WO2023247747A1 - Protective staphylococcal exotoxin vaccine - Google Patents

Protective staphylococcal exotoxin vaccine Download PDF

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WO2023247747A1
WO2023247747A1 PCT/EP2023/067094 EP2023067094W WO2023247747A1 WO 2023247747 A1 WO2023247747 A1 WO 2023247747A1 EP 2023067094 W EP2023067094 W EP 2023067094W WO 2023247747 A1 WO2023247747 A1 WO 2023247747A1
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vaccine
seb
tsst
staphylococcal
seq
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PCT/EP2023/067094
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French (fr)
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Martha M. EIBL
Günter HALLER
Andreas RÖTZER
Nina MODEL
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Biomedizinische Forschung & Bio-Produkte AG
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response

Definitions

  • Staphylococcus aureus produces a variety of exoproteins and Staphylococcal superantigens (SAG) that contribute to its ability to colonize and cause disease of varying severity in mammalian hosts ranging from superficial skin infections, such as abscesses and impetigo, to serious invasive infections such as pulmonary disease, osteomyelitis and endocarditis, as well as to the acute and potentially fatal toxic shock syndrome (TSS) and septic shock syndrome.
  • SAG Staphylococcal superantigens
  • Staphylococcal Enterotoxin B and Staphylococcal Enterotoxin C are superantigen exotoxins of staphylococci, also understood as Staphylococcal Exotoxin B (SEB) and Staphylococcal Exotoxin C (SEC), respectively.
  • SEB and SEC are commonly produced by invasive S. aureus isolates, especially methicillin-resistant strains and isolates from animal diseases.
  • Superantigens are far more potent than conventional antigens and may trigger up to 30% of the entire T cell population. They cause massive proliferation of T cells and uncontrolled release of cytokines. Superantigens activate T-cells at much lower concentrations than nominal antigens. Activation by superantigens can be detected at picogram to low nanogram concentrations, by nominal antigens at micrograms and higher. The difference, thus, is 5 to 8 log steps. Clonal expansion and upregulation of the IL-2 receptor on the cell surface are consequences of cross-linking of MHC class II on antigen presenting cells and TCR on CD4 + cells. The massive release of cytokines is the basis of toxicity of Staphylococcal superantigens.
  • Superantigen toxins can also activate different pathways of innate immunity by their mitotic effects e.g., IL-1 , CD14, IL-6 etc.
  • the effect of IL-1 leads to mitosis (polyclonal dysregulation) and numerous effector and modulatory activation of innate immunity (NK cells, toll like receptor positive lymphocytes and their binding proteins, phagocytes and their ligands etc.).
  • the SEB plays a pivotal role as a causative agent in TSS (Toxic Shock Syndrome).
  • TSS Toxic Shock Syndrome
  • Kappler et al. (J. Exp. Med. 1992, 175:387-396) describe mutations defining functional regions of the superantigen SEB.
  • Fries et al. (Microbiol Spectr. 2013,1 (2): 2013) disclose specific binding regions of SEB and provides a sequence alignment of amino acid sequences of SEB derived from S. aureus clinical isolates.
  • Woody et al. disclose SEB mutants with either N23K or F44S mutations, and describes the vaccine potential in a mouse model.
  • Jeong et al. disclose an SEB vaccine candidate with four mutations (N23A, Y90A, R110A, and F177A) showing eliminated superantigen activity. Bagnoli et al.
  • WO99/40935 (corresponding to EP1055429) discloses an SEB comprising a modification which is one or more amino acid substitutions e.g., at position 9, 23, 41 , or 44, to confer inhibitory activity on T cell activation.
  • WO201 4/205111 discloses a multivalent peptide oligopeptide including a Staphylococcus aureus antigen or mutant, fragment, variant, or derivative thereof, which may include, SEB, SECI-3, SEE, SEH, SEI, SEK, TSST-1 , SpeC, SED, or SpeA, or any mutant, fragment, variant, or derivative thereof, or any combination thereof, in any order.
  • the oligopeptide is disclosed to include a SEB mutant which is the attenuated toxoid SEB comprising the amino acid substitutions L45R/Y89A/Y94A.
  • Hu et al. (Infect Immun. 2005 Jan;73(1): 174-80) have investigated a mutant staphylococcal enterotoxin C (mSEC) comprising an N23A substitution and reduced superantigenic activity.
  • mSEC staphylococcal enterotoxin C
  • Hu et al (Microbes and Infection 2006, 8 (14-15):2841 -2848) describe a double mutant of SEC (dmSEC) comprising a N23A substitution for reducing binding to TCR, and a Y94A substitution for reducing binding to MHC class II.
  • dmSEC double mutant of SEC
  • WO201 4/205111 discloses a multivalent peptide oligopeptide including a Staphylococcus aureus antigen or mutant, fragment, variant, or derivative thereof, which may include, SEB, SECI-3, SEE, SEH, SEI, SEK, TSST-1 , SpeC, SED, or SpeA, or any mutant, fragment, variant, or derivative thereof, or any combination thereof, in any order.
  • Venkatasubramaniam et al. disclose a fusion toxoid vaccine for protection and neutralization of staphylococcal superantigens, including toxoid versions of the SAGs TSST-1 , SEB and SEA, wherein the TSST-1 comprises amino acid substitutions L30R, D27A and I46A.
  • T cell activation has been assessed by lymphocyte proliferation and IL-2 gene expression after in vivo challenge with TSST-1 and the mutant antigens; expression of the genes of proinflammatory cytokines were taken as indicators for the inflammatory reaction after the combined treatment with TSST-1 and LPS.
  • US2014199339A1 discloses compositions comprising two or more staphylococcal toxoids for inducing protective immune response against staphylococcal diseases.
  • US2003157113A1 discloses methods and compositions for treatment of neoplastic disease.
  • the methods employ conjugates comprising superantigen polypeptides, nucleic acids with other structures that preferentially bind to tumor cells and are capable of inducing apoptosis.
  • US2003032582A1 discloses nucleic acid molecules encoding membraneinfiltrating polypeptides, and truncated superantigen polypeptide encoding nucleic acid molecules as well as truncated superantigen polypeptides. These truncated superantigens are described to elicit an anti-tumor immune response without binding MHC II molecules.
  • the objective is solved by the subject of the present claims and as further described herein.
  • the invention provides for a combination of vaccine antigens which confer protective immunity, in particular targeting professional antigen presenting cells (APC) or cells of the innate and/or adaptive immune system expressing constitutively MHC Class II.
  • the vaccine comprising the combination of vaccine antigens is herein also referred to as “multivalent vaccine”.
  • the multivalent vaccine is provided as a non-pyrogenic vaccine, in particular a non-pyrogenic combination of vaccine antigens, such as obtained by a combination of Staphylococcal superantigen mutants as described herein which are non-pyrogenic Staphylococcal superantigen vaccine antigens.
  • the non-pyrogenic vaccine or combination of vaccine antigens does not trigger an abnormally high body temperature in a subject (in particular, fever) upon parenteral administration.
  • the vaccine described herein is compared to the wild type toxins, not pyrogenic, and not lymphopenic. Specifically, it does neither change chemical blood parameters nor the amount of white blood cells.
  • the non-lymphopenic vaccine or combination of vaccine antigens does not trigger an abnormally low level of lymphocytes in the blood of a subject (in particular, lymphopenia) upon parenteral administration.
  • a Staphylococcal superantigen vaccine comprising a combination of detoxified Staphylococcal superantigen vaccine antigens which are genetically modified toxins that incorporate detoxifying mutations in its T cell receptor binding region and MHC Class II binding region, wherein the combination comprises at least the vaccine antigens Staphylococcal Exotoxin B (SEB) and any one or both of Staphylococcal Exotoxin C (SEC) and Staphylococcal toxic shock syndrome toxin-1 (TSST-1).
  • SEB Staphylococcal Exotoxin B
  • SEC Staphylococcal Exotoxin C
  • TSST-1 Staphylococcal toxic shock syndrome toxin-1
  • the Staphylococcal superantigen vaccine antigens are non- pyrogenic.
  • the Staphylococcal superantigen vaccine is non-pyrogenic.
  • the targets of the combination are the respective wild-type toxins.
  • the vaccine antigens described herein are detoxified whole proteins, comprising a length of at least 95%, 96%, 97%, 98%, 99%, or 100% of the length of the respective wild-type toxin.
  • the vaccine antigens described herein comprise detoxifying mutations in the regions herein referred to as “core regions”, which particularly consists of both, the T cell receptor binding region and MHC Class II receptor binding region.
  • core regions which particularly consists of both, the T cell receptor binding region and MHC Class II receptor binding region.
  • double mutated Such vaccine antigens are herein also referred to as “double mutated”.
  • the T cell receptor (a/p) binding region and MHC Class II receptor binding region of a Staphylococcal superantigen, and in particular of any one of SEB, TSST-1 or SEC, are herein referred to as “core region”. Any other region(s) besides the core region are herein referred to as “non-core region(s)”.
  • the core region consists of the T cell receptor binding region and MHC Class II binding region.
  • the MHC Class II receptor binding region is an immunoglobulin superfamily binding region, which comprises or consists of the region binding inside or adjacent to the antigen-binding groove (and outside the antigen-binding groove).
  • the class II MHC binding site is located in the hydrophobic region of the NH2-terminal domain, and the TCR binding site is primarily in the major central groove of the COOH-terminal domain.
  • McCormick and Schlievert J Immunol August 1 , 2003, 171 (3) 1385-1392
  • JM Hurley and M. Matsumura J Exp Med. 1995 Jun 1 ; 181 (6): 2229-2235.
  • the detoxified SEB toxin sequence comprises at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:1 , or to any other wild-type SEB toxin sequence.
  • any naturally-occurring SEB, SEC or TSST-1 sequence such as SEQ ID NO:1 (as for SEB), SEQ ID NO:2 (as for SEC) and SEQ ID NO:3 (as for TSST-1 ), respectively, or any other respective wild-type toxin sequence which is a variant of the respective SEQ ID NO:1 (as for SEB), SEQ ID NO:2 (as for SEC) and SEQ ID NO:3 (as for TSST- 1), may be used to introduce respective detoxifying mutations at the core region, in particular at respective corresponding positions within the core regions, such as further described herein.
  • Naturally-occurring (i.e. , wild-type, wt) toxin sequences may originate from different S.
  • aureus sources or isolates
  • variability in core and/or non-core regions in many cases, the variability is in the non-core region only
  • Wild-type variant toxin sequences typically comprise the same core region, whereas a certain degree of variability may be found in the non-core region, such as to provide at least 95%, 96%, 97%, 98%, or 99% sequence identity to the respective wt sequences of SEB, SEC, or TSST-1 , referred to herein.
  • a number of point mutations may be comprised in wild-type variant toxin sequences.
  • an exemplary wild-type SEB sequence comprises or consists of SEQ ID NO:1.
  • the wild-type SEB toxin comprises or consists of SEQ ID NO:1 or an amino acid sequence that comprises at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:1 when comparing the whole sequence of SEQ ID NO:1.
  • Exemplary naturally-occurring wild-type variants of SEQ ID NO:1 are selected from those comprising or consisting of SEQ ID NO: 19, 21 , 23, 25, 27, 29, 31 , or wt SEB sequences which differ from any of the foregoing though polymorphisms.
  • an exemplary wild-type SEC sequence comprises or consists of SEQ ID NO:2.
  • the wild-type SEC toxin comprises or consists of SEQ ID NO:2 or an amino acid sequence that comprises at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:2 when comparing the whole sequence of SEQ ID NO:2.
  • Exemplary naturally-occurring wild-type variants of SEQ ID NO:2 are selected from those comprising or consisting of SEQ ID NO:46-50, or wt SEC sequences which differ from any of the foregoing though polymorphisms.
  • the SEB is characterized as follows.
  • the SEB is mutated to comprise at least two or at least three point mutations at amino acid positions 21-25 and 21-24, respectively (i.e., “within aa21-25” or “within aa21-24”, respectively) also referred to as “in the region of aa21-25” and “in the region of aa21-24”, respectively, in the SEB toxin sequence SEQ ID NO:1 , wherein said at least two or three point mutations comprise a deletion of any one of aa21-22, aa22-23, aa23-24, aa24-25, aa21-23, aa22-24, or aa23-25, in the SEB toxin sequence SEQ ID NO:1 , or at a corresponding region in any other wild-type SEB toxin sequence.
  • SEQ ID NO:1 or the wild-type variant SEB toxin is functional as a staphylococcal superantigen, unless being engineered to incorporate the respective detoxifying mutations.
  • the wild-type SEB toxin sequence comprises or consists of SEQ ID NO:1 , or any one of SEQ ID NO:19, 21 , 23, 25, 27, 29, 31.
  • said detoxified SEC variant sequence of SEQ ID NO:5 comprises said deletion of aa22-24 comprised in SEQ ID NO:5; and: a) one or more further point mutations in its immunoglobulin superfamily binding region, preferably within (i.e., in the region of) aa42-47 or aa43-45, and/or b) one or more further point mutations as naturally-occurring in other regions of the SEB sequence, such as in the non-core regions e.g., one or more point mutations as naturally-occurring in SEB wild-type variants, such as occurring in any one of SEQ ID NO: 19, 21 , 23, 25, 27, 29, 31 , in particular one or more of the following point mutations: A14S, K16E, L20T, E22G, V26Y, K39M, G72D, D124N, N127S, V154A, or M200I in SEQ ID NO:1 ; and/or c) at least 95%, 96%, 97%,
  • Specific embodiments comprise a SEB toxin variant sequence of SEQ ID NO:5, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective regions of SEQ ID NO:5, in particular over the whole length of all corresponding regions of SEQ ID NO:5.
  • the detoxified SEB toxin comprises or consists of SEQ ID NO:7, or a detoxified SEC variant sequence thereof, which comprises at least said deletion of amino acids within 21-25 as described herein, particularly comprising said deletion of at least two or at least three amino acids at amino acid positions 21 to 25, and which comprises at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:7.
  • SEQ ID NO:7 differs from SEQ ID NO:1 in the deletion of aa22-24 and the amino acid substitution L45R.
  • said detoxified SEB variant sequence of SEQ ID NO:7 comprises said deletion of aa22-24 comprised in SEQ ID NO:7, and said amino acid substitution L45R, and additionally: a) one or more further point mutations in its immunoglobulin superfamily binding region, preferably within aa42-47 or aa43-45, and/or b) one or more further point mutations as naturally-occurring in other regions of the SEB sequence, such as in the non-core regions e.g., one or more point mutations as naturally-occurring in SEB wild-type variants, such as occurring in any one of SEQ ID NO: 19, 21 , 23, 25, 27, 29, 31 , in particular one or more of the following point mutations: K7N, S14A, V82L, T133S, K141 E, T150I, S225F in SEQ ID NO:1 ; and/or c) at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:
  • Specific embodiments comprise a SEB toxin variant sequence of SEQ ID NO:7, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective non-core regions, in particular over the whole length of all corresponding non-core regions, compared to the corresponding regions of SEQ ID NO:7.
  • the non-core region of the detoxified SEB described herein differs from SEQ ID NO:1 only in 1 , 2, 3, 4, 5, or 6 amino acids, e.g., with an amino acid substitution at only 1 , 2, 3, 4, 5, or 6 positions, such as amino acid substitution(s) selected from those naturally-occurring in wt SEB sequences e.g., selected from the group consisting of K7N, S14A, V82L, T133S, K141 E, T150I, S225F, as compared to SEQ ID NO:1.
  • the SEB is a double mutated vaccine antigen, as described herein, which is preferably combined with any one or both of TSST-1 and SEC vaccine antigens which are also detoxified by “double mutation”.
  • Exemplary double mutated TSST-1 and SEC vaccine antigens are any of the following: a) the SEC comprises a wild-type SEC amino acid sequence that is modified to comprise deletion of at least two amino acids in the T cell receptor binding region between aa 21 to 25, and to further comprise at least one point mutation in the MHC Class II binding region, wherein said at least one point mutation comprises an amino acid substitution at a position selected from the group consisting L45, Q43, or F44, preferably L45R, Q43P, F44P, or F44S, wherein the wild-type SEC toxin amino acid sequence is of SEQ ID NO:2, or of any other wild-type SEC toxin sequence; b) the TSST-1 comprises a wild-type TSST-1 amino acid sequence that is modified to comprise at least one point mutation in the MHC Class II binding region consisting of G31 , L30, and S32, preferably comprising a deletion or substitution of G31 , and at least one point mutation in the T cell receptor binding region consisting
  • SEB, SEC and TSST-1 sequences are described in Figure 1 , which includes wt sequences, exemplary detoxifying mutations and exemplary vaccine antigens.
  • the detoxified SEB, SEC and TSST-1 sequences described herein may or may not comprise any further (though a limited number e.g., up to 6, 5, 4, 3, 2, or only 1 ) conservative amino acid substitutions in the non-core region.
  • a conservative alternative to a first amino acid is herein understood as a second amino acid which is different from the first amino acid and which has about the same properties of charge and polarity as the first amino acid.
  • Amino acids are typically grouped as follows; each of the amino acids within any such group is understood to have about the same properties of charge and polarity: a) basic (positively charged), polar: R, H, K, b) acidic (negatively charged), polar: D, E; c) hydrophobic: A, I, L, M, F, P, W, V; d) polar, uncharged: N, C, Q, G, S, T, Y;
  • a preferred amino acid substitution is with an amino acid other that the original amino acid, such original amino acid e.g., being identified in a wild-type sequence.
  • the SEC is characterized as follows.
  • SEB and SEC comprise or share a similar structure, in particular regarding the core regions.
  • the core region of SEB or SEC comprises or consists of either or both of aa21-25 and aa42-47, and optionally in addition, any one or both of aa89-91 or aa66-68. [please confirm]
  • the immunoglobulin superfamily binding region such as e.g., the MHC Class II receptor binding region, in particular the region binding inside, adjacent or outside the antigen-binding groove, comprises or consists of e.g., aa42-47 in SEQ ID N0:1 and SEQ ID N0:2, respectively, or aa89-91 in SEQ ID NO:1 and SEQ ID NO:2, respectively; or aa66-68 in SEQ ID NO:1 and SEQ ID NO:2, respectively.
  • SEB aa42-47 in SEQ ID NO:1 : “DQFLYF” (SEQ ID NO:67); identifying positions D42, Q43, F44, L45, Y46, and F47.
  • SEC aa42-47 in SEQ ID NO:2: “DKFLAH” (SEQ ID NO:68); identifying positions D42, K43, F44, L45, A46, and H47.
  • the detoxifying point mutations in SEB or SEC consist of amino acid deletions within the T cell receptor binding region of the SEC sequence, more specifically within aa21-25.
  • the detoxifying point mutations consist of a deletion of at least (or only) two or three amino acids in the region of aa21-25.
  • the detoxifying point mutations in the region of aa21-25 consist of a deletion of only 2, 3, 4, or 5 amino acids, preferably at least comprising a deletion of N23 and a deletion of one or both amino acids which are adjacent to N23.
  • the number of point mutations within aa21-25 can be 2, 3, 4, or 5, specifically wherein point mutations are of at least 2 or 3 consecutive (adjacent) aa positions, such as aa21-22, aa22-23, aa23-24, aa24-25, aa21-23, aa22-24, or aa23-25.
  • a point mutation at position aa23 is combined with one or more non-consecutive (non-adjacent) mutations, such as at aa21 and/or aa25.
  • the mutation in the region of aa21-25 is a deletion of at least (or only) two, three or four amino acids, at least including a deletion of N23 and/or E22 and/or M24, and/or M21 and/or K25, such as a deletion or substitution of at least any one of aa22-23, aa23-24, aa21-23, aa22-24, or aa23-25, in particular a deletion of aa22-24 only.
  • said point mutations at amino acid positions 21 to 25 comprise or consist of a deletion of amino acids 22-24 and/or 21-23.
  • the detoxified SEB or SEC toxin is characterized by one or more further (additional) point mutations (i.e. , other than those in the region of aa21-25), in particular in its immunoglobulin superfamily binding region, preferably in the region of aa42-47 or aa43-45.
  • the detoxified SEC toxin is of a type C1 , C2, or C3.
  • An exemplary wild-type SEC toxin comprises or consists of any one of SEQ ID NO:2 (type C1) SEQ ID NO:46 (NGA13141.1 , type C1), SEQ ID NO:47 (HAY3254229.1 , type C1), SEQ ID NO:48 (HAZ5071097.1 , type C1), SEQ ID NO:49 (WP_061047250.1 , type C2), or SEQ ID NO:50 (WP_077156035.1 , type C3).
  • accession number references are NCBI accession numbers (National Library of Medicine, Bethesda, USA).
  • the detoxified SEC toxin may comprise one or more, preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations, preferably up to 10, 9, 8, 7, 6, or 5 point mutations, preferably amino acid substitutions or a combination of amino acid substitutions as naturally-occurring in any of the wild-type SEC sequences, such as found in a wild-type SEC toxin sequence, e.g.: one or more of AMS, K16E, L20T, E22G, V26Y, K39M, G72D, D124N, N127S, V154A, or M200I, compared to SEQ ID NO:2.
  • the number of point mutations in the non-core region is limited, such that the non-core region has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2.
  • Specific embodiments comprise a SEC toxin variant sequence of SEQ ID NO:42 x, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective non-core regions, in particular over the whole length of all corresponding non-core regions, compared to the corresponding regions of SEQ ID NO:42.
  • the detoxified SEC toxin comprises or consists of SEQ ID NO:44, or a detoxified SEC variant sequence thereof, which comprises at least said deletion of amino acids within 21-25 as described herein, particularly comprising said deletion of at least two or at least three amino acids at amino acid positions 21 to 25, and which comprises at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:44.
  • SEQ ID NO:44 differs from SEQ ID NO:2 in the deletion of aa22-24 and the amino acid substitution L45R.
  • said detoxified SEC variant sequence of SEQ ID NO:44 comprises said deletion of aa22-24 comprised in SEQ ID NO:44, and said amino acid substitution L45R, and additionally: a) one or more further point mutations in its immunoglobulin superfamily binding region, preferably within aa42-47 or aa43-45, and/or b) one or more further point mutations as naturally-occurring in other regions of the SEC sequence, such as in the non-core regions e.g., one or more point mutations as naturally-occurring in SEC wild-type variants, such as occurring in any one of SEQ ID NO:46-50, in particular one or more of the following point mutations: A14S, K16E, L20T, E22G, V26Y, K39M, G72D, D124N, N127S, V154A, or M200I in SEQ ID NO:2; and/or c) at least 95%, 96%, 97%, 98%, 99%, or 99.5%
  • Specific embodiments comprise a detoxified SEC variant sequence of any one of SEQ ID NO:42 or 44, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective non-core regions of any one of the detoxified SEC toxin sequences described herein, in particular over the whole length of all corresponding non-core regions of any one of the detoxified SEC toxin sequences described herein.
  • the detoxified SEC comprises or consists of any one of SEQ ID NO:42 or 44, or a detoxified SEC variant sequence of any one of the foregoing which comprises said modifications in the T cell receptor binding region and in the MHC Class II binding region, and at least any one of 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:1 and/or to any one of SEQ ID NO:42 or 44.
  • the TSST-1 is characterized as follows.
  • SEQ ID NO:3 is the amino acid sequence of a wild-type mature TSST-1 protein.
  • toxicity was found to be greatly reduced in TSST-1 that is genetically modified by one or more detoxifying point mutations within the core region of the MHC Class II binding region, wherein the point mutations are a substitution or deletion of G31 , or a substitution and/or deletion of any two or three amino acids at positions L30 to S32, including the substitution or deletion of G31 , in a wt TSST- 1 sequence, such as the TSST-1 sequence of SEQ ID NO:3 or in any other wild-type (wt) TSST-1 sequence.
  • the amino acid substitution at position 31 is any one of G31 R, or any other conservative substitutions as an alternative to G31 R.
  • the amino acid Glycine (G), a non-polar amino acid is substituted for a basic, positively charged amino acid, like “R”, or any conservative alternative thereto, such as H or K.
  • said one or more detoxifying deletion mutations are in the MHC Class II binding region only.
  • Such TSST-1 molecules are herein also referred to as “single mutant”.
  • substitution or deletion of G31 or a substitution and/or deletion of any two or three amino acids at positions L30 to S32, including the substitution or deletion of G31 , are comprised in the MHC Class II binding region, as further described herein.
  • the TSST-1 amino acid sequence that comprises detoxifying mutations in the MHC Class II binding region only differs from a wt TSST-1 sequence only in the substitution or deletion of G31 , or in the substitution and/or deletion of any two or three amino acids at positions L30 to S32, including the substitution or deletion of G31 .
  • two or more detoxifying deletion mutations are in the MHC Class II binding region and in the T cell receptor binding region.
  • Such TSST-1 molecules are herein also referred to as “double mutant”.
  • at least the substitution or deletion of G31 , or a substitution and/or deletion of any two or three amino acids at positions L30 to S32, including the substitution or deletion of G31 are comprised in the MHC Class II binding region, and at least one point mutation e.g., an amino acid substitution is comprised in the T cell receptor binding region.
  • the G31 substitution or deletion in SEQ ID NO:3 (which results in SEQ ID NO:53) refers to the amino acid “G” at position 31 in SEQ ID NO:3.
  • the amino acid at a corresponding position is a glycine in many naturally-occurring wildtype TSST-1 sequences, such as e.g., in any one of the wt TSST-1 sequences SEQ ID NO:58-63.
  • wildtype TSST-1 sequences such as e.g., in any one of the wt TSST-1 sequences SEQ ID NO:58-63.
  • amino acid at the corresponding position is glutamate (“E”).
  • E glutamate
  • the TSST-1 core region is composed of: a) the T cell receptor binding region consisting of the amino acid region of aa132 to aa139 and aa16 in a wt TSST-1 sequence, such as e.g., E132 to Q139 and G16 in SEQ ID NO:3; b) the MHC Class II binding region consisting of aa30, aa31 , and aa32 in a wt TSST-1 sequence, such as e.g., L30, G31 and S32 in SEQ ID NO:3.
  • the number of point mutations in the core region is limited, such as consisting of: a) only one substitution or deletion of the amino acid at position 31 in a wt TSST- 1 sequence; or b) one substitution or deletion of the amino acid at position 31 in a wt TSST-1 sequence, and only one, two, three or four further point mutations in the core region, which is e.g., selected from the group consisting of: AS32, AL30, an amino acid substitution at position H135, preferably H135X, wherein X is A, D, I, Q, or R; G16V, and L137V.
  • a detoxified TSST-1 which is genetically modified by detoxifying mutations that comprise at least the substitution or deletion of the aa31 of the mature wt TSST-1 sequence, which is a mutation in the MHC Class II binding region, and an additional modification in the T cell receptor binding region of the TSST-1 sequence.
  • the detoxified TSST-1 described herein is genetically mutated to incorporate the detoxifying TSST-1 mutations in the core region as described herein.
  • the TSST-1 mutation(s) described herein are point mutations.
  • the detoxified TSST-1 described herein is at least double or triple mutated, such as comprising at least the substitution or deletion of the amino acid at position 31 in the mature TSST-1 sequence, and one or two mutation(s) in the T cell receptor binding region.
  • the number of detoxifying point mutations within the core region is at least (or only) one, 2, 3, or 4, up to 5, as compared to a wt TSST-1 amino acid sequence.
  • the point mutation(s) in the MHC Class II binding region are substitution or deletion mutations.
  • the point mutation(s) in the T cell receptor binding region, if any, are substitutions.
  • the detoxified TSST-1 is characterized by at least 2, up to 5, 4, 3, or 2 point mutations in the core region, compared to the mature wt TSST-1 sequence.
  • the detoxified TSST-1 is characterized by the core region mutations described herein, and up to 6, 5, 4, 3, 2 or only 1 point mutation in the full-length sequence, compared to the mature wt TSST-1 sequence.
  • the sequence of the detoxified TSST-1 described herein differs from a wt TSST-1 sequence in the detoxifying point mutations which comprise or consist of: a) one or more point mutations in the MHC Class II binding region, wherein one point mutation is a substitution or deletion of G31 , optionally wherein additionally any one or both of L30 and S32 are substituted or deleted; and optionally: b) one or more point mutations in the T cell receptor binding region, preferably comprising or consisting of an amino acid substitution at H135, preferably H135X, wherein X is A, D, I, Q, or R; and/or G16V, and/or L137V.
  • the detoxified TSST-1 described herein comprises a TSST-1 sequence which is any one of: a) SEQ ID NO:53, which comprises the amino acid sequence of SEQ ID NO:3, wherein G31 is deleted; b) SEQ ID NO:54, which comprises the amino acid sequence of SEQ ID NO:3, wherein G31 and L30 are deleted; c) SEQ ID NO:55, which comprises the amino acid sequence of SEQ ID NO:3, wherein G31 and S32 are deleted; d) SEQ ID NO:56, which comprises the amino acid sequence of SEQ ID NO:3, wherein L30, G31 and S32 are deleted.
  • any of the sequences SEQ ID NO:53-56 can be further modified to comprise one or more point mutations in the T cell receptor binding region, preferably comprising an amino acid substitution at any one of H135X, wherein X is A, D, I, Q, or R; and/or G16V, and/or L137V.
  • SEQ ID NO:57 comprises the TSST-1 sequence incorporating the delta G31 and H135A mutations.
  • SEQ ID NO:64 comprises the TSST-1 sequence incorporating the G31 R and H135A mutations.
  • any of the sequences SEQ ID NO:53-57 and SEQ ID NO:64 may be further modified to incorporate one or more, e.g., up to 6, 5, 4, 3, 2, or only 1 aa substitution outside the core region.
  • the detoxified TSST-1 may comprise one or more further mutations in a non-core region i.e., the region of TSST-1 which is not the core region.
  • Specific embodiments comprise a detoxified TSST-1 variant sequence of any one of SEQ ID NO:53-57 or SEQ ID NO:64, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective non-core regions of any one of the detoxified TSST-1 toxin sequences described herein, in particular over the whole length of all corresponding non-core regions of any one of the detoxified TSST-1 toxin sequences described herein.
  • the detoxified TSST-1 comprises or consists of any one of SEQ ID NO:53-57 or SEQ ID NO:64, or a detoxified TSST-1 variant sequence of any one of the foregoing which comprises said modifications in the T cell receptor binding region and in the MHC Class II binding region, and at least any one of 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:3 and/or to any one of SEQ ID NO:53-57 or SEQ ID NO:64.
  • the detoxified TSST-1 comprises or consists of the wild-type TSST-1 sequence, such as e.g., amino acid sequence of any one of SEQ ID NO:3, or SEQ ID NO:3 to 3, which is modified to incorporate said detoxifying point mutations wherein one of the point mutations is a substitution or deletion of G31 , and to incorporate an amino acid substitution H135A.
  • the TSST-1 sequence comprises any one of SEQ ID NO:53-57 or SEQ ID NO:64, or the respective detoxifying point mutations comprised in NO:53-57 or SEQ ID NO:64 as compared to SEQ ID NO:3.
  • the vaccine antigens may comprise one or more detoxified toxin molecules that are fused to another detoxified toxin molecule, optionally comprising a linker sequence.
  • exemplary combinations are fusions of atoxic or detoxified toxoid molecules, wherein at least one (or two) of said molecules is/ are the detoxified SEB comprising the point mutations as described herein.
  • said at least one detoxified SEB toxin molecule can be fused to another detoxified SEB toxin molecule, optionally comprising a linker sequence.
  • linkers suitably used are peptidic linkers such as consisting of a series of at least two, or at least three amino acids. Specifically, the fusion of molecules is in any order, by a peptide bond, with or without a linker.
  • the fusion comprises at least two identical molecules of said detoxified SEB toxin as described herein, or at least two molecules of said detoxified SEB toxin as described herein that differ in at least one of the detoxifying point mutations as described herein.
  • a fusion of at least two molecules, in particular two identical molecules, with or without a linker, is herein also referred as a “tandem fusion” or “tandem construct”.
  • An exemplary tandem fusion of SEB comprises or consists of any one of SEQ ID NO:15, 33, 35, or 37.
  • Fusion may be achieved by recombination of nucleic acid molecules encoding the respective peptide sequences, or otherwise by synthesizing the coding nucleic acid molecules or fused (poly)peptide sequences.
  • the linker can be a peptidic linker, preferably composed of a number of consecutive amino acid residues, such as selected from any of the naturally-occurring amino acids, preferably any of Gly, Ser, His, Met, Lys, Leu, and Thr.
  • Linkers can be composed of flexible residues like glycine and serine so that the adjacent peptides are free to move relative to one another.
  • short linkers are sufficient, such as a peptide linker comprising or consisting of a sequence of 2-10 amino acids, e.g., comprising or composed of two amino acids selected from the group consisting of Lys-Leu, His-Met, Gly-Thr and Gly- Gly-Gly.
  • Longer linkers such as longer than 10 amino acids can be used e.g., when necessary to ensure that two adjacent molecules do not sterically interfere with each other.
  • the antigen may comprise one or more peptide spacers in addition to linker, such as to improve the structure or stability of the polypeptide.
  • the peptide fusion described herein may comprise the peptides which are conveniently bound to each other by bioconjugation, chemical conjugation or crosslinking.
  • the antigen may comprise peptide conjugates.
  • Specific embodiments may employ multimerization domains, carriers, or devices such as nanostructures or beads that are suitably used to immobilize a series of peptides.
  • the antigen may be provided as a molecule or molecule complex composed of two or more polypeptide chains, which are associated through covalent or non- covalent linkage, or just mixed to obtain an antigenic composition.
  • Alternative combinations comprised in a vaccine antigen described herein are complexes, wherein said molecules are adsorbed, adhered or otherwise immobilized or bound to a liposomal, nanoparticle or solid carrier, such as those suitable for use in formulating vaccine preparations.
  • compositions of molecules such as provided in a vaccine formulation at a predefined ratio or dose, for example, a mixture formulated on an adjuvant compound, such as an insoluble metal salt e.g., alum, aluminum hydroxide, or aluminum phosphate.
  • an adjuvant compound such as an insoluble metal salt e.g., alum, aluminum hydroxide, or aluminum phosphate.
  • the invention further provides for a pharmaceutical preparation comprising the antigen described herein, further comprising a pharmaceutically acceptable carrier, e.g., in an immunogenic formulation.
  • the invention further provides for a vaccine comprising the vaccine antigen described herein, and a pharmaceutically acceptable carrier.
  • the vaccine is a protective vaccine, such as protective against S. aureus disease conditions.
  • the vaccine is specifically protective at a site of exposure or toxicity of the respective target toxin or SAG(s) in the respective multivalent vaccine described herein, which are targeted by a multivalent vaccine, such as at one or more of the following sites: subcutaneous, mucosal gastrointestinal, skin or intramuscular sites.
  • the vaccine comprise an adjuvant, such as suitably used in human vaccine preparations.
  • adjuvants are selected from the group consisting of an insoluble metal salt, a glucopyranosyl Lipid A adjuvant, adjuvant systems which are stimulants of innate immunity, such as AS01 , AS03, or AS04, MF59, a TCR stimulant such as a toll-like receptor agonist or CpG oligonucleotides, preferably alum, aluminum hydroxide, or aluminum phosphate.
  • the vaccine can be administered to a subject, in particular a human subject, in an effective amount employing a prime-boost strategy.
  • the combination comprises an effective amount of any one or two or three of the vaccine antigens described herein which is ranging between 0.0001 and 1 mg per administration, preferably at least any one of 100, 200, 300, 400, 500, 600, 700, 800, 900 ng, or at least any one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg, per dose.
  • the vaccine is a multivalent vaccine comprising said combinations of vaccine antigens described herein, which may additionally comprise one or more other Staphylococcal superantigen toxoid antigens, preferably selected from the group consisting of alpha-hemolysin, gamma-hemolysin, betahemolysin, staphylococcal exotoxins or enterotoxins, such as e.g., enterotoxin A (SEA), B (SEB), I (SEI), and K (SEK).
  • Staphylococcal superantigen toxoid antigens preferably selected from the group consisting of alpha-hemolysin, gamma-hemolysin, betahemolysin, staphylococcal exotoxins or enterotoxins, such as e.g., enterotoxin A (SEA), B (SEB), I (SEI), and K (SEK).
  • SEA enterotoxin A
  • SEB SEB
  • I SEI
  • the combination of vaccine antigens described herein is provided for medical, diagnostic or analytical use.
  • the invention further provides for the medical use of the combination of vaccine antigens described herein, and in particular the use of such material in a method of producing a pharmaceutical preparation, such as a vaccine, for treating a subject e.g., a human subject or patient, in particular for the prevention or therapy of specific disease conditions or diseases.
  • an immunotherapy such as an active immunotherapy.
  • Specific immunotherapies provide for the treatment of a subject afflicted with, or at risk of contracting or suffering a disease or recurrence of a disease, by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.
  • the vaccine is provided for use in the prevention, treatment against or therapy of a staphylococcal toxin or superantigen-expressing bacterial infection, and/or a disease condition directly or indirectly mediated by exposure to Staphylococcal superantigen toxins, Staphylococcus infection and/or contamination.
  • the disease condition is caused or triggered by a target Staphylococcus species.
  • a subject is immunized with the vaccine, who is at risk of staphylococcal disease condition and/or complication.
  • a subject is at risk of a staphylococcal disease condition and/or complication, where the respective disease condition or complication is possible, probable or proven.
  • the staphylococcal disease condition and/or complication is/are due to primary and/or secondary forms of inheritable (including in particular primary or genetic predisposition or disease) and/or acquired (including in particular secondary disorders) immunodeficiency disorders and/or immune-modulatory disorders.
  • Secondary immunodeficiencies can include loss of barrier function of skin and mucosal surfaces, such as e.g., where a subject is undergoing surgery, in particular plastic surgery, or where the subject is exposed to or suffering from skin injuries.
  • the subject is likely exposed to staphylococcal infection, such firefighters, medical staff, or regional populations of areas where fire, flooding, or hurricanes are frequent.
  • the subject may be a healthy subject.
  • Large, basically healthy populations in whom exposure is likely are e.g., firefighters, medical staff (nurses, aids), or regional populations of areas where fire, flooding, hurricanes are frequent.
  • the multivalent vaccine is used for any one or more of the following indications: a) a subject is immunized with a combination of at least SEB and TSST-1 , optionally combined with SEC, to prevent a sepsis condition, preferably wherein the sepsis condition is sepsis, septic shock or toxic shock syndrome (TSS); and/or b) a subject is immunized with a combination of at least SEB and TSST-1 , optionally combined with SEC, to prevent Staphylococcal wound infective disorders, preferably wherein wound infection is upon burn, injuries, or surgical treatment; c) a subject is immunized with a combination of at least SEB and SEC, optionally combined with TSST-1 , to prevent Staphylococcal enteric disorders, preferably wherein the enteric disorder is enteritis or a digestive disorder resulting from Staphylococcal food poisoning; and/or d) a subject is immunized with a combination of at least SE
  • the vaccine may be used for preventing or treating a disease condition which is a sepsis condition induced by microbial mediators of Grampositive bacterial, viral or fungal pathogens or toxins.
  • a disease condition which is a sepsis condition induced by microbial mediators of Grampositive bacterial, viral or fungal pathogens or toxins.
  • the sepsis condition is sepsis, toxic shock syndrome (TSS) or septic shock.
  • the kit provides for the combination treatment of a subject with the vaccine antigens provided as kit component e.g., simultaneously or consecutively, in a mixture, or in parallel e.g., by one, two or more administrations (or injections), such as by administration at one, two or more different sites and/or routes of administration.
  • the multivalent vaccine comprising two or three different vaccine antigens can be administered at the same site of administration, or at different sites (e.g., different injection sites), or the combination of vaccine antigens is administered in parallel or within a certain period of time (such as e.g., within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days, or within 3 or 4 weeks) before or after the vaccination with a first vaccine antigen.
  • a certain period of time such as e.g., within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days, or within 3 or 4 weeks
  • a vaccine kit of parts is used to immunize the subject with the combination of vaccine antigens, whereby the all of the individual vaccine antigens are provided by one administration, or by two or more separate administrations, preferably by administering a mixture of two or more individual vaccine antigens, or by parallel or consecutive administration of two or more individual antigens, such as at two or more different sites or routes of administration.
  • the kit components can be administered in a mixture of said components, or to provide a combination of said components in vitro or in vivo. Specifically, a mixture comprising or consisting of said kit components is administered to the subject.
  • the invention further provides for the use of the vaccine described herein in a method of producing an antibody preparation comprising antibodies specifically recognizing the respective vaccine antigens.
  • the invention further provides for the new use of the vaccine described herein, in a method of producing an antibody preparation comprising antibodies specifically recognizing the target toxin.
  • the antibody preparation may be produced by immunizing a subject to produce antibody-containing blood preparations or fractions thereof, such as an antiserum.
  • the antibody preparation may be produced by selecting respective antibody-binding sites from a library of antibodies or binding sequences, using the vaccine antigen as binding agent for selection purposes, and preparing an antibody comprising the selected antibody-binding sites or respective sequences.
  • the invention provides for a polyclonal antibody preparation obtainable or obtained by immunizing a subject with a vaccine described herein, isolating polyclonal antibodies or a fraction of polyclonal antibodies comprising the antibodies specifically recognizing the respective Staphylococcal superantigen vaccine antigens, and formulating a preparation comprising said antibodies, wherein the antibodies are cross-reactive with the respective wild-type Staphylococcal superantigen toxins.
  • the vaccine antigens described herein may be provided as recombinant proteins, such as produced by recombinant expression technologies, in particular by expressing a nucleic acid in a suitable host cell, which nucleic acid encodes the respective vaccine antigen.
  • the nucleic acid molecule may be codon-optimized, such that the risk of reverse mutations is reduced, or to improve expressing the sequence in a recombinant host organism, such as prokaryotic or eukaryotic or insect host cells, e.g., E. coli, yeast or mammalian expression systems.
  • Suitable host cells may be selected from the group consisting of bacterial host cells, such as E.
  • coli but also from mammalian, insect, or yeast cells, e.g., HEK293 cells, CHO cells, NSO cells, Sf9 cells, High Five cells, Pichia pastoris, Saccharomyces cerevisiae, among many others.
  • mammalian, insect, or yeast cells e.g., HEK293 cells, CHO cells, NSO cells, Sf9 cells, High Five cells, Pichia pastoris, Saccharomyces cerevisiae, among many others.
  • the detoxified toxins and vaccine antigens used herein are specifically designed to trigger an immune response in a subject, and particularly include one or more antigenic determinants, which can be possibly recognized by a binding site of an antibody or are able to bind to the peptide groove of HLA class I or class II molecules or other antigen presenting molecules such as CD1 and as such may serve as stimulant for specific T-cells.
  • the vaccine antigens specifically include one or more immunologically relevant epitopes, which are herein understood as structures that are recognized by the subject’s immune system and/or respective antibodies.
  • Epitopes can e.g., be B-cell epitopes or T-cell epitopes, such as CD4+ T-cell epitopes or CD8+ T-cell epitopes.
  • Neutralizing activity of an immune response or respective neutralizing antibodies can be tested in cell-based assays and in vivo.
  • Neutralizing antibodies can be determined e.g., by enumerating bacterial titers in the presence of antibodies and/or by detecting a cytopathic effect in cell-based infection assays.
  • Protective immune responses can be measured using in vivo models of Staphylococcus infection.
  • nucleic acid molecules containing a desired coding sequence of an expression product such as e.g., a detoxified SAG or a vaccine antigen as described herein, and control sequences such as e.g. a promoter in operable linkage, may be used for expression purposes.
  • Hosts transformed or transfected with these sequences are capable of producing the encoded proteins.
  • the expression system may be included in a vector; however, the relevant DNA may also be integrated into the host cell chromosome.
  • the term refers to a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.
  • Vectors used herein are defined as DNA sequences that are required for the transcription of cloned recombinant nucleotide sequences, i.e. , of recombinant genes and the translation of their mRNA in a suitable host organism.
  • An “expression cassette” refers to a DNA coding sequence or segment of DNA that code for an expression product that can be inserted into a vector at defined restriction sites.
  • the cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame.
  • foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA.
  • a segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a “DNA construct”.
  • Expression vectors comprise the expression cassette and additionally usually comprise an origin for autonomous replication in the host cells or a genome integration site, one or more selectable markers (e.g., an amino acid synthesis gene or a gene conferring resistance to antibiotics such as zeocin, kanamycin, G418 or hygromycin), a number of restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, which components are operably linked together.
  • selectable markers e.g., an amino acid synthesis gene or a gene conferring resistance to antibiotics such as zeocin, kanamycin, G418 or hygromycin
  • a number of restriction enzyme cleavage sites e.g., zeocin, kanamycin, G418 or hygromycin
  • a common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily be introduced into a suitable host cell.
  • a plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA.
  • the term “vector” or “plasmid” refers to a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
  • host cell shall refer to primary subject cells transformed to produce a particular recombinant protein, and any progeny thereof. It should be understood that not all progeny is exactly identical to the parental cell (due to deliberate or inadvertent mutations or differences in environment), however, such altered progeny is included in these terms, so long as the progeny retain the same functionality as that of the originally transformed cell.
  • host cell line refers to a cell line of host cells as used for expressing a recombinant gene to produce recombinant proteins.
  • cell line refers to an established clone of a particular cell type that has acquired the ability to proliferate over a prolonged period of time. Such host cell or host cell line may be maintained in cell culture and/or cultivated to produce a recombinant polypeptide.
  • the detoxified toxins and vaccine antigens used herein are specifically provided as isolated proteins.
  • isolated or isolated as used herein with respect to a protein shall refer to such compound that has been sufficiently separated from the environment with which it would naturally be associated, so as to exist in “purified” or “substantially pure” form. Yet, “isolated” does not necessarily mean the exclusion of artificial or synthetic fusions or mixtures with other compounds or materials, or the exclusion of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification.
  • Isolated compounds can be further formulated to produce preparations thereof, and still for practical purposes be isolated - for example, a mixture of proteins or the respective fusion proteins described herein can be mixed with pharmaceutically acceptable carriers, including those which are suitable for analytic, diagnostic, prophylactic or therapeutic applications, or excipients when used in diagnosis, medical treatment, or for analytical purposes.
  • pharmaceutically acceptable carriers including those which are suitable for analytic, diagnostic, prophylactic or therapeutic applications, or excipients when used in diagnosis, medical treatment, or for analytical purposes.
  • purified shall refer to a preparation comprising at least 50% (w/w total protein), preferably at least 60%, 70%, 80%, 90% or 95% of a compound (e.g., a chimeric antigen described herein).
  • a highly purified product is essentially free from contaminating proteins, and preferably has a purity of at least 70%, more preferred at least 80%, or at least 90%, or even at least 95%, up to 100%. Purity is measured by methods appropriate for the compound (e.g., chromatographic methods, polyacrylamide gel electrophoresis, HPLC analysis, and the like).
  • An isolated, purified protein described herein may be obtained as a recombinant product obtained by purifying from a host cell culture expressing the product in the cell culture supernatants, to reduce or remove host cell impurities or from cellular debris.
  • isolation and purification methods for obtaining a purified protein methods utilizing difference in solubility, such as salting out and solvent precipitation, methods utilizing difference in molecular weight, such as ultrafiltration and gel electrophoresis, methods utilizing difference in electric charge, such as ion-exchange chromatography, methods utilizing specific affinity, such as affinity chromatography, methods utilizing difference in hydrophobicity, such as reverse phase high performance liquid chromatography, and methods utilizing difference in isoelectric point, such as isoelectric focusing may be used.
  • difference in solubility such as salting out and solvent precipitation
  • methods utilizing difference in molecular weight such as ultrafiltration and gel electrophoresis
  • methods utilizing difference in electric charge such as ion-exchange chromatography
  • methods utilizing specific affinity such as affinity chromatography
  • methods utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography
  • methods utilizing difference in isoelectric point such as isoelectric focusing
  • non-pyrogenic compound is formulated into a pharmaceutical product such as a vaccine for parenteral administration, safety of the respective pharmaceutical product is ensured.
  • a subject can develop lifethreatening shock-like symptoms after exposure to a pyrogen.
  • Medical products which can be inhaled, injected, or infused and medical devices such as surgical tools or implanted materials pose a particular risk of pyrogenicity. Even food or nutrients can represent a risk of pyrogenicity. Pyrogen testing of various pharmaceuticals, nutrients, and medical products for parenteral application or surgery is typically performed with standard tests to ensure the safety of such products.
  • the absence of a pyrogenic effect can be determined in a suitable animal model, such as a rabbit pyrogen test.
  • a suitable animal model such as a rabbit pyrogen test.
  • the lack of significant induction of a human inflammatory response, or induction of any of the pro-inflammatory cytokines or biomarkers can be measured as an indicator of non-pyrogenicity, e.g., by standard in vitro or ex vivo assays, such as tests that determine the effect of a compound on the in vitro activation of human monocytes like in human peripheral whole blood, peripheral blood mononuclear cells (PBMCs), or monocytic cell lines, e.g., to detect the release of pyrogenic cytokines.
  • PBMCs peripheral blood mononuclear cells
  • Nucleic acid molecules encoding the detoxified toxin molecules or vaccine antigens described herein are specifically provided by mutagenesis (mutation) of naturally-occurring toxin sequences.
  • Mutagenesis to delete specific amino acids involves the deletion of one or more nucleotides.
  • Mutagenesis to substitute one specific amino acid may involve substitution of at least 1 or 2 nucleotides, wherein the codon comprising said substituted nucleotides encodes the substituted amino acid. Where nucleotides are substituted, it is preferred to exchange more than one nucleotide within a codon, to reduce the risk of undesired spontaneous reverse mutation.
  • Codonoptimization of a nucleotide sequence and molecule described herein may involve increasing the number of substituted nucleotides within a codon, such as to improve the stability of the mutants, and/or avoiding reverse mutation by only one spontaneous nucleotide exchange.
  • the point mutation at L45 is still at a position numbered aa45, though the sequence of such deletion mutant has been shortened by three amino acids.
  • the detoxified TSST-1 may comprise an extension, insertion, or deletion of one or more amino acids, the positions following any such deletion will still be numbered as in the wild-type protein.
  • the further deletion of the aa32 or the amino acid substitution at aa135 is still at the position numbered aa32 and aa135, respectively, though the sequence of such deletion mutant has been shortened by the deleted amino acid at position aa31.
  • Specific embodiments described herein refer to the production of a chimeric antigen, and the recombinant means for such production, including a nucleic acid encoding the amino acid sequence, an expression cassette, a vector or plasmid comprising the nucleic acid encoding the amino acid sequence to be expressed, and a host cell comprising any such means.
  • Suitable standard recombinant DNA techniques are known in the art and described inter alia in Sambrook et al., “Molecular Cloning: A Laboratory Manual” (1989), 2nd Edition (Cold Spring Harbor Laboratory press).
  • the vaccine preparation described herein may also be used to prevent Staphylococcus infections and/or staphylococcal complications that may arise during or following a surgical procedure, such as surgery that involves implantation of a pacemaker, artificial heart valve, a joint implant, or a prosthetic.
  • the prosthetic may be a prosthetic limb, such as an arm or a leg.
  • the prosthetic may be a hip replacement.
  • the prosthetic may be a cosmetic prosthetic, such as, but not limited to an ocular prosthetic, silicone hands, fingers, breasts, feet, toes, or a facial implant.
  • treatment refers to medical management of a subject with the intent to cure, ameliorate, stabilize, reduce the incidence or prevent a disease, pathological condition, or disorder, which individually or together are understood as “disease condition”.
  • compositions suitable for facilitating certain means of administration are well known in the art.
  • Specific embodiments refer to immunogenic formulations, which comprise a pharmaceutically acceptable carrier and/or adjuvant, which trigger a humoral (B-cell, antibody) or cytotoxic (T-cell) immune response.
  • Adjuvants may specifically be used to enhance the effectiveness of the vaccine.
  • Adjuvants may be added directly to the vaccine compositions or can be administered separately, either concurrently with or shortly before or after administration of the vaccine antigen.
  • the injectable preparations may include dosage forms for subcutaneous, intracutaneous and intramuscular injections, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the protein in a sterile aqueous medium or an oily medium conventionally used for injections.
  • Vaccine antigens were formulated as vaccine preparations using 1 mg AL(OH)s as adjuvant.
  • the multivalent vaccine was provided as a series of combination of individual vaccines targeting the respective toxins, wherein each vaccine comprises only one vaccine antigens.
  • TSST-1 vaccine antigen 1 OOpg; SEQ ID NO:64
  • TSST-1 vaccine antigen 30pg; SEQ ID NO:64
  • SEB vaccine antigen 1 OOpg; SEQ ID NO:15
  • SEB vaccine antigen 30pg; SEQ ID NO:15
  • SEB vaccine antigen 1 Opg; SEQ ID NO:15
  • SEC vaccine antigen 30pg; SEQ ID NO:44
  • SEC vaccine antigen 1 pg; SEQ ID NO:44
  • Rabbits were immunized three or four times with the individual vaccine antigens and with a combination of the vaccine antigens.
  • the antisera were characterized by ELISA determining titers of antibodies specifically recognizing the target toxins.
  • Plates were coated with 0.5 pg/ml antigen (TSST-1 , SEB, SEC) at 4°C for 16-18 hours. Plates were washed four times with 100pl/well washing puffer (1x PBS 0.1%Tween 20) and then blocked with 200 pl blocking puffer (1x PBS 0.1% Tween 20 3% BSA) at 37°C for one hour. Plates were frozen at -20°C until use. After the plates were allowed to thaw for at least 60 minutes, plates were washed and sera (50pl/well) were added in a row of twofold dilutions including negative and positive control sera. Plates were incubated for one hour at 37°C.
  • TSST-1 0.5 pg/ml antigen

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Abstract

A non-pyrogenic Staphylococcal superantigen vaccine comprising a combination of detoxified Staphylococcal superantigen vaccine antigens which are genetically modified toxins that incorporate detoxifying mutations in its T cell receptor binding region and MHC Class II binding region, wherein the combination comprises at least the vaccine antigens Staphylococcal Exotoxin B (SEB) and any one or both of Staphylococcal Exotoxin C (SEC) and Staphylococcal toxic shock syndrome toxin-1 (TSST-1).

Description

PROTECTIVE STAPHYLOCOCCAL EXOTOXIN VACCINE
FIELD OF THE INVENTION
The invention relates to a Staphylococcal superantigen vaccine comprising a combination of detoxified Staphylococcal superantigen vaccine antigens. The present invention particularly relates to a combination of vaccine antigens and multivalent vaccines inducing protective immunity against Staphylococcus aureus infections or exotoxin exposure and protection against a Staphylococcal exotoxin-induced disease condition.
BACKGROUND OF THE INVENTION
Staphylococcus aureus produces a variety of exoproteins and Staphylococcal superantigens (SAG) that contribute to its ability to colonize and cause disease of varying severity in mammalian hosts ranging from superficial skin infections, such as abscesses and impetigo, to serious invasive infections such as pulmonary disease, osteomyelitis and endocarditis, as well as to the acute and potentially fatal toxic shock syndrome (TSS) and septic shock syndrome.
Staphylococcal Enterotoxin B and Staphylococcal Enterotoxin C are superantigen exotoxins of staphylococci, also understood as Staphylococcal Exotoxin B (SEB) and Staphylococcal Exotoxin C (SEC), respectively. SEB and SEC are commonly produced by invasive S. aureus isolates, especially methicillin-resistant strains and isolates from animal diseases.
Superantigen toxins activate a large population of mononuclear cells (MNC) at very low concentrations and activate several innate immune modulator and effector cells thus leading to uncontrolled inflammation. They can be presented by the antigen presenting cell (APC) with or without intracellular processing. When processed via the intracellular pathway, antigens are presented as peptides bound to the antigen-binding groove of the major histocompatibility complex (MHC) class II and can interact with the antigen-specific region of the T cell receptor (TCR). In higher amounts, processed superantigens activate macrophages to release modulators such as tumour necrosis factor alpha (TNFa). TNFa activates MNCs, which in turn produce and secrete cytokine mediators, thereby activating effector functions (e.g., interferon gamma [IFNy])- Furthermore, antigen specific T cell lineage activation is induced.
Superantigens are far more potent than conventional antigens and may trigger up to 30% of the entire T cell population. They cause massive proliferation of T cells and uncontrolled release of cytokines. Superantigens activate T-cells at much lower concentrations than nominal antigens. Activation by superantigens can be detected at picogram to low nanogram concentrations, by nominal antigens at micrograms and higher. The difference, thus, is 5 to 8 log steps. Clonal expansion and upregulation of the IL-2 receptor on the cell surface are consequences of cross-linking of MHC class II on antigen presenting cells and TCR on CD4+ cells. The massive release of cytokines is the basis of toxicity of Staphylococcal superantigens.
Superantigens can also be presented without intracellular processing and then possess the ability to bridge APCs and T cells by binding outside the antigen binding groove of the TCR. In this case, they activate T cells via the variable V|3 region. Minute amounts of superantigens induce the release of interleukin-6 (IL-6) and mediate uncontrolled inflammation leading to T cell mitogenesis. The induction of up to 20% of T cells and activation of macrophages can trigger a cytokine storm.
Superantigen toxins cause dysregulated T-cell activation, dysregulated B-cell activation and dysregulated activation of antigen presenting cells e.g., dendritic cells, or mononuclear cells, such as containing lineages of cells of adaptive immunity (dendritic cells, T-cells and B-cells). Antigen is presented by peptides bound to MHC and interact with T-cells via the antigen binding grove and bind to receptors of Va and/or Vp on the T-cell. This is followed by uncontrolled immune cell activation.
Furthermore, superantigen toxins can also activate immune cells by numerous pathways of innate immunity (e.g. IL-1 , CD14, IL-6 etc.).
Superantigen toxins can also activate different pathways of innate immunity by their mitotic effects e.g., IL-1 , CD14, IL-6 etc. The effect of IL-1 leads to mitosis (polyclonal dysregulation) and numerous effector and modulatory activation of innate immunity (NK cells, toll like receptor positive lymphocytes and their binding proteins, phagocytes and their ligands etc.).
The sequence of these events is accompanied with diseases due to immunodeficiency, autoimmunity and immune modulatory systemic, organ-specific, and immune-regulatory conditions. Superantigen toxins may cause systemic diseases such as toxic shock syndrome (TSS). The main toxins causing TSS are TSST-1 , SEB and SEC. Examples of organspecific illnesses caused by superantigen toxins include toxic pneumonia, endocarditis and atopic dermatitis. Superantigen toxins can also affect the gastro-intestinal tract. Additionally, staphylococcal enterotoxins and exotoxins have been suggested to play a pathogenic role in inflammatory bowel diseases
The SEB plays a pivotal role as a causative agent in TSS (Toxic Shock Syndrome). Experimental application of superantigen leads to an overwhelming inflammatory response with uncontrolled release of inflammatory cytokines resulting in shock and multi organ failure (Ulrich et al. Vaccine 1998, 16(19): 1857-1864).
SEB and SEC are also a common cause of food poisoning, causing severe diarrhea, nausea and intestinal cramping. Being quite stable, the toxin may remain active even after contaminating bacteria are killed.
Based on information on the structure and functional relationship of S. aureus superantigens, recombinant mutants have been established with the objective of eradicating the toxic and superantigenic properties.
Based on information on the structure and functional relationship of S. aureus SEB, multiple mutants have been established with the objective of eradicating the toxic and superantigenic properties, while leaving specific immunogenicity and protectivity unimpaired (Lowell et al. Infection and Immunity 1996, 64(5): 1706-1713; LeClaire et al. Infection and Immunity 2002, 770(5):2278-2281).
Kappler et al. (J. Exp. Med. 1992, 175:387-396) describe mutations defining functional regions of the superantigen SEB.
Fries et al. (Microbiol Spectr. 2013,1 (2): 2013) disclose specific binding regions of SEB and provides a sequence alignment of amino acid sequences of SEB derived from S. aureus clinical isolates.
US6713284 discloses genetically attenuated superantigen toxin vaccines, and in particular an SEB wherein certain amino acid positions have been altered such that binding to the MHC Class II receptor and T cell antigen receptor is altered.
Woody et al. (Vaccine 1997, 15(2): 133-139) disclose SEB mutants with either N23K or F44S mutations, and describes the vaccine potential in a mouse model.
Jeong et al. (Acta Cryst. 2017, F73, 595-600) disclose an SEB vaccine candidate with four mutations (N23A, Y90A, R110A, and F177A) showing eliminated superantigen activity. Bagnoli et al. (PNAS 2015, 112(12):3680-3685) describe combined vaccine antigens a-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A, formulated with aluminum hydroxide or with a toll-like receptor 7-dependent (TLR7) agonist (SMIP.7-10) adsorbed to alum.
JP4571586B2 (corresponding to EP1661911) discloses a modified SEB being protease-resistant with a reduced toxicity, comprising a substitution of lysine at position 97, and a substitution of lysine at position 98, with any other amino acid. In addition, a substitution of asparagine at position 23 to tyrosine is disclosed.
WO99/40935 (corresponding to EP1055429) discloses an SEB comprising a modification which is one or more amino acid substitutions e.g., at position 9, 23, 41 , or 44, to confer inhibitory activity on T cell activation.
WO201 4/205111 discloses a multivalent peptide oligopeptide including a Staphylococcus aureus antigen or mutant, fragment, variant, or derivative thereof, which may include, SEB, SECI-3, SEE, SEH, SEI, SEK, TSST-1 , SpeC, SED, or SpeA, or any mutant, fragment, variant, or derivative thereof, or any combination thereof, in any order. In certain aspects, the oligopeptide is disclosed to include a SEB mutant which is the attenuated toxoid SEB comprising the amino acid substitutions L45R/Y89A/Y94A.
Hu et al. (Infect Immun. 2005 Jan;73(1): 174-80) have investigated a mutant staphylococcal enterotoxin C (mSEC) comprising an N23A substitution and reduced superantigenic activity.
Hu et al (Microbes and Infection 2006, 8 (14-15):2841 -2848) describe a double mutant of SEC (dmSEC) comprising a N23A substitution for reducing binding to TCR, and a Y94A substitution for reducing binding to MHC class II.
WO201 4/205111 discloses a multivalent peptide oligopeptide including a Staphylococcus aureus antigen or mutant, fragment, variant, or derivative thereof, which may include, SEB, SECI-3, SEE, SEH, SEI, SEK, TSST-1 , SpeC, SED, or SpeA, or any mutant, fragment, variant, or derivative thereof, or any combination thereof, in any order.
Venkatasubramaniam et al. (Sci Rep 2019; 9:3279) disclose a fusion toxoid vaccine for protection and neutralization of staphylococcal superantigens, including toxoid versions of the SAGs TSST-1 , SEB and SEA, wherein the TSST-1 comprises amino acid substitutions L30R, D27A and I46A.
In a rabbit model, T cell activation has been assessed by lymphocyte proliferation and IL-2 gene expression after in vivo challenge with TSST-1 and the mutant antigens; expression of the genes of proinflammatory cytokines were taken as indicators for the inflammatory reaction after the combined treatment with TSST-1 and LPS. The question as to whether the biological activities of TSST-1 , e.g., lymphocyte extravasation, toxicity and increased sensitivity to LPS, are mediated by T cell activation or activation by MHC Il-only, were addressed by studying these reactions in vivo, with two TSST-1 mutants: one mutated at the MHC binding site (G31 R) with reduced MHC binding with residual activity still present, and the other at the T cell binding site (H135A) with no residual function detectable. The mutant G31 R was reported to induce all the biological effects of the wild type TSST-1 , while the mutant with non-functional TCR binding did not retain any of the toxic effects, proving the pivotal role of T cells in this system. (Stich et al. Toxins 2010, 2, 2272-2288).
US2014199339A1 discloses compositions comprising two or more staphylococcal toxoids for inducing protective immune response against staphylococcal diseases.
US2003157113A1 discloses methods and compositions for treatment of neoplastic disease. The methods employ conjugates comprising superantigen polypeptides, nucleic acids with other structures that preferentially bind to tumor cells and are capable of inducing apoptosis.
US2003032582A1 discloses nucleic acid molecules encoding membraneinfiltrating polypeptides, and truncated superantigen polypeptide encoding nucleic acid molecules as well as truncated superantigen polypeptides. These truncated superantigens are described to elicit an anti-tumor immune response without binding MHC II molecules.
SUMMARY OF THE INVENTION
It is the objective to trigger a protective immune response and/or to improve the efficacy of preventing Staphylococcal disorders. It is a particular objective to provide a multivalent Staphylococcal superantigen vaccine, in particular with reduced side reactions or adverse events. It is a particular objective to provide a combination of vaccine antigens for appropriate immunization that effectively treats subject at risk of or suffering from Staphylococcal infections and respective disease or disorders.
The objective is solved by the subject of the present claims and as further described herein. The invention provides for a combination of vaccine antigens which confer protective immunity, in particular targeting professional antigen presenting cells (APC) or cells of the innate and/or adaptive immune system expressing constitutively MHC Class II. The vaccine comprising the combination of vaccine antigens is herein also referred to as “multivalent vaccine”.
Specifically, the multivalent vaccine is provided as a non-pyrogenic vaccine, in particular a non-pyrogenic combination of vaccine antigens, such as obtained by a combination of Staphylococcal superantigen mutants as described herein which are non-pyrogenic Staphylococcal superantigen vaccine antigens.
Specifically, the non-pyrogenic vaccine or combination of vaccine antigens does not trigger an abnormally high body temperature in a subject (in particular, fever) upon parenteral administration.
Specifically, the vaccine described herein is compared to the wild type toxins, not pyrogenic, and not lymphopenic. Specifically, it does neither change chemical blood parameters nor the amount of white blood cells.
Specifically, the multivalent vaccine is provided as a non-lymphopenic vaccine, in particular a non-lymphopenic combination of vaccine antigens, such as obtained by a combination of Staphylococcal superantigen mutants as described herein which are a non-lymphopenic Staphylococcal superantigen vaccine antigens.
Specifically, the non-lymphopenic vaccine or combination of vaccine antigens does not trigger an abnormally low level of lymphocytes in the blood of a subject (in particular, lymphopenia) upon parenteral administration.
Herein described is a Staphylococcal superantigen vaccine comprising a combination of detoxified Staphylococcal superantigen vaccine antigens which are genetically modified toxins that incorporate detoxifying mutations in its T cell receptor binding region and MHC Class II binding region, wherein the combination comprises at least the vaccine antigens Staphylococcal Exotoxin B (SEB) and any one or both of Staphylococcal Exotoxin C (SEC) and Staphylococcal toxic shock syndrome toxin-1 (TSST-1). Specifically, the Staphylococcal superantigen vaccine antigens are non- pyrogenic. Specifically, the Staphylococcal superantigen vaccine is non-pyrogenic.
The vaccine antigen herein referred to as SEB, TSST-1 or SEC, is meant to be the respective detoxified Staphylococcal superantigen vaccine antigen as further described herein. According to a specific aspect, the combination comprises or consists of any one of the embodiments: a) SEB and TSST-1 ; or b) SEB and SEC; or c) SEB, TSST-1 and SEC.
The targets of the combination are the respective wild-type toxins.
Specifically, the vaccine antigens described herein are detoxified whole proteins, comprising a length of at least 95%, 96%, 97%, 98%, 99%, or 100% of the length of the respective wild-type toxin.
Specifically, the vaccine antigens described herein comprise detoxifying mutations in the regions herein referred to as “core regions”, which particularly consists of both, the T cell receptor binding region and MHC Class II receptor binding region. Herein, such vaccine antigens are herein also referred to as “double mutated”.
The T cell receptor (a/p) binding region and MHC Class II receptor binding region of a Staphylococcal superantigen, and in particular of any one of SEB, TSST-1 or SEC, are herein referred to as “core region”. Any other region(s) besides the core region are herein referred to as “non-core region(s)”. Specifically, the core region consists of the T cell receptor binding region and MHC Class II binding region. Specifically, the MHC Class II receptor binding region is an immunoglobulin superfamily binding region, which comprises or consists of the region binding inside or adjacent to the antigen-binding groove (and outside the antigen-binding groove). The class II MHC binding site is located in the hydrophobic region of the NH2-terminal domain, and the TCR binding site is primarily in the major central groove of the COOH-terminal domain. For the denotation of the respective regions see McCormick and Schlievert (J Immunol August 1 , 2003, 171 (3) 1385-1392), and JM Hurley and M. Matsumura (J Exp Med. 1995 Jun 1 ; 181 (6): 2229-2235).
Specifically, the detoxified SEB toxin sequence comprises at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:1 , or to any other wild-type SEB toxin sequence.
There are series of wild-type SEB, SEC and TSST-1 toxin sequences which originate from a variety of S. aureus sources (or isolates), which are characterized by a certain (limited) variability in non-core regions. Specifically, the wild-type SEB or SEC toxin identified by SEQ ID NO:1 and SEQ ID NO:2, respectively, originates from S. aureus (ATCC 19095). Any naturally-occurring SEB, SEC or TSST-1 sequence, such as SEQ ID NO:1 (as for SEB), SEQ ID NO:2 (as for SEC) and SEQ ID NO:3 (as for TSST-1 ), respectively, or any other respective wild-type toxin sequence which is a variant of the respective SEQ ID NO:1 (as for SEB), SEQ ID NO:2 (as for SEC) and SEQ ID NO:3 (as for TSST- 1), may be used to introduce respective detoxifying mutations at the core region, in particular at respective corresponding positions within the core regions, such as further described herein. Naturally-occurring (i.e. , wild-type, wt) toxin sequences may originate from different S. aureus sources (or isolates), with variability in core and/or non-core regions (in many cases, the variability is in the non-core region only), and are herein also referred to as naturally-occurring wild-type variants of the exemplary sequences for wild-type toxins as referred to herein e.g., the wt sequences provided in Fig. 1 and in particular SEQ ID NO:1 (as for SEB), SEQ ID NO:2 (as for SEC) and SEQ ID NO:3 (as for TSST-1).
Wild-type variant toxin sequences typically comprise the same core region, whereas a certain degree of variability may be found in the non-core region, such as to provide at least 95%, 96%, 97%, 98%, or 99% sequence identity to the respective wt sequences of SEB, SEC, or TSST-1 , referred to herein. A number of point mutations may be comprised in wild-type variant toxin sequences. The number of point mutations within the non-core regions of the respective naturally-occurring wt sequences of SEB, SEC, or TSST-1 , referred to herein, may be only 1 , but may be more than one, such as to comprise one or more point mutations that are occurring in any other naturally- occurring wild-type variant of the respective toxin.
Specifically, an exemplary wild-type SEB sequence comprises or consists of SEQ ID NO:1. Specifically, the wild-type SEB toxin comprises or consists of SEQ ID NO:1 or an amino acid sequence that comprises at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:1 when comparing the whole sequence of SEQ ID NO:1. Exemplary naturally-occurring wild-type variants of SEQ ID NO:1 , are selected from those comprising or consisting of SEQ ID NO: 19, 21 , 23, 25, 27, 29, 31 , or wt SEB sequences which differ from any of the foregoing though polymorphisms.
Specifically, an exemplary wild-type SEC sequence comprises or consists of SEQ ID NO:2. Specifically, the wild-type SEC toxin comprises or consists of SEQ ID NO:2 or an amino acid sequence that comprises at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:2 when comparing the whole sequence of SEQ ID NO:2. Exemplary naturally-occurring wild-type variants of SEQ ID NO:2, are selected from those comprising or consisting of SEQ ID NO:46-50, or wt SEC sequences which differ from any of the foregoing though polymorphisms.
Specifically, an exemplary wild-type TSST-1 sequence comprises or consists of SEQ ID NO:3. Specifically, the wild-type SEC toxin comprises or consists of SEQ ID NO:3 or an amino acid sequence that comprises at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:3 when comparing the whole sequence of SEQ ID NO:3. Exemplary naturally-occurring wild-type variants of SEQ ID NO:3, are selected from those comprising or consisting of SEQ ID NO:58-63, or wt TSST-1 sequences which differ from any of the foregoing though polymorphisms.
Preferred detoxified SEB
According to a particularly preferred embodiment, the SEB is characterized as follows.
Specifically, the SEB comprises a wild-type SEB amino acid sequence that is modified to comprise a deletion of at least two amino acids in the T cell receptor binding region, namely between amino acid positions (aa) 21 to 25, and to further comprise at least one point mutation in the MHC Class II binding region, wherein said at least one point mutation comprises an amino acid substitution at a position selected from the group consisting L45, Q43, or F44, preferably L45R, Q43P, F44P, or F44S, wherein the wild-type SEB toxin amino acid sequence is of SEQ ID NO:1 , or of any other wild-type SEB toxin sequence.
Specifically, said deletion of at least two amino acids in the T cell receptor binding region comprises or consists of a deletion of any of aa21-22, aa22-23, aa23-24, aa24- 25, aa21 -23, aa22-24, or aa23-25, or at corresponding amino acid positions in any other wild-type SEB toxin sequence.
Specifically, the SEB is mutated to comprise at least two or at least three point mutations at amino acid positions 21-25 and 21-24, respectively (i.e., “within aa21-25” or “within aa21-24”, respectively) also referred to as “in the region of aa21-25” and “in the region of aa21-24”, respectively, in the SEB toxin sequence SEQ ID NO:1 , wherein said at least two or three point mutations comprise a deletion of any one of aa21-22, aa22-23, aa23-24, aa24-25, aa21-23, aa22-24, or aa23-25, in the SEB toxin sequence SEQ ID NO:1 , or at a corresponding region in any other wild-type SEB toxin sequence.
Specifically, SEQ ID NO:1 or the wild-type variant SEB toxin is functional as a staphylococcal superantigen, unless being engineered to incorporate the respective detoxifying mutations. Specifically, the wild-type SEB toxin sequence comprises or consists of SEQ ID NO:1 , or any one of SEQ ID NO:19, 21 , 23, 25, 27, 29, 31.
Specifically, the detoxified SEB toxin comprises or consists of SEQ ID NO:5 which differs from SEQ ID NO:1 in the deletion of aa22-24, or a detoxified SEB variant sequence thereof (i.e., a variant SEB sequence of SEQ ID NO:5) comprising said deletion of aa22-24, and at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:5.
Specifically, said detoxified SEC variant sequence of SEQ ID NO:5 comprises said deletion of aa22-24 comprised in SEQ ID NO:5; and: a) one or more further point mutations in its immunoglobulin superfamily binding region, preferably within (i.e., in the region of) aa42-47 or aa43-45, and/or b) one or more further point mutations as naturally-occurring in other regions of the SEB sequence, such as in the non-core regions e.g., one or more point mutations as naturally-occurring in SEB wild-type variants, such as occurring in any one of SEQ ID NO: 19, 21 , 23, 25, 27, 29, 31 , in particular one or more of the following point mutations: A14S, K16E, L20T, E22G, V26Y, K39M, G72D, D124N, N127S, V154A, or M200I in SEQ ID NO:1 ; and/or c) at least 95%, 96%, 97%, 98% 99%, or 99.5% sequence identity to SEQ ID NO:5, or to the corresponding non-core regions of SEQ ID NO:5.
Specific embodiments comprise a SEB toxin variant sequence of SEQ ID NO:5, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective regions of SEQ ID NO:5, in particular over the whole length of all corresponding regions of SEQ ID NO:5.
Specific embodiments comprise a SEB toxin variant sequence of SEQ ID NO:5, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective non-core regions, in particular over the whole length of all corresponding non-core regions, compared to the corresponding regions of SEQ ID NO:5.
According to a specific aspect, the detoxified SEB toxin comprises or consists of SEQ ID NO:7, or a detoxified SEC variant sequence thereof, which comprises at least said deletion of amino acids within 21-25 as described herein, particularly comprising said deletion of at least two or at least three amino acids at amino acid positions 21 to 25, and which comprises at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:7.
SEQ ID NO:7 differs from SEQ ID NO:1 in the deletion of aa22-24 and the amino acid substitution L45R.
Specifically, said detoxified SEB variant sequence of SEQ ID NO:7 comprises said deletion of aa22-24 comprised in SEQ ID NO:7, and said amino acid substitution L45R, and additionally: a) one or more further point mutations in its immunoglobulin superfamily binding region, preferably within aa42-47 or aa43-45, and/or b) one or more further point mutations as naturally-occurring in other regions of the SEB sequence, such as in the non-core regions e.g., one or more point mutations as naturally-occurring in SEB wild-type variants, such as occurring in any one of SEQ ID NO: 19, 21 , 23, 25, 27, 29, 31 , in particular one or more of the following point mutations: K7N, S14A, V82L, T133S, K141 E, T150I, S225F in SEQ ID NO:1 ; and/or c) at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:7, or to the corresponding non-core regions of SEQ ID NO:7.
Specific embodiments comprise a SEB toxin variant sequence of SEQ ID NO:7, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective regions of SEQ ID NO:7, in particular over the whole length of all corresponding regions of SEQ ID NO:7.
Specific embodiments comprise a SEB toxin variant sequence of SEQ ID NO:7, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective non-core regions, in particular over the whole length of all corresponding non-core regions, compared to the corresponding regions of SEQ ID NO:7.
Specifically, the detoxified SEB comprises or consists of any one of SEQ ID NO:5, 7, 9, 11 , 13, or a detoxified SEB variant sequence of any one of the foregoing which comprises said modifications in the T cell receptor binding region and in the MHC Class II binding region, and at least any one of 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:1 and/or to any one of SEQ ID NO:5, 7, 9, 11 , 13.
Specific embodiments comprise a detoxified SEB variant sequence of any one of SEQ ID NO:5, 7, 9, 11 , 13, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective non-core regions of any one of the detoxified SEB toxin sequences described herein, in particular over the whole length of all corresponding non-core regions of any one of the detoxified SEB toxin sequences described herein.
Specifically, the detoxified SEB toxin may comprise one or more, preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations, preferably up to 10, 9, 8, 7, 6, or 5 point mutations, preferably amino acid substitutions or a combination of amino acid substitutions as naturally-occurring in any of the wild-type SEB sequences, such as found in a wild-type SEB toxin sequence, e.g.: one or more of K7N, S14A, V82L, T133S, K141 E, T150I, S225F compared to SEQ ID NO:1.
Specifically, the non-core region of the detoxified SEB described herein differs from SEQ ID NO:1 only in 1 , 2, 3, 4, 5, or 6 amino acids, e.g., with an amino acid substitution at only 1 , 2, 3, 4, 5, or 6 positions, such as amino acid substitution(s) selected from those naturally-occurring in wt SEB sequences e.g., selected from the group consisting of K7N, S14A, V82L, T133S, K141 E, T150I, S225F, as compared to SEQ ID NO:1.
Specifically, the number of point mutations in the non-core region is limited, such that the non-core region has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.
Specifically, the SEB is a double mutated vaccine antigen, as described herein, which is preferably combined with any one or both of TSST-1 and SEC vaccine antigens which are also detoxified by “double mutation”.
Exemplary double mutated TSST-1 and SEC vaccine antigens are any of the following: a) the SEC comprises a wild-type SEC amino acid sequence that is modified to comprise deletion of at least two amino acids in the T cell receptor binding region between aa 21 to 25, and to further comprise at least one point mutation in the MHC Class II binding region, wherein said at least one point mutation comprises an amino acid substitution at a position selected from the group consisting L45, Q43, or F44, preferably L45R, Q43P, F44P, or F44S, wherein the wild-type SEC toxin amino acid sequence is of SEQ ID NO:2, or of any other wild-type SEC toxin sequence; b) the TSST-1 comprises a wild-type TSST-1 amino acid sequence that is modified to comprise at least one point mutation in the MHC Class II binding region consisting of G31 , L30, and S32, preferably comprising a deletion or substitution of G31 , and at least one point mutation in the T cell receptor binding region consisting of the amino acid region of E132 to Q139 and G16, wherein the wild-type TSST-1 toxin amino acid sequence is of SEQ ID NO:3, or of any other wild-type TSST-1 toxin sequence.
Exemplary SEB, SEC and TSST-1 sequences are described in Figure 1 , which includes wt sequences, exemplary detoxifying mutations and exemplary vaccine antigens.
Specifically, the detoxified SEB, SEC and TSST-1 sequences described herein may or may not comprise any further (though a limited number e.g., up to 6, 5, 4, 3, 2, or only 1 ) conservative amino acid substitutions in the non-core region.
A conservative alternative to a first amino acid (such as in an amino acid substitution) is herein understood as a second amino acid which is different from the first amino acid and which has about the same properties of charge and polarity as the first amino acid. Amino acids are typically grouped as follows; each of the amino acids within any such group is understood to have about the same properties of charge and polarity: a) basic (positively charged), polar: R, H, K, b) acidic (negatively charged), polar: D, E; c) hydrophobic: A, I, L, M, F, P, W, V; d) polar, uncharged: N, C, Q, G, S, T, Y;
Specifically, a preferred amino acid substitution is with an amino acid other that the original amino acid, such original amino acid e.g., being identified in a wild-type sequence.
Preferred detoxified SEC
According to a particularly preferred embodiment, the SEC is characterized as follows.
SEB and SEC comprise or share a similar structure, in particular regarding the core regions. Specifically, the core region of SEB or SEC comprises or consists of either or both of aa21-25 and aa42-47, and optionally in addition, any one or both of aa89-91 or aa66-68. [please confirm]
In a wild-type SEB or SEC toxin, the T cell receptor (a/p) binding region (aa21-25 in SEQ ID NO:1 or SEQ ID NO:2) comprises or consists of “MENMK”, SEQ ID NO:66.
In a wild-type SEB or SEC toxin, the immunoglobulin superfamily binding region such as e.g., the MHC Class II receptor binding region, in particular the region binding inside, adjacent or outside the antigen-binding groove, comprises or consists of e.g., aa42-47 in SEQ ID N0:1 and SEQ ID N0:2, respectively, or aa89-91 in SEQ ID NO:1 and SEQ ID NO:2, respectively; or aa66-68 in SEQ ID NO:1 and SEQ ID NO:2, respectively.
SEB: aa42-47 in SEQ ID NO:1 : “DQFLYF” (SEQ ID NO:67); identifying positions D42, Q43, F44, L45, Y46, and F47.
SEC: aa42-47 in SEQ ID NO:2: “DKFLAH” (SEQ ID NO:68); identifying positions D42, K43, F44, L45, A46, and H47.
Specifically, the detoxifying point mutations in SEB or SEC consist of amino acid deletions within the T cell receptor binding region of the SEC sequence, more specifically within aa21-25. Specifically, the detoxifying point mutations consist of a deletion of at least (or only) two or three amino acids in the region of aa21-25. Specifically, the detoxifying point mutations in the region of aa21-25 consist of a deletion of only 2, 3, 4, or 5 amino acids, preferably at least comprising a deletion of N23 and a deletion of one or both amino acids which are adjacent to N23. The number of point mutations within aa21-25 can be 2, 3, 4, or 5, specifically wherein point mutations are of at least 2 or 3 consecutive (adjacent) aa positions, such as aa21-22, aa22-23, aa23-24, aa24-25, aa21-23, aa22-24, or aa23-25. Alternatively, a point mutation at position aa23 is combined with one or more non-consecutive (non-adjacent) mutations, such as at aa21 and/or aa25.
According to a specific aspect, the mutation in the region of aa21-25 is a deletion of at least (or only) two, three or four amino acids, at least including a deletion of N23 and/or E22 and/or M24, and/or M21 and/or K25, such as a deletion or substitution of at least any one of aa22-23, aa23-24, aa21-23, aa22-24, or aa23-25, in particular a deletion of aa22-24 only. According to a specifically preferred aspect, said point mutations at amino acid positions 21 to 25 comprise or consist of a deletion of amino acids 22-24 and/or 21-23.
According to a specific aspect, the detoxified SEB or SEC toxin is characterized by one or more further (additional) point mutations (i.e. , other than those in the region of aa21-25), in particular in its immunoglobulin superfamily binding region, preferably in the region of aa42-47 or aa43-45.
Specifically, the detoxified SEB or SEC toxin comprises one or more additional point mutations comprising at least one amino acid substitution at a position within the MHC Class II binding region, preferably wherein said at least one point mutation comprises amino acid substitutions at any one or more of positions L45, K43, or F44, preferably L45R, K43P, F44P, or F44S.
Alternative amino acids can be used for substituting amino acids in any of such preferred L45R, K43P, F44P, or F44S point mutations.
For detoxifying the toxins by an amino acid substitution as described herein, any of the preferred amino acids may be used. Still, any other alternative, or conservative alternative amino acid may be used. The preferred amino acids at selected positions are selected from the following: a) at position 43, the amino acid Lysine (K), a basic, positively charged amino acid, is substituted for a hydrophobic amino acid, like “P”, or any conservative alternative thereto, such as A, I, L, M, F, W, or V; b) at position 44, the amino acid Phenylalanine (F), a hydrophobic amino acid, is substituted for a polar uncharged one, like “S”, or any conservative alternative thereto, such as N, C, Q, G, T, or Y; or substituted for another hydrophobic one, like “A”, or any conservative alternative thereto, such as I, L, M, P, W, or V; c) at position 45, the amino acid Leucine (L), a hydrophobic amino acid is substituted for a basic, positively charged amino acid, like “R”, or any conservative alternative thereto, such as H or K.
Specifically, the detoxified SEC toxin is of a type C1 , C2, or C3.
An exemplary wild-type SEC toxin comprises or consists of any one of SEQ ID NO:2 (type C1) SEQ ID NO:46 (NGA13141.1 , type C1), SEQ ID NO:47 (HAY3254229.1 , type C1), SEQ ID NO:48 (HAZ5071097.1 , type C1), SEQ ID NO:49 (WP_061047250.1 , type C2), or SEQ ID NO:50 (WP_077156035.1 , type C3). Unless otherwise described herein, accession number references are NCBI accession numbers (National Library of Medicine, Bethesda, USA).
Specifically, the detoxified SEC toxin may comprise one or more, preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations, preferably up to 10, 9, 8, 7, 6, or 5 point mutations, preferably amino acid substitutions or a combination of amino acid substitutions as naturally-occurring in any of the wild-type SEC sequences, such as found in a wild-type SEC toxin sequence, e.g.: one or more of AMS, K16E, L20T, E22G, V26Y, K39M, G72D, D124N, N127S, V154A, or M200I, compared to SEQ ID NO:2.
Specifically, the non-core region of the detoxified SEC described herein differs from SEQ ID NO:2 only in 1 , 2, 3, 4, 5, or 6 amino acids, e.g., with an amino acid substitution at only 1 , 2, 3, 4, 5, or 6 positions, such as amino acid substitution(s) selected from those naturally-occurring in wt SEC sequences e.g., selected from the group consisting of AUS, K16E, L20T, E22G, V26Y, K39M, G72D, D124N, N127S, V154A, or M200I, as compared to SEQ ID NO:2.
Specifically, the number of point mutations in the non-core region is limited, such that the non-core region has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2.
Specifically, the detoxified SEC toxin comprises or consists of SEQ ID NO:42 which differs from SEQ ID NO:2 in the deletion of aa22-24, or a detoxified SEC variant sequence thereof (i.e., a variant SEC sequence of SEQ ID NO:42) comprising said deletion of aa22-24, and at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:42.
Specifically, said detoxified SEC variant sequence of SEQ ID NO:42 comprises said deletion of aa22-24 comprised in SEQ ID NO:42; and: a) one or more further point mutations in its immunoglobulin superfamily binding region, preferably within (i.e., in the region of) aa42-47 or aa43-45, and/or b) one or more further point mutations as naturally-occurring in other regions of the SEC sequence, such as in the non-core regions e.g., one or more point mutations as naturally-occurring in SEC wild-type variants, such as occurring in any one of SEQ ID NO:46-50, in particular one or more of the following point mutations: A14S, K16E, L20T, E22G, V26Y, K39M, G72D, D124N, N127S, V154A, or M200I in SEQ ID NO:2; and/or c) at least 95%, 96%, 97%, 98% 99%, or 99.5% sequence identity to SEQ ID NO:42, or to the corresponding non-core regions of SEQ ID NO:42.
Specific embodiments comprise a SEC toxin variant sequence of SEQ ID NO:42, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective regions of SEQ ID NO:42, in particular over the whole length of all corresponding regions of SEQ ID NO:42.
Specific embodiments comprise a SEC toxin variant sequence of SEQ ID NO:42 x, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective non-core regions, in particular over the whole length of all corresponding non-core regions, compared to the corresponding regions of SEQ ID NO:42. According to a specific aspect, the detoxified SEC toxin comprises or consists of SEQ ID NO:44, or a detoxified SEC variant sequence thereof, which comprises at least said deletion of amino acids within 21-25 as described herein, particularly comprising said deletion of at least two or at least three amino acids at amino acid positions 21 to 25, and which comprises at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:44.
SEQ ID NO:44 differs from SEQ ID NO:2 in the deletion of aa22-24 and the amino acid substitution L45R.
Specifically, said detoxified SEC variant sequence of SEQ ID NO:44 comprises said deletion of aa22-24 comprised in SEQ ID NO:44, and said amino acid substitution L45R, and additionally: a) one or more further point mutations in its immunoglobulin superfamily binding region, preferably within aa42-47 or aa43-45, and/or b) one or more further point mutations as naturally-occurring in other regions of the SEC sequence, such as in the non-core regions e.g., one or more point mutations as naturally-occurring in SEC wild-type variants, such as occurring in any one of SEQ ID NO:46-50, in particular one or more of the following point mutations: A14S, K16E, L20T, E22G, V26Y, K39M, G72D, D124N, N127S, V154A, or M200I in SEQ ID NO:2; and/or c) at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:44, or to the corresponding non-core regions of SEQ ID NO:44.
Specific embodiments comprise a detoxified SEC variant sequence of any one of SEQ ID NO:42 or 44, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective non-core regions of any one of the detoxified SEC toxin sequences described herein, in particular over the whole length of all corresponding non-core regions of any one of the detoxified SEC toxin sequences described herein.
Specifically, the detoxified SEC comprises or consists of any one of SEQ ID NO:42 or 44, or a detoxified SEC variant sequence of any one of the foregoing which comprises said modifications in the T cell receptor binding region and in the MHC Class II binding region, and at least any one of 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:1 and/or to any one of SEQ ID NO:42 or 44.
Preferred detoxified TSST-1 According to a particularly preferred embodiment, the TSST-1 is characterized as follows.
According to a specific example, SEQ ID NO:3 is the amino acid sequence of a wild-type mature TSST-1 protein. Specifically, toxicity was found to be greatly reduced in TSST-1 that is genetically modified by one or more detoxifying point mutations within the core region of the MHC Class II binding region, wherein the point mutations are a substitution or deletion of G31 , or a substitution and/or deletion of any two or three amino acids at positions L30 to S32, including the substitution or deletion of G31 , in a wt TSST- 1 sequence, such as the TSST-1 sequence of SEQ ID NO:3 or in any other wild-type (wt) TSST-1 sequence.
According to a specific embodiment, the amino acid substitution at position 31 is any one of G31 R, or any other conservative substitutions as an alternative to G31 R.
Specifically, at position 31 , the amino acid Glycine (G), a non-polar amino acid is substituted for a basic, positively charged amino acid, like “R”, or any conservative alternative thereto, such as H or K.
According to a specific embodiment, said one or more detoxifying deletion mutations are in the MHC Class II binding region only. Such TSST-1 molecules are herein also referred to as “single mutant”. Specifically, substitution or deletion of G31 , or a substitution and/or deletion of any two or three amino acids at positions L30 to S32, including the substitution or deletion of G31 , are comprised in the MHC Class II binding region, as further described herein. Specifically, the TSST-1 amino acid sequence that comprises detoxifying mutations in the MHC Class II binding region only, differs from a wt TSST-1 sequence only in the substitution or deletion of G31 , or in the substitution and/or deletion of any two or three amino acids at positions L30 to S32, including the substitution or deletion of G31 .
According to another specific embodiment, two or more detoxifying deletion mutations are in the MHC Class II binding region and in the T cell receptor binding region. Such TSST-1 molecules are herein also referred to as “double mutant”. Specifically, at least the substitution or deletion of G31 , or a substitution and/or deletion of any two or three amino acids at positions L30 to S32, including the substitution or deletion of G31 , are comprised in the MHC Class II binding region, and at least one point mutation e.g., an amino acid substitution is comprised in the T cell receptor binding region. Specifically, the G31 substitution or deletion in SEQ ID NO:3 (which results in SEQ ID NO:53) refers to the amino acid “G” at position 31 in SEQ ID NO:3. The amino acid at a corresponding position (at aa31) is a glycine in many naturally-occurring wildtype TSST-1 sequences, such as e.g., in any one of the wt TSST-1 sequences SEQ ID NO:58-63. However, there are alternative wild-type TSST-1 sequences, where the amino acid at the corresponding position is glutamate (“E”). Hence, in specific wt TSST- 1 sequences, the corresponding detoxifying point mutation at aa31 is at amino acid E31 instead of amino acid G31.
Specifically, the TSST-1 core region is composed of: a) the T cell receptor binding region consisting of the amino acid region of aa132 to aa139 and aa16 in a wt TSST-1 sequence, such as e.g., E132 to Q139 and G16 in SEQ ID NO:3; b) the MHC Class II binding region consisting of aa30, aa31 , and aa32 in a wt TSST-1 sequence, such as e.g., L30, G31 and S32 in SEQ ID NO:3.
Specifically, the number of point mutations in the core region is limited, such as consisting of: a) only one substitution or deletion of the amino acid at position 31 in a wt TSST- 1 sequence; or b) one substitution or deletion of the amino acid at position 31 in a wt TSST-1 sequence, and only one, two, three or four further point mutations in the core region, which is e.g., selected from the group consisting of: AS32, AL30, an amino acid substitution at position H135, preferably H135X, wherein X is A, D, I, Q, or R; G16V, and L137V.
Specifically, a detoxified TSST-1 is provided which is genetically modified by detoxifying mutations that comprise at least the substitution or deletion of the aa31 of the mature wt TSST-1 sequence, which is a mutation in the MHC Class II binding region, and an additional modification in the T cell receptor binding region of the TSST-1 sequence.
According to a specific aspect, the detoxified TSST-1 described herein is genetically mutated to incorporate the detoxifying TSST-1 mutations in the core region as described herein. Specifically, the TSST-1 mutation(s) described herein are point mutations.
According to a specific aspect, the detoxified TSST-1 described herein is at least double or triple mutated, such as comprising at least the substitution or deletion of the amino acid at position 31 in the mature TSST-1 sequence, and one or two mutation(s) in the T cell receptor binding region.
Specifically, the number of detoxifying point mutations within the core region is at least (or only) one, 2, 3, or 4, up to 5, as compared to a wt TSST-1 amino acid sequence. Specifically, the point mutation(s) in the MHC Class II binding region are substitution or deletion mutations. Specifically, the point mutation(s) in the T cell receptor binding region, if any, are substitutions.
According to a specifically preferred aspect, the detoxified TSST-1 is characterized by at least 2, up to 5, 4, 3, or 2 point mutations in the core region, compared to the mature wt TSST-1 sequence.
According to a specifically preferred aspect, the detoxified TSST-1 is characterized by at least 3, up to 5, 4, or 3 point mutations in the core region, compared to the mature wt TSST-1 sequence.
According to a specifically preferred aspect, the detoxified TSST-1 is characterized by no mutation (meaning an identical sequence), or only 1 , 2, 3, 4, 5, or 6 point mutations in the non-core region, compared to the mature wt TSST-1 sequence.
According to a specifically preferred aspect, the detoxified TSST-1 is characterized by the core region mutations described herein, and up to 6, 5, 4, 3, 2 or only 1 point mutation in the full-length sequence, compared to the mature wt TSST-1 sequence.
Specifically, the sequence of the detoxified TSST-1 described herein differs from a wt TSST-1 sequence in the detoxifying point mutations which comprise or consist of: a) one or more point mutations in the MHC Class II binding region, wherein one point mutation is a substitution or deletion of G31 , optionally wherein additionally any one or both of L30 and S32 are substituted or deleted; and optionally: b) one or more point mutations in the T cell receptor binding region, preferably comprising or consisting of an amino acid substitution at H135, preferably H135X, wherein X is A, D, I, Q, or R; and/or G16V, and/or L137V.
Specifically, the detoxified TSST-1 described herein comprises a TSST-1 sequence which is any one of: a) SEQ ID NO:53, which comprises the amino acid sequence of SEQ ID NO:3, wherein G31 is deleted; b) SEQ ID NO:54, which comprises the amino acid sequence of SEQ ID NO:3, wherein G31 and L30 are deleted; c) SEQ ID NO:55, which comprises the amino acid sequence of SEQ ID NO:3, wherein G31 and S32 are deleted; d) SEQ ID NO:56, which comprises the amino acid sequence of SEQ ID NO:3, wherein L30, G31 and S32 are deleted.
Specifically, any of the sequences SEQ ID NO:53-56 can be further modified to comprise one or more point mutations in the T cell receptor binding region, preferably comprising an amino acid substitution at any one of H135X, wherein X is A, D, I, Q, or R; and/or G16V, and/or L137V.
SEQ ID NO:57 comprises the TSST-1 sequence incorporating the delta G31 and H135A mutations.
SEQ ID NO:64 comprises the TSST-1 sequence incorporating the G31 R and H135A mutations.
Specifically, any of the sequences SEQ ID NO:53-57 and SEQ ID NO:64, which may or may not include a further modification in the T cell receptor binding region, may be further modified to incorporate one or more, e.g., up to 6, 5, 4, 3, 2, or only 1 aa substitution outside the core region.
Specifically, the detoxified TSST-1 may comprise one or more further mutations in a non-core region i.e., the region of TSST-1 which is not the core region.
Specifically, the detoxified TSST-1 toxin may comprise one or more, preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations, preferably up to 10, 9, 8, 7, 6, or 5 point mutations, preferably amino acid substitutions or a combination of amino acid substitutions as naturally-occurring in any of the wild-type TSST-1 sequences, such as found in a wild-type TSST-1 toxin sequence, e.g.: one or more of T2A, D11 N, V25F, N28D, S111 N, and S111 R, as compared to SEQ ID NO:3.
Specifically, the non-core region of the detoxified TSST-1 described herein differs from SEQ ID NO:3 only in 1 , 2, 3, 4, 5, or 6 amino acids, e.g., with an amino acid substitution at only 1 , 2, 3, 4, 5, or 6 positions, such as amino acid substitution(s) selected from those naturally-occurring in wt TSST-1 sequences e.g., selected from the group consisting of T2A, D11 N, V25F, N28D, S111 N, and S111 R, as compared to SEQ ID NO:3.
Specifically, the number of point mutations in the non-core region is limited, such that the non-core region has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3. Specifically, a wild-type TSST-1 sequence may be identical to or differ from SEQ ID NO:3 in one or more amino acids e.g., up to 6, 5, 4, 3, 2, or only 1 aa, outside the core region, such as one or more point mutations as naturally-occurring in a wild-type TSST-1 sequence, e.g., one or more of the amino acid substitutions selected from the group consisting of T2A, D11 N, V25F, N28D, S111 N, and S111 R, as compared to SEQ ID NO:3. Exemplary wt TSST-1 sequences referred to herein are SEQ ID NO:3, and SEQ ID NO:58-63.
Specific embodiments comprise a detoxified TSST-1 variant sequence of any one of SEQ ID NO:53-57 or SEQ ID NO:64, which comprises the detoxifying mutations as described herein, and which is particularly characterized by at least any one of 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the respective non-core regions of any one of the detoxified TSST-1 toxin sequences described herein, in particular over the whole length of all corresponding non-core regions of any one of the detoxified TSST-1 toxin sequences described herein.
Specifically, the detoxified TSST-1 comprises or consists of any one of SEQ ID NO:53-57 or SEQ ID NO:64, or a detoxified TSST-1 variant sequence of any one of the foregoing which comprises said modifications in the T cell receptor binding region and in the MHC Class II binding region, and at least any one of 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:3 and/or to any one of SEQ ID NO:53-57 or SEQ ID NO:64.
According to a specific aspect, the detoxified TSST-1 comprises or consists of the wild-type TSST-1 sequence, such as e.g., amino acid sequence of any one of SEQ ID NO:3, or SEQ ID NO:3 to 3, which is modified to incorporate said detoxifying point mutations wherein one of the point mutations is a substitution or deletion of G31 , and to incorporate an amino acid substitution H135A.
Preferably, the TSST-1 sequence comprises any one of SEQ ID NO:53-57 or SEQ ID NO:64, or the respective detoxifying point mutations comprised in NO:53-57 or SEQ ID NO:64 as compared to SEQ ID NO:3.
According to a specific aspect, the vaccine antigens provided in the combination described herein are protective vaccine antigens. Therefore, the combination provides for the respective protective multivalent vaccine, and the use of such vaccine for medical purposes. According to a specific aspect, the combination is provided as a fusion, mixture, or a complex comprising said molecule and antigen bound to each other and/or bound to a carrier, thereby forming the complex.
Specifically, the vaccine antigens may comprise one or more detoxified toxin molecules that are fused to another detoxified toxin molecule, optionally comprising a linker sequence. Exemplary combinations are fusions of atoxic or detoxified toxoid molecules, wherein at least one (or two) of said molecules is/ are the detoxified SEB comprising the point mutations as described herein.
Specifically, said at least one detoxified SEB toxin molecule can be fused to another detoxified SEB toxin molecule, optionally comprising a linker sequence. Specific linkers suitably used are peptidic linkers such as consisting of a series of at least two, or at least three amino acids. Specifically, the fusion of molecules is in any order, by a peptide bond, with or without a linker.
According to a specific aspect, the fusion comprises at least two identical molecules of said detoxified SEB toxin as described herein, or at least two molecules of said detoxified SEB toxin as described herein that differ in at least one of the detoxifying point mutations as described herein. A fusion of at least two molecules, in particular two identical molecules, with or without a linker, is herein also referred as a “tandem fusion” or “tandem construct”.
An exemplary tandem fusion of SEB comprises or consists of any one of SEQ ID NO:15, 33, 35, or 37.
Fusion may be achieved by recombination of nucleic acid molecules encoding the respective peptide sequences, or otherwise by synthesizing the coding nucleic acid molecules or fused (poly)peptide sequences.
Specifically, the linker can be a peptidic linker, preferably composed of a number of consecutive amino acid residues, such as selected from any of the naturally-occurring amino acids, preferably any of Gly, Ser, His, Met, Lys, Leu, and Thr. Linkers can be composed of flexible residues like glycine and serine so that the adjacent peptides are free to move relative to one another. To fuse two detoxified SEC toxin molecules as described herein, short linkers are sufficient, such as a peptide linker comprising or consisting of a sequence of 2-10 amino acids, e.g., comprising or composed of two amino acids selected from the group consisting of Lys-Leu, His-Met, Gly-Thr and Gly- Gly-Gly. Longer linkers such as longer than 10 amino acids can be used e.g., when necessary to ensure that two adjacent molecules do not sterically interfere with each other.
According to a specific aspect, the antigen may comprise one or more peptide spacers in addition to linker, such as to improve the structure or stability of the polypeptide.
Specifically, the peptide fusion described herein may comprise the peptides which are conveniently bound to each other by bioconjugation, chemical conjugation or crosslinking. For example, the antigen may comprise peptide conjugates. Specific embodiments may employ multimerization domains, carriers, or devices such as nanostructures or beads that are suitably used to immobilize a series of peptides.
Yet, the antigen may be provided as a molecule or molecule complex composed of two or more polypeptide chains, which are associated through covalent or non- covalent linkage, or just mixed to obtain an antigenic composition.
Alternative combinations comprised in a vaccine antigen described herein, are complexes, wherein said molecules are adsorbed, adhered or otherwise immobilized or bound to a liposomal, nanoparticle or solid carrier, such as those suitable for use in formulating vaccine preparations.
Alternative combinations are mixtures of molecules, such as provided in a vaccine formulation at a predefined ratio or dose, for example, a mixture formulated on an adjuvant compound, such as an insoluble metal salt e.g., alum, aluminum hydroxide, or aluminum phosphate.
The invention further provides for a pharmaceutical preparation comprising the antigen described herein, further comprising a pharmaceutically acceptable carrier, e.g., in an immunogenic formulation.
The invention further provides for a vaccine comprising the vaccine antigen described herein, and a pharmaceutically acceptable carrier. Specifically, the vaccine is a protective vaccine, such as protective against S. aureus disease conditions.
The vaccine is specifically protective at a site of exposure or toxicity of the respective target toxin or SAG(s) in the respective multivalent vaccine described herein, which are targeted by a multivalent vaccine, such as at one or more of the following sites: subcutaneous, mucosal gastrointestinal, skin or intramuscular sites.
Specific embodiments of the vaccine comprise an adjuvant, such as suitably used in human vaccine preparations. Specific examples of adjuvants are selected from the group consisting of an insoluble metal salt, a glucopyranosyl Lipid A adjuvant, adjuvant systems which are stimulants of innate immunity, such as AS01 , AS03, or AS04, MF59, a TCR stimulant such as a toll-like receptor agonist or CpG oligonucleotides, preferably alum, aluminum hydroxide, or aluminum phosphate.
Specifically, the vaccine can be administered to a subject, in particular a human subject, in an effective amount employing a prime-boost strategy.
Specifically, the combination comprises an effective amount of any one or two or three of the vaccine antigens described herein which is ranging between 0.0001 and 1 mg per administration, preferably at least any one of 100, 200, 300, 400, 500, 600, 700, 800, 900 ng, or at least any one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg, per dose.
Specifically, the vaccine described herein is provided in a formulation for human use. Specific embodiments are provided in a formulation for parenteral such as intramuscular or subcutaneous, intranasal, mucosal, oral, microneedle skin, transdermal, sublingual, aerosolic or inhaled administration. Preferably, treatment comprises administration of the vaccine described herein by the respective route of administration.
According to a specific aspect, the vaccine is a multivalent vaccine comprising said combinations of vaccine antigens described herein, which may additionally comprise one or more other Staphylococcal superantigen toxoid antigens, preferably selected from the group consisting of alpha-hemolysin, gamma-hemolysin, betahemolysin, staphylococcal exotoxins or enterotoxins, such as e.g., enterotoxin A (SEA), B (SEB), I (SEI), and K (SEK).
According to a specific aspect, the combination of vaccine antigens described herein is provided for medical, diagnostic or analytical use.
The invention further provides for the medical use of the combination of vaccine antigens described herein, and in particular the use of such material in a method of producing a pharmaceutical preparation, such as a vaccine, for treating a subject e.g., a human subject or patient, in particular for the prevention or therapy of specific disease conditions or diseases.
Specifically, the medical use involves an immunotherapy, such as an active immunotherapy. Specific immunotherapies provide for the treatment of a subject afflicted with, or at risk of contracting or suffering a disease or recurrence of a disease, by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.
Specifically, the vaccine is provided for use in the prevention, treatment against or therapy of a staphylococcal toxin or superantigen-expressing bacterial infection, and/or a disease condition directly or indirectly mediated by exposure to Staphylococcal superantigen toxins, Staphylococcus infection and/or contamination. Specifically, the disease condition is caused or triggered by a target Staphylococcus species.
Specifically, a subject is immunized with the vaccine, who is at risk of staphylococcal disease condition and/or complication.
According to a specific aspect, a subject is at risk of a staphylococcal disease condition and/or complication, where the respective disease condition or complication is possible, probable or proven.
Specifically, the staphylococcal disease condition and/or complication is/are due to primary and/or secondary forms of inheritable (including in particular primary or genetic predisposition or disease) and/or acquired (including in particular secondary disorders) immunodeficiency disorders and/or immune-modulatory disorders. Secondary immunodeficiencies can include loss of barrier function of skin and mucosal surfaces, such as e.g., where a subject is undergoing surgery, in particular plastic surgery, or where the subject is exposed to or suffering from skin injuries.
Specifically, the subject is likely exposed to staphylococcal infection, such firefighters, medical staff, or regional populations of areas where fire, flooding, or hurricanes are frequent.
The subject may be a healthy subject. Large, basically healthy populations in whom exposure is likely are e.g., firefighters, medical staff (nurses, aids), or regional populations of areas where fire, flooding, hurricanes are frequent.
Depending on the type of Staphylococcus aureus strain or the situation, the multivalent vaccine can be composed in a way to provide personalized treatment.
According to a specific aspect, the subject at risk of a staphylococcal disease condition and/or complication is a patient in whom consequences of immunological disorders require surgical interventions (e.g., reconstructive/plastic surgery) like head and neck tumors, major malformations, surgery on major organs, intestines, lung, heart etc, or a patient undergoing gastrointestinal surgery (e.g., where the patient is diagnosed with autoimmune and/or auto inflammatory disease), surgery of vital organs, heart, lung or kidney, in particular where the patient cannot be treated otherwise. The 2017 IUIS Phenotypic Classification for Primary Immunodeficiencies is described by Bousfiha et al. (J Clin Immunol. 2018 Jan;38(1):129-143). The 2019 Update of the IUIS Phenotypical Classification is described by Bousfiha et al. (Journal of Clinical Immunology (2020) 40:66-81).
Specifically, the vaccine is provided for vaccinating a subject for prophylactic treatment to prevent infection with a Staphylococcus species, in particular vaccination and immunizing a subject in need thereof.
According to a specific aspect, the vaccine may be used for preventing or treating any disease, disease condition or disorder in which inhibition, reduction of the growth of a Staphylococcus species would be beneficial.
Specifically, the multivalent vaccine is used for any one or more of the following indications: a) a subject is immunized with a combination of at least SEB and TSST-1 , optionally combined with SEC, to prevent a sepsis condition, preferably wherein the sepsis condition is sepsis, septic shock or toxic shock syndrome (TSS); and/or b) a subject is immunized with a combination of at least SEB and TSST-1 , optionally combined with SEC, to prevent Staphylococcal wound infective disorders, preferably wherein wound infection is upon burn, injuries, or surgical treatment; c) a subject is immunized with a combination of at least SEB and SEC, optionally combined with TSST-1 , to prevent Staphylococcal enteric disorders, preferably wherein the enteric disorder is enteritis or a digestive disorder resulting from Staphylococcal food poisoning; and/or d) a subject is immunized with a combination of at least SEB and TSST-1 , optionally combined with SEC, wherein the subject is at risk of or suffers from genetic susceptibility to Staphylococcus aureus bacteremia; and/or e) a subject is immunized with a combination of at least SEB and TSST-1 , optionally combined with SEC, wherein the subject is at risk of or suffers from acute multisystem inflammatory disease e.g., of blood vessels or vasculitis and/or aneurism caused by Staphylococcus aureus, or Kawasaki syndrome.
Specifically, the sepsis condition or acute multisystem inflammatory disease is a systemic, severe inflammatory diseases with potentially lethal complications.
According to a specific aspect, the vaccine may be used for preventing or treating any disease, disease condition or disorder, which is associated with direct or indirect exposure to the respective target Staphylococcal toxin, such as SEB and any one of or both of SEC and TSST-1. Such treatment may be indicated in a method of prevention or therapy of poisoning with such toxins, such as may arise from food poisoning or poisoning upon contact e.g., through mucosal or skin contact, or by inhalation of Staphylococcal toxins.
According to a specific aspect, the vaccine may be used for preventing or treating a disease condition which is a sepsis condition induced by microbial mediators of Grampositive bacterial, viral or fungal pathogens or toxins. Specifically, the sepsis condition is sepsis, toxic shock syndrome (TSS) or septic shock.
Microbial toxins, such as LPS or similar toxins of Gram-positive bacterial, viral or fungal pathogens may trigger severe sepsis conditions in a subject that has a predisposition of staphylococcal complications e.g., upon prior contact with Staphylococcal toxins and/or a Staphylococcus infection.
According to a specific aspect, the vaccine may be used for immunizing a patient with the vaccine preparation, who is at risk of staphylococcal complications following a surgical intervention or invasive treatment of disease. Immunizing such patient prior to such surgical intervention or invasive treatment would reduce the risk of staphylococcal complications.
Specifically, the invention provides for a vaccine kit of parts comprising a combination of vaccine antigens which comprises the following components in separate containments:
According to a specific aspect, the invention provides for a vaccine kit of parts comprising the combination of vaccine antigens described herein, wherein one or more of the vaccine antigens are provided in separate containments. Specifically, the kit of parts comprises components, wherein one or more of the vaccine antigens are provided as separate components of the kit. Preferably, each component is provided in a separate containment. The kit of parts may additionally comprise a component comprising an adjuvant.
According to specific embodiments, the kit may comprise the following kit components, wherein each of the vaccine antigens is provided in separate containments: a) SEB and TSST-1 ; or b) SEB and SEC; or c) SEB, TSST-1 and SEC. Therefore, the invention specifically provides for a two-component kit comprising e.g. SEB and SEC or SEB and TSST-1 , or a three-component kit comprising SEB, TSST-1 and SEC.
Specifically, the kit provides for the combination treatment of a subject with the vaccine antigens provided as kit component e.g., simultaneously or consecutively, in a mixture, or in parallel e.g., by one, two or more administrations (or injections), such as by administration at one, two or more different sites and/or routes of administration. For example, the multivalent vaccine comprising two or three different vaccine antigens can be administered at the same site of administration, or at different sites (e.g., different injection sites), or the combination of vaccine antigens is administered in parallel or within a certain period of time (such as e.g., within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days, or within 3 or 4 weeks) before or after the vaccination with a first vaccine antigen.
Specifically, a vaccine kit of parts is used to immunize the subject with the combination of vaccine antigens, whereby the all of the individual vaccine antigens are provided by one administration, or by two or more separate administrations, preferably by administering a mixture of two or more individual vaccine antigens, or by parallel or consecutive administration of two or more individual antigens, such as at two or more different sites or routes of administration.
According to a specific aspect, the vaccine kit as described herein is used to immunize a subject, preferably wherein the respective kit components are used by one administration (e.g., only one administration, such as by one injection at the same injection site), or by two or more separate administrations.
According to a specific aspect, the kit components can be administered in a mixture of said components, or to provide a combination of said components in vitro or in vivo. Specifically, a mixture comprising or consisting of said kit components is administered to the subject.
According to another specific aspect, vaccine antigens described herein can be administered by parallel or consecutive administration of the respective kit components, such as by administering at two or more different sites or routes of administration.
The invention further provides for the use of the vaccine described herein in a method of producing an antibody preparation comprising antibodies specifically recognizing the respective vaccine antigens. The invention further provides for the new use of the vaccine described herein, in a method of producing an antibody preparation comprising antibodies specifically recognizing the target toxin. Specifically, the antibody preparation may be produced by immunizing a subject to produce antibody-containing blood preparations or fractions thereof, such as an antiserum. Specifically, the antibody preparation may be produced by selecting respective antibody-binding sites from a library of antibodies or binding sequences, using the vaccine antigen as binding agent for selection purposes, and preparing an antibody comprising the selected antibody-binding sites or respective sequences.
According to a specific aspect, the invention provides for a polyclonal antibody preparation obtainable or obtained by immunizing a subject with a vaccine described herein, isolating polyclonal antibodies or a fraction of polyclonal antibodies comprising the antibodies specifically recognizing the respective Staphylococcal superantigen vaccine antigens, and formulating a preparation comprising said antibodies, wherein the antibodies are cross-reactive with the respective wild-type Staphylococcal superantigen toxins.
The vaccine antigens described herein may be provided as recombinant proteins, such as produced by recombinant expression technologies, in particular by expressing a nucleic acid in a suitable host cell, which nucleic acid encodes the respective vaccine antigen. The nucleic acid molecule may be codon-optimized, such that the risk of reverse mutations is reduced, or to improve expressing the sequence in a recombinant host organism, such as prokaryotic or eukaryotic or insect host cells, e.g., E. coli, yeast or mammalian expression systems. Suitable host cells may be selected from the group consisting of bacterial host cells, such as E. coli, but also from mammalian, insect, or yeast cells, e.g., HEK293 cells, CHO cells, NSO cells, Sf9 cells, High Five cells, Pichia pastoris, Saccharomyces cerevisiae, among many others.
Exemplary coding nucleic acids are provided in Figure 1 .
The vaccine antigen may be produced by expressing the recombinant protein from a recombinant expression construct which comprising the nucleic acid molecule as described herein. Such expression construct comprises at least an expression cassette e.g., within a vector or plasmid, or be provided for chromosomal integration into the host cell genome.
According to a specific aspect, the invention further provides for a method of producing the combination of vaccine antigens as described herein, wherein each of the vaccine antigens are produced by a recombinant host cell described herein which is cultivated or maintained under conditions to produce said antigen.
The invention further provides for a method of producing the multivalent vaccine described herein, by combining the vaccine antigens and formulating the vaccine antigens with a pharmaceutically acceptable carrier, and preferably further comprising an adjuvant, in one or more packaged units e.g., in a kit of parts. The adjuvant may be comprised within said one or more packaged units. Alternatively or additionally, the adjuvant may be provided in a packaged unit which is separate from any one or more of the vaccine antigens e.g., as part of a kit of parts.
Specifically provided herein is a method of producing the combination vaccine described herein, by expressing nucleic acid sequences encoding the respective staphylococcal toxoids or superantigen toxoids in recombinant host cells, isolating and optionally purifying the respective toxoids, and combining said toxoids in a vaccine formulation comprising a pharmaceutically acceptable carrier, and preferably further comprising an adjuvant.
FIGURES
Figure 1 shows sequences referred to herein.
Figure 2: First bar represents TSST-1 titer of a rabbit serum immunized three times with 10pg TSST-1 variant. Second bar represents TSST-1 titer of a rabbit serum immunized three times with 10pg TSST-1 variant plus 100pg SEB variant. TSST-1 titer was raised 2.1 times. Titers were measured by ELISA.
Figure 3: First bar represents TSST-1 titer of a rabbit serum immunized three times with 10pg TSST-1 variant. Second bar represents TSST-1 titer of a rabbit serum immunized three times with 10pg TSST-1 variant plus 1 pg SEC variant. TSST-1 titer was raised 2.5 times. Titers were measured by ELISA.
Figure 4: First bar represents SEB titer of a rabbit serum immunized three times with 10pig SEB variant. Second bar represents SEB titer of a rabbit serum immunized three times with 10pg SEB variant plus 100pg TSST-1 variant. SEB titer was raised 4 times. Titers were measured by ELISA.
Figure 5: First bar represents SEB titer of a rabbit serum immunized three times with 30pg SEB variant. Second bar represents SEB titer of a rabbit serum immunized three times with 30pg SEB variant plus 30pg SEC variant. SEB titer was raised 1 .6 times. Titers were measured by ELISA.
Figure 6: First bar represents SEC titer of a rabbit serum immunized three times with 30pg SEC variant. Second bar represents SEC titer of a rabbit serum immunized three times with 30pg SEC variant plus 30pg SEB variant. SEC titer was raised 3.3 times. Titers were measured by ELISA.
Figure 7: First bar represents SEC titer of a rabbit serum immunized three times with 30pg SEC variant. Second bar represents SEC titer of a rabbit serum immunized three times with 30pg SEC variant plus 30pg TSST-1 variant. SEC titer was raised 1.6 times. Titers were measured by ELISA.
DETAILED DESCRIPTION OF THE INVENTION
Specific terms as used throughout the specification have the following meaning.
The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.
The term “antigen” as used herein shall in particular refer to any antigenic determinant, which can be possibly recognized by a binding site of an antibody. Specifically, preferred antigens are those molecules or structures, which have already been proven to be or are capable of being immunologically or therapeutically relevant, especially those, for which a clinical efficacy has been tested. The term as used herein shall in particular comprise molecules or structures selected from antigens comprising immunoaccessible and immunorelevant epitopes, in particular conserved antigens found in one or more species or serotype. Immunoaccessible viral epitopes are typically presented by or comprised in antigens expressed on the outer surface of a virion or on the surface of an infected cell.
Selected epitopes and peptides as described herein may trigger an immune response in vivo, so to induce neutralizing antibodies against the antigen and target bacteria, respectively. This provides for the effective protection upon active immunization with the antigen. Polypeptide antigens are preferred antigens due to their inherent ability to elicit both cellular and humoral immune responses.
Protective antigens referred to herein are understood to induce a protective immune response.
The term “protective immune response” or “protective immunity” is herein understood as follows: Protective immunity is a condition of a subject conferred by the immune response generated by immunization, such that the subject’s response is sufficient to survive or neutralize a challenging dose of the respective antigen or pathogen, even a dose that would be otherwise toxic or lethal in a non-immunized subject, or which response inhibits an undesired or uncontrolled inflammation and/or activation of immune cells. Staphylococcal toxins like the SAGs referred to herein, in particular SEB, SEC, or TSST-1 , may cause activation of antigen-presenting cells and/or T cells and uncontrolled inflammatory reactions which can damage organs e.g., gastrointestinal, respiratory, urogenital, mucosal or brain damage, or can even be lethal. The subject vaccines described herein are specifically designed to confer protective immunity to prevent or reduce such inflammatory reactions. Protective immunity can be determined by in vitro or in vivo assays determining inhibition of undesired stimulation of antigen-presenting cells and/or T cell activation. In vivo assays preferably employ suitable animal models, preferably in at least two different species, e.g., mouse and rabbit models.
The antigens described herein are understood as protein antigens specifically comprising a linear, unbranched amino acid sequence, which is naturally-occurring but modified to introduce certain detoxifying mutations, which may be further modified, e.g., by mutation or chemical derivatization, such as by phosphorylation, methylation, acetylation, amidation, formation of pyrrolidone carboxylic acid, isomerization, hydroxylation, sulfation, flavin-binding, cysteine oxidation and nitrosylation.
The detoxified toxins and vaccine antigens used herein are specifically designed to trigger an immune response in a subject, and particularly include one or more antigenic determinants, which can be possibly recognized by a binding site of an antibody or are able to bind to the peptide groove of HLA class I or class II molecules or other antigen presenting molecules such as CD1 and as such may serve as stimulant for specific T-cells. The vaccine antigens specifically include one or more immunologically relevant epitopes, which are herein understood as structures that are recognized by the subject’s immune system and/or respective antibodies. Epitopes can e.g., be B-cell epitopes or T-cell epitopes, such as CD4+ T-cell epitopes or CD8+ T-cell epitopes.
Neutralizing activity of an immune response or respective neutralizing antibodies can be tested in cell-based assays and in vivo. Neutralizing antibodies can be determined e.g., by enumerating bacterial titers in the presence of antibodies and/or by detecting a cytopathic effect in cell-based infection assays.
Protective immune responses can be measured using in vivo models of Staphylococcus infection.
The term “expression” is understood in the following way. Nucleic acid molecules containing a desired coding sequence of an expression product such as e.g., a detoxified SAG or a vaccine antigen as described herein, and control sequences such as e.g. a promoter in operable linkage, may be used for expression purposes. Hosts transformed or transfected with these sequences are capable of producing the encoded proteins. In order to effect transformation, the expression system may be included in a vector; however, the relevant DNA may also be integrated into the host cell chromosome. Specifically, the term refers to a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.
Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular polypeptide or protein. Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.
“Vectors” used herein are defined as DNA sequences that are required for the transcription of cloned recombinant nucleotide sequences, i.e. , of recombinant genes and the translation of their mRNA in a suitable host organism.
An “expression cassette” refers to a DNA coding sequence or segment of DNA that code for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a “DNA construct”.
Expression vectors comprise the expression cassette and additionally usually comprise an origin for autonomous replication in the host cells or a genome integration site, one or more selectable markers (e.g., an amino acid synthesis gene or a gene conferring resistance to antibiotics such as zeocin, kanamycin, G418 or hygromycin), a number of restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, which components are operably linked together. The term “vector” as used herein includes autonomously replicating nucleotide sequences as well as genome integrating nucleotide sequences. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily be introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Specifically, the term “vector” or “plasmid” refers to a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
The term “host cell” as used herein shall refer to primary subject cells transformed to produce a particular recombinant protein, and any progeny thereof. It should be understood that not all progeny is exactly identical to the parental cell (due to deliberate or inadvertent mutations or differences in environment), however, such altered progeny is included in these terms, so long as the progeny retain the same functionality as that of the originally transformed cell. The term “host cell line” refers to a cell line of host cells as used for expressing a recombinant gene to produce recombinant proteins. The term “cell line” as used herein refers to an established clone of a particular cell type that has acquired the ability to proliferate over a prolonged period of time. Such host cell or host cell line may be maintained in cell culture and/or cultivated to produce a recombinant polypeptide.
The detoxified toxins and vaccine antigens used herein are specifically provided as isolated proteins. The term “isolated” or “isolation” as used herein with respect to a protein shall refer to such compound that has been sufficiently separated from the environment with which it would naturally be associated, so as to exist in “purified” or “substantially pure” form. Yet, “isolated” does not necessarily mean the exclusion of artificial or synthetic fusions or mixtures with other compounds or materials, or the exclusion of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification. Isolated compounds can be further formulated to produce preparations thereof, and still for practical purposes be isolated - for example, a mixture of proteins or the respective fusion proteins described herein can be mixed with pharmaceutically acceptable carriers, including those which are suitable for analytic, diagnostic, prophylactic or therapeutic applications, or excipients when used in diagnosis, medical treatment, or for analytical purposes.
The term “purified” as used herein shall refer to a preparation comprising at least 50% (w/w total protein), preferably at least 60%, 70%, 80%, 90% or 95% of a compound (e.g., a chimeric antigen described herein). A highly purified product is essentially free from contaminating proteins, and preferably has a purity of at least 70%, more preferred at least 80%, or at least 90%, or even at least 95%, up to 100%. Purity is measured by methods appropriate for the compound (e.g., chromatographic methods, polyacrylamide gel electrophoresis, HPLC analysis, and the like). An isolated, purified protein described herein may be obtained as a recombinant product obtained by purifying from a host cell culture expressing the product in the cell culture supernatants, to reduce or remove host cell impurities or from cellular debris.
As isolation and purification methods for obtaining a purified protein, methods utilizing difference in solubility, such as salting out and solvent precipitation, methods utilizing difference in molecular weight, such as ultrafiltration and gel electrophoresis, methods utilizing difference in electric charge, such as ion-exchange chromatography, methods utilizing specific affinity, such as affinity chromatography, methods utilizing difference in hydrophobicity, such as reverse phase high performance liquid chromatography, and methods utilizing difference in isoelectric point, such as isoelectric focusing may be used. Standard methods can be used such as cell (debris) separation and wash by Microfiltration or Tangential Flow Filter (TFF) or centrifugation, protein purification by precipitation or heat treatment, protein activation by enzymatic digest, protein purification by chromatography, such as ion exchange (IEX), hydrophobic interaction chromatography (HIC), Affinity chromatography, size exclusion (SEC) or HPLC chromatography, protein precipitation of concentration and washing by ultrafiltration steps. An isolated and purified protein can be identified by conventional methods such as Western blot, HPLC, activity assay, or ELISA.
The term “non-pyrogenic” as referred to herein is meant to a substance compound or compositions which does not trigger a fever response upon administration to a subject like a human being or in a respective animal model. In particular, using a compound that is non-pyrogenic avoids a pyrogenic reaction. A fever response (also known as pyrexia) is typically meant to be an increase in body temperature above normal, e.g., of more than 1 °C above the normal (reference) value or range. There is not a single agreed- upon upper limit for normal temperature. Exemplary normal values are between 36°C and 38.3°C, or within 37°C and 38.3 C in humans, depending on the method of measuring the body temperature.
Where such non-pyrogenic compound is formulated into a pharmaceutical product such as a vaccine for parenteral administration, safety of the respective pharmaceutical product is ensured.
A compound that triggers fever has historically been referred to as a "pyrogen" or a "pyrogenic" compound, referring to the fever response which such compounds may cause. Some compounds, like Staphylococcal superantigen toxins, however, are generally pro-inflammatory and cause fever as part of the inflammatory response that they cause. Staphylococcal superantigen toxins are known to be pyrogenic and increase the body temperature by more than 1 °C up to very high body temperatures, depending on the bacterial Staphylococcus aureus strain and the situation of contact or contamination with the SAG.
In some cases, depending on the sensitivity of a subject and the type and concentration of pyrogen, the subject is exposed to, a subject can develop lifethreatening shock-like symptoms after exposure to a pyrogen. Medical products which can be inhaled, injected, or infused and medical devices such as surgical tools or implanted materials pose a particular risk of pyrogenicity. Even food or nutrients can represent a risk of pyrogenicity. Pyrogen testing of various pharmaceuticals, nutrients, and medical products for parenteral application or surgery is typically performed with standard tests to ensure the safety of such products.
Usually, compounds which act as a pyrogen do so by stimulating the production of endogenous pyrogens, such as prostaglandins and proinflammatory cytokines, in monocytes after contact with tissue, cells, or body fluids. It is these endogenously produced pyrogens which mediate the inflammatory response in the affected organism. The most important and well-known of these endogenous pyrogens are the cytokines interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor (TNF) and the low molecular weight lipid mediator prostaglandin E2 (PGE2). These compounds are routinely assayed by ELISA, or enzyme-linked immunosorbent assays (for IL-1 , IL-6, or TNF), and EIA, or enzyme immunoassay (for PGE2).
In order to determine that a compound is non-pyrogen, the absence of a pyrogenic effect can be determined in a suitable animal model, such as a rabbit pyrogen test. Alternatively, the lack of significant induction of a human inflammatory response, or induction of any of the pro-inflammatory cytokines or biomarkers can be measured as an indicator of non-pyrogenicity, e.g., by standard in vitro or ex vivo assays, such as tests that determine the effect of a compound on the in vitro activation of human monocytes like in human peripheral whole blood, peripheral blood mononuclear cells (PBMCs), or monocytic cell lines, e.g., to detect the release of pyrogenic cytokines.
The term "nucleic acid molecule” used herein refers to either DNA (including e.g., cDNA) or RNA (including e.g., mRNA) molecules comprising a polynucleotide sequence. The molecule may be a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. The term includes coding sequences, such as genes, artificial polynucleotides such as comprised in an expression construct expressing the respective polypeptide sequence.
Nucleic acid molecules encoding the detoxified toxin molecules or vaccine antigens described herein are specifically provided by mutagenesis (mutation) of naturally-occurring toxin sequences. Mutagenesis to delete specific amino acids involves the deletion of one or more nucleotides. Mutagenesis to substitute one specific amino acid may involve substitution of at least 1 or 2 nucleotides, wherein the codon comprising said substituted nucleotides encodes the substituted amino acid. Where nucleotides are substituted, it is preferred to exchange more than one nucleotide within a codon, to reduce the risk of undesired spontaneous reverse mutation. Codonoptimization of a nucleotide sequence and molecule described herein may involve increasing the number of substituted nucleotides within a codon, such as to improve the stability of the mutants, and/or avoiding reverse mutation by only one spontaneous nucleotide exchange.
A DNA or RNA molecule can be used which comprises a nucleotide sequence that is degenerate to any of the sequences or a combination of degenerate sequences, or which comprises a codon-optimized sequence to improve expression in a host. For example, an E. coli codon optimized sequence can be used. Specific RNA molecules can be used to provide a respective RNA-vaccine. Expression systems, genetic constructs or modifications described herein may employ tools, methods and techniques known in the art, such as described by J. Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2001). Expression vectors may include but are not limited to cloning vectors, modified cloning vectors and specifically designed plasmids. Preferred expression vectors described herein are expression vectors suitable for expressing of a recombinant gene in a bacterial or eukaryotic host cell and are selected depending on the host organism. Appropriate expression vectors typically comprise regulatory sequences suitable for expressing DNA encoding a recombinant protein in a eukaryotic host cell. Examples of regulatory sequences include promoter, operators, enhancers, ribosomal binding sites, and sequences that control transcription and translation initiation and termination. The regulatory sequences are typically operably linked to the DNA sequence to be expressed.
The term “naturally-occurring” as used herein shall refer to those elements, sequences or products which are found in nature, such as expressed by or found in native, wild-type organism. Specifically, the detoxified toxins or vaccine antigens described herein comprise mutations which are not naturally-occurring, i.e., which are artificial (“man-made”) but artificial mutants e.g., produced employing a mutagenesis technology.
A recombinant nucleic acid may be one that has a sequence that is not naturally- occurring or that has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques well- known in the art. For example, a nucleic acid can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization.
Mutants of a protein, such as comprising detoxifying mutations as described herein, may be provided e.g., by introducing a certain number of point mutations into a parent aa sequence. Specifically, a mutagenesis method is used to introduce one or more point mutations. The term “detoxifying mutations” as used herein shall refer to those one or more point mutation(s) in either or both of the T cell receptor binding region or the immunoglobulin superfamily (or MHC Class II) binding region of a naturally-occurring (wild-type) Staphylococcal toxin, in particular any of the SAGs described herein. Such regions are herein also referred to as “core region”. The core region may comprise critical core regions such as further described herein, which are herein understood as the specific positions that are modified by the exemplary detoxifying mutations described herein. It is preferred to genetically modify any one or more amino acids, at least within the core region, in particular the critical core region(s), to detoxify the toxin, thereby providing a toxoid. Any other regions, i.e. other than the core regionsor critical core regions, of the toxin are herein also referred to as “non-core regions”. Detoxifying mutations reduce, diminish or abolish the protein’s toxicity, thereby improving the safety upon administration to a subject.
A point mutation as described herein is typically at least one of a deletion, insertion, and/or substitution of one or more nucleotides within a nucleotide sequence to achieve the deletion, insertion, and/or substitution of one (only a single one) amino acid at a certain, defined position within the amino acid sequence encoded by said nucleotide sequence. Therefore, the term “point mutation” as used herein shall refer to a mutation of a nucleotide sequence or an amino acid sequence. Specifically, preferred point mutations are substitutions, in particular non-conservative or conservative ones. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc. In this regard, amino acids refer to twenty naturally occurring amino acids encoded by sixty-four triplet codons. These 20 amino acids can be split into those that have neutral charges, positive charges, and negative charges. Preferred point mutations refer to the exchange of amino acids of the different polarity and/or charge.
Specific mutagenesis methods provide for point mutations of one or more nucleotides in a sequence, in some embodiments tandem point mutations, such as to change at least 2, 3, 4, or 5, or even more contiguous nucleotides within a nucleotide sequence of a parent molecule. In general, mutations referred to herein which are made or identified in a sequence (e.g., an amino acid sequence as described herein) are numbered in relation to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutations. Therefore, unless explicitly described otherwise, the positions of amino acids in the detoxified SEB or SEC toxin are those of the respective wild-type toxin e.g., identified by SEQ ID NO:1 (as for SEB) and SEQ ID NO:2 (as for SEC), respectively. Although the detoxified SEB or SEC toxin may undergo modifications such as a deletion of one or more amino acids in the T cell receptor binding region, or an insertion of amino acids, such as an N-terminal extension, the positions following any such modification will still be numbered as in the wild-type protein. For example, in a deletion mutant, deleting aa22-24, the point mutation at L45 is still at a position numbered aa45, though the sequence of such deletion mutant has been shortened by three amino acids. Although the detoxified TSST-1 may comprise an extension, insertion, or deletion of one or more amino acids, the positions following any such deletion will still be numbered as in the wild-type protein. For example, in a deletion mutant deleting one amino acid at position aa31 , the further deletion of the aa32 or the amino acid substitution at aa135 is still at the position numbered aa32 and aa135, respectively, though the sequence of such deletion mutant has been shortened by the deleted amino acid at position aa31.
The term “mutagenesis” as used herein shall refer to a method of preparing or providing mutants of a nucleotide sequence and the respective protein encoded by said nucleotide sequence, e.g., through insertion, deletion and/or substitution of one or more nucleotides, so to obtain variants thereof with at least one change in the coding region. Mutagenesis may be through site directed, random, or semi-random. A mutagenesis method can encompass methods of engineering the nucleic acid or de novo synthesizing a nucleotide sequence using the respective parent sequence information as a template. For instance, a library of nucleotide sequences may be prepared by mutagenesis of a selected parent nucleotide sequence. A library of variants may be produced and a suitable mutant protein sequence be selected according to a specifically desired genotype or phenotype.
According to a specific embodiment, any of the detoxified toxins and vaccine antigens used herein may be produced as a recombinant protein, such as produced by recombinant DNA technology. As used herein, the term “recombinant” refers to a molecule or construct that does not naturally occur in a host cell. In some embodiments, recombinant nucleic acid molecules contain two or more naturally-occurring sequences that are linked together in a way that does not occur naturally. A recombinant protein refers to a protein that is encoded and/or expressed by a recombinant nucleic acid. In some embodiments, “recombinant cells” express genes that are not found in identical form within the native (i.e., non-recombinant) form of the cell and/or express native genes that are otherwise abnormally over-expressed, under-expressed, and/or not expressed at all due to deliberate human intervention. Recombinant cells contain at least one recombinant polynucleotide, polypeptide or protein. “Recombination”, “recombining”, and generating a “recombined” nucleic acid generally encompass the assembly of at least two nucleic acid fragments.
The term “recombinant” as used herein specifically means “being prepared by or the result of genetic engineering” i.e., by human intervention. A recombinant nucleotide sequence may be engineered by introducing one or more point mutations in a parent nucleotide sequence, and may be expressed in a recombinant host cell that comprises an expression cassette including such recombinant nucleotide sequence. The polypeptide expressed by such expression cassette and host cell, respectively, is also referred to as being “recombinant”. For the purpose described herein conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed. Specific embodiments described herein refer to the production of a chimeric antigen, and the recombinant means for such production, including a nucleic acid encoding the amino acid sequence, an expression cassette, a vector or plasmid comprising the nucleic acid encoding the amino acid sequence to be expressed, and a host cell comprising any such means. Suitable standard recombinant DNA techniques are known in the art and described inter alia in Sambrook et al., “Molecular Cloning: A Laboratory Manual” (1989), 2nd Edition (Cold Spring Harbor Laboratory press).
Herein the term “subject” is understood to comprise human or mammalian subjects, including livestock animals, companion animals, and laboratory animals, in particular human beings, which are either patients suffering from a specific disease condition or healthy subjects. The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. The term “patient” as used herein always includes healthy subjects.
In particular the treatment and medical use described herein applies to a subject in need of prophylaxis or therapy of a disease condition associated with a Staphylococcus infection and/or contamination. Specifically, the treatment may be by interfering with the pathogenesis of a disease condition where a Staphylococcus species is a causal agent of the condition. The subject may be a subject at risk of such disease condition or suffering from disease.
Non-limiting examples of some common disease conditions or disorders caused by Staphylococcus aureus bacteria or toxins comprise burns, cellulitis, skin necrosis, eyelid infections, food poisoning, joint infections, pneumonia, skin infections, surgical wound infection, scalded skin syndrome and toxic shock syndrome. Some of the disease conditions may relate to the direct, indirect or secondary effect of toxins on cell function and cell damage or lysis.
According to a specific aspect, the Staphylococcus species targeted by the vaccine as described herein are selected from human pathogenic Staphylococcus species, or those Staphylococcus species that cause an infection in humans. In embodiments, the Staphylococcus species comprises Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, and/or Staphylococcus saprophyticus, in particular including antibiotic resistant species such as methicillin resistant Staphylococcus aureus, and including mutant pathogenies which may evolve during an infection, or mutants that are artificially evolved.
In specific embodiment, a treatment method is provided to prevent an infection with a Staphylococcus species in a subject, and/or to ameliorate a disease condition that may arise in a subject at risk of staphylococcal complications. The vaccine preparation described herein may be specifically used to treat diseases or disorders associated with Staphylococcus infection, especially infection with antibiotic resistant species, including a skin infection, cellulitis, pneumonia, meningitis, urinary tract infection, toxic shock syndrome, endocarditis, pericarditis, osteomyelitis, bacteremia, or sepsis. The vaccine preparation described herein may also be used to prevent Staphylococcus infections and/or staphylococcal complications that may arise during or following a surgical procedure, such as surgery that involves implantation of a pacemaker, artificial heart valve, a joint implant, or a prosthetic. In certain embodiments, the prosthetic may be a prosthetic limb, such as an arm or a leg. In certain embodiments, the prosthetic may be a hip replacement. In certain embodiments, the prosthetic may be a cosmetic prosthetic, such as, but not limited to an ocular prosthetic, silicone hands, fingers, breasts, feet, toes, or a facial implant. In some embodiments, a subject is treated who has undergone, or who is planning on undergoing a surgical procedure, wherein the subject is at risk of acquiring a Staphylococcus infection and/or staphylococcal complications. Staphylococcal complications are specifically those associated with Staphylococcus aureus bacteremia or intoxication. Although most staphylococcus infections are not serious, S. aureus can cause serious infections such as bloodstream infections, pneumonia, or bone and joint infections, including e.g., acute complications (such as sepsis, toxic shock syndrome (TSS), septic shock, adult respiratory distress syndrome, disseminated intravascular coagulation, etc.), or chronic (including relapsing) diseases (such as osteomyelitis, endocarditis, infections of indwelling devices and wound infections). Patients at risk of complications of Staphylococcus aureus Infection include those receiving hemodialysis, injection drug users, patients with diabetes, and patients with nasal carriage of S. aureus, in particular methicillin-resistant S. aureus, and/or those with preexisting cardiac conditions or other comorbidities.
The term “at risk of’ a certain disease conditions, refers to a subject that potentially develops such a disease condition, e.g., by a certain predisposition, exposure to Staphylococcus bacteria or toxins, or that already suffers from a respective disease condition at various stages, particularly associated with other causative disease conditions or else conditions or complications following as a consequence of viral infection.
The term “treatment” as used herein shall always refer to treating subjects for prophylactic (i.e. , to prevent infection and/or disease status) or therapeutic (i.e. to treat diseases regardless of their pathogenesis) purposes.
The term “prophylaxis” as used herein refers to preventive measures which is intended to encompass prevention of the onset of pathogenesis or prophylactic measures to reduce the risk of pathogenesis.
The term “therapy” as used herein with respect to treating subjects refers to medical management of a subject with the intent to cure, ameliorate, stabilize, reduce the incidence or prevent a disease, pathological condition, or disorder, which individually or together are understood as “disease condition”.
The vaccine described herein specifically comprises the detoxified toxins or the vaccine antigens in an effective amount, which is herein specifically understood as “immunologically effective amount”. By "immunologically effective amount", it is meant that the administration of that amount to a subject, either in a single dose or as part of a series of doses, is effective on the basis of the therapeutic or prophylactic treatment objectives. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing a target Staphylococcus bacterial infection or a staphylococcal complication, or inhibiting diseases directly or indirectly caused by Staphylococcus bacteria or toxins. This amount will vary depending upon the health and physical condition of the subject to be treated, age, the capacity of the subject’s immune system to synthesize antibodies, the type and degree of immune response desired, the formulation of the vaccine, and other conditions.
An effective amount or dosage may range from 0.0001 to 2 mg, e.g., between 0.001 and 2 mg, of the vaccine antigen administered to the subject in need thereof, e.g., an adult human subject. For example, the effective dosage of the vaccine antigen is capable of eliciting an immune response in a subject of effective levels of antibody titer to bind and neutralize one or more target Staphylococcus bacteria or toxins, e.g., 1-3 months after immunization. The effectiveness can be assayed by the respective antibody titers in samples of blood taken from the subject.
In some embodiments, an effective amount is one that has been correlated with beneficial effect when administered as part of a particular dosing regimen, e.g., a single administration or a series of administrations such as in a “boosting” regimen. For treatment, the vaccine described herein may be administered at once, or may be divided into the individual components and/or a number of smaller doses to be administered at intervals of time. Typically, upon priming a subject by a first injection of a vaccine described herein, one or more booster injections may be performed over a period of time by the same or different administration routes. Where multiple injections are used, subsequent injections may be made, e.g., within 1 to 52 weeks of the previous injection.
The vaccine described herein may comprise the detoxified toxins or the vaccine antigens in an immunogenic formulation. Specific embodiments comprise one or more adjuvants and/or pharmaceutically acceptable excipients or carriers.
Pharmaceutical carriers suitable for facilitating certain means of administration are well known in the art. Specific embodiments refer to immunogenic formulations, which comprise a pharmaceutically acceptable carrier and/or adjuvant, which trigger a humoral (B-cell, antibody) or cytotoxic (T-cell) immune response. Adjuvants may specifically be used to enhance the effectiveness of the vaccine. Adjuvants may be added directly to the vaccine compositions or can be administered separately, either concurrently with or shortly before or after administration of the vaccine antigen.
The term “adjuvant” as used herein specifically refers to a compound that when administered in conjunction with an antigen augments and/or redirects the immune response to the antigen, but when administered alone does not generate an immune response to the antigen. Adjuvants can augment an immune response by several mechanisms including lymphocyte recruitment, stimulation of B- and/or T-cells, and stimulation of macrophages.
An “effective amount” of an adjuvant can be used in a vaccine described herein, which is specifically understood to be an amount which enhances an immunological response to the immunogen such that, for example, lower or fewer doses of the immunogenic composition are required to generate a specific immune response and a respective effect of preventing or combating bacterial infection, intoxication, or disease.
The term “pharmaceutically acceptable carrier” denotes one or more non-toxic material effectiveness of biological activity does not interfere with active ingredients, including but not limited to buffer solution, preservative, compatible carrier and optionally other additives or encapsulating substances. The term "carrier" denotes a natural or synthetic organic or inorganic ingredient and active ingredient combined with the carrier to facilitate application. Pharmaceutically acceptable carriers generally include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible with an antibody or related composition or combination provided by the invention. Specific examples of pharmaceutically acceptable carriers include sterile water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, polyethylene glycol, and the like, as well as combinations of any thereof. Additional pharmaceutically acceptable carriers are known in the art and described in, e.g., Remington: The Science and Practice of Pharmacy, 22nd revised edition (Allen Jr, LV, ed., Pharmaceutical Press, 2012). Liquid formulations can be solutions, emulsions or suspensions and can include excipients such as suspending agents, solubilizers, surfactants, preservatives, and chelating agents. Exemplary carriers are carrier proteins such as selected from tetanus toxoid, diphtheria toxoid and CRM197, or liposomes or cationic peptides; exemplary adjuvants are alum, aluminum phosphate or aluminum hydroxide, MF59 or CpG oligonucleotides. Further adjuvants include (in)complete Freund's adjuvant, B. pertussis or its toxin, or IC31.
The preferred preparation is in a ready-to-use, storage stable form, with a shelflife of at least one or two years. The invention also provides a delivery device e.g., a syringe, pre-filled with the vaccine as described herein. The vaccine described herein can be administered by conventional routes known the vaccine field, in such as to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface, via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route, or topical administration (e.g., via a patch). The choice of administration route depends upon a number of parameters, such as the adjuvant associated with the polypeptide. If a mucosal adjuvant is used, the intranasal, oral or inhaled route is preferred. If a lipid formulation or an aluminum compound is used, the parenteral route is preferred with the sub-cutaneous or intramuscular route being most preferred.
The injectable preparations may include dosage forms for subcutaneous, intracutaneous and intramuscular injections, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the protein in a sterile aqueous medium or an oily medium conventionally used for injections. As aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant e.g., polysorbate 80, HCO- 50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil), etc. As oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.
The foregoing description will be more fully understood with reference to the following examples. Such examples are, however, merely representative of methods of practicing one or more embodiments of the present invention and should not be read as limiting the scope of invention. EXAMPLES
Example 1 : Combination vaccines
The following exemplary combination vaccines were tested in a rabbit model. Vaccine antigens were formulated as vaccine preparations using 1 mg AL(OH)s as adjuvant. The multivalent vaccine was provided as a series of combination of individual vaccines targeting the respective toxins, wherein each vaccine comprises only one vaccine antigens.
A selection of individual vaccines and vaccine antigens were used in combination:
TSST-1 vaccine antigen, 1 OOpg; SEQ ID NO:64
TSST-1 vaccine antigen, 30pg; SEQ ID NO:64
TSST-1 vaccine antigen, 1 Opg; SEQ ID NO:64
SEB vaccine antigen, 1 OOpg; SEQ ID NO:15
SEB vaccine antigen, 30pg; SEQ ID NO:15
SEB vaccine antigen, 1 Opg; SEQ ID NO:15
SEC vaccine antigen, 30pg; SEQ ID NO:44
SEC vaccine antigen, 1 pg; SEQ ID NO:44
Example 2: Immunogenicity in rabbits
Rabbits were immunized three or four times with the individual vaccine antigens and with a combination of the vaccine antigens. The antisera were characterized by ELISA determining titers of antibodies specifically recognizing the target toxins.
Results are shown in Figures 2-7.
There is a surprisingly improved effect upon combination of the vaccine antigens. ELISA Titer
Plates were coated with 0.5 pg/ml antigen (TSST-1 , SEB, SEC) at 4°C for 16-18 hours. Plates were washed four times with 100pl/well washing puffer (1x PBS 0.1%Tween 20) and then blocked with 200 pl blocking puffer (1x PBS 0.1% Tween 20 3% BSA) at 37°C for one hour. Plates were frozen at -20°C until use. After the plates were allowed to thaw for at least 60 minutes, plates were washed and sera (50pl/well) were added in a row of twofold dilutions including negative and positive control sera. Plates were incubated for one hour at 37°C. After incubation plates were washed and horseradish labeled anti rabbit IgG antibody (50 pl/well) was added at a dilution of 1 :20.000. Plates were incubated for one hour at 37°C. After incubation, plates were washed and 100pl/well substrate (o-Phenylendiamine in the presence of H2O2) was added. Plates were incubated in darkness for 15 minutes. Thereafter the reaction was stopped by adding 1% H2SO4. Optical density was measured with a photometer and results were calculated by a program. Results multiplied with dilution = Titer

Claims

1. A non-pyrogenic Staphylococcal superantigen vaccine comprising a combination of detoxified Staphylococcal superantigen vaccine antigens which are genetically modified toxins that incorporate detoxifying mutations in its T cell receptor binding region and MHC Class II binding region, wherein the combination comprises at least the vaccine antigens Staphylococcal Exotoxin B (SEB) and any one or both of Staphylococcal Exotoxin C (SEC) and Staphylococcal toxic shock syndrome toxin-1 (TSST-1).
2. The vaccine of claim 1 , wherein the combination comprises or consists of any one of: a) SEB and TSST-1 ; b) SEB and SEC; or c) SEB, TSST-1 and SEC.
3. The vaccine of claim 1 or 2, wherein the SEB comprises a wild-type SEB amino acid sequence that is modified to comprise deletion of at least two amino acids in the T cell receptor binding region between amino acid positions (aa) 21 to 25, and to further comprise at least one point mutation in the MHC Class II binding region, wherein said at least one point mutation comprises an amino acid substitution at a position selected from the group consisting L45, Q43, or F44, preferably L45R, Q43P, F44P, or F44S, wherein the wild-type SEB toxin amino acid sequence is of SEQ ID NO:1 , or of any other wildtype SEB toxin sequence.
4. The vaccine of any one of claims 1 to 3, wherein: a) the SEC comprises a wild-type SEC amino acid sequence that is modified to comprise deletion of at least two amino acids in the T cell receptor binding region between aa 21 to 25, and to further comprise at least one point mutation in the MHC Class II binding region, wherein said at least one point mutation comprises an amino acid substitution at a position selected from the group consisting L45, Q43, or F44, preferably L45R, Q43P, F44P, or F44S, wherein the wild-type SEC toxin amino acid sequence is of SEQ ID NO:2, or of any other wild-type SEC toxin sequence; b) the TSST-1 comprises a wild-type TSST-1 amino acid sequence that is modified to comprise at least one point mutation in the MHC Class II binding region consisting of G31 , L30, and S32, preferably comprising deletion or substitution G31 , and at least one point mutation in the T cell receptor binding region consisting of the amino acid region of E132 to Q139 and G16, wherein the wild-type TSST-1 toxin amino acid sequence is of SEQ ID NO:3, or of any other wild-type TSST-1 toxin sequence.
5. The vaccine of any one of claims 1 to 4, comprising the vaccine antigens and a pharmaceutically acceptable carrier, preferably comprising an adjuvant.
6. The vaccine of claim 5, wherein the adjuvant is selected from the group consisting of an insoluble metal salt, a glucopyranosyl Lipid A adjuvant, adjuvant systems which are stimulants of innate immunity, such as AS01 , AS03, or AS04, MF59, a TCR stimulant such as a toll-like receptor agonist or CpG oligonucleotides, preferably alum, aluminum hydroxide, or aluminum phosphate.
7. The vaccine of any one of claims 1 to 6, wherein the combination further comprises one or more Staphylococcal toxoid antigens, preferably selected from the group consisting of alpha-hemolysin, gamma-hemolysin, beta-hemolysin, and staphylococcal exotoxins or enterotoxins, such as enterotoxin A (SEA), I (SEI), and K (SEK).
8. The vaccine of any one of claims 1 to 7, wherein the combination of vaccine antigens is a mixture, fusion, or a complex comprising said vaccine antigens bound to each other and/or bound to a carrier, thereby forming the complex.
9. A vaccine kit of parts comprising the combination of vaccine antigens set forth in any one of claims 1 to 8, wherein one or more of the vaccine antigens are provided in separate containments.
10. The vaccine of any one of claims 1 to 8, for use in the prevention, treatment against or therapy of a staphylococcal toxin or superantigen-expressing bacterial infection, and/or a disease condition directly or indirectly mediated by exposure to Staphylococcal superantigen toxins, Staphylococcus infection and/or contamination.
11. The vaccine for use according to claim 10, wherein a subject is immunized with the vaccine, who is at risk of staphylococcal disease condition and/or complications, preferably wherein a) the staphylococcal disease condition and/or complication are due to primary and/or secondary forms of inheritable and/or acquired immunodeficiency disorders and/or immune-modulatory disorders; and/or b) the subject is a subject who is likely exposed to staphylococcal infection, such firefighters, medical staff, or of regional populations of areas where fire, flooding, or hurricanes are frequent.
12. The vaccine for use according to claim 10 or 11 , wherein: a) a subject is immunized with a combination of at least SEB and TSST-1 , optionally combined with SEC, to prevent a sepsis condition, preferably wherein the sepsis condition is sepsis, septic shock or toxic shock syndrome (TSS); and/or b) a subject is immunized with a combination of at least SEB and TSST-1 , optionally combined with SEC, to prevent Staphylococcal wound infective disorders, preferably wherein wound infection is upon burn, injuries, or surgical treatment; c) a subject is immunized with a combination of at least SEB and SEC, optionally combined with TSST-1 , to prevent Staphylococcal enteric disorders, preferably wherein the enteric disorder is enteritis or a digestive disorder resulting from Staphylococcal food poisoning; and/or d) a subject is immunized with a combination of at least SEB and TSST-1 , optionally combined with SEC, wherein the subject is at risk of or suffers from genetic susceptibility to Staphylococcus aureus bacteremia; and/or e) a subject is immunized with a combination of at least SEB and TSST-1 , optionally combined with SEC, wherein the subject is at risk of or suffers from acute multisystem inflammatory disease of blood vessels or vasculitis and/or aneurism caused by Staphylococcus aureus, or Kawasaki syndrome.
13. The vaccine for use according to any one of claims 10 to 12, wherein a vaccine kit of parts is used to immunize the subject with the combination of vaccine antigens, whereby the all of the individual vaccine antigens are provided by one administration, or by two or more separate administrations, preferably by administering a mixture of two or more individual vaccine antigens, or by parallel or consecutive administration of two or more individual antigens, such as at two or more different sites or routes of administration.
14. Use of the vaccine of any one of claims 1 to 8, in a method of producing an antibody preparation comprising antibodies specifically recognizing the vaccine antigens.
15. Polyclonal antibody preparation obtainable by immunizing a subject with a vaccine of any one of claims 1 to 8, isolating polyclonal antibodies or a fraction of polyclonal antibodies comprising the antibodies specifically recognizing the Staphylococcal superantigen vaccine antigens, and formulating a preparation comprising said antibodies, wherein the antibodies are cross-reactive with wild-type Staphylococcal superantigen toxins.
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