COMPOSITIONS FOR IMMUNIZATION AGAINST STAPHYLOCOCCUS AUREUS

专利摘要:
FIG.1 L'invention propose également une OMV à partir d'une bactérie Gram négatif, où l'OMV comprend au moins un antigène de S. aureus. L'antigène/les antigènes peut/peuvent être partiellement présent (s) dans la membrane de l'OMV et partiellement présent (s) dans la lumière de l'OMV ou libres dans la lumière de l'OMV. L'invention propose également un procédé de préparation d'une OMV de l'invention. The invention relates to S. aureus antigens which may be useful for immunization when used in combination and in particular a pharmaceutical composition of at least two antigens selected from (i) an hla antigen, (ii) a antigen sta, (iii) a lukE antigen and (iv) a spa antigen as being particularly useful for immunization. The antigens can be included within outer membrane vesicles (OMV). FIG. 1 The invention also provides OMV from a Gram-negative bacterium, wherein the OMV comprises at least one S. aureus antigen. The antigen / antigens may be partially present in the OMV membrane and partially present in the OMV lumen or free in the OMV lumen. The invention also proposes a process for preparing an OMV of the invention.
公开号:BE1022875B1
申请号:E2015/5177
申请日:2015-03-24
公开日:2016-09-30
发明作者:Fabio Bagnoli；Guido Grandi
申请人:Glaxosmithkline Biologicals S.A.；
IPC主号:

专利说明:

COMPOSITIONS FOR IMMUNIZATION AGAINST STAPHYLOCOCCUS
Aureus
This application claims the priority of the European Patent Application 14161861.1 (filed March 26, 2014), the entirety of which is incorporated herein by reference for all objects.
Technical area
This invention relates to combinations of S. aureus antigens and their use in immunization.
State of the art context
Staphylococcus aureus is a gram-positive spherical bacterium. In the United States, annual mortality exceeds that of any other infectious disease, including HIV / AIDS, and S. aureus is the leading cause of infections in the bloodstream, lower respiratory tract, skin and tissues. soft. S. aureus causes a range of diseases ranging from minor skin infections to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, bacteremia, endocarditis, toxic shock syndrome, abscess organs and sepsis. The bacterium has multiple virulence factors that are differentially expressed during the different phases of its life cycle, and so a vaccine that can prevent one disease may not prevent another. An object of the invention is to provide a vaccine that protects against a disease caused by S. aureus, ideally against multiple diseases caused by S. aureus.
WO 2010/119343 discloses S. aureus antigens that can be used in vaccines to protect against diseases.
However, there remains the need to identify particular antigens and combinations of antigens for use in S. aureus vaccines. There is also a need to develop S. aureus vaccines with improved immunogenicity in mammals and methods for producing and administering such vaccines.
Disclosure of the invention
The inventors have identified particular S. aureus antigens that may be useful for immunization when used in combination. In particular, the inventors have identified combinations of at least two antigens selected from (i) a hla antigen, (ii) a stain antigen, (iii) a lukE antigen and (iv) a spa antigen as being particularly useful for immunization. Generally, the combination is at least three of these antigens, and more generally 4 antigens.
Accordingly, the invention provides a pharmaceutical composition comprising at least two antigens from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen. Generally, the combinations comprise at least three antigens from the group consisting of: (i) hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen, or more generally the four antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen. In one aspect, the pharmaceutical composition comprises a lukE antigen and an antigen selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen, and (iii) a spa antigen. At least one antigen may be included within outer membrane vesicles (OMV). When included within OMV, the antigen / antigens can be included within separate OMVs or within the same OMV. The invention also provides a pharmaceutical composition comprising at least two antigens from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen. Generally, the combinations comprise at least three antigens from the group consisting of: (i) hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally at least four of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally at least five of the group consisting of: (i) an hla antigen; (ii) a staOOδ antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally the four antigens of the group consisting of: (i) an hla antigen; (ii) a staOOδ antigen; (iii) a lukE antigen and (iv) a spa antigen, or more generally the four antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) esxAB and (iv) staOll antigen. In one aspect, the pharmaceutical composition comprises a lukE antigen and an antigen selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a spa antigen; (iv) a staOll antigen; (v) an esxA antigen and (vi) an esxB antigen. At least one antigen may be included within outer membrane vesicles (OMV). When included within OMV, the antigen / antigens can be included within separate OMVs or within the same OMV. The invention also provides an OMV from a Gram-negative bacterium, wherein the OMV comprises at least one S. aureus antigen, wherein the antigen is selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen. The OMVs may comprise at least two antigens from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or they may comprise at least three antigens from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or they may comprise at least four of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or they may comprise at least five of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or they may comprise the four antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen, or they may comprise the four of the antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) esxAB and (iv) staOll antigen. The invention also proposes a process for preparing an OMV of the invention. The invention also provides an OMV from a Gram-negative bacterium, wherein the OMV comprises at least one S. aureus antigen in its membrane where the antigen is selected from the group consisting of: (i) an antigen hla ; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen. Generally, OMV comprises at least two antigens in its membrane of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally at least three antigens in its membrane of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally at least four of the group consisting of: (i) an hla antigen; (ii) a staOOδ antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or it may comprise the four antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen, or it may comprise all four of the antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) esxAB and (iv) staOll antigen. The invention also provides a process for preparing an OMV of the invention. The invention also provides an OMV from a Gram-negative bacterium, wherein the OMV comprises at least one S. aureus antigen in its lumen, wherein the antigen is selected from the group consisting of: (i) an antigen hla ; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen. Generally, OMV comprises at least two antigens in its lumen from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally at least three antigens in its light from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally at least four of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally the four antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen, or more generally the four antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) esxAB and (iv) staOll antigen. The antigen or antigens may be partially present in the membrane and partially present in OMV lumen. Alternatively, the antigen or antigens may be free in the OMV lumen. The invention also proposes a process for preparing an OMV of the invention. The method may include the step of expressing S. aureus antigen in the periplasm of the Gram-negative bacterium. The method may include the step of expressing S. aureus antigen in the periplasm of the Gram-negative bacterium using an expression vector comprising a nucleic acid sequence encoding the antigen operably linked to a nucleic acid encoding a signal sequence of a periplasmic protein. The native signal sequence of the S. aureus antigen may be replaced by the signal sequence of a periplasmic protein. The invention also provides an OMV from a Gram-negative bacterium, wherein the OMV comprises at least one S. aureus antigen, wherein the antigen is selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, wherein the gram negative bacterium comprises a synthetic genome in which: a) one or more nonessential sequences for the production of vesicles are absent so that, compared to the same bacteria without said absent sequences, the genome is between 1% and 50% smaller, and b) one or more sequences are present such that, compared to the same bacterium without said sequences present, the bacterium produces vesicles that include a or more additional antigens. Generally, the OMV comprises at least two antigens from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally at least three antigens from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally at least four of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally the four antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen, or more generally the four antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) esxAB and (iv) staOll antigen. The antigen may be in the OMV lumen, and possibly free in the OMV lumen. The invention also proposes a process for preparing an OMV of the invention. The invention also provides a pharmaceutical composition comprising (a) at least one OMV of the invention and (b) a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise at least one OMV which comprises at least two S. aureus antigens selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, i.e. a combination of antigens is provided within the same OMV. Alternatively, the pharmaceutical composition may comprise multiple different OMVs that comprise at least one S. aureus antigen selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, i.e. a combination of antigens is provided in separate OMVs.
The pharmaceutical compositions and / or OMVs of the invention may further comprise at least one antigen selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen. The invention also provides a method of generating an immune response against a S. aureus antigen in a mammal, the method comprising administering an effective amount of an OMV of the invention, or a pharmaceutical composition of the invention, to the mammal, wherein the immune response is directed against the S. aureus antigen in OMV. The invention also provides an OMV of the invention, or a pharmaceutical composition of the invention, for use in therapy.
Combinations of S. aureus antigens The invention provides one or more S. aureus antigens selected from the group consisting of: (i) hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen. The invention may further involve one or more S. aureus antigens selected from the group consisting of: (i) a staOll antigen; (ii) esxA antigen and / or esxB antigen and (iii) antigen, clfA.
Hla The "Hla" antigen is the "precursor of alpha-hemolysin" also known as "alpha toxin" or simply "hemolysin". In strain NCTC 8325, Hla is SAOUHSC_01121 and has the amino acid sequence SEQ ID NO: 1 (GI: 88194865). In the Newman strain, it is nwmn_1073 (GI: 151221285). 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.
Hla antigens can elicit an antibody (e.g., when administered to a human) that recognizes SEQ ID NO: 1 and / or may comprise an amino acid sequence: (a) having 50% or more 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: 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, 100, 150, 200, 250 or more). These Hla proteins 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 first 26 N-terminal amino acids of SEQ ID NO: 1 can be conveniently omitted (eg, to give SEQ ID NO: 2). Truncation at the C-terminus may also be used, for example leaving only 50 amino acids (residues 27 to 76 of SEQ ID NO: 1) [1]. Other fragments omit one or more protein domains.
The toxicity of Hla can be avoided in compositions of the invention by chemical inactivation (e.g., using formaldehyde, glutaraldehyde or other cross-linking reagents). However, instead, it is preferable to use mutant forms of Hla that eliminate its toxic activity while maintaining its immunogenicity. Such detoxified mutants are already known from the state of the art. A useful Hla antigen has a mutation at residue 61 of SEQ ID NO: 1, which is the residue of the mature antigen (i.e., after omission of the first 26 N-terminal amino acids = residue Of SEQ ID NO: 2). Thus, residue 61 may not be histidine and may instead be, for example, Ile, Val or preferably Leu. His-Arg mutation at this position can also be used. For example, SEQ ID NO: 3 is the sequence of the mature Hla-H35L mutant (i.e., SEQ ID NO: 2 with an H35L mutation) and a useful Hla antigen comprises SEQ ID NO: 3. 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: 1 with a tetramer such as PSGS, as in SEQ ID NO: 4 (which also includes H35L mutation) and SEQ ID NO: 5 (which does not include the H35L mutation). Another useful mutation replaces the Y101 residue, for example with leucine (SEQ ID NO: 6). Another useful mutation replaces the residue D152, for example with leucine (SEQ ID NO: 7). Another useful mutant replaces residues H35 and Y101, for example with leucine (SEQ ID NO: 8). Another useful mutant replaces residues H35 and D152, for example with leucine (SEQ ID NO: 9). Other useful antigens of Hla are disclosed in references 2 and 3. SEQ ID NO: 10, 11 and 12 are three useful fragments of SEQ ID NO: 1 (Hla27-76 ', Hla27-89' and 'Hla27 -79 ', respectively). SEQ ID NO: 13, 14 and 15 are the corresponding fragments from SEQ ID NO: 3.
A useful sequence of Hla is SEQ ID NO: 16. It comprises an N-terminal Met, then an Ala-Ser dipeptide from the expression vector, then SEQ ID NO: 3 (from strain NCTC8325). It is encoded by SEQ ID NO: 17.
In a preferred embodiment, the Hla antigen is encoded by the nucleic acid of SEQ ID NO: 121 and / or the amino acid sequence of SEQ ID NO: 122.
StaOOô The "StaOOô" antigen is annotated as "ferrichrome binding protein", and has already been called "FhuD2" in the literature [4]. In strain NCTC 8325, StaOOδ is SAOUHSC_02554 and has the amino acid sequence SEQ ID NO: 18 (GI: 88196199). In the Newman strain, it is nwmn_2185 (GI: 151222397).
StaOOO used in the invention may elicit an antibody (e.g., when administered to a human) that recognizes SEQ ID NO: 18 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: 18, and / or (b) comprising a fragment of at least "η" consecutive amino acids of SEQ ID NO: 18, 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) . These Sta006 proteins include variants of SEQ ID NO: 18. The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 18. 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: 18 while retaining at least one epitope of SEQ ID NO: 18. The first 17 N-terminal amino acids of SEQ ID NO: 18 can be conveniently omitted (to provide SEQ ID NO: 19). Other fragments omit one or more protein domains. Mutant forms of Sta006 are reported in reference 5. A Sta006 antigen can be lipidated, for example with acylated N-terminal cysteine. A useful sequence of Sta006 is SEQ ID NO: 20, which has a Met-Ala-Ser- sequence at the N-terminus. Sta006 can exist as a monomer or oligomer (eg, dimer), with Ca ++ ions promoting oligomerization. The invention can use Sta006 monomers and / or oligomers. Sta006 can be a homodimer or a heterodimer with StaOll. The Sta006 antigen naturally has an N-terminal cysteine in its mature form. This cysteine can be deleted in the Sta006 antigen to stop dimerization and provide a protein that is easier to characterize and analyze without negatively impacting immunogenicity. The compositions containing Sta006 antigens deficient in cysteine are more stable. Thus, the Sta006 antigen may comprise an amino acid sequence that is at least 90% (eg, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and the like). %,> 98%,> 99%, 99.5%) identity with SEQ ID NO: 21, where the polypeptide does not have a free thiol group, and can elicit antibodies (for example, when is administered to a human) that recognize a wild type StaOOδ antigen (e.g., a S. aureus protein consisting of the amino acid sequence SEQ ID NO: 18). The polypeptide can not form covalent dimers via disulfide bonds. SEQ ID NO: 21 corresponds to amino acid residues 19 to 302 of SEQ ID NO: 18. Compared to SEQ ID NO: 21, SEQ ID NO: 22 has an additional amino acid residue "X" at the end N-terminal, where "X" is an amino acid that does not contain a free thiol group. Compared with SEQ ID NO: 21, SEQ ID NO: 23 has a Met-Ala-Ser- sequence at the N-terminus. Compared to SEQ ID NO: 22, SEQ ID NO: 24 has a Met-Ala-Ser- sequence at the N-terminus. A Sta006 polypeptide comprising any of SEQ ID NO: 21, 22, 23 or 24 may be used in the invention.
A useful variant form of Sta006 can include at least one point mutation that replaces, modifies or deletes the cysteine residue present in the wild-type form of the antigen. For example, a Sta006 polypeptide may comprise an amino acid sequence having SEQ ID NO: 20, wherein the cysteine residue at position 4 of SEQ ID NO: 20 is replaced, modified or deleted. Preferably, the replacement is by a serine or an alanine residue. Alternatively, the cysteine residue is deleted (e.g., providing SEQ ID NO: 23).
In a preferred embodiment, the Sta006 antigen is encoded by the nucleic acid of SEQ ID NO: 123 and / or the amino acid sequence of SEQ ID NO: 124.
lukE The "lukE" antigen is annotated as "leukotoxin lukE". In strain NCTC 8325, lukE is SAOUHSC_01955 and has the amino acid sequence SEQ ID NO: 25 (GI: 88195648).
Useful lukE antigens can elicit an antibody (e.g., when administered to a human) that recognizes SEQ ID NO: 25 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: 24; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 25, 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 lukE proteins include variants of SEQ ID NO: 25. The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 25. 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: 24 while retaining at least one epitope of SEQ ID NO: 25. Other fragments omit one or more protein domains.
In a preferred embodiment, the lukE antigen is encoded by the nucleic acid of SEQ ID NO: 127 or SEQ ID NO: 129 and / or the amino acid sequence of SEQ ID NO: 128 or SEQ ID NO. : 130. spa The "spa" antigen is labeled "protein A" or "SpA". In strain NCTC 8325, spa is SAOUHSC_00069 and has the amino acid sequence SEQ ID NO: 26 (GI: 88193885). In the Newman strain, it is nwmn_0055 (GI: 151220267). All strains of S. aureus express the structural gene for spa, a well characterized virulence factor whose cell wall-anchored surface protein product has five highly homologous immunoglobulin binding domains designated 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]. SpA is synthesized as a precursor protein with an N-terminal signal peptide and a C-terminal sorting signal [8,9]. Spa anchored to the cell wall is shown in great abundance on the staphylococcal surface [10,11]. Each of its immunoglobulin binding domains is composed of antiparallel helices that assemble into a three-helix bundle and can bind to the Fc domain of immunoglobulin G (IgG) [12,13], the heavy chain VH3 (Fab) of IgM (i.e., B cell receptor) [14], von Willebrand factor at its Al domain [15] and / or TNF-α receptor I (TNFRI) [16], which is presented on the surfaces of airway epithelia.
Useful spa antigens can elicit an antibody (e.g., when administered to a human) that recognizes SEQ ID NO: 26 and / or may include 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: 26; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 26, 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 proteins include variants of SEQ ID NO: 26. The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 26. 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: 26 while retaining at least one epitope of SEQ ID NO: 26. The final 35 C-terminal amino acids of SEQ ID NO: 26 may be omitted in a useful manner. The first 36 N-terminal amino acids of SEQ ID NO: 26 may be omitted in a useful manner. Other fragments omit one or more protein domains. Reference 17 suggests that individual IgG binding domains could be useful immunogens, alone or in combination. SEQ ID NO: 27 is a useful fragment of SEQ ID NO: 26 ('Spa37-325'). 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 domain. the most exposed of SpA. It also reduces the similarity of the antigen with human proteins. Other useful fragments may omit 1, 2, 3 or 4 of the natural 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 B-cell superantigen As reported in reference 17, other useful fragments may comprise only 1, 2, 3 or 4 of the A, B, C, D and / or E natural domains, for example, they may comprise 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 for the invention may comprise 1, 2, 3, 4 or 5 IgG binding domains, but ideally it comprises 4 or less.
If an antigen comprises only one type of spa domain (for example, only the SpA (A) or SpA (D) domain), it may include more than one copy of this domain, for example multiple SpA (D) domains. in a single polypeptide chain.
An individual domain within the antigen can be mutated at the level of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids compared to SEQ ID NO: 26 (e.g. see reference 17, disclosing mutations at residues 3 and / or 24 of domain D, at residue 46 and / or 53 at domain A, etc.). Such mutants should not eliminate the ability of the antigen to elicit an antibody that recognizes SEQ ID NO: 26, but they can eliminate antigen binding to IgG and / or other human proteins (such as human blood).
In some aspects, a spa antigen comprises (a) one or more amino acid substitutions in a SpA domain IgG Fc binding subdomain A, B, C, D and / or E, which disrupts or decreases binding to IgG Fc, and (b) one or more amino acid substitutions in a SpA domain Vh3 binding subdomain A, B, C, D and / or E, which disrupts or decreases the link to Vh3. In some embodiments, a variant SpA comprises at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more variant D domain peptides of SpA.
In some aspects, amino acid residues F5, Q9, Q10, Sil, F13, Y14, L17, N28, 131, and / or K35 of the domain D IgG Fc binding subdomain are modified or substituted. , i.e., amino acids 100, 104, 105, 106, 108, 109, 112, 123, 126 and / or 130 of SEQ ID NO: 26. In some aspects, amino acid residues Q26 , G29, F30, S33, D36, D37, Q40, N43, and / or E47 of the domain D VH3 binding subdomain are modified or substituted such that the binding to Fc or VH3 is attenuated, that is, amino acids 121, 124, 125, 128, 131, 132, 135, 138 and / or 141 of SEQ ID NO: 26. In other aspects, corresponding modifications or substitutions can be made in corresponding positions of the domain A, B, C and / or E. The corresponding positions are defined by the alignment of the amino acid sequence of the domain D with one or more of the amino acid sequences from other domains of made friends SpA IgG sound. In some aspects, the amino acid substitution may be of any of the other 20 amino acids. In another aspect, conservative amino acid substitutions may be specifically excluded from possible amino acid substitutions. In other aspects, only non-conservative substitutions are included. In any case, any substitution or combination of substitutions which reduces the binding of the domain is contemplated such that the toxicity of SpA is significantly reduced. The significance of the binding reduction refers to a variant that produces minimal toxicity or no toxicity when introduced into a subject and can be estimated using in vitro methods described herein.
In one aspect of the invention, glutamine residues at position 9 and / or 10 of SpA domain D, i.e., amino acids 104 and / or 105 of SEQ ID NO: 26 (or in other areas) are mutated. In another aspect, aspartic acid residues 36 and / or 37 of SpA domain D, i.e., amino acids 131 and 132 of SEQ ID NO: 26 (or corresponding positions in other domains) are mutated. In another aspect, residues of glutamine 9 and 10, and aspartic acid 36 and 37 of domain D of SpA, i.e., amino acids 104, 105, 131 and 132 of SEQ ID NO: 26 (or corresponding positions in other areas) are mutated.
In other aspects, the amino acid glutamine (Q) at position 9 of SpA domain D, i.e., amino acid 104 of SEQ ID NO: 26 (or its analogous amino acid in other domains of SpA) can be replaced by alanine (A), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), phenylalanine (F), glycine (G), a histidine (H), an isoleucine (I), a lysine (K), a leucine (L), a methionine (Μ), a proline (P), a serine (S), a threonine (T) ), a valine (V), a tryptophan (W), or a tyrosine (Y). In some aspects, 9-position glutamine may be substituted with arginine (R). In another aspect, glutamine at position 9 of SpA domain D, i.e., amino acid 104 of SEQ ID NO: 26, or its equivalent, may be substituted with lysine or glycine.
In another aspect, the amino acid glutamine (Q) at position 10 of domain D of SpA, i.e., amino acid 105 of SEQ ID NO: 26 (or its analogous amino acid in other SpA domains) can be replaced by an alanine (A), an asparagine (N), an aspartic acid (D), a cysteine (C), a glutamic acid (E), a phenylalanine (F), a glycine (G ), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), proline (P), serine (S), threonine (T), valine (V), tryptophan (W), or tyrosine (Y). In some aspects, glutamine at the 10-position may be substituted with arginine (R). In another aspect, glutamine at position 10 of SpA domain D, i.e., amino acid 105 of SEQ ID NO: 26, or its equivalent, may be substituted with lysine or glycine.
In some aspects, aspartic acid (D) at position 36 of SpA domain D, i.e., amino acid 131 of SEQ ID NO: 26 (or its analogous amino acid in other SpA) can be replaced by alanine (A), asparagine (N), arginine (R), cysteine (C), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), a lysine (K), a leucine (L), a methionine (M), a proline (P), a glutamine (Q), a serine (S), a threonine (T), a valine (V) ), tryptophan (W), or tyrosine (Y). In some aspects, aspartic acid at position 36 of the SpA D domain, i.e., amino acid 131 of SEQ ID NO: 26 may be substituted with glutamic acid (E). In some aspects, an aspartic acid at position 36 of the SpA D domain, i.e., amino acid 131 of SEQ ID NO: 26, or its equivalent, may be substituted with alanine or serine.
In another aspect, aspartic acid (D) at position 37 of domain D of SpA, i.e., amino acid 132 of SEQ ID NO: 26 (or its analogous amino acid in other domains of SpA) can be replaced by alanine (A), asparagine (N), arginine (R), cysteine (C), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), proline (P), glutamine (Q), serine (S), threonine (T), valine ( V), tryptophan (W), or tyrosine (Y). In some aspects, aspartic acid at position 37 of SpA domain D, i.e., amino acid 132 of SEQ ID NO: 26 may be substituted with glutamic acid (E). In some aspects, aspartic acid at position 37 of SpA domain D, i.e., amino acid 132 of SEQ ID NO: 26, or its equivalent, may be substituted with alanine or serine.
In a particular embodiment, the amino acid at position 9 of domain D of SpA, i.e., amino acid 104 of SEQ ID NO: 26 (or a similar amino acid in another field of SpA) is replaced by alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P), serine (S), or valine (V). In some aspects, the amino acid at position 9 of SpA domain D, i.e., amino acid 104 of SEQ ID NO: 26 is replaced by a glycine. In another aspect, the amino acid at position 9 of SpA domain D, i.e., amino acid 104 of SEQ ID NO: 26 is replaced with lysine.
In a particular embodiment, the amino acid at position 10 of domain D of SpA, that is, amino acid 105 of SEQ ID NO: 26 (or a similar amino acid in another field of SpA) is replaced by alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P), serine (S), or valine (V). In some aspects, the amino acid at position 10 of SpA domain D, i.e., amino acid 105 of SEQ ID NO: 26 is replaced by a glycine. In another aspect, the amino acid at position 10 of domain D of SpA, i.e., amino acid 105 of SEQ ID NO: 26 is replaced by lysine.
In a particular embodiment, the amino acid at position 36 of domain D of SpA, i.e., amino acid 131 of SEQ ID NO: 26 (or a similar amino acid in another field of SpA) is replaced by alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P), serine (S), or valine (V). In some aspects, the amino acid at position 36 of SpA domain D, i.e., amino acid 131 of SEQ ID NO: 26 is replaced with serine. In another aspect, the amino acid at position 36 of SpA domain D, i.e., amino acid 131 of SEQ ID NO: 26 is replaced by alanine.
In a particular embodiment, the amino acid at position 37 of domain D of SpA, that is, amino acid 132 of SEQ ID NO: 26 (or a similar amino acid in another SpA domain ) is replaced by alanine (A), glycine (G), isoleucine (I), leucine (L), proline (P), serine (S), or valine (V). In some aspects, the amino acid at position 37 of domain D of SpA, i.e., amino acid 132 of SEQ ID NO: 26 is replaced by serine. In another aspect, the amino acid at position 37 of domain D of SpA, i.e., amino acid 132 of SEQ ID NO: 26 is replaced by alanine.
A useful Spa variant is SpakkAA5 (SEQ ID NO: 28) which comprises the five immunoglobulin binding domains with four amino acid substitutions, corresponding to Gln9Lys, GlnlOLys, Asp36Ala and Asp37Ala of the D domain, i.e. ie amino acids 104, 105, 131 and 132 of SEQ ID NO: 26, in each of its five immunoglobulin binding domains (E, D, A, B and C).
In a preferred embodiment, the Spa antigen is encoded by the nucleic acid of SEQ ID NO: 125 and / or the amino acid sequence of SEQ ID NO: 126.
StaOll The "StaOll" antigen was originally annotated simply as "lipoprotein". In strain NCTC 8325, StaO11 is SAOUHSC_00052 and has the amino acid sequence SEQ ID NO: 41 (GI: 88193872). The known StaOll antigen has an N-terminal cysteine in its mature form, and it can be lipidized. Wild-type StaOll containing a cysteine may exist as a monomer or oligomer (eg, covalent dimer), with Ca ++ ions promoting oligomerization.
A variant form of StaOll can be used which can not form covalent dimers via disulfide bonds. In such embodiments, the polypeptide does not contain any free thiol groups (under reducing conditions). It can elicit antibodies (e.g., when administered to a human) that recognize a wild-type StaOll antigen (e.g., SEQ ID NO: 45, 53, 54 or 55). The polypeptide may comprise an amino acid sequence having 80% or more identity (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%). %, 99%, 99.5% or greater) with any of SEQ ID NO: 47 to 52. SEQ ID NO: 47 corresponds to amino acid residues 26 to 256 of SEQ ID NO. : 41. Compared with SEQ ID NO: 47, SEQ ID NO: 48 has an additional amino acid residue "X" at the N-terminus, where "X" is an amino acid that does not contain a thiol group free (for example, is Ser = SEQ ID NO: 52). SEQ ID NO: 49 contains a Met-Gly- sequence at the N-terminus of SEQ ID NO: 47. SEQ ID NO: 50 has a Met-Gly- sequence at the N-terminus of SEQ ID NO : 48. SEQ ID NO: 51 has the sequence of SEQ ID NO: 50, wherein "X" is a serine. A StaOll polypeptide comprising any of SEQ ID NOs: 47 to 52 may be used in the invention.
Alternative forms of SEQ ID NO: 41 which may also be used include, but are not limited to, SEQ ID NO: 42, 43 and 44 with various Ile / Val / Leu substitutions. Compared with SEQ ID NO: 41, SEQ ID NO: 42 a Leu-146 in place of Ile-146 and Ile-165 in place of Leu-165. Compared to SEQ ID NO: 41, SEQ ID NO: 43 has Val-146 instead of Ile-146 and Ile-165 instead of Leu-165. Compared with SEQ ID NO: 41, SEQ ID NO: 44a Leu-146 instead of Ile-146 and Val-165 instead of Leu-165. The first 23 N-terminal amino acids of SEQ ID NO: 41 to 44 (i.e., the signal peptide) can be conveniently omitted to provide SEQ ID NOs: 45, 53, 54 and 55, respectively . Thus, a StaOll polypeptide of the invention may comprise residues 26 to 256 of any of SEQ ID NO: 41 to 44, and may elicit antibodies (e.g., when administered to a human) which recognize the mature StaOll antigen (e.g., SEQ ID NO: 45, 53, 54 or 55).
A useful variant form of StaOll may include at least one point mutation that replaces, modifies or deletes the cysteine residue present in the wild-type form of the antigen. For example, a StaOll polypeptide may comprise an amino acid sequence having SEQ ID NO: 46, wherein the cysteine residue at position 3 of SEQ ID NO: 46 is substituted, modified or deleted. Preferably, the replacement is by a serine residue (eg, providing SEQ ID NO: 51) or an alanine residue. Alternatively, the cysteine residue is deleted.
In a preferred embodiment, the StaOll antigen is encoded by the nucleic acid of SEQ ID NO: 131 and / or the amino acid sequence of SEQ ID NO: 132.
EsxAB
esxA
In strain NCTC 8325, esxA is SAOUHSC_00257 and has the amino acid sequence SEQ ID NO: 56 (GI: 88194063). SEQ ID NO: 56 has no cysteine residues and contains no free thiol groups. EsxA used in the invention should also have no free thiol group.
Useful esxA antigens can elicit an antibody (e.g., when administered to a human) that recognizes SEQ ID NO: 56 and / or can include 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: 56; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 56, 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 proteins include variants of SEQ ID NO: 56. Preferred fragments of (b) include an epitope derived from SEQ ID NO: 10. 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: 56 while retaining at least one epitope of SEQ ID NO: 56. Other fragments omit one or more protein domains.
EsxA may be present as a hybrid polypeptide with EsxB as discussed below.
esxB
In strain NCTC 8325, esxB is SAOUHSC_00265 and has the amino acid sequence SEQ ID NO: 57 (GI: 88194070). The invention uses an EsxB form that can not form covalent dimers via disulfide bonds. The polypeptide does not contain any free thiol group (under reducing conditions).
Useful esxB antigens can elicit an antibody (e.g., when administered to a human) that recognizes SEQ ID NO: 57 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 greater) with any of SEQ ID NO: 57, 60-68 and 77-82; and / or (b) comprising a fragment of at least "n" consecutive amino acids of any one of SEQ ID NO: 57, 60 to 68 and 77 to 82, 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 proteins include variants of SEQ ID NO: 57. Preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 57. 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: 57 while retaining at least one epitope of SEQ ID NO: 57. Other fragments omit one or more protein domains. SEQ ID NO: 61 is the C-terminus of SEQ ID NO: 57, from amino acids 32 to 104. Compared to SEQ ID NO: 61, SEQ ID NO: 64 has an additional amino acid residue " X "at the N-terminus, where" X "is an amino acid that does not contain a free thiol group (e.g., Ala = SEQ ID NO: 10). Compared with SEQ ID NO: 57, SEQ ID NO: 66 does not contain N-terminal methionine, and has an amino acid residue "X" in place of Cys-31, where "X" is an amino acid which does not contain a free thiol group (for example, Ala = SEQ ID NO: 67). Compared to SEQ ID NO: 57, Met-1 and Cys-31 are absent in SEQ ID NO: 68. SEQ ID NO: 60 corresponds to amino acid residues 2 to 30 of SEQ ID NO: 57. Compared to SEQ ID No. NO: 60, SEQ ID NO: 62 has an additional amino acid residue "X" at the C-terminus, where "X" is an amino acid that does not contain a free thiol group (eg, Ala, to give SEQ ID NO: 63).
Useful EsxB polypeptides may include an N-terminal methionine (eg, SEQ ID NO: 77-82).
A useful EsxB may comprise at least one point mutation that replaces, modifies or deletes the cysteine residue present in the wild-type form of the antigen. For example, an EsxB polypeptide may comprise an amino acid sequence having SEQ ID NO: 57, wherein the 31 cysteine residue of SEQ ID NO: 57 is replaced, modified, or deleted. Preferably, the replacement is by a serine residue or an alanine residue (eg, providing SEQ ID NO: 80). Alternatively, the cysteine residue is deleted (e.g., providing SEQ ID NO: 78).
EsxB may be present as a hybrid polypeptide with EsxA as discussed below.
Hybrid Polypeptides EsxAB and EsxBA
An EsxAB hybrid antigen comprises both EsxA and EsxB antigens. These can be in any order, from the N-terminus to the C-terminus. SEQ ID NO: 58 which comprises SEQ ID NO: 56 and 57 ('EsxAB'); and 44 which comprises SEQ ID NO: 57 and 56 ('EsxBA') are examples of such hybrids, both of which include ASGGGS hexapeptide linkers (SEQ ID NO: 101). Another 'EsxAB' hybrid comprises SEQ ID NO: 59, where the N-terminal methionine of EsxB has been removed. The hybrid polypeptide used in the invention ideally has no free thiol group (under reducing conditions). SEQ ID NO: 69 to 76 and 83 to 98 are 'EsxAB' hybrids, with EsxA upstream from EsxB; in contrast, SEQ ID NO: 99 and 100 are 'EsxBA' hybrids, with EsxB at the N-terminus of EsxA. All of SEQ ID NOS: 69 to 76 and 83 to 100 include a hexapeptide linker ASGGGS (SEQ ID NO: 101) and no cysteine residues. SEQ ID NO: 87 to 100 include N-terminal methionine residues, whereas the 'EsxAB' hybrids of SEQ ID NOs: 69 to 76 and 83 to 86 do not include them.
Thus, a useful polypeptide comprises an amino acid sequence having 80% or more identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, 99.5% or more) with any of SEQ ID NOS: 69-76 and 84-98. These polypeptides can elicit antibodies (e.g., when administered to a human) that recognize both the wild-type staphylococcal protein comprising SEQ ID NO: 56 and the wild type staphylococcal protein comprising SEQ ID NO: 57. Thus, the immune response will recognize staphylococcal antigens both EsxA and EsxB.
Usefully, these hybrid polypeptides can elicit antibodies (e.g., when administered to a human) that recognize each of the wild-type staphylococcal proteins (e.g., as shown in the sequence listing) shown in US Pat. the hybrid, for example, which recognize both wild-type EsxA and wild-type EsxB.
In a preferred embodiment, the EsxAB antigen is encoded by the nucleic acid of SEQ ID NO: 133 and / or the amino acid sequence of SEQ ID NO: 134.
Clf A The "ClfA" antigen is annotated as "agglutination factor A". In strain NCTC 8325, clfA is SAOUHSC_00812 and has the amino acid sequence SEQ ID NO: 102 (GI: 88194572). In the Newman strain, it is nwmn_0756 (GI: 151220968).
Useful clfA antigens can elicit an antibody (e.g., when administered to a human) that recognizes SEQ ID NO: 102 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: 102; and / or (b) comprising a fragment of at least "n" consecutive amino acids of SEQ ID NO: 102, 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 clfA proteins include variants of SEQ ID NO: 102. The preferred fragments of (b) comprise an epitope derived from SEQ ID NO: 102. 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: 102. The final 368 C-terminal amino acids of SEQ ID NO: 102 may be conveniently omitted. The first 39 N-terminal amino acids of SEQ ID NO: 102 may be omitted in a useful manner. Other fragments omit one or more protein domains. SEQ ID NO: 103 is a useful fragment of SEQ ID NO: 102 ('ClfA40-559'). This fragment omits the long repetitive region towards the C-terminus of SEQ ID NO: 102.
In a preferred embodiment, the ClfA antigen has the amino acid sequence of SEQ ID NO: 135.
OMV
OMVs are well known in the state of the art and are released spontaneously into the culture medium by bacteria. "Native OMVs" ("NOMV" [18]), microvesicles (MV [19]), detergent-extracted OMVs (DOMV), OMVs derived from a mutant (m-OMV), and buds, which are protrusions on the outer membrane that remain attached to the bacteria prior to release as MV ([20]; [21]), all form a part of the invention and are collectively referred to herein as OMV.
The OMVs of the invention can be obtained from any suitable Gram negative bacterium. The Gram-negative bacterium is usually E. coli. However, in place of E. coli, it can be a different Gram negative bacterium. Preferred Gram-negative bacteria for use in the invention include bacteria that are not pathogenic in humans. For example, bacteria can be commensal in humans. However, in some embodiments, bacteria are used which are generally not found at all in human hosts.
Examples of species for use according to the invention include species in any of the genera Escherichia, Shigella, Neisseria, Moraxella, Bordetella, Borrelia, Brucella, Chlamydia Haemophilus, Legionella, Pseudomonas, Yersinia, Helicobacter, Salmonella, Vibrio, etc. In particular, the bacterium may be a species of Shigella (such as S. dysenteriae, S. flexneri, S. boydii or S.sonnei). Alternatively, it may be a Neisseria species, particularly a non-native species such as N. bacilliformis, N. cinerea, N. elongata, N. flavescens, N. lactamica, N. macacae, N. mucosa, N. polysaccharea, N. sicca or N. subflava, and in particular N. lactamica. Alternatively, a pathogenic species of Neisseria can be used, for example N. gonorrhoeae or N. meningitidis. In other examples, the bacterium may be Bordetella pertussis, Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis, Moraxella catarrhalis, Haemophilus influenzae (including non-typeable strains), Legionella pneumophila, Pseudomonas aeruginosa, Yersinia enterocolitica , Helicobacter pylori, Salmonella enterica (including serovars typhi and typhimurium, as well as serovars paratyphi and enteritidis), Vibrio cholerae, Proteus, Citrobacter, Serratia, Erwinia, Pasteurelia etc. Photosynthetic Gram negative bacteria can also be used. Generally, the bacterium is a competent strain. This character facilitates the genetic modification of the bacteria.
In a particular embodiment, the gram-negative bacterium is a "hyperbourgeonnante" strain of this bacterium. Gram-negative hyperbourgeonating bacteria from which buds can be manufactured more easily in higher yield and can be more homogeneous in nature are described in WO 02/062378. For example, the buds may be derived from bacteria selected from the group consisting of Neisseria meningitidis, Neisseria lactamica, Neisseria gonorrhoeae, Helicobacter pylori, Salmonella typhi, Salmonella typhimurium, Vibrio cholerae, Shigella spp., Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa and Moraxella catarrhalis.
The Gram-negative bacterium from which OMVs are produced may include one or more adaptation (s) for vesicle production. In particular, compared to the same bacterium without said adaptation (s), the bacterium comprises a genome in which one or more non-essential sequences for the production of vesicles are absent. The absent sequences may be totally absent from the genome, or they may be partially deleted so that they are in an inactivated form. In addition to the absence of one or more sequences, one or more other sequences that are not essential for vesicle production may be inactivated by sequence insertion. The absence of sequences means that, compared to the same bacteria without said absent sequences, i.e., compared to the same bacterium with the sequences), the bacterium generally comprises a reduced size genome. Generally, the genome is between 1% and 50% smaller (in total number of base pairs) compared to the same bacteria without the missing sequences. In particular, the genome can be between 5% and 50%, between 5% and 40%, between 5% and 30%, between 5% and 20%, between 10% and 30% or between 10% and 20% smaller . When the bacterium is any Gram-negative bacterium, it is useful for the 16S rRNA gene to be present in the genome because differences in the sequence of that gene can be used to distinguish different species of bacteria. It can also be useful to have one or more genes that make the bacteria competent. This character facilitates the genetic modification of the bacteria. Sequences not essential for the production of vesicles
The category of nonessential sequences for vesicle production may include genes for metabolic processes that are not necessary for vesicle production. For example, genes for nutrient biosynthesis may be absent when these nutrients can be artificially provided, for example, under culture conditions. As a result, the bacteria may be auxotrophic for one or more nutrients. Those skilled in the art are aware of the gene (s) that may be absent to produce a given auxotrophy. For example, the bacterium may be auxotrophic for one or more of the following amino acids, particularly when the bacterium is E. coli (for example, by deleting the indicated genes): arginine (argA); asparagine (both asnA and asnB); aspartic acid (aspC and tyrB); cysteine (cysE); glutamic acid (both gltB and gdhA); glutamine (glnA); glycine (glyA); histidine (hisB); isoleucine (ilvA); leucine (leuB); lysine (lysA); methionine (metA); phenylalanine (pheA); proline (proA); serine (serA); threonine (thrC); tryptophan (trpC); tyrosine (tyrA); and valine, isoleucine and leucine (ilvD). However, in some embodiments, the bacterium does not exhibit auxotrophy compared to the same bacteria without the absent sequences. In particular, the bacterium is able to grow on minimal media. Other metabolic processes that are not required for vesicle production may include transport routes. Therefore, genes for components of these transport pathways may be absent. For example, the genes for ABC transporters and / or components of type I, II, III, IV, V and / or VI secretory systems may be missing from the bacterium.
The category of sequences not essential for vesicle production may also include other sequences. For example, it is preferable that sequences that give rise to genetic instability are absent, for example transposable elements, including retrotransposons, DNA transposons and insertion sequences; the bacteriophage and group II introns. For example, up to 50% of the Shigella virulence plasmid is composed of insertion sequence elements; when the bacterium is a Shigella species, these insertion sequences are preferably absent. Genes that encode genes in restriction modification systems may also be absent (e.g., hsd regions in E. coli), as may genes encoding other endogenous nucleases that destroy foreign DNA. These genes are not essential for bacterial growth in controlled culture environments, that is, typical GM P conditions during growth for vaccine manufacture. However, it may be useful to maintain at least one restriction modification system such that the genome can be transformed into a restriction-restricted host cell (see below). Genes encoding bacterial flagella components may also be absent because they are not essential. Rh elements can also be absent. These are large homologous repeat regions that facilitate rearrangement of the genome through homologous recombination and can therefore lead to genomic instability. Nontranscribed regions may also be generally absent because they are not necessary for bacterial growth. Similarly, prophages and / or pseudogenes may be absent, as can the bacteriophage receptor genes.
Methods for identifying non-essential sequences are well known in the state of the art, for example, from references [22] and [23]. For example, a method for identifying sequences that are not essential for growth involves comparing the genomes of two or more strains of the bacterium and identifying sequences that are not present in all strains. These sequences are less likely to be necessary for bacterial growth and thus are candidates for an absence in the invention. However, it is possible that one sequence is essential for the growth of one strain but not another. If a sequence is essential in the particular strain used in the invention, then the sequence should be present in the genome or replaced by another sequence having a complementary function to allow the growth of the strain. To confirm that a sequence is not essential for vesicle production, the sequence can be deleted from a vesicle producing strain and the effect on vesicle production is determined. Again, it is possible that one sequence is essential for the production of vesicles in one strain but not in another. If a sequence is essential for the production of the vesicles in at least one strain, then it is preferably present in the genome of the invention.
The category of nonessential sequences for vesicle production may also include sequences that cause vesicle reactogenicity, for example, genes encoding membrane-associated components that induce undesirable immunogenic responses and / or toxicity. For example, it is preferred that the genes involved in the production of endotoxin, for example, lipopolysaccharide (LPS) or lipooligosaccharide (LOS), be absent. For example, in E. coli it is preferred that the msbB and pagA genes be absent, so that the bacterium expresses a detoxified lipopolysaccharide (penta-acylated LPS). This can be achieved using an allelic exchange procedure assisted by Red recombinases carried in pKD46 [24; 25]. pKD3 can be used as a template for PCR to generate mutated alleles containing the chloramphenicol cassette. Then, pCP20 can be used to excise the chloramphenicol cassette from the chromosomal DNA of the mutant [25]. In order to induce an internal deletion from the msb ORF and the pagP ORF, primers can be designed to carry a homologous extension sequence and the hybridization sequence for the pKD3 template [2 6]. To determine whether a sequence causes the reactogenicity of the vesicles, and therefore to identify a sequence that may be absent in the invention, the sequence may be deleted from a vesicle-producing strain and the effect on the reactogenicity of the vesicles is determined . Reactogenicity is generally determined using in vitro or animal models, for example, by measuring pyrogenicity in rabbits, production of proinflammatory cytokines by human monocytic cells, and / or the degree of inflammation at the site of infection. injection in mice and rabbits (see, for example, reference 27).
The category of nonessential sequences for vesicle production may also include sequences that suppress the production of vesicles, particularly genes that suppress the release of buds. These genes are often involved in maintaining the integrity of the outer membrane of the bacteria. For example, many Gram-negative bacteria have a Tol-Pal system which consists of TolA, TolB, TolQ, TolR and Pal proteins. The absence of one or more genes for one or more components of this system may cause the bacterium to release greater amounts of buds into its culture medium during bacterial replication. For example, at least one of the five Tol-Pal genes may be absent. Thus, the bacteria 1, 2, 3, 4 or 5 of the genes tolA, tolB, tolQ, tolR and Pal may be missing. Preferably, the tolR gene is absent, particularly in E. coli. Thus, the bacterium can be tolA + tolB + tolQ + TolR ~ Pal +. The bacteria can also release larger amounts of buds if it lacks the OmpA protein. Therefore, it is also preferable that the ompA gene be absent from the genome of the bacterium, particularly in E. coli. To determine whether a sequence suppresses the production of vesicles, and therefore to identify a sequence that may be absent in the invention, the sequence may be deleted from a vesicle-producing strain and the effect on vesicle production is determined .
In a specific embodiment, the bacterium is an ompA mutant of E. coli and / or a tolR mutant of E. coli. In some embodiments, the bacterium is selected from E. coli BL21 (DE3) AompA, E. coli BL21 (DE3) AompAAtolR, E. coli BL21 (DE3) AtolR, E. coli Δηΐρΐ, or E. coli AdegP. The Δ symbol is used herein to refer to a bacterial strain from which the coding sequence of the gene named after the Δ symbol has been deleted. Thus, a bacterial strain that is "AompA" does not include the coding sequence for the ompA gene. Similarly, a bacterial strain that is "AtolR" does not include the coding sequence for the tolR gene. The entire coding sequence can be deleted. However, the coding sequence may alternatively be partially deleted. For example, the N-terminal half or the C-terminal half can be deleted. Alternatively, the ompA and / or tolR genes may be mutated by introduction of one or more substitutions and / or insertions.
Mutant strains of E. AtolR coli and mutant strains of E. coli. AompA coli overproduce OMVs compared to wild-type E. coli. Thus, the mutation of the ompA gene and / or one or more components of the Tol-Pal complex results in the mutant bacterium producing an increased number of OMVs compared to its respective wild-type strain which carries an ompA gene and / or or a wild type Tol-Pal complex. OmpA is an integral protein of the membrane and is the most abundant protein of the outer membrane in E. coli. Therefore, it is surprising that an E. coli lacking the OmpA protein is viable. Indeed, according to Murakami et al. [28], a simple ompA mutant of E. coli can not promote the release of vesicles.
The category of nonessential sequences for vesicle production may also include genes for proteins that are not required in vesicles, depending on their intended use. For example, genes for undesirable antigens may be removed, for example genes for proteins against which it is not desired to induce an immune response (such as non-protective antigens, particularly immunodominant antigens). Proteins that are not required in the vesicles can be identified by preparing vesicles from a bacterium comprising a genome that still includes genes (not yet identified) for these proteins, and by analyzing the protein content of these vesicles using proteomics based on high throughput mass spectrometry, as described below. Once the proteins have been identified, the corresponding genes can be omitted from the genome of the bacterium of the invention and therefore absent.
Preferably, the category of sequences not essential for the production of vesicles does not include sequences which allow the vesicles to possess adjuvant properties. For example, it is useful that one or more sequences that encode TLR-2 ligands, e.g., lipoproteins, be present in the genome. Similarly, it is useful that one or more sequences that encode TLR-4 ligands, e.g., LPS components, be present in the genome. Such sequences are useful because they contribute to the effectiveness of the vesicles as immunogens, for example, for use as vaccines. To identify such sequences, the sequence can be deleted from a vesicle-producing strain and the effect on the adjuvancy of the vesicles is determined. Adjuvanticity is generally determined by comparing the immune response to an antigen in the presence or absence of the putative adjuvant, for example, by measuring the antibody titer against the antigen.
The bacterium preferably contains other adaptations for the production of vesicles. In particular, the bacterium generally comprises a genome in which one or more sequences are present such that, compared to the same bacterium without said sequence (s), the bacterium produces higher amounts of vesicles. Once again, sequences that increase vesicle production can be identified by adding or deleting the sequence to a vesicle-producing strain and determining the effect on vesicle production.
Method of making the genome
The methods for producing reduced size bacterial genomes relative to the wild-type genome are known in the art. For example, reference 29 describes various "top-down" approaches that involve the deletion of unwanted sequences (described in, for example, references 22 and 23) and "bottom-up" approaches that involve de novo genome synthesis, without the sequences undesirable (described in, for example, references 30 and 31).
Accordingly, a method of making a genome for use in a bacterium to produce OMVs comprises the following steps: (a) providing a genome from a Gram-negative bacterium; and (b) deleting one or more nonessential sequences for vesicle production so that, compared to the genome in step (a), the genome is reduced in size. The method further comprises a step (c) of inserting into the genome one or more sequences that encode a protein selected from the group consisting of (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen. Steps (b) and (c) can be performed in this order or order (c) then (b), provided that the sequence (s) inserted in step (c) remains (nt) after step (b).
Another method of making a genome for use in a bacterium for producing OMV involves synthetic genomic techniques. These techniques make it possible to prepare synthetic genomes by chemical synthesis at least in part, for example, as disclosed in reference 31. The synthesis method may involve the theoretical division of the desired DNA sequence of the genome into fragments. These fragments can again be theoretically divided into one or more times, possibly to a set of fragments which are each of a size that can be prepared by a DNA synthesis method chosen, for example, by chemistry. phosphoramidites. These fragments are then synthesized and joined to give the longer fragments from the theoretical division stage, and these longer fragments are then joined, etc. until the eventual preparation of the complete sequence. In this way, reference 31 prepared a 583 kbp genome by assembling ~ 104 50-mer oligonucleotides in various stages. The 50-mers were assembled into cassettes 5 to 7 kb in length, and these cassettes were then assembled into ~ 24 kbp fragments, which were then assembled into ~ 72 kbp fragments and then ~ 144 kbp. kpb, then giving two ~ 290 kbp constructs, which were finally joined to give the complete genome. The fragments are designed to overlap, allowing this way to assemble them in the correct order. For example, the cassettes overlapped by at least 80bp, thereby allowing them to be assembled into ~ 24kbp fragments, and so on. Thus, the method involves the synthesis of a plurality of overlapping fragments of the desired DNA molecule, so that the overlapping fragments cover the entire DNA molecule. Both ends of each fragment overlap with a neighboring 5 'or 3' fragment, with the exception of the terminal fragments of a linear molecule where no overlap is required (but to synthesize a circular molecule, the two terminal fragments will have to overlap). Fragments at each stage can be maintained as inserts in vectors, for example, in plasmids or BAC or YAC vectors. The assembly of the fragments during the synthesis process may involve in vitro and / or in vivo recombination. For in vitro methods, digestion with a 3 'exonuclease can be used to expose the protruding ends of a fragment, and the complementary protruding ends in the overlapping fragments can then be hybridized, followed by repair of the junctions (method "chew-back assembly"). For in vivo methods, the overlapping clones can be assembled using, for example, the TAR cloning method disclosed in reference 31. When prepared by these methods, a genome of the invention may include one or more sequences in "watermark". These are sequences that can be used to identify or encode information in the genome. It can be either non-coding or coding sequences. Most commonly, they encode information within the coding sequences without modifying the amino acid sequences.
Once the genome has been produced, it can be introduced into a host cell by transformation according to the method of reference 32. In one method, a restriction-deficient host cell is transformed with a genome that encodes a restriction system that is expressed in the host cell degrading the genome of the host cell. The adapted genome may include one or more sequences that are not integrated into the bacterial chromosome. This / these sequence (s) is / are present in one or more non-integrated genomic elements, for example one or more plasmids. In these embodiments, the transformation may comprise substeps, eg, transformation of the host cell with the bacterial chromosome and transformation of the host cell with the non-integrated genomic element or elements. These sub-steps can be performed in any order, although generally the host cell is transformed with the bacterial chromosome prior to any other transformation (s) with one or more non-integrated genomic elements.
OMV production processes
OMVs can be prepared from a bacterium as described above. The method comprises a step of obtaining vesicles from a culture of the bacterium. The vesicles can be obtained by bursting or budding from the outer membrane of the bacterium to form vesicles therefrom.
OMVs are artificially prepared from bacteria, and may be prepared using detergent treatment (e.g., with deoxycholate or sarkosyl), or by non-detergent means (e.g., see reference 33). Techniques for forming OMVs include treating the bacteria with a bile acid salt detergent (e.g., salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholic acid, etc., with sodium deoxycholate [34 and 35] being preferred for the treatment of Neisseria) at a pH sufficiently high not to precipitate the detergent [36]. Other techniques can be performed substantially in the absence of detergent [33] using techniques such as sonication, homogenization, microfluidization, cavitation, osmotic shock, grinding, French press, mixing, etc. Processes using little or no detergent can retain useful antigens such as NspA [33]. Thus, one method can use an OMV extraction buffer with about 0.5% deoxycholate or less, for example, about 0.2%, about 0.1%, <0.05%, or zero.
A useful method for the preparation of OMV is described in reference 37 and involves ultrafiltration on crude OMVs rather than high speed centrifugation. The process may involve an ultracentrifugation step after ultrafiltration takes place.
Antigens in OMVs
The combination of antigens selected from the group consisting of (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen may be present in a single OMV or in multiple distinct OMVs. The antigens can be targeted to and expressed in the periplasm of the Gram-negative bacterium such that the antigen is in the OMV lumen. In some embodiments, the antigen is present in the OMV membrane, i.e., only a portion of the antigen is present in the OMV lumen. In some embodiments, the antigen is free in the OMV lumen.
The term "in the light" of OMV encompasses both proteins that are membrane associated but not exposed at the surface, and proteins that are free in the OMV lumen. The antigen is generally free in OMV lumen in the present invention. By "free in the light" this means that the antigen is not integrally associated with the OMV membrane. Integral membrane association describes these proteins that require the use of a detergent or other apolar solvent to dissociate the protein from the membrane. A description of the membrane anchors for integral membrane association can be found in reference 38. A protein that is free in the OMV lumen can be associated with the membrane or an integral membrane protein through non-membrane interactions. covalent or it may not associate with the OMV membrane at all. For example, the protein may associate loosely or temporarily with the membrane, for example, via hydrophobic, electrostatic, ionic and / or other non-covalent interactions with the lipid bilayer and / or an integral protein. .
An advantage of the antigen being in the OMV lumen, rather than being associated with the membrane and exposed, is that it can be protected against degradation by proteases in vivo. This protection may in turn result in more efficient activation of B cells. OMV is able to elicit an immune response against the antigen when administered to a mammal. The immune response may be a cellular or humoral immune response. Generally, the immune response is an antibody response.
In one embodiment, the OMV of the invention is capable of eliciting an immune response against the pathogen from which the antigen is derived, i.e., S. aureus. For example, the antigen preferably triggers an immune response of T cells that can neutralize the infection and / or virulence of the pathogen from which the antigen is derived, i.e., S. aureus. The preferred antigens for a use of which the invention is thus are those which are recognized by the cellular immune system during an infection with a pathogen of interest. More preferred are those antigens that elicit a protective immune response of T cells against a pathogen of interest.
In one embodiment, the OMV of the invention is capable of eliciting antibodies that recognize a pathogen from which the antigen is derived, i.e., S. aureus. For example, the antigen preferably triggers antibodies that can bind to, and preferably neutralize, the infection and / or virulence of the pathogen from which the antigen is derived, i.e. , S. aureus. The preferred antigens for a use of which the invention is thus are those which are recognized by antisera during infection with the pathogen. More preferred are those antigens that elicit a protective immune response against a pathogen of interest. The antigen may exhibit increased immunogenicity when presented in OMV compared to when administered in purified form.
In one embodiment, the antigens of the invention are functionally active in the OMV lumen and / or upon release from the OMV lumen (e.g., by a detergent-mediated disruption OMV). Functional activity is an indicator that the antigen is correctly folded and has the same or substantially the same tertiary and quaternary structure as the same protein in its native state. By "functionally active" it means that the antigen retains at least 50% or more of at least one biological activity of the same protein when expressed in its native environment (for example, in the organism from which it is derived). For example, the antigen may be considered functionally active if it retains at least 50%, 60%, 70%, 80%, 90% or more of at least one biological activity of the same protein when expressed. in its native environment.
In embodiments in which the antigen comprises or consists of a fragment of a wild-type protein or a variant thereof, the fragment or variant may be functionally active. By "wild-type protein fragment" this means that the antigen comprises or consists of at least 7 consecutive amino acids from the wild-type protein. In some embodiments, the fragment is comprised of at least 7, 8, 9, 10, 20, 30, 40 amino acids or more from the wild-type protein. The fragment can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the wild-type protein.
Preferably, the fragment is an immunogenic fragment of the antigen. By "immunogenic fragment" this means that the fragment has at least one epitope in common with the antigen. The term "epitope" encompasses all kinds of epitopes and includes epitopes of both B cells and T cells, and both linear and discontinuous epitopes. In one embodiment, an antibody that specifically binds to the antigen also specifically binds to the immunogenic fragment, i.e. the antigen and its immunogenic fragment both contain the epitope to which that antibody is binds.
By "specifically binds", it means that the antibodies bind to the antigen of the invention with an affinity substantially greater than the BSA. Preferably, the affinity is at least 100 times, 103 times, 104 times, 105 times, 106 times, etc. superior for the antigen of the invention than for BSA.
The epitopes present in the antigens can be determined and / or predicted using all known methods of the state of the art. For example, epitope prediction software such as EpiToolKit, which is an Internet server for computer immunomics [39]. This epitope prediction software provides several methods for predicting potential T cell epitopes, both class I MHC and class II MHC binding epitopes.
The presence of B cell epitopes can also be predicted using any of the known methods of the state of the art, for example, as described in references [40, 41 and 42]. The presence of continuous linear epitopes and / or discontinuous epitopes can be predicted using the methods described therein.
By "wild type protein variant", it means that the antigen comprises or consists of a full-length protein, for example, a protein with the same number of amino acids as the wild-type protein, or a fragment of the wild-type protein that contains one or more variations in the amino acid sequence compared to the wild-type sequence. A variant may have at least 50%, 60%, 70%, 80%, 90%, 95% or more sequence identity with the wild-type protein. In some embodiments, the variant is also functionally active. The antigen may be fused to a fusion partner, i.e. the antigen may be part of a fusion protein. The fusion proteins may comprise a sequence -X-Y- or -Y-X-, wherein: -X- is an antigen as defined above, and -Y- is an additional polypeptide sequence. In a particular embodiment, -Y- is a protein marker that assists in the detection of antigen such as 6xHIS, FLAG, HA, GST, GFP or other fluorescent protein, and / or luciferase or any polypeptide suitable for the function of the antigen. When the antigen is part of a fusion protein, the entire fusion protein will be in the OMV lumen. In some embodiments, the fusion protein will be free in the OMV lumen.
Pharmaceutical Compositions The invention provides a pharmaceutical composition comprising (a) at least two of the antigens selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen and (b) a pharmaceutically acceptable carrier. The invention also provides a pharmaceutical composition comprising (a) at least one OMV comprising at least one of the antigens selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen and (b) a pharmaceutically acceptable carrier. Generally, the pharmaceutical composition comprises an OMV which comprises at least two antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally at least three antigens from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally at least four of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, or more generally the four antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen, or more generally the four antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) esxAB and (iv) staOll. The invention also provides a pharmaceutical composition comprising (a) at least two different OMVs, wherein each OMV comprises at least one of the antigens selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen in its light and (b) a pharmaceutically acceptable carrier.
The pharmaceutical composition may comprise at least one OMV which comprises at least two S. aureus antigens selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, i.e. a combination of antigens is provided within the same OMV. Alternatively, the pharmaceutical composition may comprise multiple different OMVs that comprise at least one S. aureus antigen selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, i.e. a combination of antigens is provided in separate OMVs. The pharmaceutical composition may comprise (a) at least one OMV of the invention and (b) a pharmaceutically acceptable carrier. The pharmaceutical composition can comprise at least two, at least three or at least four OMVs of the invention. The invention also provides a process for preparing such a composition, comprising the step of mixing the OMV of the invention with a pharmaceutically acceptable carrier. The invention also provides a container (e.g., a vial) or delivery device (e.g., a syringe) pre-filled with a pharmaceutical composition of the invention. The invention also provides a method of providing such a container or device comprising introducing into the container or device a composition containing vesicles of the invention.
The immunogenic composition may comprise a pharmaceutically acceptable carrier, which may be any substance which does not itself induce the production of antibodies harmful to the patient receiving the composition, and which can be administered without undue toxicity. Pharmaceutically acceptable carriers may include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also be present in such vehicles. A full discussion of the appropriate media is available in reference 43.
The bacteria can affect various areas of the body and thus the compositions of the invention can be prepared in various forms. For example, the compositions may be prepared as an injectable, 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. The composition may be prepared for topical administration, for example as an ointment, cream or powder. The composition may be prepared for oral administration, for example in the form of a tablet or capsule, or in the form of a syrup (optionally flavored). The composition may be prepared for pulmonary administration, for example, in the form of an inhaler, using a fine powder or spraying. The composition may be prepared in the form of a suppository or an egg. The composition may be prepared for nasal, atrial or ocular administration, for example in the form of drops.
A pharmaceutical carrier may include 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 44, 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-controlling 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 protecting agent may be mixed in such a way 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").
The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered, for example, between pH 6 and pH 8, generally around pH 7. The compositions of the invention may be isotonic with respect to humans.
The immunogenic compositions comprise an immunologically effective amount of immunogenic antigen or vesicles, as well as any other specified components, as needed. By "immunologically effective amount" it means 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, a non-human primate, a primate, etc.), the capacity of the system the individual's immune system to synthesize antibodies, the degree of protection desired, the vaccine formulation, the physician's estimate of the medical situation, and other relevant factors. The quantity is expected to be within a relatively wide range that can be determined through routine testing.
Previous work with vesicle-based vaccines (eg for meningococcus) provides pharmaceutical, dosage and formulation indications for the compositions of the invention. The concentration of the vesicles in the compositions of the invention will generally be between 10 and 500 μg / ml, preferably between 25 and 200 μς / ηιΐ, and more preferably about 50 μ9 / ιη1 or about 100 μ9 / ιη1 (expressed as terms of total proteins in the vesicles). A dosage volume of 0.5 ml is typical for an injection.
The composition may be administered together with other immunoregulatory agents.
Adjuvants which may be used in the compositions of the invention include, but are not limited to: A. Compositions containing minerals
The mineral-containing compositions suitable for use as adjuvants in the invention include inorganic salts, such as aluminum salts and calcium salts. The invention includes inorganic salts such as hydroxides (eg, oxyhydroxides), phosphates (eg, hydroxyphosphates, orthophosphates), sulfates, and the like. [for example, see chapters 8 &amp; 9 of 48], or mixtures of different inorganic compounds, with compounds of any suitable form (eg, gels, crystalline, amorphous, etc.), and with adsorption being preferred. The mineral-containing compositions may also be formulated as a metal salt particle.
Adjuvants known as "aluminum hydroxide" are generally aluminum oxyhydroxide salts, which are usually at least partially crystalline. Aluminum oxyhydroxide, which may be represented by the formula IAO (OH), may be distinguished from other aluminum compounds, such as Al (OH) 3 aluminum hydroxide, by infrared spectroscopy (IR) , in particular by the presence of an adsorption band at 1070 cm -1 and a strong shoulder at 3090 at 3100 cm -1 (chapter 9 of reference 48). The degree of crystallinity of an aluminum hydroxide adjuvant is reflected by the width of the mid-height diffraction band (WHH), with the low crystalline particles having higher line broadening due to smaller crystallite sizes . Specific surface area increases as WHH increases, and adjuvants with higher WHH values have been found to exhibit superior antigen adsorption capacity. 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.
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. The hydroxyphosphates generally have a molar ratio PO4 / Al between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AIPO4 by the presence of hydroxyl groups. For example, an IR spectrum band at 3164 cm-1 (for example, at 200 ° C) indicates the presence of structural hydroxyls [Chapter 9 of Reference 48].
The P04 / A13 + molar ratio of an aluminum phosphate adjuvant will generally be 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 PCh / Al molar ratio of between 0.84 and 0.92, including 0.6 mg of Al3 + / ml. Aluminum phosphate will generally be particulate (eg, plate-like morphology as observed on transmission electron micrographs). The typical diameters of the particles are in the range of 0.5 to 20 μm (for example, about 5 to 10 μπι) after the 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.
In one embodiment, an adjunct component comprises 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 reference 48; see also reference 45] (5% squalene, 0.5% Tween 80, and 0.5% Span 85, formulated in submicron particles using a microfluidizer). Freund's Complete Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) can also be used.
Various suitable 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 (s)). )) and biocompatible (s). The oil droplets in the emulsion generally have a diameter of less than 5 μπι, and advantageously the emulsion comprises oil droplets with a submicron diameter, with these small sizes being obtained with a microfluidizer to provide stable emulsions . Droplets with a size smaller than 220 nm are preferred because they can be sterilized by filtration. The invention can be used with oils such as those of animal source (such as fish) or vegetable. 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. Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Other preferred oils are tocopherols (see below). Oil-in-water emulsions comprising squalene are particularly preferred. Oil blends can be used.
Surfactants can be classified by their "BHL" (hydrophilic / lipophilic balance). The preferred surfactants of the invention have a BHL of at least 10, preferably at least 15, and more preferably at least 16. The invention may be used with surfactants comprising, but not limited to, surfactants. polyoxyethylene sorbitan ester surfactants (commonly called Tween), 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 linear EO / PO 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 phosphatidyl choline (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Preferred surfactants for inclusion in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100. As mentioned above, detergents such as Tween 80 can contribute to the thermal stability observed in the examples below.
Surfactant mixtures may be used, for example, Tween 80 / Span 85 mixtures. 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 appropriate. Another useful combination includes laureth 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol.
Preferred amounts of the surfactants (wt%) are: polyoxyethylene (such as Tween 80) sorbitan esters 0.01 to 1%, especially about 0.1%; octyl- or nonyl-phenoxypolyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1%, especially 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.
Adjuvants based on specific oil-in-water emulsions useful for the invention include, but are not limited to: a submicron emulsion of squalene, 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" [45-47], as further described in chapter 10 of reference 48 and Chapter 12 of reference 49. The MF59 emulsion advantageously comprises citrate ions, for example, 10 mM sodium citrate buffer. An emulsion comprising squalene, an α-tocopherol, and polysorbate 80. These emulsions may have from 2 to 10% of squalene, from 2 to 10% of tocopherol and from 0.3 to 3% of Tween 80, and Weight ratio of squalene / tocopherol is preferably 1 (for example, 0.90) as this provides a more stable emulsion. Squalene and Tween 80 may be present in a volume ratio of about 5/2, or in a weight ratio of about 11/5. Such an emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, and then mixing 90 ml of this solution with a mixture of (5 g of DL-α-tocopherol and 5 ml of squalene), then microfluidizing 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. • An emulsion of squalene, a tocopherol and a Triton detergent (eg Triton X-100). The emulsion may also include a 3d-MPL (see below). The emulsion may contain a phosphate buffer. An emulsion comprising a polysorbate (e.g., polysorbate 80), a Triton detergent (e.g., Triton X-100) and a tocopherol (e.g., α-tocopherol succinate). The emulsion can comprise these three components in a mass ratio of approximately 75/11/10 (for example, 750 μg / ml polysorbate 80, 110 μg / ml Triton X-100 and 100 μg / ml succinate d α-tocopherol), and these concentrations should include any contribution of these components from the antigens. The emulsion may also include squalene. The emulsion may also include a 3d-MPL (see below). The aqueous phase may contain a phosphate buffer. • An emulsion of squalane, polysorbate 80 and poloxamer 401 ("Pluronic ™ L121"). The emulsion can be formulated in phosphate buffer solution, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in adjuvant "SAF-1" [50] (0.05 to 1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without Thr-MDP, as in the adjuvant "AF" [51] (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 of less than 200 nm [52]. The emulsion may also include one or more of: an alditol; a cryoprotectant (e.g., a sugar, such as dodecylmaltoside and / or sucrose); and / or an alkylpolyglycoside. Such emulsions can be lyophilized. • 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 53, the preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipid. Submicron droplet sizes are advantageous. • An oil emulsion in submicron water of a non-metabolizable oil (such as a light mineral oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80). Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophilic conjugate (such as GPI-0100, described in reference 54, produced by addition of an aliphatic amine to a deacyl saponin via the group carboxyl of glucuronic acid), dimethyl-dioctadecylammonium bromide and / or N, N-dioctadecyl-N, N-bis (2-hydroxyethyl) propanediamine. • 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) [55]. • 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) [55]. • An emulsion in which a saponin (for example,
QuilA or QS21) and a sterol (for example, cholesterol) are associated in the form of helicoidal micelles [56].
Antigens and adjuvants in a composition will generally be in admixture at the time of administration to a patient. The emulsions may be mixed with the antigen during manufacture, or extemporaneously, at the time of administration. Thus, adjuvant and antigen can be kept separately in a packaged or dispensed vaccine, ready for final formulation at the time of use. The antigen will generally be in an aqueous form, so that the vaccine is finally prepared by mixing two liquids. The volume ratio of the two liquids for mixing can vary (for example, between 5/1 and 1/5) but is generally about 1/1. C. Saponin Formulation [Chapter 22 of Reference 48]
Saponin formulations may also be used as adjuvants in the invention. Saponins are a heterogeneous group of sterol glycosides and triterpenoid glycosides found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. The saponin from the bark of the Quillaia saponaria Molina tree has been widely studied as an adjuvant. Saponin can also be obtained commercially from Smilax ornata (sarsaparilla), Gypsophilla paniculata (bridal veil), and Saponaria officianalis (soap root). Saponin-based 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 57. The saponin formulations may also include a sterol, such as cholesterol [58].
Combinations of saponins and cholesterols can be used to form unique particles called immunostimulatory complexes (ISCOM, see Chapter 23 of Reference 48, also references 59 and 60). ISCOMs also generally include 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. Optionally, ISCOMs may lack additional detergent [61].
A description of the development of saponin-based adjuvants can be found in references 62 and 63. D. Bacterial or Microbial Derivatives
Suitable adjuvants for use in the invention include bacterial or microbial derivatives such as non-toxic enterobacterial lipopolysaccharide (LPS) derivatives, lipid A derivatives, immunostimulatory oligonucleotides, and ADP-rybosylating toxins and their detoxified derivatives.
Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of monophosphoryl lipid A 3-de-O-acylated with 4, 5 or 6 acylated chains. A preferred form of "small particles" of 3-de-O-acylated monophosphoryl lipid A is disclosed in reference 64. Such "small particles" of 3dMPL are small enough to be sterilized by filtration through a membrane of 0. 22 pm [64]. Other non-toxic LPS derivatives include monophosphoryl lipid A mimetics, such as aminoalkylglucosaminide phosphate derivatives, e.g. RC-529 [65,66].
The lipid A derivatives include lipid A derivatives from Escherichia coli such as OM-174. OM-174 is described for example in references 67 and 68.
Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing unmethylated cytosine linked by a guanosine phosphate linkage). It has also been shown that double-stranded RNAs and oligonucleotides containing palindromic or poly (dG) sequences are immunostimulatory.
CpGs may include nucleotide modifications / analogs such as phosphorothioate modifications and they may be double-stranded or single-stranded. References 69, 70 and 71 disclose possible analogous substitutions, for example, replacement of guanosine with 2'-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is further discussed in references 72 to 77.
The CpG sequence can be directed against the TLR9, such as the GTCGTT or TTCGTT motif [78]. 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 inducing a B cell response, such as a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in references 79-81. Preferably, 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 82 to 84.
A particularly useful adjuvant based around immunostimulatory oligonucleotides is known as IC-31 ™ [85-87]. Thus, an adjuvant used in the invention may comprise a mixture of (i) an oligonucleotide (e.g., between 15 and 40 nucleotides) including at least (and preferably multiple) Cpl motif (s) that is, an inosine-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 (preferably multiple) sequence (s) tripeptide (s) Lys-Arg-Lys. The oligonucleotide may be a deoxynucleotide comprising a 26-mer 5 '- (IC) 13-3' sequence (SEQ ID NO: 2). The polycationic polymer may be a peptide comprising an 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 3). This combination of SEQ ID NO: 6 and 7 provides the IC-31 ™ adjuvant.
ADP-rybosylating bacterial toxins and their detoxified derivatives can be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (heat labile enterotoxin "LT" from E. coli), cholera ("CT"), or pertussis ("PT"). The use of detoxified ADP-rybosylating toxins as mucosal adjuvants is described in reference 88 and as parenteral adjuvants in reference 89. The toxin or toxoid is preferably in the form of a holotoxin, comprising subunits of both A and B. Preferably, subunit A contains a detoxifying mutation; preferably, the subunit B is unmutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192. The use of ADP-rybosylating toxins and their detoxified derivatives, particularly LT-K63 and LT-R72, as adjuvants can be found in references 90 to 97. A useful CT mutant is CT-E29H [98] . The reference numeral for amino acid substitutions is preferably based on the A and B subunit alignments of the ADP-rybosylating toxins presented in reference 99, specifically incorporated herein by reference in its entirety. E. Human immunomodulators
Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7). , IL-12 [100], etc.) [101], interferons (e.g. interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor. A preferred immunomodulator is IL-12. F. Bioadhesives and mucoadhesives
Bioadhesives and mucoadhesives may also be used as adjuvants in the invention. Suitable bioadhesives include microspheres of esterified hyaluronic acid [102] or mucoadhesives such as crosslinked derivatives of polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides and carboxymethylcellulose. Chitosan and its derivatives can be used as adjuvants in the invention [103]. G. Microparticles
Microparticles may also be used as adjuvants in the invention. Microparticles (i.e., a particle having a diameter of ~ 100 nm to -150 nm, more preferably a diameter of -200 nm to -30 μτη, and most preferably a diameter of -500 nm to -10 μτη) formed from materials that are biodegradable and non-toxic (for example, 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) . H. Liposomes (chapters 13 and 14 of reference 48)
Examples of liposome formulations suitable for use as adjuvants are described in references 104-106. I. Imidazoquinolone Compounds
Examples of imidazoquinolone compounds suitable for use as adjuvants in the invention include Imiquamod and its homologs (e.g., "Resiquimod 3M"), further described in references 107 and 108. The invention also include combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion [109]; (2) a saponin (eg, QS21) + a nontoxic derivative of LPS (e.g., 3dMPL); (3) a saponin (eg, QS21) + a nontoxic derivative of LPS (e.g., 3dMPL) + cholesterol; (4) a saponin (eg, QS21) + 3dMPL + IL-12 (optionally + a sterol) [111]; (5) combinations of 3dMPL with, for example, QS21 and / or oil-in-water emulsions [112]; (6) SAF, containing 10% squalane, 0.4% Tween 80 ™, 5% Pluronic L121 sequenced polymer, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate an emulsion for further large particle size; (7) Ribi ™ adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components of the monophosphoryl group. lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox ™); and (8) one or more inorganic salts (such as an aluminum salt) + a nontoxic derivative of LPS (such as 3dMPL). Other substances that act as immunostimulatory agents are disclosed in Chapter 7 of Reference 48.
An aluminum hydroxide adjuvant is useful, and the antigens are generally adsorbed on this salt. Oil-in-water emulsions comprising squalene, with submicron oil droplets, are also preferred, particularly in the elderly. Useful adjuvant combinations include combinations of Th1 and Th2 adjuvants such as CpG and aluminum salt, or resiquimod and aluminum salt. A combination of an aluminum salt and 3dMPL can be used.
Immunization
In addition to providing immunogenic compositions as described above, the invention also provides a method for raising an immune response in a mammal, comprising administering an immunogenic composition of the invention to the mammal. Generally, the immune response is an antibody response. The antibody response is preferably a protective antibody response. The invention also provides compositions of the invention for use in such methods. The invention also provides a method of protecting a mammal against infection and / or bacterial disease, comprising administering to the mammal an immunogenic composition of the invention.
The invention provides compositions of the invention for use as medicaments (for example, in the form of immunogenic compositions or in the form of vaccines). It also proposes the use of the combinations or OMVs of the invention in the manufacture of a medicament for the prevention of a bacterial infection in a mammal.
The mammal is preferably a human. The human being can be an adult or, preferably, a child. When the vaccine is for prophylactic use, the human is preferably a child (e.g., a young child or an infant); when the vaccine is for therapeutic use, the human being is preferably an adult. A vaccine intended for children may also be administered to adults, for example, to estimate safety, dosage, immunogenicity, etc. The effectiveness of the therapeutic treatment can be tested by following the bacterial infection after administration of the composition of the invention. The effectiveness of the prophylactic treatment can be tested by following immune responses against immunogenic proteins in vesicles or other antigens after administration of the composition. The immunogenicity of the compositions of the invention can be determined by administering them to test subjects (e.g., children aged 12 to 16 months) and then determining conventional serological parameters. These immune responses will generally be determined about 4 weeks after administration of the composition, and compared to predetermined values prior to administration of the composition. When more than one dose of the composition is administered, more than one determination may be made after administration.
The compositions of the invention will generally be administered directly to a patient. Direct administration may be accomplished by parenteral injection (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, or interstitally in tissue), or by rectal, oral, vaginal, topical, transdermal administration, intranasal, ocular, atrial, pulmonary or other mucosal administration. Intramuscular administration in the thigh or upper arm is preferred. Injection may be by needle (e.g., hypodermic needle), but needleless injection may be used alternatively. A typical intramuscular dose is about 0.5 ml. The invention can be used to trigger systemic and / or mucosal immunity.
The dosage treatment may be 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. An awareness dose schedule may be followed by a booster dose schedule. Appropriate times between sensitization doses (eg, between 4 and 16 weeks), and between sensitization and recall, can be routinely determined. Overview
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 "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.
The term "about" in relation to a numerical value x is optional and means, for example, x ± 10%. The identity between polypeptide sequences is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using a gap search affine with the parameters, penalty of breach = 12 and gap extension penalty = 1.
Brief description of the figures
Figure 1 - Map of the pET-OmpA plasmid. pET-OmpA is derived from the plasmid pET21b and contains the nucleic acid sequence encoding the E. OmpA coli (SS) fused to a gene of interest (GOI) encoding a heterologous protein. SS OmpA SS targets the protein encoded by GOI in the light of OMVs.
Figure 2 - SDS-PAGE analysis of total bacterial lysates from strains BL21 (DE3) AompA and BL21 (DE3) AtolR expressing staphylococcal antigens. The bacteria were analyzed before (-) and after (+) induction with 1 mM IPTG for 3 hours. (A) Analysis of BL21 (DE3) AompA strains expressing staphylococcal antigens. The bands corresponding to HlaH35L, Sta006, LukE (-LP) and LukE (+ LP) are indicated by arrows. (B) Analysis of BL21 (DE3) AtolR strains expressing staphylococcal antigens. The bands corresponding to HlaH35L and Sta006 are indicated by arrows.
Figure 3 - Immunoblot analysis on strains BL21 (DE3) AompA and BL21 (DE3) AtolR expressing staphylococcal antigens. (A) Expression of HlaH35L was assessed using anti-Hla antiserum. (B) The expression of Sta006 was evaluated using anti-Sta006 antiserum. (C) Expression of SpA_DEABC was evaluated using anti-SpA antiserum. The bands corresponding to HlaH35L and SpA_DEABC are indicated by arrows.
4 - SDS-PAGE analysis of the total (T) and soluble (S) fractions of bacterial lysates from BL21 strains (DE3MompA expressing staphylococcal antigens) The fractions were prepared and analyzed after 2 hours of induction with 1 mM d IPTG The bands corresponding to Sta006, SpA_DEABC, LukE (-LP) and LukE (+ LP) are indicated by arrows.
Figure 5 - Quantification of staphylococcal antigens released in the culture medium by immunoblotting. The amount of antigen released into the culture supernatant by BL21 (DE3) ΔοιτιρΑ strains expressing staphylococcal proteins was determined by comparing the chemiluminescent signals of the supernatant samples with those from known amounts of purified recombinant protein. (A) 0.7, 7 and 70 μΐ of culture supernatant from BL21 (DE3) AompA expressing LSSOmpA-Sta006 were analyzed. 10, 20, 40, 80 and 160 ng of purified recombinant Sta006 were loaded as references. 7 μl of supernatant contained approximately 20 ng of SSOmpA-Sta006. (B) 0.7, 7 and 70 μΐ of culture supernatant from BL21 (DE3) ΔοπιρΑ expressing SSOmpA-SpA_DEABC were analyzed. 10, 20, 40, 80 and 160 ng of the purified recombinant protein SpA_DEABC were loaded as references. 7 μl of supernatant contained approximately 40 ng of SSOmpA-SpA_DEABC. (C) 7, 70 and 700 μΐ of culture supernatant from two BL21 (DE3) AompA strains expressing SSOmpA-HlaH35L were analyzed. 20, 40, 80 and 160 ng of the purified recombinant HlaH35L protein were loaded as references. In both strains, 700 μΐ of supernatant contained approximately 10 ng of SSOmpA-HlaH35L.
Embodiments of the Invention Generation of E mutants coli BL21 (DE3) AtolR and AompA knockout
BL21 (DE3) cells prone to recombination were produced using the highly efficient homologous recombination system ("red" operon) [113]. Briefly, electrocompetent bacterial cells were transformed with 5 μl of plasmid pAJD434 by electroporation (5.9 ms at 2.5 kV). The bacteria were then cultured for 1 hour at 37 ° C in 1 ml SOC broth and then plated on Luria-Bertani (LB) plates containing trimethoprim (100 μg / ml). Expression of the "red" genes carried by pAJD434 was induced by adding 0.2% L-arabinose to the medium.
Mutant strains of E. coli BL21 AtolR and AompA, which are known to spontaneously produce a large amount of OMV, were produced by replacing the coding sequences of ompA and tolR with cassettes of resistance to kanamycin (kmr) and chloramphenicol (cmr) respectively . A three-step PCR protocol was used to fuse the upstream and downstream regions of ompA and tolR to the kmr and cmr genes, respectively. Briefly, the upstream and downstream regions of the genes tolR and ompA were amplified from genomic DNA of BL21 (DE3) with the specific primer pairs tolR-1 / tolR-2 and tolR-3 / tolR -4; ompA-1 / ompA-2 and ompA-3 / ompA-4, respectively (Table 1). The kmr cassette was amplified from plasmid pUC4K using primers PUC4K-rev and PUC4K-for and the cmr cassette was amplified using CMR-for / CMR-rev primers. Finally, 100 ng of each of the three amplified fragments were fused together by mixing in a PCR containing the 1/4 primers.
Linear fragments in which the antibiotic resistance gene was flanked by the upstream and downstream regions of tolR / ompA were used to transform the E. coli strain. coli BL21 (DE3) prone to recombination, which was made electrocompetent by three washing steps in cold water. The transformation was performed by electroporation of 5.9 ms at 2.5 kV. Transformants were selected by depositing the cells on LB plates containing 30 μg / ml kanamycin or 20 μg / ml chloramphenicol. Deletion of the genes tolR and ompA was confirmed by PCR amplification of the genomic DNA using the primer pairs tolR-Ι / PUC4K-rev and PUC4K-for / tolR-4; ompA-1 / CMR-rev and CMR-for / ompA-4.
Table 1
Oligonucleotide primers
Construction of Plasmids for Expression of Staphylococcal Antigens in E. coli OMVs coli
Four antigens from Staphylococcus aureus were selected for expression in E. coli OMVs. coli: (i) HlaH35L, an inactive mutant of α-hemolysin (Hla) of Staphylococcus aureus strain NCTC8325; (ii) Sta006, an ABC transporter of iron compounds from Staphylococcus aureus strain NCTC8325; (iii) SpA_DEABC, a mutated form of protein A (SpA) from strain NCTC8325, which is incapable of binding immunoglobulins; (iv) LukE, one of two LukED leukotoxin subunits derived from the Staphylococcus aureus strain NCTC8325.
The nucleic acid coding sequence of each protein was cloned into the plasmid pET-OmpA using the incomplete primer extension cloning method by polymerase (PIPE) [114]. pETOmpA (Figure 1) is a derivative of pET21b that allows the expression of proteins in the periplasmic space of E. coli through a translational fusion between a signal sequence of E. coli (signal sequence of OmpA) and the mature form of the protein of interest.
The plasmid pET-OmpA was amplified by PCR using primers omprev / nohisflag (Table 2). HlaH35L was amplified by PCR from plasmid pET15TEV-HlaH35L using primers OmpA-HlaI and HlaR1 (Table 2), which exclude the N-terminal signal sequence of Hla predicted by SignalP 4.1 software. The OmpA-HlaH35L sequence that is produced is represented by SEQ ID NO: 121. Sta006 was amplified from plasmid pET15TEV-Sta006 using primers OmpA-Sta006 fl and Sta006 rl (Table 2), which exclude the N part. -terminal coding sequence up to the LXXC lipoboite pattern, to avoid membrane anchoring. The OmpA-Sta006 sequence that is produced is represented by SEQ ID NO: 123. SpA_DEABC was amplified by PCR from the plasmid pET15TEV-SpA_DEABC using primers OmpA-SpA_DEABC fl and SpA_DEABC rl (Table 2). The OmpA-SpA_DEABC sequence that is produced is represented by SEQ ID NO: 125. LukE was amplified by PCR from the plasmid pET15TEV-LukE in two forms: the first, excluding the N-terminal signal sequence predicted by the SignalP software 4.1 and named "LukE (-LP)" was amplified using primers OmpA-LukE fl and LukE rl (Table 2); the second, comprising the entire coding sequence and named "LukE (+ LP)", was amplified using OmpA- primers.
LukE f2 and LukE rl (Table 2). The OmpA-LukE (-LP) sequence that is produced is represented by SEQ ID NO: 127. The OmpA-LukE (+ LP) sequence that is produced is represented by SEQ ID NO: 129. StaOll was amplified by PCR from the plasmid pET15TEV-Sta011 using primers OmpA-StaOll fl and StaOll rl (Table 2). The OmpA-StaOll sequence that is produced is represented by SEQ ID NO: 131. EsxAB was amplified by PCR from plasmid pET15TEV-EsxAB using primers OmpA-EsxAB1 and EsxAB R1 (Table 2). The OmpA-EsxAB sequence that is produced is represented by SEQ ID NO: 133. ClfA was amplified from the plasmid pET15TEV-ClfA using primers OmpA-ClfA F1 and ClfA R1 (Table 2). The OmpA-ClfA sequence that is produced is represented by SEQ ID NO: 135.
In this manner, plasmids pET21-SSOmpA-HlaH35L, pET21-SSOmpA-StaOO 6, pET21-SSOmpA -SpA_DEABC, pET21-SSOmpA-LukE (-LP), pET21-SSOmpA-LukE (+ LP), pET21-SSOmpA-StaOH pET21-SSOmp-EsxAB and pET21-SSOmp-ClfA were generated.
Table 2
Expression of antigens in AompA and AtolR mutants and analysis of antigen expression in total bacterial lysates, soluble bacterial fractions and culture supernatant fractions
Mutants of E. coli BL21 (DE3) AompA were transformed with plasmids pET21-SSOmpA-HlaH35L, pET21-SSOmpA-Sta006, pET21-SS0mpA-SpA_DEABC, pET21-SS0mpA-LukE (-LP), pET21-SS0mpA-LukE (+ LP), pET21-SS0mpA-esxAB and pET21-SSOmpA-Sta011 and mutants of E. coli BL21 (DE3) AtolR were transformed with plasmids pET21-SSOmpA-HlaH35L, pET21-SSOmpA-StaOO6, pET21-S SmpA-SpA_DEABC, pET21-SSOmpA-esxAB and pET21-SSOmpA-Sta011. As a negative control, strains of E. coli BL21 (DE3) AtolR and AompA were transformed with the empty vector pET-OmpA.
All strains were grown in liquid medium to the log phase, when antigen expression was induced by the addition of 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside). To test for recombinant strains for antigen expression, total bacterial lysates were analyzed (before and after IPTG induction for 3 hours) by polyacrylamide gel electrophoresis with SDS (SDS-PAGE) (Fig. 2). ). The bands corresponding to HlaH35L, Sta006, LukE (-LP) and LukE (+ LP) were detectable in the samples induced in AompA (Figure 2A), whereas the bands corresponding to HlaH35L and Sta006 were detectable in the samples induced in AtolR (Figure 2B), indicating that the expression of these antigens was successfully induced in E. coli. The bands corresponding to staOll and EsxAB were also detectable in samples induced in AompA (not shown). The AompA and AtolR strains transformed with the empty vector pET-OmpA were used as negative controls (FIG. 2). Demonstration of SpA_DEABC expression and confirmation of expression of HlaH35L (Figure 3), Sta006 (Figure 3), EsxAB (not shown) and StaOll (not shown) in total lysates were obtained by immunoblotting).
To test whether the heterologous antigens are soluble, soluble fractions were prepared from AompA strains induced for 2 hours with IPTG and analyzed by SDS-PAGE (Figure 4). An AompA strain transformed with the empty vector pET-OmpA was used as a negative control. This analysis showed that Sta006, SpA_DEABC, LukE (-LP) and LukE (+ LP) are all soluble.
Quantification of heterologous proteins in culture supernatant fractions
In order to quantify the amount of heterologous proteins released into the culture supernatants during growth, immunoblotting analysis was performed (Figure 5). The amount of antigen released into the culture supernatant by BL21 (DE3) AompA strains expressing staphylococcal proteins was determined by comparison of the chemiluminescence signals of the supernatant samples (prepared by precipitation of the proteins with trichloroacetic acid) with those of known amounts of purified recombinant proteins. This analysis showed that 7 μΐ of supernatant from AompA strain expressing SSOmpA-StaOOδ contained approximately 20 ng of antigen (FIG. 5A), 7 μΐ of supernatant from AompA strain expressing SSOmpA-SpA_DEABC contained approximately 40 ng of antigen (FIG. 5B) and 700 μl of supernatant from AompA strain expressing SSOmpA-HlaH35L contained approximately 10 ng of antigen (FIG. 5C).
The OMVs can then be used to form a pharmaceutical composition that can be used to immunize a mammal.
Murine renal abscess model and immunization experiments
CD1 mice four or five weeks old were immunized intramuscularly (IM) with 14-day sensitization-boosting injections of 100 μM (IM) containing 10 μg of each purified protein or OMV (see details of the combination tested in Table 3) adsorbed on aluminum hydroxide adjuvant (alum, 2 mg / ml). Control mice received equal amounts of alum adjuvant alone. Immunized animals were stimulated ("challenged") on 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). The kidneys were also treated for histopathology.
Table 3 Details of compositions used for immunization experiments
The protective efficacy of candidate vaccine formulations against S. aureus infection in a murine kidney abscess model is shown in Table 4 below.
Table 4
In order to evaluate the protective efficacy of candidate vaccine formulations against S. aureus infection, a mouse abscess model was used. Mice (N = 16 per group, two separate experiments) were immunized with alum alone, or with the following vaccines formulated with alum (Composition 1), a combined vaccine containing the following purified recombinant antigens: HlaH35L , Sta006, StaOll, EsxAB (Composition 2), a mixture of OMV containing the four antigens HlaH35L, Sta006, StaOll, EsxAB (Composition 3), and a mixture of OMV containing the four antigens HlaH35L, StaOO6, LukE, SpA ( Composition 4). After being immunized, the mice were then stimulated ("challenged") with the Newman strain of S. aureus and the numbers of CFU in the kidneys were measured 4 days after the challenge. The average numbers of CFUs measured in the control group were 6.92 Log, in the mice vaccinated with the vaccine of the composition 2, the CFUs were 5.87 and in the mice which received the compositions of OMV 3 and 4 of 1.69. Importantly, most mice immunized with the OMV-containing formulation showed undetectable levels of CFU numbers. In addition, the protective efficacy generated by the OMV formulation was statistically superior to that associated with the composition 1 vaccine.
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权利要求:
Claims (28)
[1]
A pharmaceutical composition comprising at least three antigens from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen.
[2]
The pharmaceutical composition of claim 1 comprising the four of the antigens of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen.
[3]
3. Pharmaceutical composition according to claim. 1, comprising at least four of the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen.
[4]
4. Pharmaceutical composition according to any one of claims 1 and 2, wherein said at least one antigen is included in OMV from a Gram-negative bacterium.
[5]
5. OMV from a gram-negative bacterium, wherein the OMV comprises at least one S. aureus antigen, wherein the antigen is selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen.
[6]
The OMV of claim 5, wherein the OMV comprises at least two S. aureus antigens, wherein the antigens are selected from the group consisting of: (i) an hla antigen; (ii) a staOOδ antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen.
[7]
The OMV of claim 5, wherein the OMV comprises at least three S. aureus antigens, wherein the antigens are selected from the group consisting of: (i) an hla antigen; (ii) a staOOδ antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen.
[8]
The OMV of claim 5, wherein the OMV comprises four S. aureus antigens, wherein the antigens are (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen and (iv) a spa antigen.
[9]
The OMV of claim 5, wherein the OMV comprises four S. aureus antigens, wherein the antigens are (i) an hla antigen; (ii) a sta006 antigen; (iii) esxAB antigen and (iv) staOll antigen.
[10]
The OMV according to any one of claims 5 to 9, wherein the OMV further comprises at least one, at least two or all three antigens from the group consisting of (i) a stall antigen; (ii) esxAB antigen and (iii) elfA antigen.
[11]
11. Process for preparing an OMV according to any one of claims 5 to 9.
[12]
12. OMV from a Gram-negative bacterium, wherein the OMV comprises at least one S. aureus antigen in its lumen, wherein the antigen is selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen.
[13]
The OMV of claim 12, wherein the OMV comprises at least two S. aureus antigens in its lumen, wherein the antigen is selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen.
[14]
The OMV of claim 12, wherein the OMV comprises at least three S. aureus antigens in its lumen, wherein the antigen is selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen.
[15]
The OMV of claim 12, wherein the OMV comprises four S. aureus antigens, wherein the antigens are (i) an hla antigen; (ii) a staOOδ antigen; (iii) a lukE antigen and (iv) a spa antigen.
[16]
The OMV of claim 12, wherein the OMV comprises four S. aureus antigens, wherein the antigens are (i) an hla antigen; (ii) a sta006 antigen; (iii) esxAB antigen and (iv) staOll antigen.
[17]
The OMV according to any one of claims 12 to 16, wherein the OMV further comprises in its lumen at least one, at least two or all of the antigens of the group consisting of: (i) a staOll antigen; (ii) esxAB antigen and (iii) elf A antigen.
[18]
The OMV according to any one of claims 12 to 17, wherein the antigen / antigens are free in the OMV lumen.
[19]
19. Process for preparing an OMV according to any one of claims 12 to 18.
[20]
The method of claim 19 comprising the step of expressing the antigen in the periplasm of the Gram-negative bacterium.
[21]
The method of claim 20, wherein the antigen is expressed in the periplasm of the Gram-negative bacterium using an expression vector comprising a nucleic acid sequence encoding the nucleic acid-bound antigen encoding a signal sequence of a periplasmic protein.
[22]
The method of claim 21, wherein the native signal sequence of the antigen is replaced by the signal sequence of a periplasmic protein.
[23]
23. OMV from a Gram-negative bacterium, wherein the OMV comprises at least one S. aureus antigen, wherein the antigen is selected from the group consisting of: (i) an hla antigen; (ii) a sta006 antigen; (iii) a lukE antigen; (iv) a spa antigen; (v) a staOll antigen; (vi) an esxA antigen and (vii) an esxB antigen, wherein the gram negative bacterium comprises a synthetic genome in which: a) one or more nonessential sequences to the production of vesicles are absent such that, compared to even bacteria without said absent sequences, the genome is between 1% and 50% smaller; and b) one or more sequences are present such that, compared to the same bacterium without said sequences present, the bacterium produces vesicles that comprise one or more additional antigens.
[24]
An OMV or method according to any one of claims 4 to 23, wherein the gram negative bacterium is selected from the group consisting of E. coli, N. meningitidis, Salmonella sp., and Shigella sp.
[25]
An OMV or method according to any one of claims 4 to 23, wherein the Gram-negative bacterium is a hyperbourgeonating strain of the Gram-negative bacterium.
[26]
The OMV or method of claim 25, wherein the Gram-negative bacterium is a strain of E. coli. AtolR coli or a strain of E. coli. AompA coli.
[27]
27. A pharmaceutical composition comprising (a) at least one OMV according to any one of claims 4 to 9 or 12 to 17 and (b) a pharmaceutically acceptable carrier.
[28]
28. The pharmaceutical composition according to claim 27, comprising at least two different OMVs according to any one of claims 4 to 9 or 12 to 17.

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EP14161861|2014-03-26|

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EP14161861.1|2014-03-26|

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