WO2007018482A2 - Polypeptides utilises pour induire une reponse immunitaire protectrice contre le staphylococcus aureus - Google Patents

Polypeptides utilises pour induire une reponse immunitaire protectrice contre le staphylococcus aureus Download PDF

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Publication number
WO2007018482A2
WO2007018482A2 PCT/US2005/023511 US2005023511W WO2007018482A2 WO 2007018482 A2 WO2007018482 A2 WO 2007018482A2 US 2005023511 W US2005023511 W US 2005023511W WO 2007018482 A2 WO2007018482 A2 WO 2007018482A2
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polypeptide
seq
aureus
amino acid
acid sequence
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WO2007018482A3 (fr
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Donna L. Montgomery
Kathrin Ute Jansen
Annaliesa S. Anderson
William L. Mcclements
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Merck and Co Inc
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Merck and Co Inc
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Priority to EP05858353A priority Critical patent/EP1784212A4/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus

Definitions

  • Staphylococcus aureus is a pathogen responsible for a wide range of diseases and conditions.
  • diseases and conditions caused by S. aureus include bacteremia, infective endocarditis, folliculitis, furuncle, carbuncle, impetigo, bullous impetigo, cellulitis, botryomyosis, toxic shock syndrome, scalded skin syndrome, central nervous system infections, infective and inflammatory eye disease, osteomyletitis and other infections of joints and bones, and respiratory tract infections. (The Staphylococci in Human Disease, Crossley and Archer (eds.), Churchill Livingstone Inc. 1997.)
  • S. aureus vaccines target S. aureus polysaccharides and polypeptides. Targeting can be achieved using suitable 5. aureus polysaccharides or polypeptides as vaccine components. Examples of polysaccharides that may be employed as possible vaccine components include S. aureus type 5 and type 8 capsular polysaccharides. (Shinefield et ah, N. Eng. J. Med. 346:491- 496, 2002.) Examples of polypeptides that may be employed as possible vaccine components include collagen adhesin, fibrinogen binding proteins, and clumping factor. (Mamo et al., FEMS Immunology and Medical Microbiology 70:47-54, 1994, Nilsson et ah, J. Clin.
  • SEQ ID NO: 1 is a truncated derivative of a full-length S. aureus polypeptide.
  • the full-length naturally occurring polypeptide is referred to herein as full-length "ORF2331".
  • a His-tagged derivative of SEQ ID NO: 1 was found to produce a protective immune response against S. aureus.
  • a first aspect of the present invention describes a polypeptide immunogen comprising an amino acid sequence at least 85% identical to SEQ ID NO: 1, wherein the polypeptide does not contain an amino terminus provided by amino acids 1-20 of SEQ ID NO: 3 and the polypeptide provides protective immunity against S. aureus.
  • SEQ ID NO: 3 provides a full length ORF2331 polypeptide, wherein amino acids 1-20 provide the amino terminus domain containing a signal sequence.
  • an immunogen comprising an amino acid sequence that provides protective immunity against S. aureus and one or more additional regions or moieties covalently joined to the amino acid sequence at the carboxyl terminus or amino terminus, wherein each region or moiety is independently selected from a region or moiety having at least one of the following properties: enhances the immune response, facilitates purification, or facilitates polypeptide stability.
  • additional region or moiety indicates a region or moiety different from a ORF2331 region. The additional region or moiety can be, for example, an additional polypeptide region or a non-peptide region.
  • a nucleic acid comprising a recombinant gene encoding a polypeptide that provides protective immunity against 5. aureus.
  • a recombinant gene contains recombinant nucleic acid encoding a polypeptide along with regulatory elements for proper transcription and processing (which may include translational and post translational elements).
  • the recombinant gene can exist independent of a host genome or can be part of a host genome.
  • the cell comprises a recombinant gene encoding a polypeptide that provides protective immunity against 5. aureus.
  • Another aspect of the present invention describes a method of making a polypeptide that provides protective immunity against S. aureus.
  • the method involves growing a recombinant cell containing recombinant nucleic acid encoding the polypeptide and purifying the polypeptide.
  • Another aspect of the present invention describes a method of inducing a protective immune response in a patient against 5. aureus.
  • the method comprises the step of administering to the patient an immunologically effective amount of an immunogen that provides protective immunity against S. aureus.
  • reference to "or” indicates either or both possibilities. Occasionally phrases such as "and/or” are used to highlight either or both possibilities.
  • Figure 1 illustrates the amino acid sequence of SEQ ID NO: 1.
  • Figure 2 illustrate a sequence comparison between SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
  • SEQ ID NO: 2 is a His-tagged construct of SEQ ID NO: 1. The SEQ ID NO: 1 region is shown underlined.
  • SEQ ID NOs: 3, 4 and 5 are different ORF2331 sequences. Some amino acid differences between the sequences are shown in bold.
  • Figure 3 illustrates a nucleic acid sequence SEQ ID NO: 6 encoding SEQ ID NO: 2. The region encoding SEQ ID NO: 1 is shown in bold.
  • Figure 4 illustrate survival data using a SEQ ED NO: 2 polypeptide in aluminum hydroxyphosphate adjuvant (AHP).
  • the polypeptide is referred to as "SEQ 2".
  • SEQ ED NO: 1 is a His-Tag derivative of SEQ ED NO: 1. The His-Tag facilitates polypeptide purification and identification.
  • SEQ ED NO: 1 is a derivative of a full-length 5. aureus polypeptide designated full- length ORF2331 (SEQ ED NO: 3). SEQ ED NO: 1 was obtained from ORF2331 by removing the first 20 amino acids of SEQ ED NO: 3. The first 20 amino acids contain the signal sequence.
  • Polypeptides structurally related to SEQ ED NO: 1 include polypeptides containing corresponding regions present in different 5. aureus strains and derivatives of naturally occurring regions.
  • the amino acid sequence of SEQ ED NO: 1 is illustrated in Figure 1. ORF2331 Sequences
  • ORF2331 related sequences have been given different designations in different references. Examples of different designations are provided in Kuroda et al, Lancet 357: 1225-1240, 2001 (SAV0361 and SA0359); and Baba et al, Lancet 359:1819-1827, 2002 (MW0227).
  • a polypeptide sequence corresponding to an ORF2331 related sequence appears to be provided in different patent publications. (Meinke et al, International Publication Number WO 02/059148, published August 1, 2002, and Masignani et al., International Publication Number WO 02/094868, published November 28, 2002.)
  • ORF2331 sequences can be identified based on the presence of a high degree of sequence similarity or contiguous amino acids compared to a known ORF2331 sequence. Contiguous amino acids provide characteristic tags.
  • a naturally occurring ORF2331 sequence is a sequence found in a Staphylococcus, preferably S. aureus, having at least 20, at least 30, or at least 50 contiguous amino acids as in SEQ ID NO: 1; and/or having at least 85% sequence similarity or identity with SEQ ID NO: 1.
  • Sequence similarity can be determined by different algorithms and techniques well known in the art. Generally, sequence similarity is determined by techniques aligning two sequences to obtain maximum amino acid identity, allowing for gaps, additions and substitutions in one of the sequences.
  • Sequence similarity can be determined, for example, using a local alignment tool utilizing the program lalign (developed by Huang and Miller, Adv. Appl. Math. 72:337-357, 1991, for the «sim» program).
  • the options and environment variables are:-f # Penalty for the first residue a gap (-14 by default); -g # Penalty for each additional residue in a gap (-4 by default)-s str (SMATRIX) the filename of an alternative scoring matrix file.
  • PAM250 is used by default-w # (LINLEN) output line length for sequence alignments (60).
  • SEQ ID NO: 1 related polypeptides contain an amino acid sequence at least 85% identical to SEQ ID NO: 1.
  • Reference to "polypeptide” does not provide a minimum or maximum size limitation.
  • a polypeptide at least 85% identical to SEQ ID NO: 1 contains up to about 25 amino acid alterations from SEQ ID NO: 1.
  • the SEQ ID NO: 1 related polypeptide is at 90%, at least 94%, or at least 99% identical to SEQ ID NO: 1; differs from SEQ ID NO: 1 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid alterations; or consists essentially of SEQ ID NO: 1.
  • Each amino acid alteration is independently either a deletion, substitution, or addition.
  • references to "consists essentially" of indicated amino acids indicates that the referred to amino acids are present and additional amino acids may be present.
  • the additional amino acids can be at the carboxyl or amino terminus. In different embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additional amino acids are present.
  • a preferred additional amino acid is an amino terminus methionine.
  • Alterations can be made to SEQ TD NO: 1 to obtain derivatives that can induce protective immunity against S. aureus. Alterations can be performed, for example, to obtain a derivative retaining the ability to induce protective immunity against 5. aureus or to obtain a derivative that in addition to providing protective immunity also has a region that can achieve a particular purpose.
  • the sequence comparison provided in Figure 2 can be used to guide the design of potential alterations to SEQ ID NO: 1.
  • alterations can be made taking into account other ORF2331 sequences and known properties of amino acids.
  • the replacement amino acid should have one or more similar properties such as approximately the same charge and/or size and/or polarity and/or hydrophobicity.
  • valine for leucine, arginine for lysine, and asparagine for glutamine are good candidates for not causing a change in polypeptide functioning.
  • Alterations to achieve a particular purpose include those designed to facilitate production or efficacy of the polypeptide; or cloning of the encoded nucleic acid.
  • Polypeptide production can be facilitated through the use of an initiation codon (e.g., coding for methionine) suitable for recombinant expression. The methionine may be later removed during cellular processing.
  • Cloning can be facilitated by, for example, the introduction of restriction sites which can be accompanied by amino acid additions or changes.
  • Efficacy of a polypeptide to induce an immune response can be enhanced through epitope enhancement.
  • Epitope enhancement can be performed using different techniques such as those involving alteration of anchor residues to improve peptide affinity for MHC molecules and those increasing affinity of the peptide-MHC complex for a T-cell receptor. (Berzofsky et ah, Nature Review 7:209-219, 2001.)
  • the polypeptide is a purified polypeptide.
  • a "purified polypeptide” is present in an environment lacking one or more other polypeptides with which it is naturally associated and/or is represented by at least about 10% of the total protein present.
  • the purified polypeptide represents at least about 50%, at least about 75%, or at least about 95% of the total protein in a sample or preparation.
  • the polypeptide is "substantially purified.”
  • a substantially purified polypeptide is present in an environment lacking all, or most, other polypeptides with which the polypeptide is naturally associated.
  • aureus polypeptide is present in an environment lacking all, or most, other S. aureus polypeptides.
  • An environment can be, for example, a sample or preparation.
  • Polypeptide stability can be enhanced by modifying the polypeptide carboxyl or amino terminus.
  • modifications include amino terminus protecting groups such as acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl or /-butyloxycarbonyl; and carboxyl terminus protecting groups such as amide, methylamide, and ethylamide.
  • polypeptide immunogen is part of an immunogen containing one or more additional regions or moieties covalently joined to the polypeptide at the carboxyl terminus or amino terminus, where each region or moiety is independently selected from a region or moiety having at least one of the following properties: enhances the immune response, facilitates purification, or facilitates polypeptide stability.
  • Polypeptide stability can be enhanced, for example, using groups such as polyethylene glycol that may be present on the amino or carboxyl terminus.
  • Polypeptide purification can be enhanced by adding a group to the carboxyl or amino terminus to facilitate purification.
  • groups that can be used to facilitate purification include polypeptides providing affinity tags.
  • affinity tags include a six-histidine tag, trpE, glutathione and maltose-binding protein.
  • the ability of a polypeptide to produce an immune response can be enhanced using groups that generally enhance an immune response.
  • groups that can be joined to a polypeptide to enhance an immune response against the polypeptide include cytokines such as IL-2. (Buchan et al., 2000. Molecular Immunology _?7:545-552.)
  • Polypeptides can be produced using standard techniques including those involving chemical synthesis and those involving purification from a cell producing the polypeptide.
  • Techniques for chemical synthesis of polypeptides are well known in the art. (See e.g., Vincent, Peptide and Protein Drug Delivery, New York, N.Y., Decker, 1990.) Techniques for recombinant polypeptide production and purification are also well known in the art. (See for example, Ausubel, Current Protocols in
  • Obtaining polypeptides from a cell is facilitated using recombinant nucleic acid techniques to produce the polypeptide.
  • Recombinant nucleic acid techniques for producing a polypeptide involve introducing, or producing, a recombinant gene encoding the polypeptide in a cell and expressing the polypeptide.
  • a recombinant gene contains nucleic acid encoding a polypeptide along with regulatory elements for polypeptide expression.
  • the recombinant gene can be present in a cellular genome or can be part of an expression vector.
  • the regulatory elements that may be present as part of a recombinant gene include those naturally associated with the polypeptide encoding sequence and exogenous regulatory elements not naturally associated with the polypeptide encoding sequence. Exogenous regulatory elements such as an exogenous promoter can be useful for expressing a recombinant gene in a particular host or increasing the level of expression.
  • the regulatory elements that are present in a recombinant gene include a transcriptional promoter, a ribosome binding site, a terminator, and an optionally present operator.
  • a preferred element for processing in eukaryotic cells is a polyadenylation signal.
  • an expression vector in addition to a recombinant gene also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number.
  • expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids and viruses.
  • nucleic acid sequences Due to the degeneracy of the genetic code, a large number of different encoding nucleic acid sequences can be used to code for a particular polypeptide.
  • the degeneracy of the genetic code arises because almost all amino acids are encoded by different combinations of nucleotide triplets or "codons". Amino acids are encoded by codons as follows:
  • Suitable cells for recombinant nucleic acid expression of SEQ K) NO: 1 related polypeptides are prokaryotes and eukaryotes.
  • prokaryotic cells include E. col ⁇ , members of the Staphylococcus genus, such as 5. aureus; members of the Lactobacillus genus, such as L. plantarum; members of the Lactococcus genus, such as L. lactis; and members of the Bacillus genus, such as B. subtilis.
  • eukaryotic cells include mammalian cells; insect cells; yeast cells such as members of the Saccharomyces genus ⁇ e.g., S.
  • members of the Pichia genus e.g., P. pastoris
  • members of the Hansenula genus e.g., H. polymorpha
  • members of the Kluyveromyces genus e.g., K. lactis or K. fragilis
  • members of the Schizosaccharomyces genus e.g., S. pombe.
  • Codon optimization includes use of more preferred codons. Techniques for codon optimization in different hosts are well known in the art.
  • SEQ ID NO: 1 related polypeptides may contain post translational modifications, for example, N-linked glycosylation, O-linked glycosylation, or acetylation.
  • Reference to "polypeptide” or an "amino acid" sequence of a polypeptide includes polypeptides containing one or more amino acids having a structure of a post-translational modification from a host cell, such as a yeast host.
  • Post translational modifications can be produced chemically or by making use of suitable hosts. For example, in S. cerevisiae the nature of the penultimate amino acid appears to determine whether the N-terminal methionine is removed.
  • the nature of the penultimate amino acid also determines whether the N-terminal amino acid is N ⁇ -acetylated (Huang et al., Biochemistry 26: 8242-8246, 1987).
  • Another example includes a polypeptide targeted for secretion due to the presence of a secretory leader (e.g., signal peptide), where the protein is modified by N-linked or O-linked glycosylation. (Kukuruzinska et al., Ann. Rev. Biochem. 56:915-944, 1987.)
  • adjuvants can be employed to assist in the production of an immune response.
  • adjuvants include aluminum hydroxide, aluminum phosphate, or other salts of aluminum, calcium phosphate, DNA CpG motifs, monophosphoryl lipid A, cholera toxin, E.
  • a "patient” refers to a mammal capable of being infected with S. aureus.
  • a patient can be treated prophylactically or therapeutically.
  • Prophylactic treatment provides sufficient protective immunity to reduce the likelihood, or severity, of a 5. aureus infection.
  • Therapeutic treatment can be performed to reduce the severity of a 5. aureus infection.
  • Prophylactic treatment can be performed using a vaccine containing an immunogen described herein. Such treatment is preferably performed on a human.
  • Vaccines can be administered to the general population or to those persons at an increased risk of S. aureus infection.
  • aureus infection include health care workers; hospital patients; patients with a weakened immune system; patients undergoing surgery; patients receiving foreign body implants, such a catheter or a vascular device; patients facing therapy leading to a weakened immunity; and persons in professions having an increased risk of burn or wound injury.
  • Non-human patients that can be infected with 5. aureus include cows, pigs, sheep, goats, rabbits, horses, dogs, cats and mice. Treatment of non-human patients is useful in protecting pets and livestock, and in evaluating the efficacy of a particular treatment.
  • Combination Vaccines include cows, pigs, sheep, goats, rabbits, horses, dogs, cats and mice. Treatment of non-human patients is useful in protecting pets and livestock, and in evaluating the efficacy of a particular treatment.
  • SEQ ID NO: 1 related polypeptides can be used alone, or in combination with other immunogens, to induce an immune response.
  • Additional immunogens that may be present include: one or more additional S. aureus immunogens, such as those referenced in the Background of the Invention supra; one or more immunogens targeting one or more other Staphylococcus organisms such as 5. epidermidis, S. haemolyticus, S. warneri, or S.lugunensis; and one or more immunogens targeting other infections organisms.
  • the animal model system was used to evaluate the efficacy of an immunogen to produce a protective immune response against 5. aureus.
  • the animal model was a slow kinetics lethality model involving S. aureus prepared from cells in stationary phase, appropriately titrated, and intravenously administered. This slow kinetics of death provides sufficient time for the specific immune defense to fight off the bacterial infection (e.g., 10 days rather 24 hours).
  • S. aureus cells in stationary phase can be obtained from cells grown on solid medium.
  • aureus can be grown from about 18 to about 24 hours under conditions where the doubling time is about 20-30 minutes.
  • S. aureus can be isolated from solid or liquid medium using standard techniques to maintain S. aureus potency. Isolated 5. aureus can be stored, for example, at -7O 0 C as a washed high density suspension (> 10 9 colony forming units (CFU)/mL) in phosphate buffered saline containing glycerol.
  • the S. aureus challenge should have a potency providing about 80 to 90% death in an animal model over a period of about 7 to 10 days starting on the first or second day.
  • Titration experiments can be performed using animal models to monitor the potency of the stored Staphylococcus inoculum. The titration experiments can be performed about one to two weeks prior to an inoculation experiment.
  • Immunogens can be formulated and administered to a patient using the guidance provided herein along with techniques well known in the art. Guidelines for pharmaceutical administration in general are provided in, for example, Vaccines Eds. Plotkin and Orenstein, W.B.
  • Pharmaceutically acceptable carriers facilitate storage and administration of an immunogen to a patient.
  • Pharmaceutically acceptable carriers may contain different components such as a buffer, sterile water for injection, normal saline or phosphate buffered saline, sucrose, histidine, salts and polysorbate.
  • Immunogens can be administered by different routes such as subcutaneous, intramuscular, or mucosal.
  • Subcutaneous and intramuscular administration can be performed using, for example, needles or jet-injectors.
  • Suitable dosing regimens are preferably determined taking into account factors well known in the art including age, weight, sex and medical condition of the patient; the route of administration; the desired effect; and the particular compound employed.
  • the immunogen can be used in multi-dose vaccine formats. It is expected that a dose would consist of the range of 1.0 ⁇ g to 1.0 mg total polypeptide. In different embodiments of the present invention the range is 0.01 mg to 1.0 mg and
  • booster doses may subsequently be administered to maintain or boost antibody titers.
  • An example of a dosing regime would be day 1, 1 month, a third dose at either 4, 6 or 12 months, and additional booster doses at distant times as needed.
  • a SEQ ID NO: 1 related polypeptide can be used to generate antibodies and antibody fragments that bind to the polypeptide or to 5. aureus.
  • Such antibodies and antibody fragments have different uses including use in polypeptide purification, S. aureus identification, or in therapeutic or prophylactic treatment against 5. aureus infection.
  • Antibodies can be polyclonal or monoclonal. Techniques for producing and using antibodies are well known in the art. Examples of such techniques are described in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002, Harlow et al., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, and Kohler et al, Nature 256:495-497, 1975. EXAMPLES
  • the protein was designed to be expressed from the pET16 vector with the His-Tag residues encoded by the vector.
  • the vector provides the initiating Met codon followed by a GIy codon, the 10 His-Tag, and an additional 12 codons for Ser Ser GIy His He GIu GIy Arg His Met Leu GIu. (Amino acids 1-24 of SEQ ID NO: 2.)
  • the designed DNA sequence encodes a 194 amino acid altered form of mature ORF2331.
  • the ORF2331 DNA sequence was translated using Vector NTI software and the resulting amino acid sequence (SEQ ID NO: 1) was analyzed.
  • PCR primers were designed to amplify the gene starting at the 21 st residue (Asn) to remove a signal sequence and membrane binding site and ending prior to the stop codon at the terminal His residue.
  • the forward PCR primers contained an Xhol restriction site to facilitate cloning into the expression vector.
  • the reverse PCR primer included a BIpI restriction site to facilitate cloning into the expression vector and a stop codon.
  • PCR amplified sequences were digested with Xhol and BIpI then ligated into the pET16b vector (Novagen) digested with Xhol and BIpI.
  • the ligation reaction was transformed into E. coli strain NovaBlue and colonies selected by growth in LB with 100 ⁇ g/mL ampicillin. DNA minipreps were made (Qiagen), and insert integrity determined by restriction digestion and sequence confirmation. A clone was selected containing no DNA changes from the desired sequence.
  • E. coli cell paste Frozen recombinant E. coli cell paste (48 grams) was thawed and resuspended in four volumes of Lysis Buffer (50 mM sodium phosphate, pH 8.0, 0.15 M NaCl, 2 mM magnesium chloride, 10 mM imidazole, 0.1% Tween-80, and 0.02% sodium azide.
  • Lysis Buffer 50 mM sodium phosphate, pH 8.0, 0.15 M NaCl, 2 mM magnesium chloride, 10 mM imidazole, 0.1% Tween-80, and 0.02% sodium azide.
  • Benzonase EM #1.01697.0002
  • protease inhibitor cocktail was added to the cell suspension at one tablet per 15 ml (CompleteTM, EDTA-Free, Roche # 1873580).
  • a lysate was prepared with a microfluidizer.
  • the Lysate was stirred overnight at 4°C, and was clarified by centrifugation at 10,800 x g for 30 minutes at 4°C.
  • the supernatant was filtered through a glass-fiber pre-filter (Millipore) and NaCl was added to a final concentration of 0.5 M from a 5 M stock solution.
  • the Filtered Supernatant was added to Ni-NTA agarose chromatography resin (Qiagen #30250) and the slurry was mixed overnight at 4°C.
  • the slurry of chromatography resin was poured into a chromatography column and the non-bound fraction was collected by gravity from the column outlet.
  • the column was washed with five column volumes of Wash Buffer (50 mM sodium phosphate, pH 8.0, 0.3 M NaCl, 0.1% Tween-80, and 20 mM imidazole).
  • the column was eluted with Elution Buffer (50 mM sodium phosphate, pH 8.0, 0.3 M NaCl, 0.1% Tween-80, and 0.3 M imidazole).
  • Ni-MAC Product Fractions containing protein were identified by dot blot on nitrocellulose membrane with Ponceau-S staining, and fractions containing the highest protein concentrations were pooled to make the Ni-MAC Product.
  • the Ni-EVlAC Product was fractionated by SEC.
  • SEC fractions containing the product protein were identified by SDS/PAGE with Coomassie staining.
  • Product-containing SEC fractions were pooled to make the SEC Product.
  • the SEC Product was filtered with a CUNO Zeta-Plus BioCap filter to remove endotoxin. The Filtrate was sterile-filtered and adsorbed on aluminum hydroxyphosphate adjuvant at a final concentration of 0.2 mg/ml.
  • mice Twenty BALB/c mice were immunized with three doses of a SEQ ID NO: 2 polypeptide
  • mice 311-321, 2000. 30 mice were vaccinated with three doses of AHP alone as an adjuvant control group.
  • mice were challenged with S. aureus (7 X 10 8 CFU/ml) and evaluated against the control set of 30 mice that had just been immunized with AHP. The mice were monitored over an 11 day period for survival. At the end of the experiment 8 of 20 (40%) mice survived the SEQ ID NO: 2 immunized group compared to 6 of 30 (20%) surviving in the AHP control group.

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Abstract

La présente invention concerne des polypeptides comprenant une séquence d'acides aminés structurellement associée à SEQ ID NO: 1 ainsi que des utilisations desdits polypeptides. La SEQ ID NO: 1 est un dérivé tronqué d'un polypeptide de S. aureus pleine longueur. Le polypeptide d'origine naturelle pleine longueur est, dans l'invention, appelé 'ORF2331' pleine longueur. On a découvert qu'un dérivé à marquage HIS de SEQ ID NO: 1 produit une réponse immunitaire protectrice contre le S. aureus.
PCT/US2005/023511 2004-07-06 2005-07-01 Polypeptides utilises pour induire une reponse immunitaire protectrice contre le staphylococcus aureus Ceased WO2007018482A2 (fr)

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