022359B1|2016-03-25|IMMUNIZATION AGAINST STAPHYLOCOCCAL INFECTIONS OF BONES AND JOINTS

---

JP2015536929A|2015-12-24|Staphylococcus aureus SDRECNAB domain and its use for vaccination

---

US20150191513A1|2015-07-09|Stabilised proteins for immunising against staphylococcus aureus

同族专利:

---

公开号 | 公开日

---

KR20190040101A|2019-04-16|

---

PL3122378T3|2020-06-01|

---

CA2942450A1|2015-10-01|

---

IL282104D0|2021-05-31|

---

KR20160132109A|2016-11-16|

---

AU2015238512A1|2016-10-13|

---

IL247328D0|2016-09-29|

---

HUE047808T2|2020-05-28|

---

US20180064800A9|2018-03-08|

---

WO2015144653A1|2015-10-01|

---

AU2015238512B2|2018-02-01|

---

EA201691502A1|2017-04-28|

---

BE1022857A1|2016-09-27|

---

ES2769647T3|2020-06-26|

---

BR112016022011A2|2017-10-24|

---

MX2016012538A|2016-12-14|

---

EP3122378B1|2019-12-11|

---

US10328140B2|2019-06-25|

---

KR102027429B1|2019-10-01|

---

PT3122378T|2020-03-04|

---

BE1022744A1|2016-08-29|

---

IL247328A|2021-04-29|

---

JP6894237B2|2021-06-30|

---

RS59971B1|2020-03-31|

---

DK3122378T3|2020-03-02|

---

WO2015144655A1|2015-10-01|

---

JP2017511312A|2017-04-20|

---

CN106103469B|2020-11-27|

---

CN112521464A|2021-03-19|

---

LT3122378T|2020-02-10|

---

EA037818B1|2021-05-25|

---

KR102297357B1|2021-09-02|

---

EP3122378A1|2017-02-01|

---

EP3639850A1|2020-04-22|

---

SI3122378T1|2020-03-31|

---

CN106103469A|2016-11-09|

---

US20170143816A1|2017-05-25|

---

JP2021035950A|2021-03-04|

---

HRP20200217T1|2020-05-15|

---

KR20190110655A|2019-09-30|

---

SG11201606887WA|2016-09-29|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

WO2011005341A2|2009-04-03|2011-01-13|University Of Chicago|Compositions and methods related to protein a variants|

---

WO2010119343A2|2009-04-14|2010-10-21|Novartis Ag|Compositions for immunising against staphylococcus aureus|

---

WO2011127032A1|2010-04-05|2011-10-13|University Of Chicago|Compositions and methods related to protein a antibodies as an enhancer of immune response|

---

WO2012034067A1|2010-09-09|2012-03-15|The University Of Chicago|Methods and compositions involving protective staphylococcal antigens|

---

WO2013030378A1|2011-09-01|2013-03-07|Novartis Ag|Adjuvanted formulations of staphylococcus aureus antigens|

---

CN103645318A|2013-12-09|2014-03-19|重庆原伦生物科技有限公司|Detection method and detection kit of antigen-specific IgG antibody of staphylococcus aureus SpA5 mutant|

---

CN103694322A|2013-12-09|2014-04-02|重庆原伦生物科技有限公司|Staphylococcus aureus SpA5 mutant, as well as preparation method and application thereof|

---

SE8205892D0|1982-10-18|1982-10-18|Bror Morein|IMMUNOGENT MEMBRANE PROTEIN COMPLEX, SET FOR PREPARATION AND USE THEREOF|

---

EP0114787B1|1983-01-25|1991-09-25|Ciba-Geigy Ag|Peptide derivatives|

---

IL73534A|1983-11-18|1990-12-23|Riker Laboratories Inc|1h-imidazoquinoline-4-amines,their preparation and pharmaceutical compositions containing certain such compounds|

---

US4680338A|1985-10-17|1987-07-14|Immunomedics, Inc.|Bifunctional linker|

---

US5057540A|1987-05-29|1991-10-15|Cambridge Biotech Corporation|Saponin adjuvant|

---

US5238944A|1988-12-15|1993-08-24|Riker Laboratories, Inc.|Topical formulations and transdermal delivery systems containing 1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine|

---

US4929624A|1989-03-23|1990-05-29|Minnesota Mining And Manufacturing Company|Olefinic 1H-imidazoquinolin-4-amines|

---

WO1990014837A1|1989-05-25|1990-12-13|Chiron Corporation|Adjuvant formulation comprising a submicron oil droplet emulsion|

---

US4988815A|1989-10-26|1991-01-29|Riker Laboratories, Inc.|3-Amino or 3-nitro quinoline compounds which are intermediates in preparing 1H-imidazo[4,5-c]quinolines|

---

US5389640A|1991-03-01|1995-02-14|Minnesota Mining And Manufacturing Company|1-substituted, 2-substituted 1H-imidazo[4,5-c]quinolin-4-amines|

---

AT229943T|1991-03-01|2003-01-15|Minnesota Mining & Mfg|INTERMEDIATE PRODUCTS FOR THE PRODUCTION OF 1-SUBSTITUTED, 2-SUBSTITUTED-1H-IMIDAZO CHINOLIN-4-AMINES|

---

US5268376A|1991-09-04|1993-12-07|Minnesota Mining And Manufacturing Company|1-substituted 1H-imidazo[4,5-c]quinolin-4-amines|

---

US5266575A|1991-11-06|1993-11-30|Minnesota Mining And Manufacturing Company|2-ethyl 1H-imidazo[4,5-ciquinolin-4-amines|

---

IL105325A|1992-04-16|1996-11-14|Minnesota Mining & Mfg|Immunogen/vaccine adjuvant composition|

---

US5395937A|1993-01-29|1995-03-07|Minnesota Mining And Manufacturing Company|Process for preparing quinoline amines|

---

EP0812593B8|1993-03-23|2010-11-10|SmithKline Beecham Biologicals S.A.|Vaccine compositions containing 3-0 deacylated monophosphoryl lipid a|

---

CA2167042A1|1993-07-15|1995-01-26|Kyle J. Lindstrom|Imidazo[4,5-c]pyridin-4-amines|

---

US5352784A|1993-07-15|1994-10-04|Minnesota Mining And Manufacturing Company|Fused cycloalkylimidazopyridines|

---

WO1995011700A1|1993-10-29|1995-05-04|Pharmos Corp.|Submicron emulsions as vaccine adjuvants|

---

GB9326174D0|1993-12-22|1994-02-23|Biocine Sclavo|Mucosal adjuvant|

---

US6239116B1|1994-07-15|2001-05-29|University Of Iowa Research Foundation|Immunostimulatory nucleic acid molecules|

---

US6429199B1|1994-07-15|2002-08-06|University Of Iowa Research Foundation|Immunostimulatory nucleic acid molecules for activating dendritic cells|

---

US6207646B1|1994-07-15|2001-03-27|University Of Iowa Research Foundation|Immunostimulatory nucleic acid molecules|

---

AUPM873294A0|1994-10-12|1994-11-03|Csl Limited|Saponin preparations and use thereof in iscoms|

---

US5482936A|1995-01-12|1996-01-09|Minnesota Mining And Manufacturing Company|Imidazo[4,5-C]quinoline amines|

---

DE69637254T2|1995-04-25|2008-06-19|Glaxosmithkline Biologicals S.A.|Vaccines containing a saponin and a sterol|

---

WO1998040100A1|1997-03-10|1998-09-17|Ottawa Civic Loeb Research Institute|USE OF NUCLEIC ACIDS CONTAINING UNMETHYLATED CpG DINUCLEOTIDE AS AN ADJUVANT|

---

US6818222B1|1997-03-21|2004-11-16|Chiron Corporation|Detoxified mutants of bacterial ADP-ribosylating toxins as parenteral adjuvants|

---

WO1998043670A2|1997-04-01|1998-10-08|Ribi Immunochem Research, Inc.|Aqueous immunologic adjuvant compositions of monophosphoryl lipid a|

---

US6080725A|1997-05-20|2000-06-27|Galenica Pharmaceuticals, Inc.|Immunostimulating and vaccine compositions employing saponin analog adjuvants and uses thereof|

---

US6562798B1|1998-06-05|2003-05-13|Dynavax Technologies Corp.|Immunostimulatory oligonucleotides with modified bases and methods of use thereof|

---

US6110929A|1998-07-28|2000-08-29|3M Innovative Properties Company|Oxazolo, thiazolo and selenazolo [4,5-c]-quinolin-4-amines and analogs thereof|

---

GB9817052D0|1998-08-05|1998-09-30|Smithkline Beecham Biolog|Vaccine|

---

CN100558401C|1998-10-16|2009-11-11|史密丝克莱恩比彻姆生物有限公司|Adjuvant system and vaccine|

---

AU6007699A|1998-10-22|2000-05-08|Yosuke Naito|Showerhead|

---

US20030130212A1|1999-01-14|2003-07-10|Rossignol Daniel P.|Administration of an anti-endotoxin drug by intravenous infusion|

---

US6551600B2|1999-02-01|2003-04-22|Eisai Co., Ltd.|Immunological adjuvant compounds compositions and methods of use thereof|

---

DE60013773T2|1999-02-03|2005-11-10|Biosante Pharmaceuticals, Inc.|Methods for the preparation of therapeutic calcium phosphate particles|

---

US6331539B1|1999-06-10|2001-12-18|3M Innovative Properties Company|Sulfonamide and sulfamide substituted imidazoquinolines|

---

KR100865706B1|2000-09-26|2008-10-28|이데라 파마슈티칼즈, 인코포레이티드|Modulation of immunostimulatory activity of immunostimulatory oligonucleotide analogs by positional chemical changes|

---

US6664265B2|2000-12-08|2003-12-16|3M Innovative Properties Company|Amido ether substituted imidazoquinolines|

---

US6677348B2|2000-12-08|2004-01-13|3M Innovative Properties Company|Aryl ether substituted imidazoquinolines|

---

US6667312B2|2000-12-08|2003-12-23|3M Innovative Properties Company|Thioether substituted imidazoquinolines|

---

ES2260323T3|2000-12-08|2006-11-01|3M Innovative Properties Company|ETERES OF IMIDAZOQUINOLINE SUBSTITUTED WITH UREA AND PHARMACEUTICAL COMPOSITIONS THAT UNDERSTAND THE SAME.|

---

US6660735B2|2000-12-08|2003-12-09|3M Innovative Properties Company|Urea substituted imidazoquinoline ethers|

---

US6664260B2|2000-12-08|2003-12-16|3M Innovative Properties Company|Heterocyclic ether substituted imidazoquinolines|

---

US6664264B2|2000-12-08|2003-12-16|3M Innovative Properties Company|Thioether substituted imidazoquinolines|

---

US6660747B2|2000-12-08|2003-12-09|3M Innovative Properties Company|Amido ether substituted imidazoquinolines|

---

US6677347B2|2000-12-08|2004-01-13|3M Innovative Properties Company|Sulfonamido ether substituted imidazoquinolines|

---

WO2003035836A2|2001-10-24|2003-05-01|Hybridon Inc.|Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5' ends|

---

US6677349B1|2001-12-21|2004-01-13|3M Innovative Properties Company|Sulfonamide and sulfamide substituted imidazoquinolines|

---

WO2003101949A2|2002-05-29|2003-12-11|3M Innovative Properties Company|Process for imidazo[4,5-c]pyridin-4-amines|

---

RU2347586C2|2004-04-05|2009-02-27|Пфайзер Продактс Инк.|Microfluidised oil-in-water emulsions and vaccine compositions|

---

US20060228369A1|2005-04-11|2006-10-12|Program For Appropriate Technology In Health|Stabilization and preservation of temperature-sensitive vaccines|

---

US7691368B2|2005-04-15|2010-04-06|Merial Limited|Vaccine formulations|

---

US8703095B2|2005-07-07|2014-04-22|Sanofi Pasteur S.A.|Immuno-adjuvant emulsion|

---

TWI382019B|2005-08-19|2013-01-11|Array Biopharma Inc|Aminodiazepines as toll-like receptor modulators|

---

GB0526038D0|2005-12-21|2006-02-01|Glaxosmithkline Biolog Sa|Immunogenic composition|

---

PT2086582E|2006-10-12|2013-01-25|Glaxosmithkline Biolog Sa|Vaccine comprising an oil in water emulsion adjuvant|

---

FR2896162B1|2006-01-13|2008-02-15|Sanofi Pasteur Sa|EMULSION OIL IN THERMOREVERSIBLE WATER|

---

US20090053235A1|2006-06-12|2009-02-26|Nabi Biopharmaceuticals|Use of alpha-toxin for treating and preventing staphylococcus infections|

---

SI2038290T1|2006-07-07|2014-01-31|Gilead Sciences, Inc.|Modulators of toll-like receptor 7|

---

US20090007256A1|2007-06-28|2009-01-01|Microsoft Corporation|Using a trusted entity to drive security decisions|

---

WO2009029831A1|2007-08-31|2009-03-05|University Of Chicago|Methods and compositions related to immunizing against staphylococcal lung diseases and conditions|

---

PE20091236A1|2007-11-22|2009-09-16|Astrazeneca Ab|PYRIMIDINE DERIVATIVES AS IMMUNOMODULATORS OF TLR7|

---

SI2268618T1|2008-03-03|2015-09-30|Novartis Ag|Compounds and compositions as tlr activity modulators|

---

MX344330B|2008-03-24|2016-12-13|4Sc Ag|Novel substituted imidazoquinolines.|

---

PL2313111T3|2008-08-01|2014-08-29|Ventirx Pharmaceuticals Inc|Toll-like receptor agonist formulations and their use|

---

EP2208787A1|2009-01-19|2010-07-21|Université de Liège|A recombinant alpha-hemolysin polypeptide of Staphylococcus aureus, having a deletion in the stem domain and heterologous sequences inserted|

---

WO2011027222A2|2009-09-02|2011-03-10|Novartis Ag|Immunogenic compositions including tlr activity modulators|

---

EA201390341A1|2010-09-01|2013-08-30|Новартис Аг|ADSORPTION OF IMMUNOTE POTENTIATORS ON INSOLUBLE SALTS OF METALS|

---

EP2793939A1|2011-12-23|2014-10-29|Novartis AG|Stable compositions for immunising against staphylococcus aureus|

---

LT2822947T|2012-03-07|2016-10-10|Glaxosmithkline Biologicals Sa|Arginine salts of a tlr7 agonist|

---

US9375471B2|2012-03-08|2016-06-28|Glaxosmithkline Biologicals Sa|Adjuvanted formulations of booster vaccines|

---

JP2015528456A|2012-08-31|2015-09-28|ノバルティス アーゲー|Stabilized protein for immunization against STAPHYLOCOCUSAUREUS|

---

WO2014033193A1|2012-08-31|2014-03-06|Novartis Ag|Stabilised proteins for immunising against staphylococcus aureus|

---

CN104619336A|2012-08-31|2015-05-13|诺华股份有限公司|Stabilised proteins for immunising against staphylococcus aureus|

---

CN104736165A|2012-08-31|2015-06-24|诺华股份有限公司|Stabilised proteins for immunising against Staphylococcus aureus|WO2013030378A1|2011-09-01|2013-03-07|Novartis Ag|Adjuvanted formulations of staphylococcus aureus antigens|

---

GB201802339D0|2018-02-13|2018-03-28|Glaxosmithkline Biologicals Sa|Immunogenic composition|

---

CN109526985B|2018-11-19|2021-03-12|黑龙江省农垦科学院|Application of geobacillus protein EsxA as biological rice blast preventing and treating agent|

---

EP3777884A1|2019-08-15|2021-02-17|GlaxoSmithKline Biologicals S.A.|Immunogenic composition|

---

WO2021067785A1|2019-10-02|2021-04-08|Janssen Vaccines & Prevention B.V|Staphylococcus peptides and methods of use|

法律状态:
2019-12-05| MM| Lapsed because of non-payment of the annual fee|Effective date: 20190331 |

优先权:

---

申请号 | 申请日 | 专利标题

---

EP14161861|2014-03-26|

---

EP14161861.1|2014-03-26|

---

EP14192913|2014-11-12|

---