Patent Grant
7189398
The invention relates to peptides structurally related to protein domains in pyrogenic exotoxins, capable of antagonizing activation of T cells mediated by said exotoxins and of eliciting protective immunity against toxic shock induced by said exotoxins. The invention further relates to pharmaceutical preparations containing the said peptides, for the treatment or prophylaxis of toxic shock and to vaccines containing the said peptides, capable of eliciting protective immunity against toxic shock induced by said exotoxins.
A family of pyrogenic exotoxins, also known as superantigenic toxins, is produced by Staphylococcus aureus and Streptococcus pyogenes . The exotoxins comprised of the S. aureus enterotoxins (SEs) cause the majority of human food poisoning cases manifested by vomiting and diarrhea after ingestion [Schlievert, J Infect Dis 167:997 (1993)]. S. aureus is found widespread in nature, often in association with humans. Among the 5 major serological types within the family of SEs (labeled SEA to SEE and SEG), SEB is the most prominent [Marrack and Kappler, Science 248:705 (1990)]. SEB has also been recognized as a leading cause of human cases of non-menstrual toxic shock syndrome that can accompany surgical or injurious wound infections, as well as viral infections of the respiratory tract of influenza patients to which children are especially vulnerable [Schlievert (1993) ibid.; Tseng et al., Infect Immun 63:2880 (1995)]. Toxic shock syndrome, in its most severe form, causes shock and death [Murray et al., ASM News 61:229 (1995); Schlievert (1993) ibid.]. More generally, members of the staphylococcal exotoxin family, including SEA to SEE and toxic shock syndrome toxin 1 (TSST-1), have been implicated in toxic shock syndrome, in atopic dermatitis [Schlievert (1993) ibid.] and in Kawasaki's syndrome [Bohach et al., Crit Rev Microbiol 17:251 (1990)].
Because of the potential for causing lethal shock in humans after aerosol exposure and because of the relative ease with which SEB can be produced in large amounts, there is concern that SEB could be used as a biological weapon [Lowell et al., Infect Immun 64:1706 (1996)]. SEB is thought to be a biological weapon mainly in view of its lethal potential. However, through its exquisite ability to induce vomiting and diarrhea, SEB is also an incapacitating agent that could severely impair the effectiveness of a fighting force, even temporarily, thereby enhancing vulnerability to conventional military means. Needless to say, the harmful effects of SEB need to be generally attacked, and not only in connection with the military aspect.
SEB is a toxic mitogen that triggers a paradoxical response in the infected organism: a vast stimulation of the immune system on one hand side and, on the other hand, a profound immunosuppression that may allow the multiplication of the infecting bacteria, unimpeded by an immune response [Hoffman, Science 248:685 (1990); Smith and Johnson J Immunol 115:575 (1975); Marrack et al, J Exp Med 171:455 (1990); Pinto et al., Transplantation 25:320 (1978)]. During the cellular immune response, a dynamic interplay is induced, by antigens or mitogens, between activation of Th1 type cytokine gene expression on one hand, exemplified by interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-β (TNF-β), and on the other hand, its cell-mediated suppression by CD8 cells and other cell subsets [Ketzinel et al., Scand J Immunol 33:593 (1991); Arad et al., Cell Immunol 160:240 (1995)], and by the inhibitory cytokines from Th2 cells, IL-4 and IL-10 [Mosmann and Coffman, Annu Rev Immunol 7:145 (1989)].
SEB is a member of the family of pyrogenic exotoxins [Herman et al., Ann Rev Immunol 9:745 (1991)] that comprises bacterial exotoxins and Mls proteins. These agents stimulate a 20,000-fold greater proportion of rodent or human T cells than do ordinary antigens. Thus, SEB activates 30–40% of all T cells in some mice to divide and produce cytokines [Marrack and Kappler (1990) ibid.]. Indeed, expression of the toxicity of SEB requires T cells; mice that lack T cells or SEB-reactive T cells are not affected by doses of SEB that cause weight loss and death in normal animals [Marrack et al. (1990) ibid.; Marrack and Kappler (1990) ibid.]. Unlike normal antigens, SEB and related toxic mitogens do not require processing and antigen presentation [Janeway et al., Immunol Rev 107:61 (1989)] but activate the T cell by binding at a specific site in the variable portion of the β chain (V-β) of the T-cell receptor [Choi et al., Nature 346:471 (1990)]. The crucial region for T-cell receptor interaction with toxin lies on the outer face of the V-β domain, a region not involved in conventional antigen recognition [Choi et al., Proc Natl Acad Sci U.S.A. 86:8941 (1989)]. Simultaneously, pyrogenic exotoxins bind directly to MHC class II molecules [Scholl et al., Proc Natl Acad Sci U.S.A. 86:4210 (1989)] and thus affect primarily CD4+ T cells, although CD8+ cells are also activated [Fleischer and Schrezenmeier, J Exp Med 167:1697 (1988); Fraser, Nature 339:221 (1989); Misfeldt, Infect Immun 58:2409 (1990)]. The current consensus is that pyrogenic exotoxins activate T cells so effectively because they bypass the ordinary interaction of antigen with class II MHC and T-cell receptor [Janeway, Cell 63:659 (1990)]. An alternative view is that pyrogenic exotoxins act as coligands that facilitate and thus greatly exaggerate the effect of minute amounts of ordinary antigens [Janeway (1990) ibid.].
The toxicity of SEB and related exotoxins is thought to be related to the capacity of these molecules to stimulate the rapid and excessive production of cytokines, especially of IL-2, IFN-γ and tumor necrosis factors (TNFs). IL-2, IFN-γ, and TNF-β are secreted from activated T helper type 1 (Th1cells while TNF-α is secreted by Th1 cells, monocytes and macrophages. High levels of these cytokines, suddenly produced, have been implicated as a central pathogenic factor in toxin-related toxicity [Schad et al., EMBO J 14:3292 (1995)] and are thought to cause a rapid drop in blood pressure leading to toxic shock.
While investigation has produced a plausible explanation for the vast stimulation of T cells by SEs, it is not yet clear why these toxins are also strongly immunosuppressive. They induce a decline in both primary T and B cell responses, including the production of antibodies and the generation of plaque-forming cells [Hoffman (1990) ibid.; Smith and Johnson (1975) ibid.; Marrack et al. (1990) ibid.; Pinto et al., (1978) ibid.; Ikejima et al., J Clin Invest 73:1312 (1984); Poindexter and Schlievert, J Infect Dis 153:772 (1986)].
The sensitivity of humans to staphylococcal toxins exceeds that of mice by a factor of 100. Thus, the toxic shock syndrome toxin 1, TSST-1, another pyrogenic exotoxin from Staphylococcus aureus, stimulates human T cells to express the key is cytokines, IL-2, IFN-γ and TNF-β at <0.1 pg/ml, while murine cells require approximately 10 pg/ml [Uchiyama et al., J Immunol 143:3173 (1989)]. Mice may have developed relative resistance to toxic mitogens by deleting from their T cell repertoire those cells that display the most highly reactive V-β chains or by eliminating these V-β genes [Marrack and Kappler (1990) ibid.]. Such deletions have not been detected in humans, making them far more vulnerable.
The incapacitating and potentially lethal effects for humans of SEB (and of exotoxins of the same family of superantigens), whether exerted on civilians or military personnel, create a need for prophylaxis against SEB, for treatment of SEB-exposed individuals and for a safe SEB vaccine.
Despite the urgency of this need, methods of protection or treatment have been lacking. Thus, in D-galactosamine-sensitized murine models of SEB intoxication, one based on intramuscular challenge with SEB toxin and the other on intranasal challenge using mucosal SEB exposure, it was possible to protect mice with proteosome-SEB toxoid vaccines in which the SEB toxoid component was prepared by a 30-day formalin treatment of the biologically active, intact SEB protein molecule [Lowell et al. (1996) ibid.]. As detailed below, however, the inventors have now found that antibodies raised against certain peptide domains within the SEB molecule enhance the ability of SEB to stimulate human T cells, rather than protecting them against the toxin. This finding limits the use of SEB toxoids as vaccine, in view of the danger of eliciting certain SEB-sensitizing antibodies that could not only fail to confer protective immunity but would lead to significant exacerbation of the toxic responses in SEB-exposed persons.
Other investigators sought recourse in the use of fragments rather than the complete SEB protein molecule, through the synthesis of a series of overlapping SEB peptides, in the order of 30 amino acids each in length [Jett et al., Infect Immun 62:3408 (1994)]. These peptides were used to generate antisera in rabbits whose ability to inhibit the SEB-induced proliferation of a mixture of human T cells and macrophages was then tested. That effort failed to yield an effective or specific inhibitory response. Thus, peptide pSEB(113–144), containing amino acids 113 to 144 of the SEB protein molecule, as well as peptides covering amino acids 130–160, 151–180, and 171–200 each elicited antisera that inhibited the SEB-induced lymphocyte proliferation weakly, by up to 2.5-fold [Jett et al. (1994) ibid.].
A number of investigators attempted the create peptide vaccines. Thus, Mayordomo et al. [J Exp Med 183:1357 (1996)] used a mutant peptide derived from p53 as vaccine for therapy of murine tumors. Hughes and Gilleland [Vaccine 13:1750 (1995)] used synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa to afford protection against P. aeruginosa infection in a murine acute pneumonia model. In an attempt to use peptide immunization in humans Brander et al. [Clin Exp Immunol 105:18 (1996)] showed that a combined CD8+/CD4+ T cell-targeted vaccine restimulated the memory CD4+ T cell response but failed to induce cytotoxic T lymphocytes.
Major sources of exotoxins are, as already mentioned, S. Aureus and S. Pyogenes. The flesh-eating bacteria, S. Pyogenes, produce a family of different toxins with closely similar mode of action: excessive activation of T cells. S. Aureus produces next to SEB as major component, also SEA, SECs, SEE and TSST-1 (toxic shock syndrome toxin 1) and S. Pyogenes produces SPE A as major toxin, as well as other pyrogenic exotoxins. Hence, in staphylococcal food poisonings and, more seriously, in biological warfare or in toxic shock caused by S. pyogenes, mixtures of toxins are encountered. The composition of such mixtures cannot be anticipated with certainty. The worst scenarios of biological warfare entail not the use of a single, purified pyrogenic exotoxin, as favored for immunological studies, but rather a readily attainable, crude natural mixtures of such toxins, as produced, for example, by culturing S. Aureus.
Clearly, this complexity demands the development of broad-spectrum antagonists of pyrogenic exotoxins as well as broad-spectrum vaccines.
There exists, therefore, a long-felt need to design a SEB vaccine that is free of sensitizing potential, yet is capable of protecting test animals or humans against lethal doses of the toxin. Even greater value would be inherent in a vaccine that can afford protection not only against SEB, but also against a wider spectrum of the SE toxin family, including, for example, SEA.
Moreover, currently, there is no prophylaxis available against SEB or any other pyrogenic exotoxin, nor treatment of exposed persons. There exists, therefore, also a long-felt need to design agents that antagonize the action of SEB, as well as any other pyrogenic exotoxin. Such antidotes will have great value, both in the medical treatment of acute food poisoning and in saving lives in cases of toxic shock and related pathological conditions.
There exists therefore a need, on the one hand, for an antagonist against pyrogenic exotoxins, for use in immediate treatment, or short term prevention and rapid prophylaxis, of acute toxic shock and of the harmful effects of such toxins which may be due to, for example, accidental food poisoning, and on the other hand, for a vaccine for immunization against intoxication by pyrogenic exotoxins, for long term protection thereagainst.
In addition, currently there is no way by which to assess the efficacy of vaccination of humans against pyrogenic toxins, since humans cannot be challenged with the toxin in order to check whether they have been conferred the desired immunity. There exists therefore a need for a clinical test for assessing the efficacy of vaccination of humans against pyrogenic toxins which employs surrogate markers.
The present invention relates to peptides comprising an amino acid sequence substantially homologous to the amino acid sequence of a fragment of a pyrogenic exotoxin, and derivatives of said peptides, capable of eliciting protective immunity against toxic shock induced by a pyrogenic exotoxin or by a mixture of pyrogenic exotoxins.
The present invention also relates to peptides comprising an amino acid sequence substantially homologous to the amino sequence of a fragment of a pyrogenic exotoxin, and derivatives of said peptides, capable of antagonizing toxin-mediated activation of T cells.
Also within the scope of this invention are peptide comprising an amino acid sequence substantially homologous to the amino acid sequence of a fragment of a pyrogenic exotoxin, and derivatives thereof, which are capable of protecting against toxic shock induced by a pyrogenic exotoxin or by a mixture of pyrogenic exotoxins.
The peptides of the invention comprise an amino acid sequence substantially homologous to the amino sequence of a fragment of Staphylococcal aureus enterotoxin B (SEB).
The pyrogenic exotoxin is a bacterial exotoxin produced by Staphylococcus aureus or Streptococcus pyogenes.
According to the invention, the peptides may be further linked through its N-terminus to a lauryl-cysteine (LC) residue and/or through its C-terminus to a cysteine (C) residue, or to other residue/s suitable for linking said peptide to adjuvant/s for immunization.
Further, the peptides may be in the form of a dimer, a multimer or in a constrained conformation, in which the constrained conformation is obtained by internal bridges, short-range cyclizations, extension or other chemical modification.
The peptides of the invention are capable of inhibiting expression of pyrogenic toxin-induced mRNA encoded by the IL-2, IFN-γ or TNF-β genes.
In addition, the peptides of the invention are capable of eliciting, in the presence of a suitable immunization adjuvant, in immunized individuals, the production of antibodies that block T-cell pyrogenic toxin-mediated activation.
In a second aspect of the invention a pharmaceutical composition is provided, for the treatment or short term prophylaxis of toxin-mediated activation of T cells, comprising as active ingredient a therapeutically effective amount of at least one peptide according to the invention or derivative thereof. The peptides of the invention are capable of protecting against toxic shock induced by a pyrogenic exotoxin or by a mixture of pyrogenic exotoxins.
Additionally, the invention provides a vaccine for conferring long term immunity against toxic shock induced by at least one pyrogenic exotoxin, comprising as active ingredient an immunologically effective amount of at least one peptide according to the invention, or derivative thereof.
Also provided by the invention is a method for the immediate treatment or short term prevention of acute toxic shock and of the harmful effects which may be due to, for example, accidental food poisoning induced by at least one pyrogenic exotoxin, comprising administering to a patient in need of such treatment a therapeutically effective amount of the pharmaceutical composition of the invention or of a therapeutically effective amount of at least one peptide of the invention.
Additionally, the invention provides a method for conferring long term immunity to toxic shock induced by a pyrogenic exotoxin or a mixture of pyrogenic exotoxins, comprising administering to a patient an effective immunizing amount of a vaccine of the invention.
In addition, the invention relates to antibodies directed against a peptide according to the invention which are capable of blocking T-cell activation by a pyrogenic exotoxin or a mixture of pyrogenic exotoxins, which may be mono- or polyclonal antibodies.
In yet a further aspect, the invention relates to an antiserum containing antibodies directed against at least one peptide of the invention, which peptide can elicit the production of the antibodies. The antiserum is capable of alleviating toxic shock induced by a pyrogenic exotoxin.
In addition, the invention provides for a method for assessing the efficacy of a vaccine for conferring immunity against one or more pyrogenic toxins comprising determining the ability of serum from an immunized individual to antagonize toxin-mediated activation of T cells. A kit for assessing the efficacy of a vaccine for conferring immunity against one or more pyrogenic toxins comprising determining the ability of serum from an immunized individual to antagonize toxin-mediated activation of T cells by the method of the invention is also provided.
, ◯, ▴) except a control group of 20 mice (▪). Thirty minutes later, each mouse received 5 μg of TSST-1 by intraperitoneal administration. An additional intravenous injection of 25 μg per mouse of p12 was administered at 3 hr post-challenge (◯) or at 3 and 18 hr post-challenge (▴). Survival in each group was determined thereafter, at the times in hours indicated. Surviving animals showed no signs of malaise and were indistinguishable from normal controls in behavior; they survived for as long as monitored, two weeks.
In search for efficient agents for long and short term treatment and prophylaxis of toxic shock induced by pyrogenic exotoxins, a series of short peptides related to certain domains within the SEB protein molecule has been designed. The biological properties of different synthetic SEB-related peptides were then evaluated by the following several criteria:
The inventors have indeed obtained peptides that meet each of these five criteria. In a specific embodiment of the invention, a SEB-related dodecapeptide antagonist was designed. This peptide blocks the action of SEB as well as other pyrogenic exotoxins on the human immune response in vitro, severely inhibiting SEB-mediated induction of IL-2, IFN-γ and TNF-β mRNA. It is clear that this peptide could be used for treatment of acute toxic shock and of harmful effects which may be due to, for example, accidental food poisoning induced by pyrogenic exotoxins.
In addition, this peptide elicits antibodies that protect human lymphoid cells against SEB, SEA, and TSST-1, indicating that it may confer wider protective immunity against pyrogenic toxins. However, antibodies raised against peptides derived from certain other SEB protein domains actually enhanced the response of human PBMC to SEB and SEA, as expressed by greater induction of IL-2 and IFN-γ mRNA, rather than protecting against the toxins. Immunization of mice with the SEB antagonist peptide elicited protection against lethal doses of SEB, resulting in survival of test animals. It is clear that the peptide may also be used for conferring long-term immunity against toxic shock induced by pyrogenic exotoxins.
Thus, in a first aspect, the present invention relates to peptides comprising an amino acid sequence substantially homologous to the amino acid sequence of a fragment of a pyrogenic exotoxin, and to functional derivatives of such peptides, capable of eliciting protective immunity against toxic shock induced by the exotoxins. The terms derivatives and functional derivatives used herein mean peptides with any insertions, deletions, substitutions and modifications that are capable of eliciting protective immunity against toxic shock induced by the exotoxins and/or of antagonizing toxin-mediated activation of T cells (hereafter referred to as “derivative/s”).
In a second aspect the invention relates to peptides comprising an amino acid sequence substantially homologous to the amino sequence of a fragment of a pyrogenic exotoxin, and to derivatives of such peptides, capable of antagonizing toxin-mediated activation of T cells. The peptides of the invention are capable of protecting against toxic shock induced by a pyrogenic exotoxin or by a mixture of pyrogenic exotoxins.
The pyrogenic exotoxin is usually a bacterial exotoxin, specifically an exotoxin produced by Staphylococcus aureus or by Streptococcus pyogenes.
In a preferred embodiment of both said aspects of the invention, the invention relates to a peptide comprising an amino acid sequence substantially homologous to the amino sequence of a fragment of Staphylococcal aureus enterotoxin B (SEB).
In a specifically preferred embodiment the invention relates to peptides comprising the amino acid sequence shown in SEQ ID NO:1 (positions 150 to 161 of the sequence of the naturally occurring SEB protein shown in SEQ ID NO:12), and functional derivatives thereof, capable of eliciting protective immunity against toxic shock induced by at least one pyrogenic exotoxin and/or of antagonizing toxin-mediated activation of T cells. This peptide is also capable of protecting against toxic shock induced by a pyrogenic exotoxin or by a mixture of pyrogenic exotoxins. These peptides can therefore be used for both immediate treatment of acute toxic shock and of the harmful effects which may be due to, for example, accidental food poisoning, induced by the pyrogenic exotoxins and for conferring long-term immunity against such toxic shock.
A particular example may be a peptide having the amino acid sequence shown in SEQ ID NO:1 (hereafter also referred to as pSEB(150–161)) and functional derivatives thereof. This peptide is capable of eliciting protective immunity against toxic shock induced by at least one pyrogenic exotoxin and/or of antagonizing toxin-mediated activation of T cells.
In an alternative embodiment the invention relates to peptides comprising the amino acid sequence shown in SEQ ID NO:2 and to derivatives thereof, capable of eliciting protective immunity against toxic shock induced by at least one pyrogenic exotoxin and/or of antagonizing toxin-mediated activation of T cells. Also these peptides can be used for both immediate treatment of acute toxic shock and of the harmful effects which may be due to, for example, accidental food poisoning induced by the pyrogenic exotoxins and for conferring long-term immunity against such toxic shock. A particular example may be a peptide having the amino acid sequence shown in SEQ ID NO:2 (hereinafter also referred to as p12(150–161)) and derivatives thereof capable of eliciting protective immunity against toxic shock induced by at least one pyrogenic exotoxin and/or of antagonizing toxin-mediated activation of T cells.
In a further embodiment the invention relates to peptides comprising the amino acid sequence shown in SEQ ID NO:3 (positions 152 to 161 of the sequence of the naturally occurring protein shown in SEQ ID NO:12) and to functional derivatives thereof, capable of eliciting protective immunity against toxic shock induced by at least one pyrogenic exotoxin and/or of antagonizing toxin-mediated activation of T cells. Also these peptides can be used for both immediate treatment of acute toxic shock and of the harmful effects which may be due to, for example, accidental food poisoning induced by the pyrogenic exotoxins and for conferring long-term immunity against such toxic shock.
Particular examples may be a peptide having the amino acid sequence shown in SEQ ID NO:3 (hereinafter also referred to as pSEB(152–161)) and derivatives thereof capable of eliciting protective immunity against toxic shock induced by a pyrogenic exotoxin or a mixture of pyrogenic exotoxins and/or of antagonizing toxin-mediated activation of T cells.
In addition, the invention relates to peptides comprising the amino acid sequence shown in SEQ ID NO:4 and to functional derivatives thereof, capable of eliciting protective immunity against toxic shock induced by at least one pyrogenic exotoxin and/or of antagonizing toxin-mediated activation of T cells. Also these peptides can be used for both immediate treatment of acute toxic shock and of the harmful effects which may be due to, for example, accidental food poisoning induced by the pyrogenic exotoxins and for conferring long-term immunity against such toxic shock.
A particular example may be a peptide having the amino acid sequence shown in SEQ ID NO:4 (hereinafter also referred to as p10(152–161)) and functional derivatives thereof, capable of eliciting protective immunity against toxic shock induced by a pyrogenic exotoxin or a mixture of pyrogenic exotoxins and/or of antagonizing toxin-mediated activation of T cells.
As mentioned, pSEB(150–161) corresponds to the natural amino acid sequence in SEB between positions 150–161, while p12(150–161) differs from the corresponding natural amino acid sequence of SEB in 3 out of the 12 positions, yet possesses even greater toxin antagonist activity. It is thus possible to design a pyrogenic exotoxin antagonist and/or vaccine through use of short peptides related, but not necessarily identical to, domains within the SEB toxin molecule.
The lack of structure of linear peptides on one hand renders them vulnerable to proteases in human serum and on the other hand acts to reduce their affinity for target sites, because only few of the possible conformations may be active. Therefore, it is desirable to optimize antagonist peptide structure.
Thus, the peptides of the invention can be coupled through their N-terminus to a lauryl-cysteine (LC) residue and/or through their C-terminus to a cysteine (C) residue, or to other residue/s suitable for linking the peptide to adjuvant/s for immunization, as will be described in more detail hereafter.
Accordingly, in a further embodiment, the invention relates to a peptide having the amino acid sequence shown in SEQ ID NO:5 (hereinafter also referred to as pSEBLC(150–161)) and functional derivatives thereof, capable of eliciting protective immunity against toxic shock induced by pyrogenic exotoxins and/or of antagonizing toxin-mediated activation of T cells. These peptides can also be used for both immediate treatment of acute toxic shock and of the harmful effects which may be due to, for example, accidental food poisoning induced by the pyrogenic exotoxins and for conferring long-term immunity against such toxic shock.
In yet another embodiment, the invention relates to a peptide having the following amino acid sequence shown in SEQ ID NO:6 (hereinafter also referred to as p12LC(150–161)) and functional derivatives thereof capable of eliciting protective immunity against toxic shock induced by pyrogenic exotoxins and/or of antagonizing toxin-mediated activation of T cells. Evidently, such peptides can also be used for both immediate treatment of acute toxic shock and of the harmful effects which may be due to, for example, accidental food poisoning induced by the pyrogenic exotoxins and for conferring long-term immunity against such toxic shock.
The peptides of the invention, as well as the derivatives thereof may all be positively charged, negatively charged or neutral and may be in the form of a dimer, a multimer or in a constrained conformation.
A constrained conformation can be attained by internal bridges, short-range cyclizations, extension or other chemical modification.
Peptides in the form of a dimer or trimer can have, for example, the amino acid sequences shown in SEQ ID NOs. 7 and 8, respectively (hereinafter also referred to as Dimer and Trimer, receptively) and functional derivatives thereof, capable of eliciting protective immunity against toxic shock induced by pyrogenic exotoxins and/or of antagonizing toxin-mediated activation of T cells. As with other peptides of the invention, these peptides can also be used for both immediate treatment of acute toxic shock and of the harmful effects caused thereby and for conferring long-term immunity against such toxic shock.
Further, the peptides of the invention may be extended at the N-terminus and/or C-terminus thereof with various identical or different amino acid residues. As an example for such extension, the peptide may be extended at the N-terminus and/or C-terminus thereof with identical or different hydrophobic amino acid residue/s which may be naturally occurring or synthetic amino acid residue/s. A preferred synthetic amino acid residue is D-alanine.
A particular example for a peptide extended with synthetic amino acid residues is the peptide having the amino acid sequence shown in SEQ ID NO:10 (hereinafter also referred to as D-Ala) and functional derivatives thereof, capable of eliciting protective immunity against toxic shock induced by pyrogenic exotoxins and/or of antagonizing toxin-mediated activation of T cells.
An additional example for such an extension is provided by peptides extended both at the N-terminus and/or C-terminus thereof with a cysteine residue. Naturally, such an extension may lead to a constrained conformation due to Cys—Cys cyclization resulting from the formation of a disulfide bond. A particular such peptide has the amino acid sequence shown in SEQ ID NO:9 (hereinafter also referred to as Cys-p12(150–162)) and functional derivatives thereof, capable of eliciting protective immunity against toxic shock induced by pyrogenic exotoxins and/or of antagonizing toxin-mediated activation of T cells. According to one embodiment of the invention, the Cys-p12(150–162)) is a cyclic peptide having a disulfide bond via the terminal cysteine residues. Nevertheless, the Cys-p12(150–162) peptide may be linear.
In addition the peptide may be extended by aromatic amino acid residue/s, which may be naturally occurring or synthetic amino acid residue/s. A preferred aromatic amino acid residue is tryptophan. Alternatively, the peptides can be extended at the N-terminus and/or C-terminus thereof with amino acids present in corresponding positions of the amino acid sequence of the naturally occurring pyrogenic exotoxin.
Nonetheless, according to the invention, the peptides of the invention may be extended at the N-terminus and/or C-terminus thereof with various identical or different organic moieties which are not a naturally occurring or synthetic amino acids. As an example for such extension, the peptide may be extended at the N-terminus and/or C-terminus thereof with an N-acetyl group. A particular example for such an extension is the peptide having the amino acid sequence shown in SEQ ID NO:11 (hereinafter also referred to as Ac-p12(150–161)) and functional derivatives thereof, capable of eliciting protective immunity against toxic shock induced by pyrogenic exotoxins and/or of antagonizing toxin-mediated activation of T cells.
These extended peptides, as other peptides of the invention, can also be used for both immediate treatment of acute toxic shock and of the harmful effects caused thereby and for conferring long-term immunity against such toxic shock.
The peptides of the invention are capable of inhibiting expression of pyrogenic toxin-induced mRNA encoded by the IL-2, IFN-γ or TNF-β genes, as will be shown in the following Examples.
In addition, the peptides of the invention are capable of eliciting the production of antibodies that block T-cell activation in immunized individuals. The production of antibodies will be enhanced in the presence of a suitable immunization adjuvant. Preferred adjuvants may be keyhole lympet hemocyanin (KLH), proteosomes or alum.
As will be shown in the Examples, peptide p12(150–161) and the specific derivatives thereof, i.e. the Dimer form, the Cys form (Cys-p12(150–161)) and D-Ala form exhibit antagonist activity against SEB as well as against other pyrogenic exotoxins. The amino acid sequence of these synthetic peptides having the sequence shown in SEQ ID NOs.:2, 7, 9 and 10, differ in several positions from the corresponding sequence in SEB, T N K K K V T A Q E L D, found in peptide pSEB(150–161). The K K K and Q E L D motifs, features shared by peptides p12(150–161) (and its derivatives) and pSEB(150–161), are spaced equally in both and may be important for antagonist activity, with the triple-lysine motif K K K conferring 3 positive charges. Residues T150, K152, E159 and D161 of this SEB domain are conserved among all staphylococcal enterotoxins [Swaminathan et al. (1992) ibid.]. Indeed, the 150–161 domain of SEB is highly conserved among pyrogenic toxins in general, with 10/12 identities for SEA, SEC1, SEC2, and S. pyogenes exotoxm A (SPEA) and 9/12 for SEE [Bohach and Schilevert, Mol Gen Genet 209:5 (1987); Couch et al., J Bacteriol 170:2954 (1988); Bohach and Schijevert, Infect Immun 57:2249 (1989)]. All of these toxins contain the residues underlined above, including the first 2 lysine residues (KK) and the QELD [Swaminathan et al. (1992) ibid.; Bohach and Schlievert (1987) ibid.; Couch et al. (1988) ibid.; Bohach and Schlievert (1989) ibid.]. The superantigen, pep M5 protein, also contains a region with limited homology (<50%) to pSEB(150–161) located near its C-terminus [Wang et al., J Inimunol 151:1419 (1993)].
Conservation of SEB domains among the pyrogenic toxin family is, however, not unique for amino acids 150–161. SEB domains covering amino acids 76–86, 113–124, 151–168 and 213–226 all show extensive conservation within this family [Hoffmann et al., Infect Immun 62:3396 (1994)]. Moreover, the relevance of the conservation of the above-mentioned motifs corresponding to part of the sequence in pSEB(150–161) is not clear, since this region does not contribute to mitogenicity [Wang et al. (1993) ibid.].
In a further aspect, the invention relates to pharmaceutical compositions for the treatment or prophylaxis of toxin-mediated activation of T cells, comprising as active ingredient a therapeutically effective amount of at least one peptide according to the invention or derivative thereof. As described above, the peptides comprise an amino acid sequence substantially homologous to the amino acid sequence of a fragment SEB. The pharmaceutical compositions of the invention are also useful in protecting against toxic shock induced by a pyrogenic exotoxin or by a mixture of pyrogenic exotoxins.
The term toxin-mediated activation as used throughout this application can mean activation of T cells mediated by a single pyrogenic exotoxin or a mixture of such toxins.
Examples 4, 5, 6 and most remarkably the in vivo murine experiments described in Example 9 and
The pharmaceutically ‘effective amount’ for purposes herein is that determined by such considerations as are known in the art. The amount must be sufficient to antagonize toxin-mediated activation of T cells.
The pharmaceutical composition of the invention can be prepared in dosage units forms and may be prepared by any of the methods well-known in the art of pharmacy. In addition, the pharmaceutical compositions of the invention may further comprise pharmaceutically acceptable additives such as pharmaceutical acceptable carrier, excipient or stabilizer, and optionally other therapeutic constituents. Naturally, the acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed.
The magnitude of therapeutic dose of the composition of the invention will of course vary with the group of patients (age, sex, etc.), the nature of the condition to be treated and with the route administration and will be determined by the attending physician.
In yet a further embodiment, the invention relates to vaccines for conferring immunity against toxic shock induced by pyrogenic exotoxins, comprising as active ingredient an immunologically effective amount of at least one peptide according to the invention or derivatives thereof and may contain mixtures of such peptides and derivatives.
By the term ‘immunologically effective amount’ is meant any amount sufficient to enhance the production of antibodies that block T cell activation induced by pyrogenic exotoxins, and confer immunity against toxic shock induced by a pyrogenic exotoxin or by a mixture of pyrogenic exotoxins.
The vaccines of the invention may optionally further comprise suitable immunization adjuvants or mixtures thereof. Suitable adjuvants may be proteosomes, KLH and alum, as well as combinations of proteosomes with alum and of KLH with alum.
As will be shown in the following Examples, the vaccines according to the invention are capable of enhancing production of antibodies that block T-cell activation induced by pyrogenic exotoxins.
Efforts to develop antidotes for use against toxic shock symptoms have concentrated on blocking downstream phenomena in the toxicity cascade, mainly by inhibiting the action of TNF with monoclonal antibodies or soluble receptors. The high levels of cytokines produced in response to toxins render this approach ineffective. The present Invention shows that it is possible to block the action of a pyrogenic exotoxin by an altogether different strategy, using antagonists that inhibit toxin action at the top of the toxicity cascade, before activation of T cells takes place.
The Examples describe in detail molecular methods, analysis of pyrogenic exotoxin-mediated activation of the human cellular immune response through expression of IL-2, IFN-γ, and TNF-β genes in PBMC, to evaluate toxin antagonist activity.
Studies in human PBMC were combined with animal tests to evaluate immunogenic properties and vaccine efficacy and it will be shown that these methods are applicable in devising agents that counteract or protect human PBMC also against other members of the family of pyrogenic exotoxins.
Because humans are far more sensitive to pyrogenic exotoxins than mice, while primate models poses other limitations such as cost, there is a need for a human in vitro system, capable of analyzing the mechanisms of toxin-mediated activation and suppression of the immune response. The present invention provides such a system, which offers major advantages:
Thus, the invention also relates to a method for treating toxic shock induced by a pyrogenic exotoxin or a mixture of pyrogenic exotoxins. The method comprises administering to a patient in need of such treatment a therapeutically effective amount of the pharmaceutical composition of the invention or a therapeutically effective amount of at least one peptide of the invention, or functional derivative thereof.
In a further embodiment there is provided a method for preventing toxic shock induced by a pyrogenic exotoxin or a mixture of pyrogenic exotoxins comprising administering to a patient in need of such treatment a therapeutically effective amount of a composition of the invention or of a therapeutically effective amount of at least one peptide of the invention or functional derivative thereof.
The invention also relates to a method for immunizing a patient against toxic shock induced by pyrogenic exotoxin, comprising administering to a patient an effective immunizing amount of the vaccine of the invention or of at least one peptide of the invention or functional derivative thereof.
The magnitude of therapeutic dose of the peptide or of the vaccine of the invention will of course vary with the group of patients (age, sex etc.), the nature of the condition to be treated and with the route administration and will be determined by the attending physician.
The peptides and the vaccines of the invention can be administered and dosed in accordance with good medical practice. In particular, the immunization method of the invention comprises a single administration of the peptides or vaccines of the invention. Administration may be carried out in various ways, including intravenous, intramuscular or subcutaneuos injection. However, other methods of administration such as intranasal administration are also possible.
As shown hereafter, antibodies raised against peptides derived from certain SEB protein domains actually enhance the response of human PBMC to SEB and SEA, as expressed by induction of IL-2 and IFN-γ mRNA, rather than protect against these toxins. This would provide a tool for detecting such potential exacerbation by any pyrogenic exotoxin vaccine even before trials with humans are conducted.
Design of pyrogenic exotoxin antagonist peptides as described herein may find novel applications not only in hitherto neglected areas, prophylaxis against pyrogenic exotoxins and treatment of toxin-exposed individuals but also may facilitate the development of a safer pyrogenic exotoxin vaccine. A defined peptide vaccine, free of exacerbating properties, would be superior to a toxoid vaccine.
Thus, the invention also relates to antibodies directed against a peptide of the invention, which are capable of blocking T-cell activation by a pyrogenic exotoxin or a mixture of pyrogenic exotoxins, which may be mono- or polyclonal antibodies.
In addition, the invention also relates to antisera containing antibodies directed against peptides of the invention. For example, peptides such as pSEBLC(150–161) or p12LC(150–161) can be linked through the lauryl cysteine residue to proteosomes. Alternatively, peptides such as those coupled through their C-terminus to a cysteine (C) residue, as described above, can be linked through the cysteine residue to KLH. KLH and proteosomes are known adjuvants for immunization and the peptides so linked are capable of eliciting the production of antibodies.
It is also known in the art that aluminum hydroxide (alum) may be used as an immunization adjuvant directly, with non-linked peptides, or after linking a peptide to proteosomes or to KLH [Lowell et al., (1996) ibid.] or other suitable adjuvants. Therefore, the invention also relates to antisera containing antibodies directed against peptides of the invention, or functional derivatives thereof. The antisera of the invention are capable of alleviating toxic shock induced by a pyrogenic exotoxin. An antiserum according to the invention can be a domestic animal antiserum, for example rabbit, sheep, bovine, equine, porcine or goat antiserum.
For some purposes, for example the in vitro use mentioned above, human sera from immunized individuals can be used.
As already mentioned above, at present there exists no tool for assessing the efficacy of a vaccine against pyrogenic toxins in conferring immunity thereagainst. The present invention provides for such a method and thus, in a further aspect the invention relates to a method for assessing the efficacy of a vaccine for conferring immunity against one or more pyrogenic toxins comprising determining the ability of serum from an immunized individual to antagonize toxin-mediated activation of T cells.
The ability of serum from an immunized individual to antagonize toxin-mediated activation of T cells can be determined by measuring the inhibition of expression of pyrogenic toxin-induced mRNA encoded by the IL-2, IFN-γ, or TNF-β genes. Reference may be made to Example 7 and
A kit for assessing the efficacy of a vaccine for conferring immunity against one or more pyrogenic toxins comprising determining the ability of serum from an immunized individual to antagonize toxin-mediated activation of T cells by the method of the invention is also provided.
Cell Culture and Induction of Human Cytokine Gene Expression
PBMC from healthy human donors were separated on Ficoll Paque (Pharmacia), washed twice with 50 ml of RPMI 1640 medium, resuspended at a density of 4×106/ml and cultured in this medium supplemented with 2% fetal calf serum, 2 mM glutamine, 10 mM MEM nonspecific amino acids, 100 mM Na-pyruvate, 10 mM Hepes pH 7.2, 5×10−
RNase Protection Analysis
Total RNA was extracted with guanidinium isothiocyanate [Chomczynski and Sacchi, Anal Biochem 162:156 (1987)]. RNase protection analysis was done [Arad et al. (1995) ibid.] using genomic antisense RNA probes transcribed with [α-32P]UTP in vitro from DNA inserted into pBS (Promega). The IL-2 probe (600 nucleotides (nt)), transcribed from the T7 promoter, is complementary to the third exon and a portion of the third intron of the IL-2 gene; in 8 M urea-polyacrylamide gels, it yields an RNA fragment of 117 nt protected by IL-2 mRNA. The IFN-γ probe (274 nt), transcribed from the T3 promoter, is complementary to the third exon and a portion of the third intron of the IFN-γ gene and yields an RNA fragment of 183 nt protected by IFN-γ mRNA. The TNF-β probe (700 nt), transcribed from the T3 promoter, is complementary to part of exon 1, exon 2, exon 3, and portions of intron 3 and exon 4; TNF-β mRNA protects 2 fragments of 274 and 263 nt. Sense RNA transcripts yielded no detectable signal upon hybridization. Antisense RNA probes for 18S rRNA (protecting 90 nt) or β-actin (protecting 415 nt) served as loading controls.
Quantitative Dot Blot Hybridization of IL-2 and IFN-γRNA
PBMC from 1-ml cultures were collected and lysed in 7.5 M guanidinium-HCl. RNA, precipitated overnight in ethanol at −20° C., was dissolved into formaldehyde and incubated for 15 min at 60° C. Four serial 2-fold dilutions, made in 10× saline sodium citrate, were applied in duplicate to nitrocellulose sheets, using a 96-well dot blot apparatus. After baking in a vacuum oven at 80° C., sheets were hybridized separately with 32P-labeled antisense RNA probes for human IL-2 and IFN-γ, respectively. Exposed autoradiograms were scanned at 630 nm in an ELISA reader. RNA levels are expressed in units of A630. Serial twofold dilutions of a given RNA sample yield a linear optical density response over a 200-fold range of intensities of gene expression that is proportional to the concentration of specific RNA present in each sample [Arad et al. (1995) ibid.; Gerez et al., Clin Immunol Immunopathol 58:251 (1991); Kaempfer et al., J Clin Oncol 14:1778 (1996)].
Synthesis of SEB-related Peptides
Peptides were synthesized in the Peptide Synthesis Unit of The Hebrew University-Hadassah Medical School, Jerusalem. tBOC chemistry (tertiary butoxycarbonyl protection of amino termini) was used to prepare one batch of peptides, used for
Proteosome or KLH Coupling of Peptides
Outer membrane proteosome preparations were stored at −70° C. after purification, as described [Mallett et al., Infect Immun 63:2382 [1995)], from group B type 2 Neisseria meningitidis by extraction of a paste of phenol-killed bacteria with a solution of 6% Empigen BB (Albright and Wilson, Whitehaven, Cumbria, UK) in 1 M calcium chloride, followed by precipitation with ethanol, solubilization in Tris-buffered saline with EDTA and 1% Empigen BB, reprecipitation with ammonium sulfate, and then resolubilization in the Tris buffer with 1% Empigen BB [Lowell et al. (1996) ibid.]. LC-peptides were coupled to proteosomes as described by Lowell et al. [Lowell et al. (1996) ibid.]. Maleimide-activated keyhole lympet hemocyanin (KLH) was coupled to C-terminal C residues following instructions of the supplier (Pierce).
Immunization
Rabbits were immunized intramuscularly with 100 μg of proteosome- or KLH-coupled peptides, at 0 and 3 weeks. Three bleeds were performed; data for the third bleed are shown in Table 2. BALB/c mice were immunized with 50–100 μg of proteosome- or KLH-coupled peptides, via the parenteral (intramuscular) or intranasal routes as described by Lowell et al. [Lowell et al. (1996) ibid.]. Blood was collected periodically from rabbits from the ear vein, and from mice from the retroorbital plexus at 3 weeks before challenge with SEB. SEB-specific IgG was assayed by ELISA as [Lowell et al., (1996) ibid.].
SEB Challenge Assays of Vaccine Efficacy in Vivo
Lot 14–30 SEB was used for challenge. For parenteral (IM) or intranasal (IN) challenge, D-galactosamine sensitization was used in conjunction with SEB, as described [Lowell et al. (1996)].
Peripheral blood mononuclear cells (PBMC) are used to provide a quantitative measure of the primary response of human T cells to an exotoxin, through expression of the Th1 type cytokine genes encoding IL-2, IFN-γ, and TNF-β. Gene expression is analyzed within hours after stimulation, providing a more direct and immediate measure of the action of SEB than cell proliferation which results from a complex series of events.
Induction of IL-2, IFN-γ and TNF-β Gene Expression by SEB
Exposure of PBMC to SEB leads to induction of IL-2 and IFN-γ mRNA, shown by quantitative dot blot hybridization (FIGS 1A–1C) and RNase protection analysis with a genomic antisense RNA probe (
Design of SEB-Related Peptides
Fourteen peptides representing particular SEB domains were synthesized (Table 1; peptides were >95% purity by HPLC) and assayed for the ability to block SEB-mediated induction of IL-2, IFN-γ or TNF-β gene expression. Identification of such a peptide could be useful for developing a peptide vaccine against SEB and more directly, for preventing the harmful effects of SEB on the immune response. Multiple, widely separated regions within SEB interact with the TCR on one hand and with the MHC class II molecule on the other [Swaminathan et al. (1992) ibid.; Jardetzky et al., Nature 368:711 (1994)]. Domains chosen consist of amino acid residues 13–33, 41–61, 81–93 and 208–218, essential for binding to TCR and MHC class II; residues 21–29 and 48–61, essential for binding to the TCR; and residues 13–17 and 44–52, essential for binding to MHC class II [Swaminathan et al. (1992) ibid.]. A 12-amino acid SEB domain, made up of residues 150–161, is thought not to be involved in binding to TCR or MHC class II but forms a central turn starting within β-strand 7 and connecting it, via short βstrand 8, to α-helix 4 (Table 1 and
To allow study of their immunogenicity and ability to elicit protective immunity against SEB, many of the peptides were synthesized also with an extra lauryl-cysteine residue at their N-terminus (LC-) or with an extra cysteine residue at their C-terminus (-C), as indicated in Table 1.
Lack of SEB Agonist Activity of SEB-derived Peptides
SEB agonist activity of peptides was examined by the ability to induce expression of IL-2 and IFN-γ genes. Even when present in 200-fold higher molar amounts than SEB, no peptide exhibited significant SEB agonist activity, defined as ≧2-fold increase in RNA over basal level (
Peptide p12(150–161) is an SEB Antagonist
Antagonist activity of SEB-related peptides was defined by the ability to block SEB-mediated induction of IL-2, IFN-γ, and/or TNF-β gene expression in PBMC. A short, unstructured peptide would be expected to compete poorly with intact SEB whose binding is stabilized by multiple interactions with the TCR and MHC class II molecule [Swaminathan et al. (1992) ibid.; Jardetzky et al. (1994) ibid.]. However, an appropriate SEB-related peptide might compete with SEB for one of its cognate sites, preventing thereby a cooperative interaction with multiple sites.
Ability to antagonize induction of IL-2 or IFN-γ gene expression was assayed by exposing PBMC populations to SEB in the presence of a 100- to 200-fold molar excess of an individual peptide. The resulting hybridization patterns for IL-2 and IFN-γ RNA are shown and quantitated in
PBMC cultured with either pSEB(150–161) or p12(150–161) showed undiminished viability, as judged by trypan blue exclusion analysis and recovery of total cellular RNA. The SEB antagonist activity of these peptides thus does not result from a cytotoxic effect. Both peptides reproducibly failed to inhibit PHA-mediated induction of IL-2 and IFN-γ genes (not shown).
The natural homolog of p12(150–161), pSEB(150–161), was less active than the variant peptide as SEB antagonist (
None of the peptides homologous to toxin domains involved in the interaction with T cell receptor and/or MHC class II molecule was able to inhibit the SEB-mediated induction of human IL-2, IFN-γ, and TNF-β genes. By contrast, the inventors have identified 12-mer p12(150–161), resembling a region well removed from these active sites which has the capacity to completely block expression of these cytokine genes upon their induction by SEB. The sequence of this potent antagonist peptide is man-made, deviating at various positions from the corresponding sequence in SEE; indeed, when a peptide with the natural SEE sequence was used, pSEB(150–161), it was less effective as antagonist. Antagonist activity decreased upon removal of 2 N-terminal amino acids. Despite its high degree of conservation, the charge of the corresponding sequence in SEA is neutral whilst that of pSEB(150–161) or of p12(150–161), is positive. Indeed, although SEE is 68% homologous with SEC. it shows only 27% homology with SEA [Betley and Mekalanos, J Bacteriol 170:34 (1995)].
The region covering amino acids 150–161 overlaps partially with a larger, 31-amino acid peptide, pSEB(130–160). When conjugated to KLH, pSEB(130–160) inhibited the SEB-induced proliferation of mixed cultures of human peripheral blood monocytes and lymphocytes by 2- to 4-fold but was not unique in this property, since peptides overlapping with other SEB domains, covering amino acids 1–30, 61–92, 93–112, 151–180, 171–200 and 191–220, had a similar inhibitory effect [Jett et al. (1994) ibid.]. Although pSEB(130–160) was able to inhibit binding of SEB to human lymphocytes [Jett et al. (1994) ibid., Komisar et al., Infect Immun 62:4775 (1994)] it was shown that a smaller overlapping peptide, pSEB(150–162), failed to inhibit binding of SEB to HUT-78 cells, a human T cell line, as studied by fluorescence, whereas another peptide, pSEB(90–114), inhibited by 2-fold. Wang et al. [(1993) ibid.] showed that a synthetic peptide encoding the carboxy-terminal 41 amino acids of the superantigenic pep M5 protein of S. pyogenes inhibited pep M5-mediated T cell proliferation. They commented with respect to pSEB(152–160) that “most studies seem to indicate that this region does not contribute to mitogenicity” while emphasizing in this context that “immunologic function is not determined solely by the primary amino acid structure of a particular region but is influenced by the context in which it is located” and indicating that the longer amino acid sequence may contribute to α-helix amphi-philicity. The lack of inductive activity of pSEB(150–161) or p12(150–161) is shown in
Enhancement of SEB Antagonist Activity
With PBMC populations from occasional healthy donors, it was observed that p12(150–161) was only weakly or not inhibitory to SEB. Such an experiment is illustrated in
A similar rise in efficacy was observed when p12(150–161) was cyclized with terminal cysteines (Cys-p12(150–161)) (
Broad-spectrum Pyrogenic Exotoxin Antagonist Activity
The SEB 150–161 domain is conserved among pyrogenic toxins. The sequence of p12(150–161), shown in SEQ ID NO: 2, differs in several positions from the corresponding sequence in SEB, TNKKKVTAQELD found in pSEB(150–161), but shared KKK and QELD motifs are spaced equally in both peptides. Residues T150, K152, E159 and D161 of this SEB domain are conserved among all staphylococcal enterotoxins [Swaminathan et al. (1992) ibid.]. Indeed, domain 150–161 of SEB is highly conserved among pyrogenic toxins, with 10/12 identities for SEA, SEC1, SEC2, and SPE A and 9/12 for SEE [Bohach and Schlievert (1987) ibid.; Couch et al. (1988) ibid.; Bohach and Schlievert (1989) ibid.]. All of these toxins contain the residues underlined above [Swaminathan et al. (1992) ibid.; Bohach and Schlievert (1987) ibid.; Couch et al. (1988) ibid.; Bohach and Schlievert (1989) ibid.].
The prediction that SEB antagonist peptides may have wider antagonist activity was tested.
The results of
These results would lead a man of the art to the conclusion that the SEB antagonist activity of the peptides examined does not extend to two less related toxins, TSST-1 and SEA.
Nevertheless, in contrast to the results of
Induction of IL-2 mRNA by SPE A for another PMBC population is depicted in
The results of
Dodecamer antagonist peptide p12(150–161) elicits, in rabbits, antibodies that protect human T cells, capable of expressing IL-2 and IFN-γ genes, from activation not only by SEB but also by SEA and TSST-1. This finding indicates that used as vaccine, the peptide has the potential to confer broad-spectrum protective immunity.
Antiserum Against p12(150–161) Blocks the Action of SEB, SEA, SPE A and TSST-1
Antibodies raised against a SEB-related peptide might bind to a pyrogenic exotoxin, for example SEB and modulate its action. To allow study of their immunogenicity and ability to elicit protective immunity against pyrogenic exotoxins, most peptides were synthesized also with an added N-terminal lauryl-Cys or C-terminal Cys (Table 1), to permit their linkage to proteosomes or KLH adjuvant, respectively [as described by Lowell et al. (1996) ibid.], in order to facilitate generation of antibodies. In addition, alum may be used as an immunization adjuvant directly with non-linked peptide, or after linking a peptide to proteosomes or to KLH [Lowell et al. (1996) ibid.].
Sera from rabbits immunized with individual peptides were titrated for their ability to bind SEB. Most of the peptides in Table 1 proved immunogenic by this parameter but greatly differed in titer of serum anti-SEB IgG achieved. Thus, serum against pSEB(13–33)C had a titer of 102,400 while corresponding titers for pSEB(81–93), pSEBLC(41–61) and p12LC(150–161) were 50, 1,600 and 1,600, respectively (Table 2). On the basis of the results in Table 2, one would not select p12(150–161) for use as a peptide vaccine but instead, prefer to use pSEB(13–33)C.
None of the antisera raised against individual SEB-related peptides had any SEB agonist activity, defined by ability to induce IL-2 or IFN-γ mRNA (not shown). Ability to inhibit SEB-mediated induction of these genes is examined in
Antisera against pSEB(81–93) and pSEBLC(41–61), which had low titers of anti-SEB IgG, not only failed to inhibit expression of IL-2 and IFN-γ mRNA but significantly stimulated their SEB-mediated induction (
There was a striking lack of correlation between the ability of anti-peptide sera to bind SEB and to block SEB action. Antisera against pSEBLC(41–61) and p12LC(150–161) showed identical anti-SEB IgG titers yet affected induction of IL-2 and IFN-γ genes by SEB in an opposite manner, stimulation vs. complete inhibition. Apparently, certain antibodies effectively potentiate SEB action while others block it. As judged by serial dilution, inhibition of SEB-mediated gene induction was also up to 30-fold more sensitive for detecting blocking antibodies than binding of IgG to SEB.
In an earlier study, rabbit sera raised against SEB peptides 113–144, 130–160, 151–180 and 171–200 each reduced SEB-induced lymphocyte proliferation weakly (≦2.5-fold), apparently in a nonspecific manner [Jett et al. (1994) ibid.].
Antibodies against p12(150–161) have broad-spectrum toxin blocking activity. In
Antibodies raised against p12(150–161) and pSEB(21–33)C likewise inhibited the action of the even less related toxin, TSST-1 (
Hence, as judged from inhibition of IL-2 and/or IFN-γ gene expression analysis, protective activity of antibodies elicited by p12(150–161) is not narrowly restricted in range of pyrogenic exotoxins (
Protective Effect of SEB-related Peptides as SEB Vaccine in Mice
The finding that antiserum against p12(150–161) blocks the action of SEB in human PBMC suggested its potential as peptide vaccine. To examine this point, the D-galactosamine-treated mouse model was used [Lowell et al. (1996a) ibid.]. Mice were repeatedly immunized with individual peptides and then challenged with a lethal dose of SEB. Table 3 details the results of 3 separate trials.
In the first, formalin-inactivated intact SEB toxoid protected 20% of the mice against a lethal dose of SEB, either upon parenteral (i.m.) or intranasal vaccination. The fact that SEB toxoid did not afford complete protection as reported [Lowell et al. (1996a) ibid.] shows that the conditions of toxin challenge were more severe in the present experiment. In this trial, proteosome-coupled p12(150–161) yielded 10% protection. Relative to SEB toxoid, p12(150–161) thus did show protective activity.
In the second trial, the ability of proteosome-coupled pSEB(150–161) and p12(150–161) to provide immune protection against SEB challenge was compared to that of several larger, synthetic peptides [Jett et al. (1994)] derived from the SEB amino acid sequence, including pSEB(130–160) which overlaps almost completely with pSEB(150–161) and p12(150–161). None of the larger peptides showed detectable protective activity against intranasal challenge with SEB, yet both pSEB(150–161) and p12(150–161) afforded protection, evident from 22–29% survival (Table 3). pSEB(150–161) also elicited protective immunity (22%) when vaccination was by the intramuscular route (trial 3) rather than intranasally (trial 2). By contrast, two larger peptides, pSEB(130–160) and pSEB(151–180) that overlap in part with pSEB(150–161), failed to give protection. Peptide pSEB(13–33)C, moreover, though able to elicit in rabbits a high titer of IgG that inhibit the action of SEB, SEA and TSST-1 on human T cells, capable of expressing IL-2 and IFN-γ genes, in vitro (Table 2 and
Despite the severity of challenge with SEB toxin in these early trials, two SEB-related peptides exhibiting SEB antagonist activity, pSEB(150–161) and p12(150–161), also showed activity as SEB vaccine. Protective effect of SEB-related peptides tended to correlate with SEB antagonist activity on human PBMC in vitro (
These examples show that it is possible to design an effective pyrogenic exotoxin antagonist. It is surprising that a linear, unstructured dodecapeptide, such as p12(150–161) or pSEB(150–161), can compete effectively with the intact, folded 239-amino acid SEB protein chain which interacts at multiple domains with regions in the MHC Class II molecule and in the T cell receptor. Such concerted interaction would lead to far higher affinity of binding for intact SEB vis-a-vis the peptides. Yet, as shown clearly in
Subtle changes in peptide composition had marked effects on its SEB antagonist activity (
For example, the amino acid sequence of these dodecapeptides could also be extended stepwise at their N- or C-termini, or both, either with the natural SEB amino acid sequence, or with a D-amino acid that will tend to render the peptide less amenable to digestion by L-amino-acid-specific proteases (as shown for D-Ala in
There are few examples of short peptides able to bind with sufficiently high affinity to a receptor in order to mimic the binding of the full-length ligand or to block its action. A T-cell receptor antagonist peptide of 4 amino acids was able to inhibit clinical disease progression in experimental allergic encephalomyelitis mediated by a diverse T cell repertoire [Kuchroo et al., J. Immunol. 153:3326 (1994)]. Peptides derived from the predicted helical region of MHC class II molecules may interact directly with T cell receptors: one such a peptide, a 16-mer, appeared capable of modulating immune responses in a physiologically significant manner [Williams et al., Immunol Res 11:11, (1992)]. In another example, a synthetic, 22-amino-acid segment of the human IFN-γ receptor was found to antagonize the action of IFN-γ [Seelig et al., J Biol Chem 270:9241 (1995)]. The linear forms of peptides generally lack a stable conformation in solution. Interaction with the cognate binding site on a receptor may induce folding of the peptide to mimic conformation in the native protein. Significant improvement in binding affinity can be achieved by generating dimers or multimeric forms of the peptide (
Methods for generating multimeric or cyclic forms of peptides exist, as by direct synthesis (
The experimental approach employed here used expression of human cytokine genes in vitro as a tool for designing both a broad-spectrum pyrogenic toxin antagonist and a prototypical peptide vaccine. This molecular approach is far more rapid than conventional methods that are based solely on animal tests, allowing evaluation of the biological properties of a candidate peptide well before moving on to animal studies for analysis of vaccine efficacy, prophylactic and therapeutic activity. Specifically, analysis of pyrogenic toxin antagonist activity on human T cells can be used to direct effective vaccine development, even before ability to elicit antibodies is studied in animals.
Efficacy of potential vaccine candidates can be evaluated not only by their pyrogenic toxin antagonist activity but also by their ability to elicit production of antibodies in rabbits (shown for SEB in Table 2) that can block the harmful action of pyrogenic exotoxins on human lymphoid cells (
In order to vaccinate humans effectively against one or more pyrogenic toxins, there is a need to assess the efficacy of vaccination. This requirement is independent of the nature of the vaccine, whether a toxoid, a mutant toxin or a peptide. However, unlike experimental animals (Table 3), humans cannot be challenged with toxin to test if the vaccination was effective. Therefore, it is desirable to use a surrogate marker instead, preferably one based on human T cell activation. Such a marker is provided through the method used in
SEB-related peptides with demonstrated SEB antagonist activity, p12(150–161) and pSEB(150–161), carry sequence elements that are highly conserved amongst pyrogenic toxins. Through improved peptide design, including introduction of structural constraints, peptides may thus be generated that exhibit broader antagonist activity (
Rabbit sera raised against p12(150–161) were equally able to block the ability of SEB, SEA, SPE A or TSST-1 to induce expression of IL-2 and IFN-γ genes (
An unexpected finding, shown in
Y N K K K A T V Q E L D
@Only the -C form was studied
See Table 1 for explanation of SEB peptides. For immunization, peptides carrying LC at their N-terminus were coupled to proteosomes; peptides carrying C at their C-terminus were coupled to KLH. Sera were assayed for IgG able to bind SEB.
Groups of mice were vaccinated with SEB-related peptides or SEB toxoid as indicated. SEB toxoid [Lowell et al., (1996) ibid.], peptides pSEB(93–112), pSEB(130–160), pSEB(151–180), pSEB(191–220) and pSEB(191–220)C [Jett et al., (1994) ibid. ] and preparation of LC-derivatives for coupling to proteosomes [Lowell et al., (1996) ibid.], were described elsewhere. In trial 1, 2 vaccinations were given 2 weeks apart, with SEB challenge after 1 month. In trials 2 and 3, 3 vaccinations were given at 2-week intervals, followed by a boost after 6 weeks and SEB challenge 2 weeks later. IgG were determined 3 weeks before challenge. IM, intramuscular; IN, intranasal.
Table 4 lists for each of the SEQ ID NOs' of the sequence listing, the corresponding alternative notation used in the specification.
Inhibition of Action of SEB and TSST-1 on Human Peripheral Blood Mononuclear Cells by p12(150–161) Carrying a D-Ala Residue at Both N- and C-termini
While 100% of mice exposed to SEB lethal challenge died within 1–2 days in the control group, 100% survived lethal challenge with SEB when p12(150–161) carrying a D-Ala residue at both N- and C-termini was administered intravenously half an hour before challenge, and 70% when it was administered intraperitoneally (
To extend this result to other toxins and to demonstrate broad-spectrum protective activity of this peptide, lethal challenge with TSST-1 was tested. Within the superantigen toxin family, TSST-1 is most remote from SEB, showing only 6% amino acid homology for the whole toxin molecule. TSST-1 kills more slowly than SEB, requiring a longer toxin exposure (
The p12(150–151) peptide carrying D-Ala residues at both its N- and C-termini, which is SEB-related, afforded good antagonist activity not only against SEB intoxication, but also against the remotely homologous TSST-1. Thus, p12(150–151) peptide carrying D-Ala residues at both its N- and C-termini can be used as an effective broad-spectrum antagonist against pyrogenic exotoxins.
This is a continuation-in-part application of International Patent Application PCT/IL97/00438, filed Dec. 30, 1997, which claims priority to Israeli Patent Application No. 119938, filed Dec. 30, 1996.
The U.S. Government has a nonexciusive, nontransferable, irrevocable paid-up license to practice or have practiced this invention for or on its behalf as provided for by the terms of Contract No. DAMD17-93-C-3108 awarded by the U.S. Department of the Army.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5310874 | Tamura et al. | May 1994 | A |
| 5407609 | Tice et al. | Apr 1995 | A |
| 5545716 | Johnson et al. | Aug 1996 | A |
| 5705151 | Dow et al. | Jan 1998 | A |
| Number | Date | Country |
|---|---|---|
| 499 681 | Aug 1992 | EP |
| 61178661 | Aug 1986 | JP |
| 9110680 | Jul 1991 | WO |
| 9112818 | Sep 1991 | WO |
| 9640235 | Dec 1996 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20020028211 A1 | Mar 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IL97/00438 | Dec 1997 | US |
| Child | 09150947 | US |
