EP1127132A1 - Geeignete peptide zur reduzierung des toxischen schock syndroms und von septischem schock - Google Patents

Geeignete peptide zur reduzierung des toxischen schock syndroms und von septischem schock

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Publication number
EP1127132A1
EP1127132A1 EP99970123A EP99970123A EP1127132A1 EP 1127132 A1 EP1127132 A1 EP 1127132A1 EP 99970123 A EP99970123 A EP 99970123A EP 99970123 A EP99970123 A EP 99970123A EP 1127132 A1 EP1127132 A1 EP 1127132A1
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EP
European Patent Office
Prior art keywords
group
amino acid
peptide
seq
independently selected
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99970123A
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English (en)
French (fr)
Inventor
Jason D. Bannan
Kumar Visvanathan
John B. Zabriskie
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Rockefeller University
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Rockefeller University
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Priority claimed from US09/335,581 external-priority patent/US7115268B1/en
Application filed by Rockefeller University filed Critical Rockefeller University
Publication of EP1127132A1 publication Critical patent/EP1127132A1/de
Withdrawn legal-status Critical Current

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to compositions and methods for protecting against, or reducing the severity, of toxic shock syndrome and septic shock from bacterial infections. More particularly it relates to peptides, which may be polymeric, and carrier-conjugates thereof, derived from homologous sequences of the family of staphylococcal and streptococcal pyrogenic toxins .
  • the peptides of the invention are useful to prevent, treat, or protect against the toxic effects of bacterial toxins, including most, if not all, of the staphylococcal and streptococcal pyrogenic toxins. These are also useful to induce serum antibodies and may also be useful in diagnostic assays.
  • the invention also relates to antibodies induced by the peptides and/or carrier-conjugates and their use to o prevent, treat, or protect against the toxic effects of bacterial toxins, including most, if not all, of the staphylococcal and streptococcal pyrogenic toxins.
  • the invention also relates to compositions and methods to protect against, or ameliorate the effects of, autoimmune diseases which are associated with, or are the result of, the presence of staphylococcal or streptococcal toxins .
  • the invention also relates to diagnostic assays and O kits to detect the presence of staphylococcal and streptococcal pyrogenic toxins, or antibodies thereto.
  • the invention also relates to isolated and purified nucleic acids encoding the peptides of the invention and transformed host cells containing those nucleic acids. 5
  • the pyrogenic exotoxins of Group A streptococci and the enterotoxins of Staphylococcus aureus, which are 0 also pyrogenic exotoxins, constitute a family of structurally related toxins which share similar biological activities (11, 13).
  • the staphylococcal and streptococcal pyrogenic exotoxins also share significant amino acid homology throughout their sequences (11, 19, 5
  • This pyrogenic exotoxin family contains nine main toxin types, and several allelic variants (subtypes) have been described.
  • Several studies have shown that the toxins share common motifs based on immunologic 0 cross reactivity between the toxins (26, 27) . They stimulate CD4 + , CD8 + and ⁇ + T cells by a unique mechanism. These toxins share the ability to bind the ⁇ chain variable region (V ⁇ ) elements on the lateral face 5 of the T cell receptor (TCR) and simultaneously bind to o the lateral face of the class II major histocompatibility complex (MHC) of antigen presenting cells (Figure 1) , causing an aberrant proliferation of specific T-cell subsets (3, 4, 12) .
  • This property of the toxins has labeled them as "superantigens" (36) since they do not interact with the MHC and TCR molecules in the manner of conventional antigens (14, 18) and produce a massive proliferation of T cells.
  • TCR TCR with MHC-II molecules by 5 superantigens causes a profound blastogenesis of lymphocytes and antigen-presenting cells.
  • the resulting stimulation of leukocytes leads to a significant increase in cytokine production.
  • Monocytes stimulated with bacterial superantigens produce the Thl cytokines IL-2 and IFN- ⁇ and the anti- inflammatory cytokines IL-10 and IL-1 receptor antagonist.
  • T cells activated by superantigen 5 stimulation produce IL-12.
  • Whole preparations of peripheral blood mononuclear cells containing lymphocytes and antigen-presenting cells elicited a wide range of inflammatory cytokines in significant amounts.
  • the generation of monocyte cytokines such as IL-1, IL-6, 0 TNF- ⁇ , and TNF- ⁇ was dependent on the presence of T cells. (73) .
  • Costimulatory molecules important in conventional immune responses also play a significant role in the 5 o response of immune cells to superantigens.
  • the costimulatory T cell antigen, CD28, and its corresponding ligand on MHC- II -bearing cells, B7 contribute to superantigen mitogenicity . (74, 75) .
  • Other costimulatory molecules, such as LFA-l/ICAM-1 and VLA-4/VCAM-1 also contribute to the activation of immune cells by superantigens. (76, 77) .
  • These immunostimulatory activities of superantigens are crucial to their ability to cause injury to the host. O The bacterial toxins cause a variety of syndromes in humans .
  • Staphylococcal enterotoxins have been implicated in staphylococcal food poisoning (26) , as well as toxic shock like syndromes (1) .
  • SEPE staphylococcal enterotoxins
  • the streptococcal pyrogenic exotoxins have been implicated in causing the symptoms of scarlet 0 fever and toxic shock like syndrome (8, 20, 30) .
  • the sequences of three members of this family are known: SPEA, SPEC, and SSA (5, 23, 35) .
  • Toxic shock syndrome toxin (TSST-1) from S . aureus shares similar biological activity with the SE's and 5 SPE's, however amino acid sequences of this toxin are significantly different from these two classes of toxins (2) .
  • Structural analysis suggests that, despite the differences in amino acid composition, the overall 0 topology of TSST-1 and the SE/SPE family of toxins is similar (41) .
  • the molecular structure of SE's and SPE's has been determined by various methods . Reviews concerning the molecular structures are available (19, 62) .
  • Molecular evolution studies of the SE/SPE family of toxins suggests that the toxins can be grouped into two main clades(34) .
  • TSST-1 soluble proteins of approximately 230 amino acids and have a central disulphide loop. In contrast TSST-1 has only 194 amino acids and does not possess any cysteines .
  • Residues determining TCR V ⁇ specificity appear to be located within the carboxy-terminus of the SE/SPE toxins (59) , while residues critical for MHC-II binding appear to be located in the amino-terminal region, and the central portion of the molecule near the disulfide loop
  • the disulfide loop and adjacent highly conserved sequences contribute to the structural integrity of the toxins, and serve to bring the TCR and MHC binding regions in functional proximity to each other (65) .
  • the SEs are named for their ability to induce gastrointestinal illnesses upon oral intake of a few micrograms of the toxin. The clinical effect appears in 2 to 4 hours and is manifested by nausea and diarrhea. These symptoms appear to be caused by leukotrienes and histamine released from mast cells. Additionally, both the staphylococcal and streptococcal exotoxins are implicated in gram-positive shock.
  • superantigen-related septic shock appears to be primarily mediated by tumor necrosis factor (TNF) - ⁇ and interleukin (IL) 12, the contribution of other cytokines cannot be discounted. (78, 79, 80).
  • the physiologic response to superantigens is similar to septic shock induced by gram-negative endotoxin (lipopolysaccharide, LPS) .
  • LPS and superantigens can work synergistically to produce lethal toxic shock. (81, 82, 83) .
  • Toxic shock syndrome can be exacerbated by the synergistic effects of TSST-1 with the SE/SPE family of toxins. (84, 85) .
  • Superantigen stimulation of immune cells can exacerbate autoimmune syndromes by causing the expansion of autoreactive T cell subsets, upregulation of MHC-II expression, and the potentiation of cytotoxic T cell response (86, 87, 88, 89, 90, 91) .
  • Toxic shock syndrome is a specific syndrome caused by either the Stapylococcal or Streptococcal organisms. It is specifically caused by the toxins produced by these bacteria. Clinically it often occurs in young women and children and is characterized by a raised temperature, low blood pressure, a rash that eventually leads to skin loss especially on the palms and the soles and multi-organ involvement.
  • Septic Shock on the other hand involves both gram negative as well as gram positive organisms, occurs in all groups of patients especially the elderly and post- surgical. It has similar symptoms except for the lack of a skin losing rash. Both diseases have a high mortality-however there are many more cases of septic shock as compared to toxic shock.
  • the term "septic shock” is used herein to describe hypotension and organ failure associated with bacterial infections.
  • Toxic shock like syndrome is the term previously used to describe the syndromes caused by staphyloccal and streptococcal pyrogenic bacterial exotoxins other than toxic shock syndrome toxin (TSST-1) from S . aureus .
  • TSST-1 toxic shock syndrome toxin
  • toxic shock syndrome is used to describe the syndromes caused by TSST-1 and the other pyrogenic exotoxins, and is the terminology used hereinafter.
  • Toxic shock syndrome can be exacerbated by the synergistic effects of TSST-1 with the enterotoxin/pyrogenic toxin family of toxins (9, 25). Gram negative bacterial endotoxin and the pyrogenic o toxins can work synergistically to produce intractable shock (17, 30) .
  • LPS lipopolysaccharide
  • IL-1 Interleukin-1
  • Necrosis Factor- ⁇ (TNF- ⁇ ) (49) . Accordingly, therapeutic strategies for septic shock have centered on the neutralization of LPS or LPS-induced cytokines (64) . Unfortunately, trials using either monoclonal antibodies directed against part of the LPS molecule or the use of
  • CD14 soluble receptors have not been very promising (45) .
  • the reasons for these failures might be: 1. The type of patient selected (many were already in irreversible shock) . 2. The monoclonal antibody did not
  • Toxic shock syndrome and septic shock are still among the most life threatening syndromes affecting humans. It is estimated that approximately 20,000 cases
  • LPS and peptidoglycan interact with macrophages.
  • the superantigens interact with T cells.
  • target cells are induced to release large amounts of cytokines .
  • Exposure to gram- negative endotoxin produces a state of macrophage hyperesponsiveness on subsequent stimulation (92) .
  • a similar state is seen with monocytes in septic shock.
  • the present invention relates to the identification of consensus sequences derived from two conserved regions of the staphylococcal enterotoxins and streptococcal pyrogenic toxins (hereinafter called “region 1" and “region 2”) and the discovery that compositions comprising amino acid sequences based on these two conserved regions of the staphylococcal enterotoxins and streptococcal pyrogenic exotoxins are capable of inducing antibodies which react with a variety of staphylococcal and streptococcal pyrogenic exotoxins and are also capable of ameliorating or preventing diseases related to the deleterious effects of these toxins .
  • the invention also relates to compositions and methods for preventing and treating diseases related to the release of certain pyrogenic exotoxins from bacteria .
  • This invention provides peptides comprising amino acid sequences which reduce, inhibit or eliminate the deleterious effects of bacterial toxins and/or are capable of inducing antibodies that reduce, inhibit or eliminate the deleterious effects of bacterial toxins, such as those of staphylococcus and a variety of streptococci.
  • Antibodies may be induced by administration of a pharmaceutical composition and/or vaccine containing a composition comprising a peptide derived from one or both of the two conserved regions described herein, or a structurally and/or immunologically related antigen.
  • amino acid sequences provided by this invention are sufficiently common to all members of this family of pyrogenic exotoxins to be useful for eliciting antibodies which are cross-reactive with toxins derived from various bacteria.
  • the amino acid sequences provided by this invention are also useful for new methods of preventing and treating symptoms associated with the bacterial release of the staphylococcal enterotoxins and the streptococcal pyrogenic exotoxins.
  • Such methods include, for example, administering to an individual who is suspected of having an infection or developing and/or having a toxic or septic reaction, a compound comprising at least one of the consensus amino acid sequences of this invention in an amount sufficient to inhibit superantigen stimulation of T-cells, preferably an amount sufficient to reduce, inhibit or eliminate the deleterious effects of the exotoxins.
  • Such methods also include administering to an individual at risk of infection or developing a toxic reaction to the exotoxins at least o one of the consensus amino acid sequences of this invention in an amount sufficient to elicit the production of antibodies to the exotoxins.
  • an individual at risk for developing toxic or septic shock syndrome or an individual with symptoms of toxic shock syndrome or septic shock may be treated by administering to such individual a composition comprising at least one of the peptides of this invention and/or carrier- j 0 conjugate thereof.
  • an individual at risk for developing toxic shock syndrome or septic shock may be treated
  • compositions of this invention by administering to such individual antibodies which have been generated in a mammal immunized with at least one of the compositions of this invention.
  • Vaccines and pharmaceutical compositions comprising
  • At least one of the consensus amino acid sequences and a physiologically acceptable carrier and optionally an adjuvant are also part of this invention.
  • Another object of the invention is to provide antibodies induced by the peptides and carrier-
  • antibodies may be used to prevent, treat, or protect against the toxic effects of most, if not all, of the staphylococcal and streptococcal pyrogenic exotoxins.
  • the antibodies may be used to prevent, treat, or protect against the toxic effects of most, if not all, of the staphylococcal and streptococcal pyrogenic exotoxins.
  • the antibodies may be used to prevent, treat, or protect against the toxic effects of most, if not all, of the staphylococcal and streptococcal pyrogenic exotoxins.
  • the antibodies may be used to prevent, treat, or protect against the toxic effects of most, if not all, of the staphylococcal and streptococcal pyrogenic exotoxins.
  • the antibodies may be used to prevent, treat, or protect against the toxic effects of most, if not all, of the staphylococcal and streptococcal pyrogenic exotoxins.
  • antibodies are also useful in diagnostic assays 35 and kits to detect the presence of staphylococcal and streptococcal pyrogenic exotoxins and to aid in the diagnosis of diseases related to the presence of those toxins .
  • Another object of the invention is to provide isolated and purified nucleic acids encoding the amino acid sequences of the invention, as well as suitable expression systems, vector components and transformed host cells containing those nucleic acids.
  • FIG. 1 Schematic diagram of the interaction between a T cell receptor, superantigen, and a class II MHC molecule.
  • Superantigens bind to common sequences in class II MHC molecules and T cell receptors that lie outside the normal antigen-binding sites. T cell activation by superantigens is not limited by the antigenic specificity of the T cell.
  • Figure 2. Diagram of the "two hit" model of septic shock.
  • FIG. 3 Comparison of the synthetic peptide sequences to conserved regions 1 and 2 of the staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE, and SEH) , and streptococcal pyrogenic exotoxins (SPEA, SPEC, and SSA) .
  • Staphylococcal toxic shock syndrome toxin 1 (TSST-1) was compared with the region 2 peptide. Numbers represent the residue positions as a reference to where these regions exist in the whole toxin molecules. Sequences are from either the Swiss protein or GenBank databases under the following accession numbers. Swiss protein: SPEA, P08095; SPEC, P13380;
  • SEA P13163; SEB, P01552; SEC, P01553; SED, P20723; SEE, P12993.
  • GenBank SEH, U11702; SSA, L29565; TSST1, J02615.
  • FIG. 4 ELISA titers of antibodies from rabbits immunized with polymeric peptide #6348.
  • the peptide was diluted so that it was delivered to each well to give a final concentration of 2 ⁇ g/100 ⁇ l .
  • the serum was then diluted to 1:1,000; 1:10,000; 1:100,000; 1:500,000; and 1:1,000,000 and 100 ⁇ l of each dilution of serum was placed in each well. Experiments were run in triplicate for each dilution of serum. Note the 1 log higher titers of rabbit #443 serum as compared to rabbit #442 serum. Cut off readings were at O.D. 0.6.
  • FIG. 5 12% SDS PAGE gel immunoblot of a variety of staphylococcal and streptococcal toxins developed with the anti-peptide 6348 antibody. Note bands of correct molecular weight (M.W.) of each toxin identified by the anti-peptide antibody. Lane 1: SPEA, lane 2: SEA, lane 3: SEB, lane 4: SED, lane 5: SEE, lane 6: SEC and lane 7 TssT-1. Note bands at appropriate M.W. in lanes 1-4. Fainter bands are seen in lanes 5 and 7.
  • FIG. 6 Bar graphs of blastogenesis assays of human mononuclear cell populations stimulated by various toxins in the presence of normal rabbit serum and anti-peptide 6348 serum. Note the marked inhibition of SEB, SEC, SEE, SPEA and SPEC by the anti-peptide antibody. Less, but definite, inhibition of SEA by the anti-peptide antibody was also seen.
  • Figure 7 Bar graphs of blastogenesis assays of human mononuclear cell populations stimulated by SEB in the presence of (A) peptide 6343 (i.e., CMYGGVTEHEGN, SEQ ID N0:3),(B) peptide 6346 (i.e., CGKKNVTVQELDYKIRKYLVDNKKLYGC , SEQ ID NO: 6)) and (C) peptide 6348 (i.e.,
  • PBMC peripheral blood mononuclear cells
  • Figure 9 Inhibition of SPEG, SPEH, and SPEZ toxin blastogenesis of peripheral blood mononuclear cells (PBMC) by the 6343 peptide.
  • 2 x 10 5 PBMC were stimulated with either 2 ⁇ g of the indicated toxin or a combination of 2 ⁇ g of the toxin with the indicated amount of the 6343 peptide. These were incubated for 72 hours and the results were measured via tritiated thymidine incorporation. CPM represents counts per minute. Normal represents normal media. Note that the single peptide (6343) inhibited the superantigens SPEG, SPEH and SPEZ. Figure 10.
  • (A) Binding of peptide 6343 to the
  • Binding of peptide 6343 is indicated by the green color.
  • Binding of anti-MHC peptide is indicated by the red color.
  • C Combined picture showing stippled pattern of red and green color. Binding of peptide 6343 is indicated by the green color and binding of anti-MHC peptide is indicated by the red color.
  • the first consensus sequence (“GCG consensus #1”) identified by the Motifs program has the amino acid sequence YGG (LIV) TXXXXN, which is rewritten herein as YGGX X TX 2 X 3 X 4 X 5 N (SEQ ID NO:l), wherein X ⁇ is selected from the group consisting of L, I, or V; and X 2 , X 3 , X 4 and X 5 are each independently selected from the group consisting of any amino acid.
  • This pattern is present in the staphylococcal enterotoxins and streptococcal pyrogenic exotoxins, but not in TSST-1. The sequence begins immediately at the COOH-terminal side of the cysteine loop.
  • the second consensus sequence (“GCG consensus #2”) identified by the Motifs program has the amino acid sequence KXX(LIV)XXXX(LIV)DXXXRXXLXXXXX(LIV) Y, rewritten herein as KX 6 7 X 8 9 X l ⁇ X ll l2 X l3 DX l 4Xl5X l 6RX l 7 l 8l J X l 9X2 ⁇ 2 -X 2 2 23 X24Y ( SEQ
  • X 8 , X 13 and X 24 are each independently selected from the group consisting of L, I and V, and Xs / X7 / X9 / XlO / ll / Xl2 / Xl4 / Xl5 / lS / Xl7 , Xl8 / l9 i X 2 0
  • X 21 , X 22 and X 23 are each independently selected from the group consisting of any amino acid. This pattern is present in the staphylococcal enterotoxins, streptococcal pyrogenic exotoxins, and TSST-1.
  • One object of the invention is to provide compositions comprising peptides comprising amino acid sequences based on these two conserved regions of the staphylococcal enterotoxins and streptococcal pyrogenic toxins. These peptides may be used for eliciting an immunogenic response in mammals, including responses which provide protection against, or reduce the severity, of toxic shock or septic shock from staphylococcal or streptococcal infections.
  • peptides may also be useful to protect against, or ameliorate the effects of, autoimmune diseases which are associated with, or are the result of, the presence of staphylococcal or streptococcal pyrogenic exotoxins .
  • These peptides are also useful in diagnostic assays and kits to detect the presence of antibodies to staphylococcal and streptococcal pyrogenic exotoxins and to aid in the diagnosis of diseases related to the presence of those toxins .
  • the peptides of the invention are those derived from either one or both of the following two consensus sequences :
  • YGGX ⁇ TX 2 X 3 X 4 X 5 N (SEQ ID NO:l), wherein Xi is selected from the group consisting of L, I, or V; and X 2 , X 3 , X 4 and X 5 are each independently selected from the group consisting of any amino acid.
  • X 2 ⁇ , X 22 and X 23 are each independently selected from the group consisting of any amino acid.
  • a preferred consensus sequence of the invention from Region 1 has the amino acid sequence X 25 X 26 YGGX 1 TX 2 X 3 X 4 X 5 N (SEQ ID NO: 28), wherein X x is selected from the group consisting of L, I, and V; X 2 X 4 and X 5 are each independently selected from the group consisting of any amino acid; and X 3 , X 25 and X 2 6 are each independently selected from the group consisting of any amino acid and of no amino acid; but preferably Xi is selected from the group consisting of I and V; X 2 is selected from the group consisting of L, E, K, P and N; X 3 is selected from the group consisting of H and A and no amino acid; X 4 is selected from the group consisting of D, N, E, Q, and H; X 5 is selected from the group consisting of N, G, S, and R; X 25 is selected from the group consisting of C and Y and no amino acid; and
  • a preferred consensus sequence of the invention f rom region 2 ( consensus #2 a ) has the amino acid sequence : KX s X 7 X 8 X 9 X 1 oX ⁇ Xi2X ⁇ 3 DX 1 4Xi5X ⁇ sRXi7Xi8X27X ⁇ 9 X2o 2i X 22 X 23 X 24 Y ( SEQ ID NO : 29 ) , wherein X 8 , X 13 and X 24 are each independent ly se lected f rom the group cons i st ing of L , I and V ; Xg , X7 , Xg , X10 , Xll , Xl 2 , Xl4 , Xl5 / Xl6 / Xl7 r Xl8 Xl9 /
  • X20 X21J X22/ and X 23 are each independently selected from the group consisting of any amino acid; and X 27 is selected from the group consisting of L and Y; but preferably X e is selected from the group consisting of K and D; X 7 is selected from the group consisting of N, K, S, E, M, I and Q; X 8 is selected from the group consisting of L and V; X 9 is selected from the group consisting of T and A; X 10 is selected from the group consisting of V, A, L, F and I; X lx is selected from the group consisting of Q and S; X i2 is selected from the group consisting of E and T; X 13 is selected from group consisting of L and I; X i4 is selected from the group consisting of L, Y, I, A, F and C; X 15 is selected from the group consisting of Q, L, K and E; X 16 is selected from the group consisting of A, T, I and V;
  • Table 1 lists the amino acids that are found at each of the variable positions in the sequences shown in Figure 3, and the number of times they appear at that position:
  • X 13 and X 4 may each independently be selected from the group consisting of L, I and V; X 2 , X 3 , X 4 , X 5 , X s , X 7 ,
  • X 23 , X 25 and X 26 may each independently be any amino acid; X 3 , X 25 and X 26 may also each independently be no amino acid; and X 27 is selected from the group consisting of L and Y.
  • the amino acids present at the positions Xi to X 27 in the toxins shown in Figure 3 (and listed in Table 1) are preferred for those positions, and the amino acids present most often at those positions in the toxins shown in Figure 3 (and listed in Table 1) are more preferred.
  • H histidine
  • A alanine
  • inosine (I) is used at position X 16 instead of the more frequently found alanine (A) .
  • the preferred consensus is larger (consensus #la) , and usually includes a C in the first position (X 5 ) .
  • the second residue (X 26 ) is most often a M, but this can vary.
  • H is the most highly conserved.
  • the eleventh residue (X 5 ) is most often a G.
  • the preferred consensus (consensus #2a) is much more highly conserved than suggested by the GCG program, especially if one excludes TSST-1 sequences from consideration, as follows:
  • the second position (X e ) is more highly conserved than suggested, being almost exclusively a K;
  • the fourth residue (X 8 ) is always a V followed exclusively by a T in the fifth position (X 9 ) ;
  • the sixth position (Xj. 0 ) is somewhat variable; but the seventh position (X n ) is always a Q, followed by E (X i2 ) •
  • the next position is almost always an L (X ⁇ 3 ) , and the second to last position (X 4 ) is almost always an L.
  • X ⁇ is V or I, preferably V;
  • X 2 is L, E, K, P or N, preferably E or L;
  • X 4 is D , N, E , Q or H, preferably E .
  • Consensus #2b is D , N, E , Q or H, preferably E .
  • X 7 is N, K, S , E , M, I or Q , preferably N;
  • Xio is V, A, L, F or I , preferably V;
  • X 14 is L , Y, I , A, F or C , preferably Y ;
  • Xi 5 is Q, L, K or E, preferably K;
  • X 16 is A, T, I or V, preferably I,
  • Xi 7 is R, H, N or K, preferably K;
  • X 18 is Y, F, I, L or Q, preferably Y;
  • X19 is Q, V, I, H, S, T or M, preferably V;
  • X 20 is E, K, N, D, G, S or Q, preferably D;
  • X 21 is K, N, D, R or I, preferably N;
  • X 22 is Y, K, L, F or H, preferably K;
  • X 23 is N, K, G or Q, preferably K.
  • X 27 is L or Y, preferably L.
  • Peptides exemplified herein are CMYGGVTEHEGN (SEQ ID NO: 3), CMYGGVTEHEGNGC* (SEQ ID NO: 5), KKNVTVQELDYKIRKYLVDNKKLY (SEQ ID NO : 4), CGKKNVTVQELDYKIRKYLVDNKKLYGC* (SEQ ID NO: 6),
  • CMYGGVTEHEGNKKNVTVQELDYKIRKYLVDNKKLY (SEQ ID NO : 7) and CMYGGVTEHEGNKKNVTVQELDYKIRKYLVDNKKLYGC* (SEQ ID NO : 8), wherein an asterisk indicates that the peptide is a randomly cross-linked polymer.
  • the exemplified polymer o peptides are at least 6,000 to 8,000 daltons .
  • the average size of the exemplified polymer peptides is about 12,000 to 15,000 daltons. Small peptides and/or contaminants may be removed by dialysis or other methods available in the art. Similarly, larger aggregates may be removed using, e.g., a 0.25 micron filter, which can also be used to sterilize the peptides.
  • the preferred peptides of the invention are those
  • the preferred peptides of this invention are not toxic, but toxic peptides maybe useful in this invention, for example, in eliciting antibodies in a non-human system.
  • the most preferred peptides of the invention do not
  • the present invention encompasses monomers of the peptides derived from either one or both of the two
  • These monomers may comprise one or more sequences derived from either region 1 or region 2 or both, such as consensus sequences #1 and #2, preferably consensus sequences #la and/or #2a, more preferably consensus sequences #lb and/or #2b, most preferably one or more of the exemplified consensus sequence peptides. If the monomer contains more than one consensus sequence, these sequences may be immediately adjacent to each other or separated by a linker. In addition, different orientations of the peptides are within the scope of this invention. Furthermore, the order of the consensus peptides within the full peptide may be variable.
  • the present invention also encompasses homogeneous or heterogeneous polymers of the peptides disclosed herein (e.g., concatenated, cross-linked and/or fused identical peptide units or concatenated, cross-linked and/or fused diverse peptide units) , and mixtures of the peptides, polymers, and/or conjugates thereof.
  • homogeneous or heterogeneous polymers of the peptides disclosed herein e.g., concatenated, cross-linked and/or fused identical peptide units or concatenated, cross-linked and/or fused diverse peptide units
  • Linkers useful in the invention may, for example, be simply peptide bonds, or may comprise amino acids, including amino acids capable of forming disulfide bonds, but may also comprise other molecules such as, for example, polysaccharides or fragments thereof.
  • sequences derived from consensus region 1 and consensus region 2 may be immediately adjacent to each other, linked by peptide bonds, (see, e.g., SEQ ID NO: 7) and/or connected via amino acid linkers capable of forming di-sulfide bonds via cysteine residues (see, e.g., SEQ ID NO: 8) .
  • the sequences of region 1 and region 2 are separated by about 27 amino acids.
  • the linkers are additional amino acids, they are most preferably 1 to 27 amino acids in length, although longer linkers may also be used in accordance with this invention.
  • linkers for use with this invention may be chosen so as to contribute their own immunogenic effect which may be either the same, or different, than that elicited by the consensus sequences of the invention.
  • linkers may be bacterial antigens which also elicit the production of antibodies to infectious bacteria.
  • the linker may be a protein or protein fragment of an infectious bacteria, or a bacterial polysaccharide or polysaccharide fragment .
  • a peptide of the invention includes any substituted analog or chemical derivative of a peptide derived from one or both of the two consensus regions described herein, most preferably of the exemplified peptides described herein, so long as the peptide is capable of inhibiting binding of staphylococcal and streptococcal pyrogenic exotoxins to the MHC complex; inhibiting blastogenesis of human mononuclear cells in the presence of any one of the toxins; eliciting the production of antibodies capable of binding to most of the staphylococcal and streptococcal pyrogenic exotoxins; or reacting with (i.e., specifically binding to) antibodies that react with most of the staphylococcal and streptococcal pyrogenic exotoxins.
  • a peptide can be subject to various changes that provide for certain advantages in its use.
  • D amino acids can be substituted for L amino acids to increase in vivo stability of the peptides, while still retaining biological activity. See, e.g., Senderoff et al . (1998)
  • peptides having at least one D amino acid on the amino terminal and/or carboxy terminal end of the molecule and which retain biological activity are considered part of the invention.
  • retro- inverso peptides which contain one or more of the amino acid sequences of the invention and which retain biological activity are also considered part of the invention.
  • the peptides of the invention are useful for providing active immunization for the prevention of disease related to the deleterious effects of staphylococcal and streptococcal pyrogenic exotoxins and for preparation of antibodies as a passive immunization therapy.
  • the peptides When used to prepare antibodies, the peptides are designed to induce antibodies which react with a variety of staphylococcal and streptococcal pyrogenic exotoxins (preferably with at least two, more preferably with at least four, and most preferably with at least seven of the pyrogenic exotoxins) for use in therapy to increase resistance to, prevent and/or treat toxic shock syndrome and septic shock.
  • staphylococcal and streptococcal pyrogenic exotoxins preferably with at least two, more preferably with at least four, and most preferably with at least seven of the pyrogenic exotoxins
  • the peptides may also be useful to protect against, or ameliorate the effects of, autoimmune diseases which are associated with, or are the result of, the presence of staphylococcal or streptococcal exotoxins.
  • the peptides of the invention will also be useful in diagnostic tests for detecting antibodies to staphylococcal and streptococcal pyrogenic exotoxins.
  • the peptide may be mixed with an adjuvant.
  • the peptide also may be bound to a non-toxic non-host protein carrier to form a conjugate or it may be bound o to a saccharide carrier and/or a non-toxic non-host protein carrier to form a conjugate.
  • the molecular weight of the peptide monomers having one consensus sequence of the invention range from about 1000 to 5000 daltons. Such lower molecular weight species of the invention are useful themselves to inhibit superantigen induced T cell proliferation and/or reduce, inhibit or eliminate the deleterious effects of bacterial exotoxins in vivo, either when used alone or
  • J0 in combination with another form of therapy, e.g., anticytokine antibodies.
  • another form of therapy e.g., anticytokine antibodies.
  • Such lower molecular weight species of the invention may also be useful as immunogens themselves or, more preferably, may be used as haptens conjugated
  • a carrier molecule such as, for example, a protein.
  • a carrier molecule such as, for example, a protein.
  • the molecular weight of the peptide alone, or when conjugated to a carrier, or in the presence of an adjuvant, is related to its
  • the peptide may vary in molecular weight in order to enhance its antigenicity or immunogenicity.
  • the molecular weight of the peptide, in polymeric form is greater than about 6000 to 8000 daltons, with an average
  • the total size of the peptide is only limited to its ability to be physiologically tolerated.
  • the invention also relates to isolated and purified
  • nucleic acid molecules which code for the peptides of the invention to produce the encoded peptides.
  • the encoded peptides may be monomers, polymers or linked to other peptide sequences (e.g., they may be fusion proteins) .
  • Other features of the invention include vectors which comprise the nucleic acid molecules of the invention operably linked to promoters, as well as cell lines, such as prokaryotic (e.g., E_;_ coli) and eukaryotic (e.g., CHO and COS) cells transfected with the nucleic acid molecules of the invention.
  • Vectors and compositions for enabling production of the peptides in vivo, i.e., in the individual to be treated or immunized, are also within the scope of this invention.
  • the nucleic acids encoding the peptides of the invention can be introduced into a vector such as a plasmid, cosmid, phage, virus or mini -chromosome and inserted into a host cell or organism by methods well known in the art.
  • the vectors containing these nucleic acids can be utilized in any cell, either eukaryotic or prokaryotic, including mammalian cells (e.g., human (e.g., HeLa), monkey (e.g., COS), rabbit (e.g., rabbit reticulocytes) , rat, hamster (e.g., CHO and baby hamster kidney cells) or mouse cells (e.g., L cells), plant cells, yeast cells, insect cells or bacterial cells (e.g., E ⁇ coli) .
  • mammalian cells e.g., human (e.g., HeLa), monkey (e.g., COS), rabbit (e.g., rabbit reticulocytes) , rat, hamster (e.g., CHO and baby hamster kidney cells) or mouse cells (e.g., L cells), plant cells, yeast cells, insect cells or bacterial cells (e.g., E ⁇ coli) .
  • mammalian cells
  • the vectors which can be utilized to clone and/or express these nucleic acids are the vectors which are capable of replicating and/or expressing the nucleic acids in the host cell in which the nucleic acids are desired to be replicated and/or expressed. See, e.g., F. Ausubel et al . , Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley- Interscience (1992) and Sambrook et al . (1989) for examples of appropriate vectors for various types of host cells. Strong promoters compatible with the host into which the gene is inserted may be used. These promoters may be inducible. The host cells containing these nucleic acids can be used to express large amounts of the protein useful in pharmaceuticals, diagnostic reagents, vaccines and therapeutics .
  • the nucleic acids could be used, for example, in the production of peptides for diagnostic reagents, vaccines and therapies for pyrogenic exotoxin related diseases.
  • vectors expressing high levels of peptide can be used in immunotherapy and immunoprophylaxis, after expression in humans.
  • Such vectors include retroviral vectors and also include direct injection of DNA into muscle cells or other receptive cells, resulting in the efficient expression of the peptide, using the technology described, for example, in Wolff et al . , Science 247:1465-1468 (1990), Wolff et al . , Human Molecular Genetics 1(6) :363-369 (1992) and Ulmer et al . , Science 259:1745-1749 (1993). See also, for example, WO 96/36366 and WO 98/34640.
  • antibodies ar e provided which react with peptides of the invention, as well as a variety of staphylococcal and streptococcal pyrogenic exotoxins (preferably with at least two, more preferably with at least four, and most preferably with at least seven of the pyrogenic exotoxins) .
  • staphylococcal and streptococcal pyrogenic exotoxins preferably with at least two, more preferably with at least four, and most preferably with at least seven of the pyrogenic exotoxins.
  • These antibodies will be useful for passive immunization therapy to increase resistance to or prevent toxic shock syndrome or septic shock or other diseases related to the presence of bacterial pyrogenic exotoxin.
  • the antibodies may also be useful to protect against, or ameliorate the effects of, autoimmune diseases which are associated with, or are the result of, the presence of staphylococcal or streptococcal pyrogenic exotoxins.
  • the antibodies of the invention will also be useful in diagnostic tests and kits for detecting the presence of staphylococcal and streptococcal pyrogenic exotoxins . These uses are discussed in more detail below.
  • the peptides of the invention may be prepared by synthetic methods or by recombinant DNA methods, as known in the art and as described herein.
  • compositions of this invention contain a pharmaceutically and/or therapeutically effective amount of at least one peptide and/or carrier thereof, antibody, or nucleic acid encoding a peptide of this invention.
  • the effective amount or peptide per unit dose is an amount sufficient to inhibit T-cell proliferation by staphylococcal and/or streptococcal pyrogenic exotoxins.
  • the effective amount of peptide per unit dose is an amount sufficient to prevent, treat or protect against the toxic effects of bacterial toxins, including diarrhea and/or cardiopulmonary depression or lethal shock.
  • the effective amount of peptide per unit dose depends, among other things, on the species of mammal inoculated, the body weight of the mammal and the chosen inoculation regimen, as is well known in the art.
  • inocula for a human or similarly sized mammal typically contain peptide concentrations of 100 to 500 mgs/kg, body weight of the mammal per inoculation dose.
  • the route of inoculation of the peptide will be subcutaneous or intravenous.
  • the dose is administered at least once.
  • the pharmaceutical composition contains an effective, immunogenic, amount of peptide of the invention.
  • the effective amount of peptide per unit dose sufficient to induce an immune response depends, among other things, on the species of mammal inoculated, the body weight of the mammal and the chosen inoculation regimen, as well as the presence or absence of an adjuvant, as is well known in the art.
  • Inocula typically contain peptide concentrations of about 1 microgram to about 1000 micrograms per inoculation (dose) , preferably about 3 micrograms to about 100 micrograms per dose, most preferably about 5 micrograms to 50 micrograms. The use of higher amounts is 5 envisaged.
  • dose micrograms per inoculation
  • Example 1 rabbits were injected twice with 500 micrograms of polymeric peptide in the presence of adjuvant.
  • Example 5 an example in which the peptide is administered directly to prevent toxic or 0 septic shock, which may not be dependent on the production of antibodies, mice were injected twice with 1.5 mg of monomer peptide for a total of 3 mgs . Standard procedures to determine dose response relationships known to those skilled in the art may be 5 used to determine optimum doses of peptide to be used either to prevent or treat toxic or septic shock, or to raise antibodies for its prevention or treatment.
  • unit dose refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of active material (e.g., peptide, antibody or nucleic acid) calculated to produce the desired immunogenic effect in association with the required diluent.
  • active material e.g., peptide, antibody or nucleic acid
  • Inocula are typically prepared as a solution in a physiologically acceptable carrier such as saline, phosphate-buffered saline and the like to form an aqueous pharmaceutical composition.
  • a physiologically acceptable carrier such as saline, phosphate-buffered saline and the like to form an aqueous pharmaceutical composition.
  • the peptides of the invention are generally administered with a physiologically acceptable carrier or vehicle therefor.
  • a physiologically acceptable carrier is one that does not cause an adverse physical reaction upon administration and one in which the antibodies are sufficiently soluble and retain their activity to deliver a therapeutically effective amount of the compound.
  • the therapeutically effective amount and method of administration of a peptide of the invention may vary based on the individual patient, the indication being treated and other criteria evident to one of ordinary skill in the art.
  • a therapeutically effective amount of a peptide of the invention is one sufficient to attenuate the dysfunction without causing significant side effects such as non-specific T cell lysis or organ damage.
  • the route (s) of administration useful in a particular application are apparent to one or ordinary skill in the art.
  • Routes of administration of the peptides include, but are not limited to, parenteral, and direct injection into an affected site.
  • Parenteral routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal and subcutaneous.
  • the route of inoculation of the peptides of the invention is typically parenteral and is preferably intramuscular, sub-cutaneous and the like.
  • compositions of the peptides described above suitable for parenteral administration including, but not limited to, pharmaceutically acceptable sterile isotonic solutions.
  • pharmaceutically acceptable sterile isotonic solutions include, but are not limited to, saline and phosphate buffered saline for nasal, intravenous, intramuscular, intraperitoneal, subcutaneous or direct injection into a joint or other area.
  • a system for sustained delivery of the peptides of the invention may also be used.
  • a delivery system based on containing a peptide in a polymer matrix of biodegradable microspheres may be used (57) .
  • One such polymer matrix includes the polymer poly (lactide- co-glycolide) (PLG) .
  • PLG is biocompatible and can be given intravenously or orally.
  • the encapsulated protein is released by a complex process involving hydration of the particles and drug dissolution. The duration of the release is mainly governed by the type of PLG polymer used and the release of modifying excipients (44) .
  • the dose is administered at least once.
  • at least one booster dose may be administered after the initial injection, preferably at about 4 to 6 weeks after the first dose, in order to increase the antibody level .
  • Subsequent doses may be administered as indicated.
  • antibody titers may be determined. In most instances it will be sufficient to assess the antibody titer in serum or plasma obtained from such an individual. Decisions as to whether to administer booster inoculations or to change the amount of the composition administered to the individual may be at least partially based on the titer.
  • the titer may be based on either an immunobinding assay which measures the concentration of antibodies in the serum which bind to a specific antigen, i.e. peptide or toxin; or bactericidal assays which measure the ability of the antibodies to participate with complement in killing bacteria.
  • an immunobinding assay which measures the concentration of antibodies in the serum which bind to a specific antigen, i.e. peptide or toxin
  • bactericidal assays which measure the ability of the antibodies to participate with complement in killing bacteria.
  • the ability to neutralize in vitro and in vivo biological effects of the pyrogenic exotoxins may also be assessed to determine the effectiveness of the treatment. See, e.g., the examples herein.
  • antibody molecules is used herein to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fab', F(ab') 2 and F (v) as well as chimeric antibody molecules.
  • An antibody of the present invention is typically produced by immunizing a mammal with an immunogen or vaccine containing one or more peptides of the invention, or a structurally and/or antigenically related molecule, to induce, in the mammal, antibody molecules having immunospecificity for the immunizing peptide or peptides.
  • the peptide (s) or related molecule (s) may be monomeric, polymeric, conjugated to a carrier, and/or administered in the presence of an adjuvant.
  • the antibody molecules may then be collected from the mammal if they are to be used in immunoassays or for providing passive immunity.
  • the antibody molecules of the present invention may be polyclonal or monoclonal. Monoclonal antibodies may be produced by methods known in the art . Portions of immunoglobulin molecules may also be produced by methods known in the art .
  • the antibody of the present invention may be contained in various carriers or media, including blood, plasma, serum (e.g., fractionated or unfractionated serum), hybridoma supernatants and the like.
  • the antibody of the present invention is isolated to the extent desired by well known techniques such as, for example, by using DEAE Sephadex, or affinity chromatography.
  • the antibodies may be purified so as to obtain specific classes or subclasses of antibody such as IgM, IgG, IgA, IgG ⁇ , IgG 2 , IgG 3 , IgG 4 and the like.
  • Antibody of the IgG class are preferred for purposes of passive protection.
  • the presence of the antibodies of the present invention, either polyclonal or monoclonal can be determined by various assays. Assay techniques include, but are not limited to, immunobinding, immunofluorescence (IF), indirect immunofluorescence, immunoprecipitation, ELISA, agglutination and Western blot techniques .
  • the antibodies of the present invention have a number of diagnostic and therapeutic uses.
  • the antibodies can be used as an in vitro diagnostic agent to test for the presence of various staphylococcal and streptococcal pyrogenic exotoxins in biological samples in standard immunoassay protocols and to aid in the diagnosis of various diseases related to the presence of bacterial pyrogenic exotoxins.
  • the assays which use the antibodies to detect the presence of bacterial pyrogenic exotoxins in a sample involve contacting the sample with at least one of the antibodies under conditions which will allow the formation of an immunological complex between the antibody and the toxin that may be present in the sample. The formation of an immunological complex if any, indicating the presence of the toxin in the sample, is then detected and measured by suitable means.
  • Such assays include, but are not limited to, radioimmunoassays, (RIA) , ELISA, indirect immunofluorescence assay, Western blot and the like.
  • the antibodies may be labeled or unlabeled depending on the type of assay used.
  • Labels which may be coupled to the antibodies include those known in the art and include, but are not limited to, enzymes, radionucleotides, fluorogenic and chromogenic substrates, cofactors, biotin/avidin, colloidal gold and magnetic particles.
  • Modification of the antibodies allows for coupling by any known means to carrier proteins or peptides or to known supports, for example, polystyrene or polyvinyl microliter plates, glass tubes or glass beads and chromatographic supports, such as paper, cellulose and cellulose derivatives, and silica.
  • Such assays may be, for example, of direct format (where the labelled first antibody reacts with the antigen) , an indirect format (where a labelled second antibody reacts with the first antibody) , a competitive format (such as the addition of a labelled antigen) , or a sandwich format (where both labelled and unlabelled antibody are utilized) , as well as other formats described in the art.
  • the biological sample is contacted to antibodies of the present invention and a labelled second antibody is used to detect the presence of staphylococcal and streptococcal pyrogenic exotoxins, to which the antibodies are bound.
  • the antibodies of the present invention are also useful as therapeutic agents in the prevention and treatment of diseases caused by the deleterious effects of staphylococcal and streptococcal pyrogenic exotoxins.
  • the antibodies are generally administered with a physiologically acceptable carrier or vehicle therefor.
  • a physiologically acceptable carrier is one that does not cause an adverse physical reaction upon administration and one in which the antibodies are sufficiently soluble and retain their activity to deliver a therapeutically effective amount of the compound.
  • the therapeutically effective amount and method of administration of the antibodies may vary based on the individual patient, the indication being treated and other criteria evident to one of ordinary skill in the art.
  • a therapeutically effective amount of the antibodies is one sufficient to inhibit superantigen stimulation of T-cells and/or attenuate the dysfunction caused by the presence of bacterial toxins without causing significant side effects such as non-specific T cell lysis or organ damage.
  • the route (s) of administration useful in a particular application are apparent to one or ordinary skill in the art.
  • Routes of administration of the antibodies include, but are not limited to, parenteral, and direct injection into an affected site.
  • Parenteral routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal and subcutaneous .
  • compositions of the antibodies described above suitable for parenteral administration including, but not limited to, pharmaceutically acceptable sterile isotonic solutions.
  • pharmaceutically acceptable sterile isotonic solutions include, but are not limited to, saline and phosphate buffered saline for nasal, intravenous, intramuscular, intraperitoneal, subcutaneous or direct injection into a joint or other area.
  • Antibodies for use to elicit passive immunity in humans are preferably obtained from other humans previously inoculated with compositions comprising one or more of the consensus amino acid sequences of the invention. Alternatively, antibodies derived from other species may also be used. Such antibodies used in therapeutics suffer from several drawbacks such as a limited half-life and propensity to elicit an immune response. Several methods have been proposed to overcome these drawbacks. Antibodies made by these methods are encompassed by the present invention and are included herein. One such method is the "humanizing" of non-human antibodies by cloning the gene segment encoding the antigen binding region of the antibody to the human gene segments encoding the remainder of the antibody.
  • the dosage of administered antibodies will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history and the like.
  • IVIG intravenously
  • IM intramuscularly
  • IVIG Intravenous immunoglobulin
  • IVIG can generally be given with a loading dose of 200 mg/kg, with monthly injections of 100 mg/kg.
  • High-dose IVIG may be given at 400-800 mg/kg, for antibody-deficient patients. See, e.g., The Merck Manual of Diagnosis and Therapy, 16 th Edition, (Berkow R and Fletcher AJ, Eds.), Merck Research Laboratories, Rahway, NJ (1992) .
  • the peptides and/or antibodies of the present invention are intended to be provided to the recipient subject in an amount sufficient to prevent, or attenuate the severity, extent or duration of the deleterious effects of staphylococcal and streptococcal pyrogenic exotoxins .
  • the administration of the agents including peptide and antibody compositions of the invention may be for either "prophylactic” or "therapeutic” purpose.
  • the agents are provided in advance of any symptom.
  • the prophylactic administration of the agent serves to prevent or ameliorate any subsequent deleterious effects of staphylococcal and streptococcal pyrogenic exotoxins .
  • the agent is provided at (or shortly after) the onset of a symptom of infection with bacteria expressing staphylococcal or streptococcal pyrogenic exotoxins.
  • the agent of the present invention may, thus, be provided either prior to the anticipated exposure to bacteria expressing staphylococcal or streptococcal pyrogenic exotoxin (so as to attenuate the anticipated severity, duration or extent of disease symptoms) or after the initiation of the infection.
  • the agent may also be provided to individuals at high risk for getting an infection with bacteria expressing staphylococcal or streptococcal pyrogenic exotoxins.
  • therapies based upon vectors, such as viral vectors containing nucleic acid sequences coding for the peptides described herein. These molecules, developed so that they do not provoke a pathological effect, will stimulate the immune system to respond to the peptides.
  • the peptide of the invention alone or linked to a carrier, as well as antibodies and other necessary reagents and appropriate devices and accessories may be provided in kit form so as to be readily available and easily used.
  • kits may contain a solid support, such as a membrane (e.g., nitrocellulose) , a bead, sphere, test tube, rod, and so forth, to which a receptor such as an antibody specific for the target molecule will bind.
  • a solid support such as a membrane (e.g., nitrocellulose)
  • a bead e.g., nitrocellulose
  • Such kits can also include a second receptor, such as a labelled antibody.
  • kits can be used for sandwich assays to detect toxins. Kits for competitive assays are also envisioned.
  • EXAMPLE 1 Peptides whose sequences are based on the two highly conserved regions of the staphylococcal and streptococcal pyrogenic exotoxins described herein were constructed. The sequences were based on alignments of the streptococcal pyrogenic exotoxins with the staphylococcal enterotoxins, and the amino acids used in positions with possible degeneracy were the amino acids most frequently found in these positions. Three of the peptides were then catenated and polymerized to produce peptides of greater than 8000 daltons (i.e., peptides
  • peptide 6348 was used to immunize rabbits, which produced high titer antibodies to this peptide. These antibodies were tested for the ability to recognize the streptococcal and staphylococcal pyrogenic exotoxins. Immunological assays (immunoblots) revealed that these antibodies recognized regions common to all the pyrogenic exotoxins. These antibodies were also tested for the ability to neutralize in vitro and o in vivo biological activity of the pyrogenic exotoxins. These antibodies protected against the biological T-cell proliferation of these toxins in an in vitro blastogenesis assay using human mononuclear cell populations.
  • Peptides were constructed by solid phase synthesis (20) using the modifications described by Houghton (10) .
  • GCG Consensus #1 YGGX 1 TX 2 X 3 X 4 X 5 N (SEQ ID NO:l) peptide #1 CMYGGVTEHEGN ( SEQ ID NO : 3 ) 5
  • synthetic peptides #1 and #2 are not native peptides, i.e., their sequences differ from those found in native toxins. Variations of these 5 peptides have also been constructed in order to generate concatenated polymers of the peptides. These polymers were constructed by the addition of glycine and of additional cysteine residues to the amino- and/or carboxyl- termini of the initial 2 peptides, thus facilitating concatenation via disulfide bond formation
  • polystyrene resin (37, 38, 39) .
  • the polymerized molecules were then dialyzed to remove molecules with molecular weights less than 6000-8000 daltons.
  • One polymeric construct is 5 composed of the monomer: CMYGGVTEHEGNGC (SEQ ID NO: 5) .
  • An additional polymer is composed of the peptide: CGKKNVTVQELDYKIRKYLVDNKKLYGC (SEQ ID NO : 6 ) .
  • consensus region #1 precedes consensus region #2 by 27 amino acid residues (e.g. [consensus region 1] x27 [consensus region 2]) .
  • consensus region #2 by 27 amino acid residues (e.g. [consensus region 1] x27 [consensus region 2]) .
  • CMYGGVTEHEGNKK VTVQELDYKIRKYLVDNKKLY SEQ ID NO: 7
  • this peptide is representative of the two consensus regions joined together in the proper order (region 1 in the N terminal half, and region 2 in the C-terminal half of the molecule) , however they are not separated by an additional 27 residues as they are in the native toxins.
  • concatenated polymers based on the monomer: CMYGGVTEHEGNKKNVTVQELDYKIRKYLVDNKKLYGC (SEQ ID NO: 8) .
  • Peptides with an (*) are cross-linked polymers composed of the described sequence. It is expected that monomers of these peptides will also be useful in the present invention. Generation of anti-peptide sera.
  • New Zealand White rabbits were immunized by subcutaneous injection with 500 ⁇ g of peptide in complete Freund's adjuvant. Additional booster injections of 500 ⁇ g in incomplete adjuvant was administered 4 weeks after the primary injections. Ten days after booster injections, the rabbits were bled, and the anti-peptide titers were determined by ELISA.
  • Staphylococcal enterotoxins TSST-1, and streptococcal pyrogenic exotoxins were purchased from Toxin Technology Inc. (Sarasota, FL) . Immunoblots
  • Each of the staphylococcal and streptococcal pyrogenic exotoxins were electrophoresed through 10% SDS PAGE gels (16) and transferred to nitrocellulose for western blots (33) .
  • the western blots were developed using the rabbit anti-peptide 6348 serum (anti-pep 6348 or AP6348) diluted 1:5000, followed by goat anti-rabbit (IgG) alkaline phosphate conjugate (Sigma) . Inhibition of blastogenesis
  • PBMC peripheral blood mononuclear cell
  • mice Female New Zealand White rabbits >lyr old were obtained from Hazelton Dutchland Labs, Inc. (Denver, PA) . Rabbits were challenged with staphylococcal or streptococcal toxins at doses ranging from 50 to 100 ⁇ g/kg, as previously described (24). Briefly, pyrogenic toxins were incubated with either 200 ul of normal rabbit serum or 200 ul of anti-pep #6348 serum for one hour prior to challenge. Toxin-serum mixtures were j 0 administered intravenously through the marginal ear veins. Normal control rabbits were treated in an identical manner, with isotonic saline substituted for the pyrogenic toxin.
  • rabbits were given a sub-lethal dose (5 ⁇ g/kg) of endotoxin (E ⁇ _ coli LPS, List Biological Laboratories, Inc., Campbell, CA) . Rabbits were monitored 72 h for clinical signs of toxic shock. These included elevated temperature, diarrhea, cardiopulmonary distress, and conjunctival injection.
  • endotoxin E ⁇ _ coli LPS, List Biological Laboratories, Inc., Campbell, CA
  • AP6348 Since recognition of the toxins by AP6348 was successful, we tested this serum for the ability to inhibit the biological effects of these pyrogenic toxins. AP6348 was capable of inhibiting in vitro blastogenesis of human PBMCs by many of the pyrogenic toxins (e.g., SEA, SEB, SEC, SEE, SPEA, and SPEC).
  • SEA SEA
  • SEB SEB
  • SEC SEC
  • SEE SEE
  • SPEA SPEA
  • SPEC SPEC
  • AP6348 was also able to provide passive in vivo protection of animals challenged with lethal doses of SEB and SPEA. These animals developed fever, however the fever returned to normal within 30 hours and remained normal. Rabbits appeared to be fully recovered within days of challenge.
  • antibodies prepared against this peptide may be administered during the early stages of toxic shock or septic shock irrespective of the toxin causing the symptoms and (ii) the peptide may be used as an immunogen to block the toxic effects of this family of superantigens.
  • PBMCs were isolated via Ficoll-Hypaque Solution.
  • Figure 7A showed more inhibitory activity of SEB than peptide 6346 (i.e., CGKKNVTVQELDYKIRKYLVDNKKLYGC, SEQ ID NO : 6 ) ( Figure 7B) or peptide 6348 (i.e., 5 CMYGGVTEHEGNKKNVTVQELDYKIRKYLVDNKKLYGC, SEQ ID NO: 8) ( Figure 7C) .
  • EXAMPLE 3 o PBMCs were isolated via Ficoll-Hypaque Solution.
  • CMYGGVTEGEGN nonpolymeric 6343 peptide
  • 2xl0 5 cells 150 ⁇ g of nonpolymeric 6343 peptide (i.e., CMYGGVTEGEGN, SEQ ID NO: 3) and 2xl0 5 cells in 200 ⁇ L of RPMI solution was plated in each well. The cells were incubated for one hour at 37 degrees Centigrade, with mild agitation 5 every 15 minutes. After one hour, 2 ⁇ g of either SEB, SEC, SED, SPEC, SPEA, or TSST-1 was added to each well. The PBMCs were incubated for 72 hours and the results were measured via tritiated thymidine incorporation. 0 The cells were collected and read on a beta counter.
  • mice were sensitized with 0.001 ⁇ g Lipopolysaccharide (LPS) and 20 mg of D-Galactosamine via intraperioneal injection. The results are shown in Table 4. After six hours, saline or 1.5 mg of the nonpolymeric peptide 6343 was administered to the experimental mice by subcutaneous injection. One hour later, the mice were injected again with either saline or 1.5 mg peptide (3.0 mg total) . One hour later, all mice were challenged with 0.02 ⁇ g SEB, SPEA or TSST-1 (via intraperitoneal injection) and the mice were observed overnight.
  • LPS Lipopolysaccharide
  • TSST-1 via intraperitoneal injection
  • PBMCs were isolated via Ficoll-Hypaque Solution. Either O ⁇ g, 75 ⁇ g, lOO ⁇ g or 150 ⁇ g of nonpolymeric 6343 peptide (i.e., CMYGGVTEGEGN, SEQ ID NO: 3) and 2xl0 5 cells in 200 ⁇ L of RPMI solution was plated in each well. The cells were incubated for one hour at 37 degrees Centigrade, with mild agitation every 15 minutes. After one hour, 2 ⁇ g of the recombinant forms of either SPEG, SPEH, SPEZ, which were provided by Dr. Fraser of the University of Auckland, New Zealand, was added to each well.
  • CMYGGVTEGEGN nonpolymeric 6343 peptide
  • the PBMCs were incubated for 72 hours and the results were measured via tritiated thymidine incorporation.
  • the cells were collected and read on a beta counter. The results are shown in Figure 9. Note that peptide 6343 inhibited blastogenesis of PBMCs by the bacterial superantigens SPEG, SPEH and SPEZ.
  • EXAMPLE 7 To determine the nature of the inhibition of superantigen stimulation by the inhibitory peptides and antigens, purified MHC class II molecules (obtained from J. Strominger, Harvard University) were used in a competitive ELISA to determine binding to this molecule. The purified MHC were immobilized on a 96 well plate and 4 ⁇ g SEB was added. After appropriate washing a rabbit monoclonal anti-SEB antibody (Toxin Tech) was added to the ELISA followed by a colorimetric reagent. The plate was read on an ELISA plate reader.
  • Toxin Tech rabbit monoclonal anti-SEB antibody
  • the peptide can be used repeatedly in the same individual without raising anti-peptide antibodies .
  • Streptococcal pyrogenic exotoxin 0 type A (scarlet fever toxin) is related to staphylococcus aureus enterotoxin B. Molecular and General Genetics. 203:354-356.
  • V beta-specific superantigen staphylococcal enterotoxin B stimulation of mature T cells and clonal deletion in neonatal mice. Cell. 56:27-35.
  • Blankson JN, Morse SS The CD28/B7 pathway cosimulates the response of primary murine T cells to superantigens as well as to conventional antigens. Cell Immunol 157:306-312, 1994.
  • SEB 140 CMYGGVTEHNGN 151 SEQ ID NO: :10
  • MOLECULE TYPE PEPTIDE
  • SEQUENCE DESCRIPTIONS SEQ ID NO: 4:
  • Tyr Lys lie Arg Lys Tyr Leu Val Asp Asn Lys Lys 15 20
  • Tyr lie Tyr Gly Gly lie Thr Pro Ala Gin Asn Asn
  • MOLECULE TYPE PEPTIDE
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 21 Lys Lys Asn Val Thr Val Gin Glu Leu Asp Ala Gin

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EP99970123A 1998-10-07 1999-09-24 Geeignete peptide zur reduzierung des toxischen schock syndroms und von septischem schock Withdrawn EP1127132A1 (de)

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CA2924155C (en) 2007-01-03 2020-02-11 Philip M. Sass High affinity antibodies that neutralize staphylococcus enterotoxin b

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WO1996040930A1 (en) * 1995-06-07 1996-12-19 Regents Of The University Of Minnesota Mutants of streptococcal toxin a and methods of use
US6075119A (en) * 1997-04-07 2000-06-13 The Rockefeller University Peptides useful for reducing symptoms of toxic shock syndrome

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