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WO2005108419A1 - Proteines mutantes de cytolysine liant le cholesterol - Google Patents

Proteines mutantes de cytolysine liant le cholesterol Download PDF

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Publication number
WO2005108419A1
WO2005108419A1 PCT/GB2005/001774 GB2005001774W WO2005108419A1 WO 2005108419 A1 WO2005108419 A1 WO 2005108419A1 GB 2005001774 W GB2005001774 W GB 2005001774W WO 2005108419 A1 WO2005108419 A1 WO 2005108419A1
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Prior art keywords
protein
mutant
wild type
amino acids
cytolysin
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PCT/GB2005/001774
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English (en)
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Lea-Ann Kirkham
Timothy John Mitchell
Graeme James Macfarlane Cowan
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Lea-Ann Kirkham
Timothy John Mitchell
Graeme James Macfarlane Cowan
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Priority claimed from GB0410221A external-priority patent/GB0410221D0/en
Application filed by Lea-Ann Kirkham, Timothy John Mitchell, Graeme James Macfarlane Cowan filed Critical Lea-Ann Kirkham
Publication of WO2005108419A1 publication Critical patent/WO2005108419A1/fr

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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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]

Definitions

  • the present invention relates to immunogenic compositions comprising mutant cholesterol-binding cytolysin proteins.
  • the invention further relates to such proteins and nucleic acids encoding these proteins.
  • Streptococcus pneumoniae is an important pathogen, causing invasive diseases such as pneumonia, meningitis and bacteraemia. Even in regions where effective antibiotic therapy is freely available, the mortality rate from pneumococcal pneumonia can be as high as 19% in hospitalised patients. In developing countries, in excess of 3 million children under the age of 5 years die each year from pneumonia, of which S. pneumoniae is the commonest causative agent. S . pneumoniae also causes less serious, but highly prevalent infections such as otitis media and sinusitis, which have a significant impact on health-care costs in developed countries. Otitis media is especially important in young children, while sinusitis affects both children and adults.
  • the vaccine comprises seven purified Streptococcus capsular polysaccharides (serotypes 4, 6B, 9V, 14, 18C, 19F and 23F) out of a possible 90 (Kalin, 1998), each conjugated to a carrier protein. Preparation of such a vaccine is described in US Patent 4,673,574 (Anderson).
  • the protein used for conjugation of the capsular polysaccharides is a diphtheria toxoid, CRM ⁇ 97 , offering an increase in the immunogenicity of the vaccine in infants (Blum et al, 2000; Katkocin, 2000) .
  • each serotype of S . pneumoniae has a structurally distinct capsular polysaccharide, such that immunization with one serotype tends not to confer protection against the majority of the other serotypes, although some cross-protection does occur against vaccine-related serotypes (Whitney et al . , 2003) .
  • Complementary approaches to serotype-specific immunization are being investigated.
  • PLY Pneumolysin
  • S. pneumoniae the 53kDa toxin produced by all invasive strains of S. pneumoniae (Paton et al, 1993) .
  • PLY could be used alone or as a carrier protein conjugated to the polysaccharides in Prevnar®, offering increased efficacy.
  • Alexander et al (1994) demonstrated that immunisation of mice with a PLY toxoid conferred immune protection upon challenge with 9 different serotypes of S. pneumoniae. PLY has been shown to stimulate an immune response similar to that of S.
  • PLY belongs to the group of Cholesterol-binding Cytolysins (CBCs) that bind to the cholesterol of host cell membranes prior to formation of large 30-50mer ring structures that create lytic pores (Palmer, 2001; Jedrzejas, 2001). The mechanism of pore-formation is not fully understood and there is much debate over the sequence of events (Bonev et al, 2000; Shepard et al, 1998) .
  • CBCs Cholesterol-binding Cytolysins
  • PLY native PLY is highly toxic, which is a problem in terms of the development of immunogenic compositions.
  • conjugation process used in production would render PLY non-toxic, it would be more favourable to start with a non-toxic form. Further, a toxic form would be difficult to use in preparation of unconjugated immunogenic compositions.
  • the toxicity of PLY can be significantly reduced by site-directed mutagenesis to create PLY toxoids, known as Pneumolysoids (Paton, 1996) .
  • Pneumolysoids Pieric acid, a variety of such toxoids exist and have been shown to give immune protection, either independently or when conjugated to polysaccharides, to mice in response to a challenge with virulent type 2 D39 S.
  • the mutant PFO has a Y ⁇ 8 ⁇ to A ⁇ 8 ⁇ mutation.
  • the mutant protein apparently has a reduced pore formation activity when compared with the non-mutant form of PFO.
  • a further problem with PLY is that it aggregates upon large-scale production, a problem which must be solved in order for PLY to be used in immunogenic compositions. It is believed that the aggregation of PLY is related to the oligomerisation of PLY involved in pore formation.
  • the present invention thus attempts to reduce or eliminate PLY-PLY interaction (oligomerisation) , such that the chance of aggregation during large-scale production will decrease, thereby creating an easily purified form of PLY.
  • Toyos et al (1996) describe the raising of monoclonal antibodies (mAbs) to various regions of PLY, and probing of the whole toxin and a ⁇ proteinase K nicked' form. Proteinase K cuts PLY into a 37kDa and 15kDa fragment.
  • Antibody mAb PLY 4 only recognised whole PLY, and neither of the fragments, indicating that the epitope on PLY for this mAb is within the nicked region.
  • Monoclonal antibody PLY 4 has been further characterised by Suarez-Alvarez et al (2003) and they suggest that the epitope for mAb PLY 4 is further downstream than the N 143 region initially proposed by Toyos et al in 1996.
  • the site of recognition now appears to be conformation dependent and within amino acids E ⁇ 5 ⁇ - Y 247 and not within the N 143 region.
  • Previously a N 1 2 N 1 3 deletion and N 143 D substitution within PLY were created by the present inventors as initial steps to understanding this region and its role in oligomerisation.
  • the present invention relates broadly to immunogenic compositions comprising mutant bacterial cytolysin proteins.
  • the invention further relates to such proteins and nucleic acids encoding these proteins.
  • the invention is directed to an isolated mutant cytolysin protein, wherein the mutant cytolysin protein differs from the wild type cytolysin protein by the presence of a mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin (PLY) sequence, such that the toxicity of the mutant cytolysin is reduced relative to that of the wild-type cytolysin protein.
  • the mutation may be located within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin (PLY) sequence .
  • the mutant cytolysin is a mutant perfringolysin comprising a substitution or deletion at Y , an d in particular, a Y 181 to A ⁇ 8 ⁇ , substitution
  • the mutant comprises a further mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin (PLY) sequence, wherein the further mutation is capable in isolation of reducing the toxicity of the wild type sequence.
  • PLY pneumolysin
  • the mutation may be a deletion or substitution of one or more amino acids within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin (PLY) sequence.
  • the mutant PLY protein may differ from the wild type protein by the substitution or deletion of one or more amino acids within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin (PLY) sequence.
  • the mutant cytolysin is not a mutant perfringolysin having a substitution at position Y 181 .
  • the mutant cytolysin protein may differ from the wild type protein by the substitution or deletion of two adjacent amino acids within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin (PLY) sequence, as exemplified by the deletion of amino acids corresponding to valine 144 and proline 145, alanine 146 and arginine 147, methionine 148 and glutamine 149, or tyrosine 150 and glutamic acid 151.
  • Any of the foregoing mutant cytolysin proteins may further comprise at least one amino acid substitution or deletion in at least one of the regions corresponding to amino acids 257-297, 367-397 or 424-437 of the wild type pneumolysin sequence .
  • the isolated mutant cytolysin protein has reduced toxicity to mammals. This is typically a consequence of having reduced pore-forming activity, which may be associated with reduced haemolytic activity and/or reduced oligomerisation activity, as compared with wild type protein. Desirably, although not necessarily, the mutant cytolysin protein has reduced oligomerisation activity to facilitate purification and subsequent manipulation.
  • the invention is directed to an immunogenic conjugate comprising: (a) a saccharide, oligosaccharide, polysaccharide, peptide, polypeptide or protein; and (b) an isolated mutant cytolysin protein, wherein the mutant cytolysin protein differs from the wild type cytolysin protein by the presence of a mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin (PLY) sequence, such that the toxicity of the mutant is reduced relative to that of the wild-type protein.
  • the mutation may be located within the region of amino acids corresponding to 144 to 151 of the wild type pneumolysin sequence .
  • the mutation may be a substitution or deletion of one or more amino acids within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin sequence.
  • the mutant cytolysin protein of the immunogenic conjugate may differ from the wild type cytolysin protein by the substitution or deletion of one or more amino acids within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin sequence.
  • the mutant cytolysin protein of the immunogenic conjugate differs from the wild type cytolysin protein by the substitution or deletion of two adjacent amino acids within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin sequence, as exemplified by the deletion of amino acids corresponding to valine 144 and proline 145, alanine 146 and arginine 147, methionine 148 and glutamine 149, or tyrosine 150 and glutamic acid 151 of the wild type pneumolysin sequence.
  • any of the foregoing mutant cytolysin proteins of the immunogenic conjugate may further comprise at least one amino acid substitution or deletion in at least one of the regions corresponding to amino acids 257-297, 367-397 or 424-437 of the wild type pneumolysin sequence.
  • the saccharide, oligosaccharide or polysaccharide of the immunogenic conjugate may be bacterial in origin, and may be derived from the same species, e.g. the same strain, as the cytolysin.
  • the invention provides an isolated and purified nucleic acid sequence comprising a nucleic acid sequence a) encoding a mutant cytolysin protein, wherein the mutant cytolysin protein differs from the wild type cytolysin protein by the presence of a mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin sequence, such that the toxicity of the mutant cytolysin is reduced relative to that of the wild-type cytolysin protein; or b) which is complementary to a nucleic acid sequence defined in a) .
  • the mutant cytolysin is a mutant perfringolysin comprising a substitution or deletion at Y ⁇ 8 ⁇ , and in particular, a Y 181 to A 181 substitution
  • the mutant comprises a further mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin (PLY) sequence, wherein the further mutation is capable in isolation of reducing the toxicity of the wild type sequence.
  • PLY pneumolysin
  • the mutation may be located within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin sequence.
  • the mutation may be a substitution or deletion of one or more amino acids within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin sequence.
  • the nucleic acid sequence may encode a mutant cytolysin protein which differs from the wild type protein by the substitution or deletion of one or more amino acids within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin sequence.
  • the nucleic acid sequence may encode a mutant cytolysin protein which differs from the wild type protein by the substitution or deletion of two adjacent amino acids within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin sequence, as exemplified by the substitution or deletion of amino acids corresponding to valine 144 and proline 145, alanine 146 and arginine 147, methionine 148 and glutamine 149, or tyrosine 150 and glutamic acid 151 of the pneumolysin sequence.
  • the mutant cytolysin proteins encoded by the nucleic acid sequence may be any of the foregoing, and may further comprise at least one amino acid substitution or deletion in at least one of the regions corresponding to amino acids 257-297, 367- 397 or 424-437 of the wild type pneumolysin sequence.
  • the invention provides a recombinant expression vector which comprises any of the foregoing isolated and purified nucleic acid sequences encoding a mutant cytolysin protein, as well as a recombinant host cell transformed, transfected or infected with such a recombinant expression vector.
  • the invention provides a method of producing an isolated mutant cytolysin protein of the invention, wherein the mutant cytolysin protein differs from the wild type protein by the presence of a mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin sequence, such that the toxicity of the mutant is reduced relative to that of the wild-type cytolysin protein, the method comprising: a) transforming, transfecting or infecting a host cell with a recombinant expression vector as described above and culturing the host cell under conditions which permit the expression of said mutant cytolysin protein by the host cell; and b) recovering the mutant cytolysin protein from the culture.
  • an immunogenic composition which comprises: a) an isolated mutant cytolysin protein, in unconjugated form or as part of an immunogenic conjugate as described above, wherein the mutant cytolysin protein differs from the wild type protein by the presence of a mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin sequence, such that the toxicity of the mutant is reduced relative to that of the wild-type cytolysin protein; and b) one or more of a physiologically acceptable adjuvant, diluent or carrier.
  • the mutation may be located within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin sequence.
  • the mutation may be a substitution or deletion of one or more amino acids within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin sequence.
  • the isolated mutant cytolysin protein of the composition may differ from the wild type cytolysin protein by the substitution or deletion of one or more amino acids within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin sequence.
  • the mutant cytolysin protein may differ from the wild type protein by the substitution or deletion of two adjacent amino acids within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin sequence, as exemplified by the substitution or deletion of amino acids corresponding to valine 144 and proline 145, alanine 146 and arginine 147, methionine 148 and glutamine 149, or tyrosine 150 and glutamic acid 151 of the pneumolysin sequence.
  • the immunogenic composition may contain any of the foregoing mutant cytolysin proteins, in unconjugated form or as part of an immunogenic conjugate as described above, which further comprises at least one amino acid substitution or deletion in at least one of the regions of amino acids corresponding to 257-297, 367-397 or 424-437 of the wild type pneumolysin sequence.
  • the immunogenic composition may comprise: a) an immunogenic conjugate comprising: (i) a saccharide, oligosaccharide or polysaccharide, which may be bacterial, and may be derived from the same species, e.g.
  • the composition may comprise saccharides, oligosaccharides or polysaccharides from a plurality of bacterial species and/or strains.
  • the invention is directed to a method of prophylaxis for a mammal, the method comprising the step of administering to a subject mammal an immunogenic composition which comprises: a) an isolated mutant cytolysin protein, wherein the mutant cytolysin protein differs from the wild type cytolysin protein by the presence of a mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin sequence, such that the toxicity of the mutant is reduced relative to that of the wild-type cytolysin protein; and b) one or more of a physiologically acceptable adjuvant, diluent or carrier.
  • an immunogenic composition which comprises: a) an isolated mutant cytolysin protein, wherein the mutant cytolysin protein differs from the wild type cytolysin protein by the presence of a mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin sequence, such that the toxicity of the mutant is reduced relative to that of the wild-type
  • the immunogenic composition may comprise: a) an immunogenic conjugate comprising: (i) a saccharide, oligosaccharide, polysaccharide, peptide, polypeptide or protein; and (ii) an isolated mutant cytolysin protein as described herein.
  • the invention is directed to the use of any of the isolated mutant cytolysin proteins or immunogenic conjugates of the invention in the preparation of an immunogenic composition.
  • the invention also provides a method of preparing an immunogenic composition, comprising the step of admixing a mutant protein or immunogenic conjugate of the invention with a pharmaceutically acceptable carrier.
  • the immunogenic compositions of the invention may be used for the prophylaxis or treatment of bacterial infection.
  • the present invention provides an isolated mutant cytolysin protein or immunogenic conjugate of the invention for use in a method of medical treatment.
  • the invention is directed to a method of preparation of an immunogenic composition, the method comprising the steps of: providing an isolated mutant cytolysin protein as described herein; and conjugating the mutant protein to a saccharide, oligosaccharide, polysaccharide, peptide, polypeptide or protein.
  • the mutant protein in the foregoing method may be conjugated to a polysaccharide derived from the same species as the cytolysin.
  • the method may comprise the further step of admixing the conjugate thus obtained with a pharmaceutically acceptable carrier.
  • the invention is directed to a method of screening candidate mutant cytolysin proteins for suitability for use in immunogenic compositions, the method comprising the steps of: providing a mutant cytolysin protein; testing the mutant protein for haemolytic activity; testing the mutant cytolysin protein for oligomerisation activity; and comparing the haemolytic and oligomerisation activity of the mutant cytolysin protein with those of a non- mutant protein, e.g. a wild type protein.
  • the cytolysin may be from any suitable species.
  • cytolysins examples include Pneumolysin from Streptococcus pneumoniae; Perfringolysin 0 from Clostridium perfringens ; Intermedilysin from Streptococcus intermedius; Alveolysin from Bacillus alvei; Anthrolysin from Bacillus anthracis; Putative Cereolysin from Bacillus cereus; Ivanolysin 0 from Listeria ivanovii; Pyolysin from Arcanobacterium pyogenes; Seeligeriolysin 0 from Listeria seeligeri ; Streptolysin 0 from S.
  • the wild type cytolysin may be any cytolysin other than pneumolysin.
  • Preferred embodiments of all aspects of the invention include mutants of perfringolysin, intermedilysin or anthrolysin. They (or mutants of the other cytolysins described here) may comprise a mutation (e.g. a substitution or deletion) at a position corresponding to amino acid A146 of wild type PLY.
  • Preferred embodiments of all aspects of the invention include mutant perfringolysin, intermedilysin or anthrolysin comprising deletions or substitutions at positions equivalent to A146 and R147 of wild type pneumolysin, i.e.
  • the mutant cytolysin is a mutant perfringolysin comprising a substitution or deletion at Y , and in particular, a Y 181 to A 181 substitution
  • it may comprise a further mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin (PLY) sequence, wherein the further mutation is capable in isolation of reducing the toxicity of the wild type sequence.
  • PLY pneumolysin
  • Figure 1 shows the amino acid sequences of wild-type cytolysin polypeptides .
  • A Pneumolysin from Streptococcus pneumoniae; B. Perfringolysin 0 from Clostridium perfringens ; C. Intermedilysin from Streptococcus intermedius ; D. Alveolysin from Bacillus alvei; E. Anthrolysin from Bacillus anthracis; F. Putative Cereolysin from Bacillus cereus; G. Ivanolysin 0 from Listeria ivanovii ; H. Pyolysin from Arcanobacterium pyogenes; I. Seeligeriolysin 0 from Listeria seeligeri; J.
  • Streptolysin 0 from S. pyogenes; K. Suilysin from Streptococcus suis; L . Tetanolysin from Clostridium tetani; M. Listeriolysin 0 from Listeria monocytogenes . All accession numbers are derived from NCBI-GenBank Flat File Release 141.0, April 15 2004.
  • Figure 2 shows a western blot of PLY deletion mutants detected by mAb PLY4;
  • Figure 3 shows the results of a quantitative haemolytic assay comparing WT PLY to ⁇ 6 PLY mutant;
  • Figure 4 shows the results of a cytotoxicity assay comparing WT PLY to ⁇ 6 PLY mutant;
  • Figure 5 shows electron micrographs of WT PLY treated erythrocyte membranes;
  • Figure 6 shows IL-6 levels in lung tissue after treatment with WT PLY or ⁇ 6 PLY;
  • Figure 7 shows IL-6 levels in lung lavage after treatment with WT PLY or ⁇ 6 PLY;
  • Figure 8 shows total protein levels in bronchoalveolar lavage after treatment with WT PLY or ⁇ 6 PLY;
  • Figure 9 shows anti-PLY antibody levels in response to immunization of mice with WT PLY or ⁇ 6 PLY;
  • Figure 10 shows the degree of haemolysis in relation to toxin concentration in SRBC (sheep red
  • Figure 11 is a Western blot showing that polyclonal ⁇ -PLY antibodies also recognise PFO.
  • Figure 12 shows the degree of haemolysis in relation to toxin concentration in SRBC (sheep red blood cell) treated with ⁇ 6 PFO and wild type PFO.
  • Figure 13 shows the degree of haemolysis in relation to toxin concentration in SRBC (sheep red blood cell) treated with ⁇ 6 ILY and wild type ILY.
  • Figure 14 shows the degree of haemolysis in relation to toxin concentration in human erythrocytes treated with ⁇ 6 ALO and wild type ALO;
  • Figure 15 compares the haemolytic activity of WT PLY and the mutants PLY W433F, ⁇ 6 PLY, ⁇ 7 PLY, ⁇ 8 PLY and ⁇ A146 PLY;
  • Figure 16 shows the cytotoxicity to murine L929 fibroblasts of WT PLY and the mutants PLY W433F, ⁇ 6 PLY, ⁇ 7 PLY, ⁇ 8 PLY and ⁇ A146 PLY;
  • Figure 17 shows that ⁇ A146 PLY does not cause degranulation of RBL-2H3 mast cells, while WT PLY does;
  • Figure 18 shows analysis of core body temperature following treatment with wild type PLY or ⁇ A146 PLY.
  • FIG. 1 The amino acid sequences of a number of wild type cholesterol-binding cytolysins (CBCs) are shown in Figure 1.
  • Figure 1 also indicates the GenBank identification number for each sequence.
  • the invention is not restricted to mutants of the cytolysins shown in Figure 1, but encompasses mutants of any cholesterol-binding cytolysin.
  • a cholesterol-binding cytolysin is a molecule which, in the wild type state, is capable of binding to membrane cholesterol molecules, and has the ability to oligomerise and form pores in cholesterol- containing membranes.
  • the wild type cytolysin may contain an amino acid sequence which has at least 33% amino acid identity, and preferably 40%, 50%, 60%, 70%, 80%, 90% or more amino acid identity to the region corresponding to amino acids 144 to 161 of the wild type pneumolysin protein, which has the consensus sequence VPARMQYEKITAHSMEQL (see Figure 1) .
  • the corresponding region of the cytolysin may be immunologically cross-reactive with amino acids 144 to 161 of the wild type pneumolysin protein. That is to say, antibodies reactive with the pneumolysin sequence will also bind to the corresponding cytolysin sequence, and vice versa .
  • the present invention relies on the identification of a number of cytolysin forms having a mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin protein which have reduced toxicity as compared to the wild type cytolysin sequence, as reflected by a reduction in haemolytic activity and/or oligomerisation.
  • the present invention relates to mutant cytolysin proteins which differ from the wild type protein by the mutation within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin sequence.
  • the consensus sequence of this region is as follows: VPARMQYE (see Figure 1) .
  • the corresponding sequences for the cytolysins shown in Figure 1 are listed below.
  • the mutant may have a substitution or deletion of one or more amino acids within the region corresponding to amino acids 144 to 161, e.g. 144 to 151.
  • the mutant cytolysin may have a mutation, e.g. a substitution or deletion, at one or more of the amino acid residues corresponding to amino acids 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161 of wild type pneumolysin.
  • the invention further relates to mutant cytolysin proteins which differ from the wild type protein by the substitution or deletion of two adjacent amino acids within the region corresponding to amino acids 144 to 151 of the wild type pneumolysin sequence.
  • double mutants are those which contain substitutions or deletions of amino acids corresponding to valine 144 and proline 145, alanine 146 and arginine 147, methionine 148 and glutamine 149, or tyrosine 150 and glutamic acid 151, i.e. the corresponding amino acids shown in the table above.
  • These mutant cytolysin proteins are used per se in immunogenic compositions, together with one or more of a physiologically acceptable adjuvant, diluent or carrier.
  • these mutant cytolysin proteins are conjugated to a saccharide, oligosaccharide, polysaccharide, peptide, polypeptide or protein, from the same or a heterologous organism as the cytolysin, to form conjugates which are used in immunogenic compositions, together with one or more of a physiologically acceptable adjuvant, diluent or carrier.
  • a physiologically acceptable adjuvant diluent or carrier.
  • the saccharide, oligosaccharide, polysaccharide, peptide, polypeptide or protein to which the mutant protein is conjugated may be from Streptococcus, e.g. Streptococcus pneumoniae, Streptococcus intermedius , Streptococcus suis , S. pyogenes, S.
  • Clostridium e.g. Clostridium perfringens, Clostridium novyi, Clostridium septicum, Clostridium tetani, Clsotridium botulinum, Clostridium chauvoei , Clostridium bifermentans , Clostridium sordelli , Bacillus, e.g. Bacillus alvei, Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis , Bacillus laterosporus , Listeria, e.g.
  • the saccharide, oligosaccharide, polysaccharide, peptide, polypeptide or protein may be derived from the bacterial capsule .
  • the mutant cytolysin proteins contained in immunogenic compositions are used in prophylaxis or therapy.
  • the mutant cytolysin proteins may further contain at least one amino acid substitution or deletion in at least one of the regions corresponding to amino acids 257-297, 367-397 or 424-437 of the wild type pneumolysin sequence. These further substitutions or deletions are described in Paton et al . published International Patent Application WO 90/06951.
  • an immunogenic composition comprising an isolated mutant cytolysin protein as described herein.
  • the mutant cytolysin proteins may retain immunogenic activity in mammals.
  • immunogenic in mammals is meant that mammalian immune systems will produce antibodies to the mutant protein, and that these antibodies will also recognise wild type protein.
  • mammalian antibodies to the wild type protein will also recognise the mutant protein.
  • the mutant protein is immunogenic in humans.
  • the mutant protein will stimulate the mammalian immune system to produce antibodies which bind to the wild type sequence corresponding to VPARMQYEKITAHSMEQL or VPARMQYE.
  • the mutation is in the region of the cytolysin protein involved in oligomerisation of the wild type protein. Without being bound by theory, it is believed that the mutant protein has reduced toxicity as a result of reduced pore-formation activity compared with wild type protein. This is believed to be associated with reduced oligomerisation activity and/or reduced haemolytic activity. Toxicity may be measured directly.
  • one or more of pore formation, oligomerisation and haemolysis may be measured to provide an indication of likely toxicity.
  • Deletions and substitutions are examples of mutations which may be used to provide the mutant proteins of the invention with reduced toxicity.
  • Non-conservative substitutions may be particularly suitable for reducing toxicity of the mutant, as a mutant having a non-conservative mutation is less likely to retain wild-type levels of function than one having a conservative substitution.
  • a conservative substitution may be defined as a substitution within an amino acid class and/or a substitution that scores positive in the BLOSUM62 matrix as shown below, thus a non-conservative substitution maybe defined as a substitution between amino acid classes, or which does not score positive in the BLOSUM62 matrix.
  • the amino acid classes are acidic, basic, uncharged polar and nonpolar, wherein acidic amino acids are Asp and Glu; basic amino acids are Arg, Lys and His; uncharged polar amino acids are Asn, Gin, Ser, Thr and Tyr; and non-polar amino acids are Ala, Gly, Val, Leu, lie, Pro, Phe, Met, Trp and Cys .
  • the amino acid classes are small hydrophilic, acid/acidamide/hydrophilic, basic, small hydrophobic and aromatic, wherein small hydrophilic amino acids are Ser, Thr, Pro, Ala and Gly; acid/acidamide/hydrophilic amino acids are Asn, Asp, Glu and Gin; basic amino acids are His, Arg and Lys; small hydrophobic amino acids are Met, lie, Leu and Val; and aromatic amino acids are Phe, Tyr and Trp.
  • Conservative substitutions which score positive in the BLOSUM62 matrix, are as follows:
  • Amino acid insertions within the region of amino acids 144 to 161, e.g. 144 to 151, may also be used to reduce toxicity of the PLY mutant. For example, insertions of 1, 2, 3, 4, 5, 10, 15, 20 or more amino acids may be used. However, deletions and substitutions are generally preferred to insertions as they are less likely to disrupt the wild type epitope; such disruption could reduce the immunogenicity of the mutant protein, which may be undesirable in an immunogenic composition.
  • the mutant cytolysin protein of the invention preferably has at least 80% amino acid identity with the corresponding wild type sequence, e.g. as shown in Figure 1. The mutant may have at least 85% identity, at least 90% identity, or at least 95% identity with the wild type sequence.
  • Percent (%) amino acid sequence identity with respect to a reference sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • % identity values may be determined by WU- BLAST-2 (Altschul et al . , Methods in Enzymology, 266:460-480 (1996) ) .
  • a % amino acid sequence identity value is determined by the number of matching identical residues as determined by WU-BLAST-2, divided by the total number of residues of the reference sequence (gaps introduced by WU-BLAST-2 into the reference sequence to maximize the alignment score being ignored) , multiplied by 100. Percent (%) amino acid similarity is defined in the same way as identity, with the exception that residues scoring a positive value in the BLOSUM62 matrix are counted. Thus, residues which are non-identical but which have similar properties (e.g. as a result of conservative substitutions) are also counted. References in this specification to an amino acid of a first sequence "corresponding to" an amino acid of a second sequence should be construed accordingly.
  • the mutant protein is conjugated to a saccharide, oligosaccharide, polysaccharide, peptide, polypeptide or protein to form an immunogenic conjugate.
  • the mutant cytolysin protein may retain its immunogenicity, or that immunogenicity may be ablated. In either event, the mutant cytolysin protein serves to enhance the immunogenicity of the saccharide, oligosaccharide, polysaccharide, peptide, polypeptide or protein in the conjugate.
  • Such saccharides, oligosaccharides, polysaccharides, peptides, polypeptides or proteins are each conjugated to the mutant protein in any suitable manner, including, but not limited to: (1) direct coupling via protein functional groups (e. g. , thiol-thiol linkage, amine-carboxyl linkage, amine- aldehyde linkage; enzyme direct coupling); (2) homobifunctional coupling of amines ( e . g.
  • contemplated are heterobifunctional "non-covalent coupling" techniques such the Biotin-Avidin interaction.
  • conjugation techniques see Aslam and Dent (1998) , incorporated hereinafter by reference in its entirety.
  • Further methods of conjugating a peptide, polypeptide or protein to a protein are described in U.S. provisional patent applications 60/530,480 and 60/530,481, both filed December 17, 2003, and both incorporated by reference in their entirety.
  • US Patent 5,565,204 to Kuo et al. described a method for conjugating such polysaccharides to the wild type PLY protein; that method is also suitable for conjugating such polysaccharides to the mutant cytolysin proteins of this invention.
  • the immunogenic compositions of the present invention may be conjugated immunogenic compositions.
  • Each immunogenic composition may comprise one or more saccharides, oligosaccharides, polysaccharides, peptides, polypeptides or proteins, which may be derived from the source organism of the wild type cytolysin protein. In non-limiting examples, such components may be derived from the capsule of the organism.
  • the saccharides, oligosaccharides or polysaccharides are derived from more than one serotype of the source organism; the particular serotypes will depend on the intended use for the immunogenic composition and the prevalence of these serotypes in the target population.
  • the saccharides, oligosaccharides, polysaccharides, peptides, polypeptides or proteins are derived from a heterologous organism (that is, an organism other than that from which the cytolysin molecule is derived) .
  • a heterologous organism that is, an organism other than that from which the cytolysin molecule is derived
  • multiple serotypes may be obtained from, without limitation, Neisseria meningitidis (for example, from serotypes A, C, Y and W135) , Staphylococcus aureus and Haemophilus influenzae .
  • the mutant cytolysin protein is conjugated to another peptide, polypeptide or protein of the same species or strain of organism.
  • the mutant cytolysin protein is conjugated to a peptide, polypeptide or protein from a heterologous organism, including a human.
  • the mutant protein is conjugated to another peptide, polypeptide or protein, which is from a pathogenic virus, bacterium, fungus or parasite, or (2) from a cancer cell or tumor cell, or (3) from an allergen so as to interfere with the production of IgE so as to moderate allergic responses to the allergen, or (4) from amyloid precursor protein (APP) so as to prevent or treat disease characterized by amyloid deposition in a vertebrate host.
  • APP amyloid precursor protein
  • the moiety of APP which is conjugated to the mutant protein may be the ⁇ -amyloid peptide (also referred to as A ⁇ peptide), which is an internal, 39-43 amino acid fragment of (APP) , which is generated by processing of APP by the ⁇ and ⁇ secretase enzymes.
  • a ⁇ peptide also referred to as A ⁇ peptide
  • a ⁇ l-42 peptide which has the following amino acid sequence: Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He Gly Leu Met Val Gly Gly Val Val He Ala
  • the A ⁇ component may be further administered in the form of a fragment conjugated to the mutant protein.
  • Non-limiting examples of such fragments include A ⁇ l-3, 1-4, 1-5, 1-6, 1-7, 3-7, 3-8, 3-9, 3-10, 3-11, 1-10 and 1-12.
  • a further aspect of the present invention provides a method of prophylaxis or treatment for a mammal, the method comprising the step of administering to a subject mammal an immunogenic composition comprising an isolated cytolysin protein having a mutation as described herein, where the mutant cytolysin protein is unconjugated or conjugated as described herein.
  • the method is intended for prophylaxis or treatment of infection by one or more species or strains of bacteria having a cholesterol-binding cytolysin which is immunologically cross-reactive with the relevant wild-type cytolysin.
  • the mode of administration of an immunogenic composition of the invention may be by any suitable route which delivers an immunoprotective amount of the protein to the subject.
  • One such route is the parenteral route, such as by intramuscular or subcutaneous administration.
  • the immunogenic composition will usually be presented as a pharmaceutical formulation including a physiologically acceptable carrier or excipient, for example, sterile water or sterile isotonic saline, as well as any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans.
  • a physiologically acceptable carrier or excipient for example, sterile water or sterile isotonic saline, as well as any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans.
  • the appropriate carrier will be evident to those skilled in the art and will depend in large part upon the route of administration.
  • the immunogenic composition of the present invention may also include a physiologically acceptable diluent such as sterile water or sterile isotonic saline.
  • a physiologically acceptable diluent such as sterile water or sterile isotonic saline.
  • the formulation may be prepared by conventional means. It will be understood, however, that the specific dose level for any particular recipient mammal will depend upon a variety of factors including age, general health, and sex; the time of administration; the route of administration; synergistic effects with any other drugs being administered; and the degree of protection being sought. Of course, the administration can be repeated at suitable intervals if necessary.
  • the mammal may be a human, or may be a non-human mammal.
  • the immunogenic composition may be administered in any convenient manner; for example, those described above.
  • the immunogenic composition of the present invention may include one or more physiologically acceptable adjuvants.
  • a substance that enhances the immune response when administered together with an immunogen or antigen is known as an adjuvant.
  • a number of cytokines or lymphokines have been shown to have immune modulating activity, and thus may be used as adjuvants, including, but not limited to, the interleukins 1- ⁇ , 1- ⁇ , 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Patent No.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • adjuvants useful in this invention include a chemokine, including without limitation, MCP-1, 'MlP-l ⁇ , MIP- l ⁇ , and RANTES.
  • Adhesion molecules such as a selectin, e.g., L-selectin, P-selectin and E-selectin may also be useful as adjuvants.
  • Still other useful adjuvants include, without limitation, a mucin-like molecule, e.g., CD34, GlyCAM-1 and MadCAM-1, a member of the integrin family such as LFA-1, VLA- 1, Mac-1 and pl50.95, a member of the immunoglobulin superfamily such as PECAM, ICAMs, e.g., ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3, co-stimulatory molecules such as CD40 and CD40L, growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, B7.2, PDGF, BL-1, and vascular endothelial growth factor, receptor molecules including Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6.
  • a mucin-like molecule e
  • Still another adjuvant molecule includes Caspase (ICE) .
  • ICE Caspase
  • Suitable adjuvants used to enhance an immune response further include, without limitation, MPLTM (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT) , which is described in U.S. Patent No. 4,912,094, which is hereby incorporated by reference.
  • MPLTM 3-O-deacylated monophosphoryl lipid A
  • Corixa Corixa, Hamilton, MT
  • AGP aminoalkyl glucosamine phosphate compounds
  • derivatives or analogs thereof which are available from Corixa (Hamilton, MT) , and which are described in United States Patent No.
  • AGP 2-[(R)-3- Tetradecanoyloxytetradecanoylamino] ethyl 2-Deoxy-4-0- phosphono-3-O- [ (R) -3-tetradecanoyoxytetradecanoyl] -2- [ (R) -3- tetradecanoyloxytetradecanoyl-amino] - ⁇ -D-glucopyranoside, which is also known as 529 (formerly known as RC529) .
  • This 529 adjuvant is formulated as an aqueous form or as a stable emulsion.
  • Still other adjuvants include mineral oil and water emulsions, aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, etc., Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronic polyols, muramyl dipeptide, killed Bordetella, saponins, such as StimulonTM QS- 21 (Antigenics, Framingham, MA.), described in U.S. Patent No.
  • WO 93/13302 and WO 92/19265 are also useful as adjuvants.
  • cholera toxins and mutants thereof including those described in published International Patent Application number WO 00/18434 (wherein the glutamic acid at amino acid position 29 is replaced by another amino acid (other than aspartic acid) , preferably a histidine) .
  • Similar CT toxins or mutants are described in published International Patent Application number WO 02/098368 (wherein the isoleucine at amino acid position 16 is replaced by another amino acid, either alone or in combination with the replacement of the serine at amino acid position 68 by another amino acid; and/or wherein the valine at amino acid position 72 is replaced by another amino acid) .
  • CT toxins are described in published International Patent Application number WO 02/098369 (wherein the arginine at amino acid position 25 is replaced by another amino acid; and/or an amino acid is inserted at amino acid position 49; and/or two amino acids are inserted at amino acid positions 35 and 36) .
  • the haemolytic activity of the mutant cytolysin protein may be determined in any suitable manner.
  • One particular protocol as used in the present invention is as follows. Toxin was prepared in serial dilutions in 1.5ml 1 x PBS (Oxoid) .
  • the mutant cytolysin protein is non-haemolytic at concentrations of more than 1 ⁇ g/ml; more preferably at concentrations of more than 5 ⁇ g/ml; still more preferably at more than 10 ⁇ g/ml, at more than 25 ⁇ g/ml, or at more than 35 ⁇ g/ml; and most preferably at more than 50 ⁇ g/ml.
  • Determination of haemolysis may be carried out as described above .
  • Determination of pore-forming activity may be determined in any suitable manner; a preferred protocol relies on visual inspection of SRBC or HRBC membranes by means of electron microscopy; this allows the number of pores to be visualised. This protocol is described in more detail below.
  • oligomerising activity of pore-forming toxins can be analysed by analytical ultracentrifugation, as described by Morgan et al (1993), to study oligomerisation of toxins in solution.
  • a sucrose density gradient can be applied to toxin bound erythrocytes in which oligomers are observed in the high molecular weight fractions and separated from other erythrocyte membrane proteins (Bhakdi et al, 1985; Saunders et al, 1989) .
  • One particular method of comparing the oligomerisation activity of mutant cytolysin in solution with that of wild type protein is to use a fluorescence assay conducted in a similar manner to that described by Search (2002) .
  • ANS (8-anilino-l-napthalene-sulphonic acid) (Kodak Ltd.) binds as an extrinsic fluor to the toxin.
  • ANS has weak fluorescence at 490nm (read with JASCO FP-750 spectrofluorometer) but in a hydrophobic environment ANS fluorescence increases. This phenomenon allows the movement of ANS bound monomers to be tracked in solution.
  • Sodium deoxycholate (BDH Laboratory supplies) can be used to induce oligomerisation.
  • the toxicity of the proteins and compositions of the invention may be determined directly, by administering the mutant to a non-human test mammal, e.g. a rodent.
  • the toxicity of the mutant may be compared with that of the wild type protein.
  • Suitable indicators of toxicity include survival, animal behaviour, and inflammation (which may be determined by measuring inflammatory cytokine production, e.g. in bronchoalveolar lavage) . Suitable protocols are described below in the Examples.
  • the present invention further provides a method of preparation of an immunogenic composition, the method comprising the steps of: providing an isolated mutant cytolysin protein with the mutations described herein and having reduced haemolytic activity compared with wild type cytolysin protein, the mutant protein being antigenic in mammals; and conjugating the mutant protein to a saccharide, oligosaccharide, polysaccharide, peptide, polypeptide or protein.
  • an isolated and purified nucleic acid sequence comprising an isolated nucleic acid sequence encoding a mutant cytolysin protein wherein the mutant cytolysin protein differs from the wild type cytolysin protein by the presence of a mutation within the region corresponding to amino acids 144 to 161 of the wild type pneumolysin sequence, such that the toxicity of the mutant is reduced relative to that of the wild-type protein, or which is complementary to such a nucleic acid sequence, the mutant protein being immunogenic in mammals.
  • Further aspects of the invention provide nucleic acid sequences which are complementary to such sequences . Nucleic acid sequences can be derived from protein sequences based on the degeneracy of the genetic code.
  • Nucleic acid sequences of the present invention may comprise additional regulatory sequences, for example, promoters or repressors.
  • the nucleic acid sequences may be comprised in an expression vector, for example, plasmids, artificial chromosomes, expression cassettes and the like.
  • a recombinant host cell transformed, transfected or infected with a recombinant expression vector comprising an isolated and purified nucleic acid sequence expressing a mutant cytolysin protein as described herein.
  • the cell is a prokaryotic cell .
  • a method of screening candidate mutant cytolysin proteins for suitability for use in immunogenic compositions comprising the steps of: providing a mutant cytolysin protein; testing the mutant protein for haemolytic activity; testing the mutant protein for oligomerisation activity; and comparing the haemolytic and oligomerisation activity of the mutant protein with those of a non-mutant (e.g. a wild type) protein.
  • a non-mutant e.g. a wild type
  • the method may further comprise the step of testing the mutant protein for immunogenic activity in a target mammal.
  • This may comprise the step of contacting the mutant protein with an antibody to the non-mutant protein.
  • This may be performed in vivo or in vitro .
  • the invention described herein relates to deletion mutants of cytolysins which exhibit reduced toxicity, haemolysis and pore formation. The data demonstrate that at 7 ⁇ g/dose, ⁇ 6 PLY is not detrimental to mice compared to 2 ⁇ g/dose of WT PLY. Nevertheless, various mutations created by the present inventors surrounding the PLY N 143 residue and described herein are still recognised by Western blotting with mAb PLY 4, indicating that this highly antigenic site on PLY has not been altered.
  • the site of mutation has been shown to be highly antigenic by epitope scanning and is recognised by both human sera and rabbit hyper-immune sera (Salo et al, 1993) . All PLY mutants created were confirmed to be forms of pneumolysin. The fact that mAb PLY4 recognises the mutants indicates that the epitope has not been altered to such an extent that it is no longer specific for this antibody. Larger deletions within this area should create mutants that are not recognised by mAbPLY4. As this region has been identified as being highly immunogenic (Salo et al, 1993) it is useful that the site remains intact in the deletions that we have created in terms of use in immunogenic compositions.
  • non-toxic mutants described herein are within the site proposed to be involved in oligomerisation (de los Toyos et al, 1996) .
  • haemolytic assays with ⁇ 6 PLY.
  • the haemagglutination effect observed in haemolytic assays of ⁇ 6 PLY with SRBC suggests that ⁇ 6 PLY monomers still bind to host cell membranes.
  • a labelled form of ⁇ 6 PLY was created which allows visualisation of binding to host cell membranes. From binding assays (data not shown) it was confirmed that ⁇ 6 PLY did bind to the host cell membrane. There may be a weak affinity between the ⁇ 6 PLY monomers, allowing cross-linking of monomers but not formation of true oligomers.
  • PLY has previously been implicated in the disruption of tight junctions (Rayner et al, 1995) , allowing host proteins to ⁇ leak f into the airways via the disruption of the capillary/airway barrier.
  • a low inflammatory response and no disruption to the lungs by ⁇ 6 PLY correlate with ⁇ 6 PLYs inability to create pore-forming oligomers in host cell membranes .
  • Table 2 Bases deleted for each mutation within the PLY gene and amino acids deleted.
  • Wild type (WT) and mutant PLY was expressed in Escherichia coli and harvested as described previously (Mitchell et al, 1989) .
  • Cells were disrupted using the benchtop cell disrupter (Constant Systems Ltd) and cytoplasmic proteins obtained by centrifugation at 13,000 rpm for 30 minutes.
  • Hydrophobic Interaction Chromatography with a phenyl ether matrix (PE20, Applied Biosystems) was used to purify PLY with the BioCAD (RTM) 700E Perfusion Chromatography Workstation (Applied Biosystems) . Eluted fractions were run on SDS-PAGE and coomassie stained using standard protocol and fractions containing pure PLY were pooled.
  • Example 3 Quantitative Haemolytic Assay Haemolytic activity of purified protein was assessed using an assay based on that reported by Walker et al . , (1987) using a 2% (vol/vol) sheep red blood cell (SRBC) (E & 0 laboratories) or human red blood cell (HRBC) solution in 1 x Phosphate Buffered Saline (PBS) (Oxoid) . Pooled fractions were concentrated using inicon B15 clinical sample concentrators (Millipore) . Toxin was prepared in serial dilutions in 1.5ml 1 x PBS (Oxoid) .
  • SRBC sheep red blood cell
  • HRBC human red blood cell
  • Example 5 Western Blotting PLY mutants created by site-directed mutagenesis were detected in Western blots using standard techniques. Blots were incubated with polyclonal anti-PLY serum from rabbit or monoclonal PLY 4 anti-PLY serum from mouse (de los Toyos et al, 1996) and then incubated with the relevant HRP-linked antibody (Amersham Life Sciences) and developed. Of the eight double amino acid deletions created, all were recognised by Western blotting with polyclonal anti-PLY serum (not shown) and by mAb PLY 4 (Fig. 2) prepared by de los Toyos et al (1996) .
  • L929 Killing Assay L929 murine fibroblasts (ECACC, no.85011425) were cultured in RPMI 1640 media + 10% Foetal Bovine Serum (FBS) (Gibco) , passaged and transferred to a 96-well plate and incubated for 24h at 37 °C, 5% C0 2 . Serial dilutions of purified WT PLY and mutant ⁇ 6 PLY toxin were prepared in RPMI 1640 media from a stock concentration of 0.05mg/ml and added to the L929 fibroblasts.
  • FBS Foetal Bovine Serum
  • MTT 3- [4, 5-Dimethylthiazol-2-yl] -2, 5- diphenyltetrzolium bromide
  • Optical density was read at 540nm with an MRX plate reader (Dynatech Laboratories) .
  • Cytotoxicity assays with L929 murine fibroblasts were run to assess the toxicity of mutant ⁇ 6 PLY compared to WT PLY (Fig. 4) . At concentrations of 30 ⁇ g/ml, ⁇ 6 PLY was not toxic to fibroblasts, whereas ⁇ 500 pg/ml of WT PLY was cytotoxic.
  • mice were monitored to a 24h end-point. Serum, bronchoalveolar lavage and lung tissue samples were recovered and processed as described previously (Kerr, et al . 2002). Cytokine levels were measured with commercial cytokine ELISA kits for Interleukin (IL)-6, Interferon (IFN)- ⁇ (Pharmigen) and Tumor Necrosis Factor (TNF)- ⁇ (R&D systems, UK). Total protein levels in the lavage were measured using standard Bradford Assay. Non-parametric analysis by Mann-Whitney U test was used to measure cytokine and total protein levels where p ⁇ 0.05 was considered statistically significant.
  • IL-6 Interleukin-6
  • IFN Interferon
  • TNF Tumor Necrosis Factor
  • mice were treated with ⁇ 6 PLY and saline recovered from the anaesthetic quicker than mice given WT PLY. Behaviour of ⁇ 6 PLY treated mice was similar to that of the saline control but WT PLY treated mice exhibited piloerection, laboured breathing and a hunched stance over a 6- hour period, recovering within the 24-hour time scale. Next, an inflammatory cytokine analysis was performed. IL-6 production was measured as a marker of toxicity of PLY to the host. There was a greater -than 10-fold increase in IL-6 levels in the bronchoalveolar lavage of WT PLY treated mice
  • Fig. 7 compared to ⁇ 6 PLY treatment (p ⁇ 0.05) and the saline control (p ⁇ 0.05).
  • Treatment with WT PLY induces inflammation in the host airways whereas treatment with ⁇ 6 PLY does not.
  • the median IL-6 level in WT treated bronchoalveolar lavage was 416pg/ml (range of 335-2225pg/ml) whereas the background IL-6 level was low (59pg/ml) with no increase in mice treated with ⁇ 6 PLY (36pg/ml) (see Table 3 below) .
  • An increase in IL-6 levels was observed in lung tissue of WT treated mice (p ⁇ 0.05) compared to the saline control (Fig. 6) . There was no significant IL-6 increase in lung tissue of ⁇ 6 treated mice compared to the saline treatment. Measurements of IFN- ⁇ and TNF- ⁇ were not significant between treatments 24h post- administration (data not shown) .
  • Table 3 IL-6 median (min-max) levels in bronchoalveolar lavage 24h post treatment
  • Total protein levels (Fig. 8) were measured in the bronchoalveolar lavage to assess lung integrity. Increases in protein levels were not observed for ⁇ 6 PLY treated mice compared to healthy lavage samples. Airways of WT PLY treated mice had large amounts of protein (3.57mg/ml) in them compared to a background total protein level of 0.23mg/ml for the saline control group (Fig. 8).
  • Example 8 Mouse Immunogenicity Studies A mouse immunogenicity study was performed to compare responses of the wild type PLY protein to the ⁇ 5, 6 and 7 mutant proteins. All immunogenic compositions were prepared at 5 ⁇ g rPLY/dose in the presence of a combination of adjuvants, A1P0 4 (0.2 mg) and MPL-SE (50 ⁇ g) . A1P0 4 (0.2 mg) and MPL-SE (50 ⁇ g) in phosphate-buffered saline (PBS) was used as a negative control . Groups of 5 female, CD-I mice, age 6-8 weeks, were immunized intraperitoneally and received 2 booster doses at 2 week intervals. Blood was collected retro-orbitally at weeks 0, 2, 4, and 6.
  • Example 9 Generation of anti-PLY Antibodies
  • Levels of anti-PLY antibodies raised in mice immunized with wild type PLY or ⁇ 6 PLY were determined by immunizing MF- 1 mice with an initial subcutaneous injection with 20 ⁇ g WT PLY or ⁇ 6 PLY, each with lOO ⁇ g Alum/ lOO ⁇ l dose. Mice were then boosted twice with the same dosage. Serum was collected on day 47 of the immunization protocol and analysed for anti-PLY IgG antibody. Antibody dilution curves are displayed in Figure 9 as the group mean OD 490 nm ⁇ SEM against the serum serial dilution. An initial dilution of serum to 1/1000 was used as more concentrated samples resulted in complete saturation of the substrate.
  • Figure 9 demonstrates that high levels of antibodies were produced in response to both ⁇ 6 PLY + Alum and WT PLY+ Alum, but not to the Alum only control group.
  • Anti-PLY antibodies in the ⁇ 6 PLY and wild type PLY treated groups were observed to completely neutralise 2.5 Haemolytic Units (HU) of PLY to a titre of 1000-2400 in a haemolytic assay (where neutralising ability is expressed as the reciprocal of the antibody dilution that completely neutralises 2.5HU of PLY) (data not shown) .
  • ⁇ 6 PFO and ⁇ 6 ILY are both are non-haemolytic in comparison to their wild type derivatives (Fig. 12 and Fig. 13) .
  • the 34 amino acid signal sequence of anthrolysin 0 from B. anthracis was removed to give the mature protein (mALO) , starting with the sequence ETQ.
  • a mutant of the mature anthrolysin 0 polypeptide was then constructed having a deletion equivalent to ⁇ 6 of PLY.
  • ⁇ 6 mALO alanine 156 and arginine 157 (corresponding to A190 and R191 in full-length wild type ALO) are deleted (see Fig. 1C) .
  • ⁇ 6 mALO was found to be non-haemolytic against human erythrocytes as compared to the mature wild-type protein ( Figure 14) .
  • Haemolytic activity of PLY mutants in comparison to PLY W433F The haemolytic activity of the deletion mutants ⁇ 6, ⁇ 7, ⁇ 8 PLY and ⁇ A146 PLY against human erythrocytes was compared with that of WT PLY and PLY mutant carrying the -substitution W433F, which has previously been described to possess only 1% of the haemolytic activity of WT PLY (see WO90/06951) .
  • Figure 15 shows that, as expected, the W433F mutant shows -1% of the haemolytic activity of wild type PLY. However the deletion mutants do not cause lysis of human erythrocytes at all.
  • Electron microscopy was performed as described above (Example 4) for negatively stained horse erythrocyte membranes treated with 0.2mg/ml wild type pneumolysin, 0.2mg/ml W433F PLY, and 0.2mg/ml ⁇ A146 PLY.
  • ⁇ 6 PLY retains the membrane-binding properties of wild type PLY but does not form pores in cell membranes.
  • the cytotoxicity of WT PLY, PLY W433F, and deletion mutants ⁇ 6, ⁇ 7, ⁇ 8 PLY and ⁇ A146 PLY against murine L929 fibroblasts was determined as described in Example 6.
  • human erythrocytes was compared with that of WT PLY and The W433F PLY mutant was found to be cytotoxic at 10 ⁇ g/ml and above, whereas the deletion mutants were non-toxic in this assay ( Figure 16) .
  • cytotoxicity of ⁇ A146 PLY against rat RBL-2H3 mast cells was assessed using a degranulation assay.
  • the assay was carried out as described by Stassen et al (2003) using 10 4 cells/well, incubated with wild type PLY or ⁇ A146 PLY for 90 minutes.
  • mice were implanted with telemetry chips which enable acquisition of core body temperature (Tc) .
  • Mice were treated with l ⁇ g wt PLY, l ⁇ g ⁇ A146 PLY, or saline solution alone.
  • Treatment with WT PLY resulted in a severe hypothermic response with Tc dropping to 28°C.
  • This Tc was sustained for 6 hours after which there was an increase in Tc by ⁇ 0.6°C/hour and by 24 hours this was similar to the Tc of the control group, though still statistically significant.
  • Treatment with ⁇ A146 PLY did not result in hypothermia and the median Tc was comparable to the saline control group.
  • treatment of mice with WT PLY resulted in a sustained hypothermic response that was not observed following treatment with the same amount of ⁇ A146 PLY.

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Abstract

L'invention concerne des compositions immunogènes comprenant des protéines mutantes de cytolysine. L'invention concerne en outre de telles protéines et des acides nucléiques codant ces protéines. Selon des formes d'exécution particulières, l'invention concerne une protéine mutante de cytolysine isolée, la protéine mutante différant de la protéine de type sauvage, par délétion d'un ou de plusieurs amioacides dans la région correspondant aux aminoacides 144 à 161 de la séquence pneumolysine de type sauvage. Dans certaines formes d'exécution, la protéine mutante diffère de la protéine de type sauvage par la délétion d'un ou de plusieurs aminoacides dans la région correspondant aux aminoacides 144 à 151 de la séquence pneumolysine de type sauvage. Dans d'autres formes d'exécution, la protéine mutante diffère de la protéine de type sauvage par la délétion de deux aminoacides adjacents dans la région correspondant aux aminoacides 144 à 151 de la séquence pneumolysine de type sauvage.
PCT/GB2005/001774 2004-05-07 2005-05-09 Proteines mutantes de cytolysine liant le cholesterol WO2005108419A1 (fr)

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008121402A1 (fr) * 2007-03-30 2008-10-09 President And Fellows Of Harvard College Procédés et compositions pour le traitement de maladies prolifératives
WO2009014560A1 (fr) * 2007-07-20 2009-01-29 President And Fellows Of Harvard College Procédés et compositions pour le traitement de maladies pathogènes
WO2007144647A3 (fr) * 2006-06-15 2009-02-05 Timothy John Mitchell Compositions d'adjuvants innovantes
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US10562941B2 (en) 2014-11-21 2020-02-18 The Board Of Regents Of The University Of Oklahoma Pneumolysin mutants and methods of use thereof
US10864278B2 (en) 2014-02-06 2020-12-15 Oncomatryx Biopharma, S.L. Antibody-drug conjugates and immunotoxins
US11207396B2 (en) 2013-05-17 2021-12-28 The Board Of Regents Of The University Of Texas System Immunization to protect against adverse cardiac events relating to pneumococcal infection
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