WO2008130381A2 - Compositions d'acide polyglutamique et leur utilisation comme immunogènes - Google Patents

Compositions d'acide polyglutamique et leur utilisation comme immunogènes Download PDF

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WO2008130381A2
WO2008130381A2 PCT/US2007/022567 US2007022567W WO2008130381A2 WO 2008130381 A2 WO2008130381 A2 WO 2008130381A2 US 2007022567 W US2007022567 W US 2007022567W WO 2008130381 A2 WO2008130381 A2 WO 2008130381A2
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substituted
compound
protein
cys
nac
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WO2008130381A3 (fr
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Bret Richard Sellman
Viliam Pavliak
Thushari Bombuwala
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Wyeth LLC
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Wyeth LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Gram-positive bacteria
    • C07K16/1271—Micrococcaceae (F); Staphylococcaceae (F), e.g. Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/07—Bacillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Gram-positive bacteria
    • C07K16/1278—Bacillus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker

Definitions

  • compositions comprising poly- ⁇ -D,L-glutamic acid
  • PGA PGA-peptide conjugates and uses thereof to raise an immune response in a subject for preventing or treating nosocomial infections caused by Staphylococcus and Bacillus.
  • Staphylococcus epidermidis is a coagulase-negative staphylococcus pathogen that has become one of the most prevalent pathogens involved in nosocomial infections. While relatively innocuous when resident on the human skin, S. epidermidis can cause severe infections in subjects after penetration of the epidermal protective barriers of the human body, hi the United States alone, S. epidermidis infections is a leading type of infection for patients with in-dwelling medical devices (e.g., catheters, prosthetic heart valves, prosthetic joints), costing the public approximately $1 billion per year. Additionally, S. epidermidis is frequently resistant to common antibiotics, making patient treatment more challenging.
  • in-dwelling medical devices e.g., catheters, prosthetic heart valves, prosthetic joints
  • in-dwelling devices When in-dwelling devices are placed in a subject, they become coated with host proteins such as fibrinogen, fibronectin, and vitronectin. These proteins serve as ligands for the specific binding of S. epidermidis surface proteins including SdrG, AtIE, GehD, and Emb. Following binding, the bacteria divide and initiate intercellular interactions facilitated by both carbohydrates and proteins (e.g. , PNAG and AAP), thereby forming a complex three-dimensional bacterial structure called a biofilm. These infections are difficult to treat because bacteria that form biofilms show reduced sensitivity to antibiotics, and the biofilm itself is thought to protect the microorganism from the host's immune response. Consequently, compositions and methods for preventing S. epidermidis infections and treating patients with S. epidermidis infections are needed.
  • aspects of the invention are directed to compounds and compositions for use in treating and preventing nosocomial infections such as those caused by Staphylococcus and Bacillus species.
  • a compound of formula :
  • X is an amino acid
  • Z is a number from 0 to 3
  • Rc is a capping group
  • n is 0, 1 or 2
  • R L is an amino acid covalently bound to LQ
  • L G is a linker
  • Pc is a carrier protein
  • N represents the N- terminus
  • C represents the C-terminus
  • D,L can be either a D stereoisomer, or a L stereoisomer, or a combination of D and L stereoisomers
  • Y is an integer between 5 and 50.
  • Pc is a carrier protein selected from the group consisting Of CRMi 97 , enzymatically inactive streptococcal C5a peptidase (SCP), a bacterial toxin, a bacterial outer membrane protein, a bacterial heat shock protein, S. epidermidis SdrG protein, S. epidermidis SitC protein, S. epidermidis ferrochrome binding protein, Staphylococcus aureus CIfA protein, S. aureus CIfB protein, S.
  • SCP enzymatically inactive streptococcal C5a peptidase
  • aureus FnbA protein ovalbumin, keyhole limpet hemocyanin (KLH), glutathione S- transferase (GST), bovine serum albumin (BSA), galactokinase (galK), ubiquitin, ⁇ - galactosidase, influenza NSl protein, tetanus toxoid, purified protein derivative of tuberculin (PPD), a virus-like particle of rotavirus VP6, and a virus-like particle of bacteriophage Q ⁇ ;
  • the carrier protein is CRMi 97 ;
  • R L is Cys;
  • R L comprises a side-chain that is covalently bound to LQ, more particularly the side- chain is an alkylthio group, an alkylamino group, an alkoxy group or an alkylcarbonyl group;
  • LQ is a covalent bond or a divalent group of the formula: -i ⁇ l ⁇ l ⁇ , wherein L 1 is oxo, S(O) q , amino, substituted amino, carbonyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkenyl, substituted cycloalkenyl, heterocyclyl, substituted heterocyclyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl; L 2 is absent or oxo, S(O) q , amino, substituted amino, carbonyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted heteroaryl, substituted heteroaryl;
  • L 2 is absent or oxo, S(O) q , amino
  • LQ is 4-(2,5-dioxopyrrolidin-l-yl)cyclohexanecarbonyl;
  • Y is an integer from 10 to 20; (1) D,L is D;
  • Rc is an alkyl group, a substituted alkyl group (e.g. alkylcarbonyl), an aryl or substituted aryl group, such as benzyl or PMB (wherein n is 2), imide, such as phthalimide, dithiosuccinamide, azide, an alkyne, or a hydroxyl or alkoxy group;
  • Rc is a carbonyl or substituted carbonyl group;
  • Rc is an acetyl, propionyl, formyl, an ester (i.e. forms a carbamate), particularly methyl formate or ethyl formate, or aminocarbonyl (i.e.
  • Rc is acetyl and n is 1 ;
  • X is an amino acid selected from Ala, Arg, Asn, Asp, Cys, GIu, GIn, GIy, His, He,
  • R L is an amino acid selected from Ala, Arg, Asn, Asp, Cys, GIu, GIn, GIy, His, He,
  • Another aspect of the invention provides an immununogenic composition
  • a carrier protein comprising: a carrier protein; and a compound of formula:
  • each X is an amino acid
  • Z is a number from 0 to 3
  • Rc is a carbonyl or substituted carbonyl group
  • n is 0 or 1
  • N represents the N-terminus
  • C represents the C-terminus
  • D,L can be either a D stereoisomer, or a L stereoisomer, or a combination of D and L stereoisomers
  • Y is an integer between 5 and 50.
  • composition further comprises an adjuvant;
  • the compound is a Staphylococcus or Bacillus poly- ⁇ - D,L-glutamic acid.
  • the carrier protein is conjugated to the compound at X either directly or through a linker, specifically, LQ is described above.
  • Another aspect of the invention provides a method of treating a nosocomial infection in a mammal comprising administering a compound or composition as described herein.
  • the nosocomial infection is a Staphylococcus or Bacillus infection.
  • Another embodiment thereof further comprises the step of administering an antibiotic in a therapeutically effective amount to treat said mammal, wherein said mammal has a S. epidermidis infection.
  • Another aspect of the invention provides a method of preparing an immuno therapeutic composition against nosocomial infections comprising the steps of:
  • the mammal is a human.
  • the nosocomial infection is a Staphylococcus infection.
  • Another aspect of the invention provides a hyperimmune globulin containing antibodies directed against a poly- ⁇ -D,L- glutamic acid, a poly- ⁇ -D,L-glutamic acid conjugated to a carrier protein, a poly- ⁇ -D,L-glutamic acid-peptide, a poly- ⁇ -D,L glutamic acid-peptide conjugated to a carrier protein, or a combination thereof, wherein the poly- ⁇ -D,L glutamic acid is a Staphylococcus or Bacillus poly- ⁇ -D,L glutamic acid.
  • a further embodiment thereof provides method of inducing passive immunity to a nosocomial infection in a mammal in need thereof comprising administering to said mammal in need thereof an immunogenic amount of the hyperimmune globulin.
  • Another aspect of the invention provides for a synthetic compound of formula: NAc-CyS-X z -Y-D 5 L-GIU Y -COOH or HOOC-Cys-X z - ⁇ - D,L-G1U Y -NAC, wherein X is any amino acid; Z is a number from 0 to 3; D,L can be either a D stereoisomer, or a L stereoisomer, or a combination of D and L stereoisomers; and Y is an integer between 5 and 50.
  • the synthetic compound can further be conjugated to a carrier protein.
  • X is a glycine, alanine, or serine
  • Z is an integer from 1 to 3.
  • Y is an integer from 10 to 20.
  • compounds are:
  • NAc-Y-D-GIu 20 -GIy 3 -L-CyS-COOH SEQ ID NO:4
  • NAc-L-Cys-Gly 3 - ⁇ -L-GluiQ-COOH SEQ ID NO:5
  • NAc-L-Cys-Gly 3 - ⁇ -L-Glu 20 -COOH SEQ ID NO:6;
  • the carrier protein selected from the group consisting of CRMi 97 , enzymatically inactive streptococcal C5a peptidase (SCP), a bacterial toxin, a bacterial outer membrane protein, a bacterial heat shock protein, S. epidermidis SdrG protein, S. epidermidis SitC protein, S. epidermidis ferrochrome binding protein, Staphylococcus aureus CIfA protein, S. aureus CIfB protein, S.
  • SCP enzymatically inactive streptococcal C5a peptidase
  • aureus FnbA protein ovalbumin, keyhole limpet hemocyanin (KLH), glutathione S- transferase (GST), bovine serum albumin (BSA), galactokinase (galK), ubiquitin, ⁇ - galactosidase, influenza NSl protein, tetanus toxoid, purified protein derivative of tuberculin (PPD), a virus-like particle of rotavirus VP6, and a virus-like particle of bacteriophage Q ⁇ ;
  • the carrier protein is CRMig 7 ;
  • a sulfhydryl group on the Cys residue binds the carrier protein;
  • X is glycine, alanine, or serine, and Z is an integer from 1 to 3;
  • the carrier protein is linked to the synthetic compound by a divalent group of the formula: -L'-L ⁇ L 3 -, wherein L 1 is oxo, S(O) q , amino, substituted amino, carbonyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkenyl, substituted cycloalkenyl, heterocyclyl, substituted heterocyclyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl; L 2 is absent or oxo, S(O) q , amino, substituted amino, carbonyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkenyl, substituted cycloalkenyl, heterocyclyl, substituted heterocyclyl
  • the linker is an acetyl group or 4-(2,5-dioxopyrrolidin-l-yl)cyclohexanecarbonyl;
  • Y is an integer from 10 to 20;
  • D,L is D;
  • D,L is L;
  • D,L is both D and L;
  • X is an amino acid selected from Ala, Arg, Asn, Asp, Cys, GIu, GIn, GIy, His, He,
  • R L is an amino acid selected from Ala, Arg, Asn, Asp, Cys, GIu, GIn, GIy, His, He,
  • the synthetic compound can have D,L as D; alternatively D,L can be L; or D,L can be both D and L isomers and the synthetic compound is conjugated to a carrier protein, and the carrier protein is CRM 197 , enzymatically inactive streptococcal C5a peptidase (SCP), a bacterial toxin, a bacterial outer membrane protein, a bacterial heat shock protein, S. epidermidis SdrG protein, S. epidermidis SitC protein, S. epidermidis ferrochrome binding protein, Staphylococcus aureus CIfA protein, S. aureus CIfB protein, S.
  • SCP enzymatically inactive streptococcal C5a peptidase
  • aureus FnbA protein ovalbumin, keyhole limpet hemocyanin (KLH), glutathione S-transferase (GST), bovine serum albumin (BSA), galactokinase (galK), ubiquitin, ⁇ -galactosidase, influenza NSl protein, purified protein derivative of tuberculin (PPD), a virus-like particle of rotavirus VP6, or a virus-like particle of bacteriophage Q ⁇ .
  • KLH keyhole limpet hemocyanin
  • GST glutathione S-transferase
  • BSA bovine serum albumin
  • galactokinase galK
  • ubiquitin ubiquitin
  • influenza NSl protein purified protein derivative of tuberculin (PPD)
  • PPD purified protein derivative of tuberculin
  • PPD virus-like particle of rotavirus VP6, or a virus-like particle of bacteriophage Q ⁇ .
  • an immunogenic composition comprising: (a) a synthetic compound of NAc-Cys-X z - ⁇ -D,L-Glu ⁇ -COOH or HOOC-Cys-X z - ⁇ -
  • D,L-Glu ⁇ -NAc wherein X is any amino acid; Z is a number from 0 to 3; D,L can be either a D stereoisomer, or a L stereoisomer, or a combination of D and L stereoisomers; and Y is an integer between 5 and 50; and
  • the synthetic compound of the immunogenic composition is conjugated to a carrier protein.
  • the carrier protein can be any of the carrier proteins listed herein or above.
  • the synthetic compound includes synthetic compounds, wherein X is glycine, alanine, or serine, and Z is an integer from 1 to 3; and/or wherein Y is an integer from 10 to 20; and/or wherein D,L is D, L or D and L.
  • the immunogenic composition can include a synthetic compound of:
  • NAC-L-CyS-GIy 3 -Y-D-GIu 20 -COOH SEQ BD NO:2
  • NAC-Y-D-GIu 10 -GIy 3 -L-CyS-COOH SEQ ID NO:3
  • NAc-L-CyS-GIy 3 -Y-L-GlUi O -COOH SEQ ID NO:5
  • Q NAC-L-CyS-GIy 3 -Y-L-GIu 20 -COOH SEQ ID NO:6
  • NAc- ⁇ -L-Glu,o-Gly 3 -L-Cys-COOH SEQ ID NO:7
  • NAC-Y-L-GIu 20 -GIy 3 -L-CyS-COOH SEQ ID NO:8.
  • the immunogenic composition comprises
  • the synthetic compound of the immunogenic composition has X as a glycine, alanine, or serine, and Z is an integer from 1 to 3; and/or Y is an integer from 10 to 20; and/or D,L is D, L or D and L.
  • the carrier protein of this immunogenic composition is CRM 19 - 7 , enzymatically inactive streptococcal C5a peptidase (SCP), a bacterial toxin, a bacterial outer membrane protein, a bacterial heat shock protein, S. epidermidis SdrG protein, S. epidermidis SitC protein, S. epidermidis ferrochrome binding protein, Staphylococcus aureus CIfA protein, S. aureus CIfB protein, S.
  • SCP enzymatically inactive streptococcal C5a peptidase
  • aureus FnbA protein ovalbumin, keyhole limpet hemocyanin (KLH), glutathione S-transferase (GST), bovine serum albumin (BSA), galactokinase (galK), ubiquitin, ⁇ -galactosidase, influenza NSl protein, purified protein derivative of tuberculin (PPD), a virus-like particle of rotavirus VP6, or a virus-like particle of bacteriophage Q ⁇ .
  • KLH keyhole limpet hemocyanin
  • GST glutathione S-transferase
  • BSA bovine serum albumin
  • galactokinase galK
  • ubiquitin ubiquitin
  • influenza NSl protein purified protein derivative of tuberculin (PPD)
  • PPD purified protein derivative of tuberculin
  • PPD virus-like particle of rotavirus VP6, or a virus-like particle of bacteriophage Q ⁇ .
  • Yet another aspect provides for a method of preventing or treating a nosocomial infection in a mammal comprising administering any of the above immunogenic compositions in an effective amount.
  • the nosocomial infection is a Staphylococcus or Bacillus infection.
  • the method further can comprise a step of administering an antibiotic in a therapeutically effective amount to treat said mammal, wherein said mammal has a S. epidermidis infection.
  • Another aspect provides for a method of preparing an immunotherapeutic composition against nosocomial infections comprising the steps of:
  • the immunized mammal can be a human or other animal discussed herein.
  • the composition can be used to treat a Staphylococcus infection.
  • a hyperimmune globulin containing antibodies directed against a poly- ⁇ -D,L- glutamic acid, a poly- ⁇ -D,L-glutamic acid conjugated to a carrier protein, a poly- ⁇ -D,L-glutamic acid-peptide, a poly- ⁇ -D,L glutamic acid-peptide conjugated to a carrier protein, or a combination thereof, wherein the poly- ⁇ -D,L glutamic acid is a Staphylococcus or Bacillus poly- ⁇ -D,L glutamic acid.
  • Another aspect provides for using the hyperimmune globulin in a method of inducing passive immunity to a nosocomial infection in a mammal in need thereof comprising administering to said mammal in need thereof an immunogenic amount of the hyperimmune globulin.
  • the nosocomial infection can be a Staphylococcus or Bacillus infection.
  • a hyperimmune globulin for the preparation of a medicament for treating a nosocomial infection in a subject in need thereof.
  • S. epidermidis possesses a poly- ⁇ -D,L-glutamic acid capsule that has been shown to be similar in composition to the poly- ⁇ -D-glutamic acid capsule of Bacillus anthracis, an etiologic agent of anthrax.
  • B. anthracis synthesizes only the D stereoisomer of poly- ⁇ -glutamic acid, while other bacilli produce both conformations.
  • Polyglutamate is found in various bacteria. However, polyglutamate displays different functions depending on the bacterial species and their environment. Thomas Candela et al., CapE, A 47 -Amino Acid Peptide, Is Necessary for Bacillus anthracis Polyglutamate Capsule Synthesis, 187 J. BACTERIOL. 7765 (2005).
  • Capsule purified from Bacillus or poly- ⁇ -D,L-glutamic acid peptides conjugated to a protein carrier have been shown to elicit an antibody response that recognizes the capsular polypeptide and is opsonic in vitro. Schneerson et al. (2003). However, PGA alone generally has a low immunogenicity. Stanislava Kocianova et al., Key Role of Poly-y-DL-glutamic Acid in Immune Evasion and Virulence of Staphylococcus Epidermidis, 115 J. CLIN. INVEST. 688 (2005).
  • the immunogenic compositions described herein provide, in one example, a subunit bacterial composition using the capsular PGA of any S. epidermidis or synthetic PGA-peptides.
  • PGA-peptide conjugates were prepared against S. epidermidis. PGA-peptides (10- and 20-mers) were synthesized with active groups at the amino or carboxy terminus and conjugated to a carrier protein, CRMi 97 . Uchida, et. al. Diphtheria toxin and related proteins. I. Isolation and properties of mutant proteins serologically related to diphtheria toxin, 248 J. BlOL. CHEM. 3838-44 (1973). The compositions were used to immunize animals and were shown to reduce the level of infection in animals.
  • CRMi 97 diphtheria toxoid cross-reactive material 197 (gly52glu)
  • GMBS N-fg-maleimidobutyiyloxyJsulfosuccinimide ester
  • IFN interferon e.g. , IFN- ⁇
  • IL interleukin e.g. , IL-2, IL- 1
  • MDP muramyl dipeptide also known as N-acetyl-muramyl-L-alanyl-D- isoglutamine MPLTM 3-O-deaylated monophosphoryl lipid A
  • PLA homo- and co-polymers of lactic acid
  • Poly rA Poly rU poly-adenylic acid-poly-uridylic acid complex POP-POE polyoxypropylenepolyoxyethylene
  • an "immunogenic composition” is a preparation containing an immunogen, including, e.g., a protein, a peptide, a PGA, a PGA-peptide conjugate, or a polysaccharide, or combination thereof, administered to stimulate the recipient's humoral and cellular immune systems to one or more of the antigens present in the immunogenic composition.
  • Immunization is the process of administering an immunogenic composition and stimulating an immune response to an antigen in a host.
  • Preferred hosts are mammals, such as primates (e.g., humans) as well as veterinary animals and agricultural animals.
  • an “immune response” refers to the activities of the immune system, including activation and proliferation of specific cytotoxic T-cells and B-cells resulting in antigen-specific antibody production, after contact with an antigen.
  • An "antigen” is any agent, e.g., a protein (or immunogenic fragments of proteins such as a fragment of an adhesion protein), a peptide or peptide conjugate, immunogen, or a polysaccharide, that elicits an immune response.
  • the antigen is a S. epidermidis antigen.
  • the immunogenic composition can comprise one or more S. epidermidis antigens or immunogens. For example, it can contain a Staphylococcus PGA, Bacillus PGA, or PGA- peptides in combination with another S. epidermidis subunit or protein ⁇ e.g., an adhesion protein).
  • an "immunogen” can be a purified, natural poly- ⁇ -glutamic acid or a synthetic poly- ⁇ - glutamic acid (either D- or L-stereoisomers).
  • the PGA can be from Staphylococcus or Bacillus.
  • the PGA or PGA-peptide can further be conjugated to a carrier protein, such as KLH or
  • the linkage can be through a sulfhydryl group from a cysteine or another moiety.
  • the immunogen of PGA can be a D - or L - stereoisomer.
  • the immunogen can be PGA alone, a PGA conjugated to a carrier protein, a PGA peptide conjugated to a carrier protein, or a combination thereof.
  • PGA polyglutamate
  • poly- ⁇ -glutamic acid as used with reference to the materials and methods for treating and preventing Staphylococcus and Bacillus infections are meant to include produced synthetically or purified from Staphylococcus or Bacillus.
  • the PGA can be in a composition comprising both L- and D- stereoisomers, or one stereoisomer alone; the PGA can be a purified or synthetic poly-y-L-glutamic acid or poly- ⁇ -D - glutamic acid.
  • a possible motif for PGA can be: Cys-Xz- ⁇ -D,L-Glu ⁇ , wherein "X” is any amino acid. "Z” is a number from 0 to 3. "Y” is a number from 5 to 50, and any integer there between.
  • the sulfhydryl providing cysteine can be at the carboxy terminus or at the amino terminus.
  • D,L can be either D-or L-or any combination of D 5 L. Examples of different types of PGA are provided herein and in the illustrated examples.
  • a "unit dose” is a defined and predetermined concentration or amount of the immunogenic composition that is safe and effective to elicit an immune response in the recipient of the composition.
  • terapéutica as used herein means a treatment and/or prophylaxis.
  • a therapeutic effect is obtained by suppression, remission, or eradication of a disease state caused by Staphylococcus or Bacillus.
  • an immunogenic composition includes, e.g., subcutaneous endothelial infections (e.g., S. aureus and S. epidermidis infections), wherein it is effective to provide measurable relief in treated subjects, such as exhibiting improvements including, but not limited to, improved survival rate, more rapid recovery, improvement or elimination of symptoms, reduction of post infectious complications and, where appropriate, antibody titer or increased titer against the infectious agent, and other measurements as known to those skilled in the art (e.g., less bacteria in blood sample).
  • "Parenteral" administration of an immunogenic composition includes, e.g. , subcutaneous
  • “Pharmaceutically acceptable” means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the PGA conjugate or other composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • adjuvant is meant a substance that serves to enhance the immunogenicity of an immunogenic composition. Thus, adjuvants are often given to boost the immune response and are well known in the art.
  • Staphylococcus is meant to include species that cause nosocomial infections such as
  • Staphylococcus aureus and Staphylococcus epidermidis are meant to include all strains of S. epidermidis including but not limited to laboratory strains, clinical stains, strains isolated from patients with S. epidermidis infections, and coagulase negative Staphylococcus (e.g., RP62A, ATCC 12228, 0-47, and 1457).
  • Staphylococcus aureus is also meant to include all strains of S. aureus, including but not limited to laboratory strains, clinical strains, and strains isolated from patients with S. aureus infections and coagulase negative Staphylococcus.
  • Treating” or “treatment” of a disease in a patient refers to 1) preventing the disease from occurring in a patient that is predisposed or does not yet display symptoms of the disease, such as through the administration of an immunogenic composition; 2) inhibiting the disease or arresting its development; or 3) ameliorating or causing regression of the disease.
  • Alkyl refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CH 3 ) 2 CH-), w-butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl ((CH 3 ) 2 CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), t-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH 2 CH 2 CH 2 CH 2 -), and neopentyl ((CH 3 ) 3 CCH 2 -).
  • Substituted alkyl refers to an alkyl group having from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, amino sulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio
  • Alkoxy refers to the group -O-alkyl wherein alkyl is defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, w-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and w-pentoxy.
  • Substituted alkoxy refers to the group -O-(substituted alkyl) wherein substituted alkyl is defined herein.
  • Amino refers to the group -NH 2 .
  • Substituted amino refers to the group -NR'R" where R' and R" are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -alkenyl, -SO 2 -substituted alkenyl, -SO 2 - cycloalkyl, -SO 2 -substituted cylcoalkyl, -SO 2 -cycloalkenyl, -SO 2 -substituted cylcoalkyl, -
  • R' is hydrogen and R" is alkyl
  • the substituted amino group is sometimes referred to herein as alkylamino.
  • R' and R" are alkyl
  • the substituted amino group is sometimes referred to herein as dialkylamino.
  • a monosubstituted amino it is meant that either R' or R" is hydrogen but not both.
  • a disubstituted amino it is meant that neither R' nor R" are hydrogen.
  • Aryl or “Ar” refers to a monovalent aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl) which condensed rings may or may not be aromatic (e.g., 2-benzoxazolinone, 2H- 1,4- benzoxazin-3(4H)-one-7-yl, and the like) provided that the point of attachment is at an aromatic carbon atom.
  • Preferred aryl groups include phenyl and naphthyl.
  • Substituted aryl refers to aryl groups which are substituted with 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloal
  • Alkenyl refers to alkenyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of alkenyl unsaturation. Such groups are exemplified, for example, by vinyl, allyl, and but-3-en-l-yl.
  • Substituted alkenyl refers to alkenyl groups having from 1 to 3 substituents, and preferably 1 to 2 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio,
  • Alkynyl refers to alkynyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of alkynyl unsaturation.
  • Substituted alkynyl refers to alkynyl groups having from 1 to 3 substituents, and preferably 1 to 2 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, amino sulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester,
  • Carboxyl or “carboxy” refers to -COOH or salts thereof.
  • Cycloalkyl refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems.
  • suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl.
  • Substituted cycloalkyl and “substituted cycloalkenyl” refers to a cycloalkyl or cycloalkenyl group having from 1 to 5 or preferably 1 to 3 substituents selected from the group consisting of oxo, thione, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, amino thiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl, carb
  • Heteroaryl refers to an aromatic group of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur within the ring.
  • Such heteroaryl groups can have a single ring (e.g., pyridinyl or furyl) or multiple condensed rings (e.g. , indolizinyl or benzothienyl) wherein the condensed rings may or may not be aromatic and/or contain a heteroatom provided that the point of attachment is through an atom of the aromatic heteroaryl group.
  • the nitrogen and/or the sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N- »O), sulfinyl, or sulfonyl moieties.
  • Preferred heteroaryls include pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
  • Substituted heteroaryl refers to heteroaryl groups that are substituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of the same group of substituents defined for substituted aryl.
  • Heterocycle or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl” refers to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, from 1 to 10 carbon atoms and from 1 to 4 hetero atoms selected from the group consisting of nitrogen, sulfur or oxygen within the ring wherein, in fused ring systems, one or more the rings can be cycloalkyl, aryl or heteroaryl provided that the point of attachment is through the non-aromatic ring.
  • the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, sulfinyl, sulfonyl moieties.
  • “Substituted heterocyclic” or “substituted heterocycloalkyl” or “substituted heterocyclyl” refers to heterocyclyl groups that are substituted with from 1 to 5 or preferably 1 to 3 of the same substituents as defined for substituted cycloalkyl.
  • heterocycle and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4- tetrahydroisoquinoline, 4,5,6,
  • capping group with respect to amine groups, hydroxyl groups and sulfhydryl groups refers to forms of these functionalities which are protected from undesirable reaction with a protecting group known to those skilled in the art, such as those set forth in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999) which can be added or removed using the procedures set forth therein.
  • Examples of protected or capped hydroxyl groups include, but are not limited to, silyl ethers such as those obtained by reaction of a hydroxyl group with a reagent such as, but not limited to, t- butyldimethyl-chlorosilane, trimethylch ' lorosilane, triisopropylchlorosilane, triethylchlorosilane; substituted methyl and ethyl ethers such as, but not limited to methoxymethyl ether, methythiomethyl ether, benzyloxymethyl ether, t-butoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ethers, 1-ethoxyethyl ether, allyl ether, benzyl ether; esters such as, but not limited to, benzoylformate, formate, acetate, trichloroacetate, and trifluoracetate.
  • a reagent such
  • Examples of protected or capped amine groups include, but are not limited to, amides such as, formamide, acetamide, trifluoroacetamide, and benzamide; carbamates; BOC; imides, such as phthalimide, and dithiosuccinimide; and others.
  • Examples of protected or capped sulfhydryl groups include, but are not limited to, thioethers such as S-benzyl thioether, and S-4-picolyl thioether; substituted S-methyl derivatives such as hemithio, dithio and aminothio acetals; and others.
  • the immunogenic composition can contain combinations of D- and L- forms of PGA either alone or linked to a carrier protein.
  • the ratio of the D- to L- isomers can be up to 50:50 of each stereoisomer.
  • the immunogenic composition could comprise 50% ofNAc-L-Cys-Gly 3 - ⁇ - L-GlUi 0 -COOH and 50% NAc-Y-D-GlUi 0 -GIy 3 -D-CyS-COOH.
  • Exemplary conventional carrier proteins that can be used with the PGA and PGA- peptides are described herein.
  • Carrier proteins are preferably proteins that are non-toxic and non- reactogenic and obtainable in sufficient amount and purity. Carrier proteins should be amenable to standard conjugation procedures.
  • CRMi 97 is used as the carrier protein.
  • CRMi 97 (Wyeth, Sanford, NC) is a non-toxic variant (i.e., toxoid) of diphtheria toxin isolated from cultures of Corynebacterium diphtheria strain C7 ( ⁇ l97) grown in casamino acids and yeast extract-based medium.
  • CRMi 97 is purified through ultra-filtration, ammonium sulfate precipitation, and ion-exchange chromatography. Another method of obtaining CRMi 97 is described in U.S. Patent No. 4,925,792.
  • CRMi 97 is prepared recombinantly in accordance with U.S. Patent No. 5,614,382.
  • Other diphtheria toxoids are also suitable for use as carrier proteins.
  • a carrier protein of the invention is an enzymatically inactive streptococcal C5a peptidase (SCP) (e.g., one or more of the SCP variants described in U.S. Patent Nos. 6,270,775; 6,355,255; and 6,951,653).
  • SCP streptococcal C5a peptidase
  • Suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (e.g., CT E29H, described in International PCT Publication No. WO2004/083251), E. coli LT, E. coli ST, E. coli DnaK protein, and exotoxin A from Pseudomonas aeruginosa.
  • Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin toxin (e.g., U.S. Patent No.
  • pneumolysin toxoid e.g., International PCT Publication No. WO 2005/108580
  • pneumococcal surface protein A PspA
  • pneumococcal adhesin protein PsaA
  • Haemophilus influenzae protein D can also be used.
  • Bacterial heat shock proteins such as mycobacterial hsp- 70 can also be used.
  • Other proteins such as Staphylococcus epidermidis proteins SdrG, SitC and ferrochrome binding proteins, and Staphylococcus aureus proteins CIfA, CIfB and FnbA can also be used.
  • Still other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), glutathione S-transferase (GST), bovine serum albumin (BSA), galactokinase (galK), ubiquitin, ⁇ -galactosidase, influenza NSl protein, or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins.
  • Virus-like particles for example from rotavirus VP6 or from bacteriophage Q ⁇ , can also be used.
  • CRMi 97 One carrier protein for use with the PGA and PGA-peptide discussed herein is CRMi 97 . Briefly, CRMi 97 was activated with a sulfo-maleimide group.
  • Additional cross linkers for -SH to -NH 2 linkages include but are not limited to: sulfo-LC-SMPT; sulfo-LC-SMPT (4- sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamido]hexanoate)); sulfo-KMUS (N-[k- maleimidoundecanoyloxy]sulfosuccinimide ester); sulfo-LC-SPDP (sulfosuccinimidyl 6-(3'-[2- pyridyldithio]-propionamido)hexanoate) which cleaves by thiols; sulfo-SMPB (sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate); sulfo-SIAB (N-sulfosuccinimidyl[4- i
  • Peptides of different lengths (10- or 20-mers) were synthesized with a reactive sulhydryl group at either the amino- or carboxy- terminus and conjugated via thio-ether linkages.
  • 2.2 Immunization Dosages of 0.1, 1.0 or 10 ⁇ g of purified conjugates in 100 ⁇ g AlPO 4 were used to immunize uninfected female BALB/c mice at 0, 3, and 6 wks. The animals were challenged at week (wk) 8 by intraperitoneal injection of 3 x 10 8 cfu S. epidermidis strain 0-47. Immunization with 10 ⁇ g of a PGA 10-mer conjugated at the carboxy terminus resulted in a statistically significant reduction in the bacteria recovered from the bloodstream.
  • Adjuvants have various uses including but not limited to increasing the amount of an antibody and the number of effector T-cells produced; reducing the quantity of antigen needed; reducing the time to protection; reducing frequency of treatment; retaining the antigen locally near the site of administration to produce a depot effect, thereby facilitating a slow, sustained- release of antigen to cells of the immune system; promoting and attracting accumulation of immunoreactive cells at the site of injection and/or the antigen depot; stimulating immunoreactive cells to elicit immune responses; maintaining serum antibody levels; modifying activities of cells that are concerned with generating and maintaining the immune response; modifying the presentation of antigen to the immune system; activating host macrophages, dendritic cells, B-cells or T-cells; increasing antigen uptake; up-regulating antigen processing; stimulating cytokine release; stimulating B-cell switching and maturation; and eliminating immunosuppressive cells.
  • Desirable characteristics of adjuvants include: (1) lack of toxicity; (2) ability to stimulate a long-lasting immune response; (3) simplicity of manufacture and stability in long-term storage; (4) ability to elicit both cell-mediated immunity (CMI) and humoral immune response (HIR) to antigens administered by various routes, if required; (5) synergy with other adjuvants; (6) capability of selectively interacting with populations of antigen presenting cells; (7) ability to specifically elicit appropriate ThI or Th2 cell-specific immune responses; and (8) ability to selectively increase appropriate antibody isotype levels (e.g., IgA) against antigens.
  • CRI cell-mediated immunity
  • HIR humoral immune response
  • the immunogenic compositions as described herein also comprise, in certain embodiments, one or more adjuvants.
  • An adjuvant is a substance that enhances the immune response when administered together with an immunogen or antigen.
  • a number of cytokines or lymphokines have been shown to have immune modulating activity, and thus are useful 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. 5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant forms); the interferons- ⁇ , - ⁇ and - ⁇ ; granulocyte-macrophage colony stimulating factor (GM-CSF) (see, e.g., U.S.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • chemokines including without limitation, MCP-I, MIP- l ⁇ , MIP- l ⁇ , and RANTES; adhesion molecules, such as a selectin, e.g., L-selectin, P-selectin and E-selectin; mucin- like molecules, e.g., CD34, GlyCAM-1 and MadCAM-1; a member of the integrin family such as LFA-I, VLA- 1, Mac-1 and pi 50.95; a member of the immunoglobulin superfamily such as PECAM, ICAMs, e.g., ICAM-I, ICAM-2 and ICAM-3, CD2 and LFA-3; co-stimulatory molecules such as
  • 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. Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa (Hamilton, MT), and which are described in United States Patent No. 6,113,918.
  • MPLTM 3-O-deacylated monophosphoryl lipid A, Corixa, Hamilton, MT
  • AGP aminoalkyl glucosamine phosphate compounds
  • AGP is 2-[(R)-3-Tetradecanoyloxytetradecanoylamino] ethyl 2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3- tetradecanoyloxytetradecanoyl-aminoJ-b-D-glucopyranoside, which is also known as 529 (formerly known as RC529).
  • This 529 adjuvant is formulated as an aqueous form (AF) or as a stable emulsion (SE).
  • Still other adjuvants include muramyl peptides, such as N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(l '-2' dipalmitoyl-s/j-glycero-3- hydroxyphosphoryloxy)-ethylamine (MTP-PE); oil-in-water emulsions, such as MF59 (International PCT Publication No.
  • muramyl peptides such as N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(l '-2' dipalmitoyl-s/j-glycero-3- hydroxyphosphoryloxy)-ethylamine (MTP-PE); oil-in-water emul
  • WO 90/14837 (containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a micro fluidizer such as Model HOY micro fluidizer (Micro fluidics, Newton, MA)), and SAF (containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, either micro fluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion); incomplete Freund's adjuvant (IFA); aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate; Amphigen; Avridine; Ll 21 /squalene; D-lactide-polylactide/glycoside; pluronic polyols; killed Bordetella; saponins, such as StimulonTM QS-21 (Antigenics, Fra
  • 1,296,713 and 1,326,634 a pertussis toxin (PT) or mutant thereof, a cholera toxin or mutant thereof (, e.g., International PCT Publication Nos. WO 00/18434, WO 02/098368 and WO 02/098369); or an E. coli heat-labile toxin (LT), particularly LT-K63, LT- R72, PT-K9/G129; see, e.g., International PCT Publication Nos. WO 93/13302 and WO 92/19265.
  • PT pertussis toxin
  • cholera toxin or mutant thereof e.g., International PCT Publication Nos. WO 00/18434, WO 02/098368 and WO 02/098369
  • LT E. coli heat-labile toxin
  • LT heat-labile toxin
  • adjuvants include but are not limited to those listed in Frederick R. Vogel et al., Compendium of Vaccine Adjuvants and Excipients (2 nd ed.) [online] [retrieved on June 27, 2006] as retrieved from the Internet: URL: ⁇ http://www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf>.
  • compositions disclosed can be administered in a variety of ways. It should be noted that the pharmaceutical composition containing the immunogen(s) can be administered alone or in combination with one or more pharmaceutically acceptable carriers, stabilizers, preservatives, colorants, flavorants, and excipients.
  • Aqueous formulations are prepared in sterile form, and when intended for delivery by routes other than oral administration, generally are isotonic. All formulations optionally contain excipients such as those provided for example in the HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (5 th ed., Raymond C. Rowe et al., eds., 2006). Excipients include ascorbic acid and other antioxidants, chelating agents ⁇ e.g., EGTA and EDTA), carbohydrates ⁇ e.g., dextrin), hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid, and the like. The pH of the formulations ranges from about 3 to about 11, but is ordinarily about 7 to about 10.
  • physiologically acceptable carriers for routes of administration other than oral administration include but are not limited to saline solutions ⁇ e.g., normal saline, Ringer's solution, PBS (phosphate-buffered saline); polysorbate 80; L-arginine; polyvinylpyrrolidone; ⁇ - D-glucopyranosyl; ⁇ -D-glucopyranoside (trehalose); and combinations, thereof.
  • trehalose can be present in the composition in an amount from about 2 to about 10% weight/volume of the composition.
  • trehalose and polysorbate 80 are both present in the composition
  • trehalose can be present in the amount of about 4 to about 6% wt./vol.
  • the polysorbate 80 can be present in the amount of about 0.001 to 0.01% (wt./vol.) and generally mixtures of various physiologically compatible salts including potassium and phosphate salts with or without sugar additives (e.g., glucose).
  • Suitable excipients for use in the immunogenic formulations are, for example, water, saline, dextrose, glycerol, and ethanol.
  • Non-toxic auxiliary substances, such as wetting agents, buffers, stabilizers, or emulsif ⁇ ers can also be added to the composition.
  • Sterile injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • the PGA or PGA-peptides conjugated to a carrier protein can further be placed in combination with other Staphylococcus immunogenic components and the combination utilized in an immunogenic composition administered to a subject.
  • Other immunogenic components include but are not limited to a protein, a peptide, polysaccharide, teichoic acid, and/or a coat protein of Staphylococcus.
  • the PGA or PGA-peptides conjugated to a carrier protein can be admixed with other Staphylococcus antigens, thereby forming other immunogenic compositions.
  • One aspect contemplates the prophylactic administration of a subject prior to surgery or prior to other medical treatment, or to any at risk population such as an immunodeficient population, to prevent or inhibit Staphylococcal infections in hospitals, geriatric centers and retirement communities, or veterinary clinics.
  • the composition can be administered preferably 3 months prior to hospitalization. However, the composition can be administered 2 days to 4 weeks prior to hospitalization. For example, the patient can be administered the composition upon entry to the hospital. In emergencies, the patient can be administered the composition once the patient's condition has been stabilized.
  • Another aspect of the invention is to administer the composition to health care workers (e.g., doctors, nurses, orderlies, medical technicians, veterinary workers) to reduce their risk of acquiring a staphylococcal (e.g., S. epidermidis) infection and/or to prevent them from carrying the microorganism and thereby infecting a patient.
  • health care workers e.g., doctors, nurses, orderlies, medical technicians, veterinary workers
  • staphylococcal infection e.g., S. epidermidis
  • the total amount of the composition necessary can be derived from protocols for immunization.
  • the exact amount of such immunogenic compositions required may vary from subject to subject, depending on the species, age, weight, and general condition of the subject, its mode of administration, whether it is administered with another Staphylococcus antigen, and the like. Generally, dosage will approximate that which is typical for the administration of other immunogenic compositions.
  • the recipient is a mammal (e.g., a cat, dog, horse, cow, pig, sheep, goat, primate, or human). Although human use is preferred, veterinary use of the invention is also contemplated.
  • the immunogenic composition is typically administered as a sterile composition.
  • the immunogenic composition can be administered by any suitable means, e.g. , parenteral (including subcutaneous, intramuscular, intravenous, intradermal, perilymphatic, intranasal, intraplenic, intrapulmonary, intrathecal, and epidural) or orally. Other routes include rectal, nasal, intranasal, topical (including buccal and sublingual), and vaginal. It is appreciated that the preferred route can vary with, for example, the condition of the recipient.
  • the first dose can be administered at the elected date and a second dose can follow several weeks to several months from the first dose. Additional booster doses of the original immunogenic composition or modified forms can be administered as necessary (e.g., annually). While it is possible for the active ingredients to be administered alone, it can be preferable to present them as immunogenic formulations.
  • the formulations both for veterinary and for human use, include at least one active ingredient together with an acceptable carrier, excipients, adjuvants and/or, optionally, other non-active and/or active ingredients.
  • the formulations include those suitable for the foregoing administration routes.
  • the formulations can conveniently be presented in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier, which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the oil phase of the emulsions of this invention can be constituted from known ingredients in a known manner. While the phase can comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier, which acts as a stabilizer. It is also preferred to include both an oil and a fat.
  • Emulients and emulsion stabilizers suitable for use in the formulation of the invention include but are not limited to Tween® 60 (as well as other polyoxyethylene sorbitan ester surfactants), Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono- stearate, and sodium lauryl sulfate.
  • Formulations suitable for nasal administration have a particle size, for example, in the range of 0.1 to 500 microns (including particle sizes in a range between 0.1 and 500 microns in incremental microns such as 0.5, 1, 30, 35, etc.), which is administered by rapid inhalation through the nasal passage.
  • Suitable formulations include aqueous or oily solutions of the active ingredient.
  • Squalene is an exemplary carrier.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic, sterile injection solutions that can contain antioxidants, buffers, bacteriostats, and solutes, which render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents and thickening agents.
  • formulations are presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injection, immediately prior to use.
  • sterile liquid carrier for example, water for injection
  • injection solutions and suspensions are prepared from sterile powders, and granules of the kind previously described.
  • compositions described herein further provide for veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier excipient, adjuvant, and/or stabilizer.
  • Veterinary carriers are materials useful for the purpose of administering the composition and can be solid, liquid or gaseous materials, which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions can be administered orally, parenterally, or by any other desired route.
  • a formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicles, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as polymer matrices, liposomes, and microspheres.
  • An implant suitable for use in the present invention can take the form of a pellet, which slowly dissolves after being implanted, or a biocompatible delivery module well known to those skilled in the art. Such well-known dosage forms and modules are designed such that the active ingredients are slowly released over a period of several days to several weeks.
  • the compounds and compositions can also be used in combination with other active ingredients.
  • compositions and compounds described herein can also be administered in conjunction with other conventional agents used to treat S. epidermidis infections, such as antibiotics, antipyretics, and analgesics. These compounds and compositions can be administered together with, or in the same course of, therapy with the compounds and compositions described herein.
  • the individual components of the combination can be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.
  • compositions and compounds disclosed herein can be given once or given daily over several days (e.g., daily for one to two weeks). While it is possible that, for use in therapy, a compound of the invention can be administered as the raw chemical, it is preferable to present the active ingredient as a pharmaceutical formulation.
  • compositions comprising an immunotherapeutic agent against nosocomial infections can be prepared and administed to the subject animal.
  • the plasma from the immunized animal is then collected, and a hyperimmune globulin harvested from the plasma that contains anti-PGA and anti-carrier protein antibodies.
  • the hyperimmune globulin can be used for inducing passive immunity to nosocomial infections.
  • the immunogenic compositions are administered to a subject to induce a humoral immune response.
  • the recipient then acts as a source of immunoglobulin (i.e., hyperimmune immunoglobulin) that is produced in response to the immunogenic composition.
  • the immunized subject donates plasma, from which the hyperimmune globulin is then obtained via, for example, conventional plasma fractionation technology.
  • the fraction comprising anti-PGA and anticarrier protein antibodies can then be administered to another subject in order to impart resistance against or to treat a nosocomial infection.
  • an immunogenic composition as described herein in combination with conventional therapies, such as antibiotic treatment.
  • Traditional antibiotics that can be used in combination with the compositions disclosed herein include but are not limited to dicloxacillin (250-500 mg, p.o. q 6 h for 7-10 days), cephalexin (250-500 mg, p.o. q. 6 h for 7-10 days), erythromycin (250-500 mg, p.o. q. 6 h,) clarithromycin (500 mg, p.o. q. 12 h), azithromycin (500 mg, p.o. on the first day then 250 mg, p.o. q 24 h), clindamycin (300 mg, p.o.
  • nafcillin 1.0-2.0 g, i.v. q 4-6h
  • cefazolin 1.0 g, i.v. q 8 h
  • clindamycin 600 mg, i.v. q. 8 h
  • vancomycin 15 mg/kg, q 12 h or 15 mg/kg i.v. q 12 h) or linezolid (600 mg, i.v. q. 12 h)
  • quinupristin and dalfopristin 7.5 mg/kg, q 8 h
  • daptomycin 4 mg/kg, q 24 h.
  • Combinations used may vary depending on the needs of the patient as determined by the medical practitioner. For example, penicillin allergic patients would not receive dicloxacillin and methicillin-resistant strains may receive vancomycin.
  • CRMi 97 Methods of making CRMi 97 are discussed, in for example U.S. Patent No. 5,614,382.
  • CPvMiQ 7 was bromoacetylated in 0.15 M borate buffer (pH 8.9) as follows. Frozen CRMi 97 was thawed and stirred in an ice bath. While stirring, bromoacetic acid N-hydroxy succinimide ester (1 :1 w/w in DMSO 20 mg/mL) was added dropwise slowly, thereby allowing the reaction mixture to admix well. The pH was adjusted to pH 8.9 with 0.1 M borate buffer (pH 8.9) and was incubated for 90 minutes in the ice-water bath (4°C). It was purified by diafiltration on Centriprep YM 10 (molecular weight cut-off at 1OK, Millipore), using 0.15 M borate buffer and purging with N 2 .
  • Centriprep YM 10 molecular weight cut-off at 1OK, Millipore
  • the bromoacetylated CRMi 97 was then stored at about 4°C.
  • the CRMi 97 was then conjugated to peptides.
  • the following method produces both L- linked PGA, as well as D-linked PGA.
  • PGA-peptide (2 mg) was dissolved in 0.1 M borate buffer (0.1 M, pH 8.9) to make the final concentration of the reaction mixture 2.5 mg/mL.
  • Peptide solution was added dropwise to bromoacetylated CRMiQ 7 with stirring on an ice bath 4 0 C overnight for about 14 to 16 hours.
  • the unreacted bromoaceytated lysines were capped by adding 5 mg of N-acetyl cysteamine (Sigma-Aldrich) with incubation for 3 hours at 4°C.
  • the capped conjugates were dialyzed using Slide A-lyzer cassettes (molecular weight cut off of 3,500, Pierce) against 4 Liters of 10 mM PBS buffer (pH 7.4) with 5 times buffer replacement.
  • the conjugation reaction is presented below:
  • PGA-peptide conjugates were prepared to determine the efficacy of capsular conjugates as immunogenic compositions against bacterial infections.
  • PGA PGA peptides (10- and 20-mers) were synthesized (Quality Controlled Biochemicals, Hopkinton, MA) with a cysteine at either the amino or carboxy terminus. The following PGA- peptides were prepared:
  • NAc-L-CyS-GIy 3 -Y-D-PGA 10 -COOH (Cys-Gly 3 - ⁇ -D-PGA 10 -C) (SEQ ID NO: 1)
  • NAc-L-Cys-Gly 3 - ⁇ -D-PGA 20 -COOH (Cys-Gly 3 - ⁇ -D-PGA 20 -C) (SEQ ID NO:2)
  • NAc- ⁇ -D-PGA 10 -Gly 3 -L-Cys -COOH N- ⁇ -D-PGA 10 -Gly 3 -Cys-C) (SEQ ID NO:3)
  • NAc- ⁇ -D-PGA 20 -Gly 3 -L-Cys -COOH N- ⁇ -D-PGA 20 -Gly 3 -Cys-C) (SEQ ID NO:4)
  • the synthesized PGA-peptides were then conjugated to CRMig 7 .
  • the cysteine provides the free sulfhydryl group required for reactivity with the activated CRM i 97 carrier protein, as discussed herein.
  • a spacer consisting of three glycine residues (indicated by GIy 3 ) separated the cysteine from the glutamic acid residues.
  • CRM !97 was activated using Sulfo-SMCC as shown in the reaction below and described as follows:
  • CRMi 97 (52.4 mg) was diluted to 5 mg/mL in ice-cold 0.1 M borate buffer, pH 8.9 and stored on ice.
  • Sulfo-SMCC (18 mg) was dissolved in dimethylsulfoxide (DMSO) (200 ⁇ L) and slowly added to the cold CRM i ⁇ >7 solution over 3 min with slow swirling. The mixture was then incubated for 1 hr at 4°C; unreacted Sulfo-SMCC was removed by passage through a Sephadex G-25 column (GE Healthcare Life Sciences, Uppsala, Sweden) equilibrated with 0.01 M borate buffer. Fractions containing CRMi 97 were pooled and concentrated using YM-10 filtration tubes (Millipore, Corp., Billerica, MA).
  • PGA-peptides were conjugated to the activated CRM is> 7 via a free N- or C-terminal sulfhydryl as depicted below and as follows:
  • PGA-peptide (4 mg) dissolved in 0.1 M borate buffer (pH 8.9) was added drop-wise to ice-cold activated CRMiQ 7 (1 :1 peptide:CRMi 97 ratio) and incubated overnight at 4°C.
  • a mock conjugate was prepared using the same activated CRM i 97 and N-Ac-cysteamine (1 mg each). Unreacted activated lysines on CRM i 97 were capped with N-Ac-cysteamine (10 mg).
  • the conjugate and the mock conjugate were purified by dialysis using Slide A-Lyzer cassettes (MWCO 10,000, Pierce) in PBS (pH 7.4). Conjugates were analyzed by SDS-PAGE (Coomassie) and amino acid analysis as discussed above. Concentration of the conjugates was assessed by Lowry assay.
  • Moles of peptides conjugates (moles of X in mock conjugate - moles of X in PGA conjugate).
  • N-terminal acetylated PGA peptides can be coupled to amino groups of Lysine residues of protein carrier using a two-step carbodiimide reaction with l-ethyl-3(3-dimethylamino-propyl) carbodiimide-HCl (EDC) and of N-hydroxysulfosuccinimide as follows:
  • Azides and terminal alkynes undergo a cycloaddition reaction in the presence of a copper catalyst.
  • the azide and alkyne moieties are directly conjugated to the PGA and/or CRM i 97 (carrier protein) or can be bound through a linker, such as an alkyl or substituted alkyl group.
  • the cycloaddition reaction in the scheme is conducted using the methods described, for example, Sharpless et al. (U.S. Patent Application Publication No. 20050222427, published October 6, 2005, PCT /US03/17311; Lewis W G, et al., Angewandte Chemie-Int'l Ed.
  • the reaction is generally known as "click chemistry".
  • the reaction is conducted in the presence of a metal catalyst and a reducing agent.
  • a reducing agent such as, but not limited to, ascorbate, metallic copper, quinone, hydroquinone, vitamin Ki, glutathione, cysteine, Fe 2+ , Co 2+ , and an applied electric potential.
  • Further preferred reducing agents include metals selected from the group consisting of Al, Be, Co, Cr, Fe, Mg, Mn, Ni, and Zn.
  • Other metals that may catalyze this type of cycloaddition reaction include, for example, Au, Ag, Hg, Cd, Zr, Ru, Fe, Co, Pt, Pd, Ni, Rh, and W. Those of ordinary skill in the art would be able to determine the appropriate metal to use for the intended ligand.
  • PGA-peptide conjugates were administered to mice as follows. Five week-old female BALB/c mice were immunized by subcutaneous injection at 0, 3, and 6 wks with 0.1, 1.0, or 10 ⁇ g of purified poly- ⁇ -D-glutamic acid conjugates in 100 ⁇ g AlPO 4 (adjuvant). The animals were bled on week (wk) 8 and challenged two days later by intraperitoneal injection of ⁇ 3 x 10 8 cfu S. epidermidis strain 0-47. Twenty-four hours post infection, the animals were sacrificed. Bacteria in the blood and spleen were enumerated.
  • a secondary antibody goat anti -mouse peroxidase conjugated (Promega, Madison, WI)
  • ABTS 2,2'-azino-bis-(3- ethyl-
  • mice were immunized with the ⁇ -D-PGA conjugates to determine if the conjugates elicit an antibody response that is protective in a mouse model of infection. All of the D-conjugates elicited high titers against B. licheniformis PGA at all doses tested. The response was dose dependent, from 0.1 to 1.0 to 10.0 ⁇ g of conjugate as in Tables 2-5 below. The titers were still increasing at the 10 ⁇ g dose. It is possible that increasing the dose beyond 10 ⁇ g will result in even higher titers.
  • Tables 2-5 demonstrate the effectiveness of the conjugates of Table 1 in each group of mice (10 mice/group).

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Abstract

L'invention porte sur des composés et des compositions contenant des fractions d'acide poly-γ-D,L-glutamique (PGA) et sur leur utilisation dans la prévention ou le traitement d'infections nosocomiales provoquées par Staphylococcus et Bacillus.
PCT/US2007/022567 2006-10-24 2007-10-24 Compositions d'acide polyglutamique et leur utilisation comme immunogènes Ceased WO2008130381A2 (fr)

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CN118240201A (zh) * 2024-05-22 2024-06-25 成都金开生物工程有限公司 利用γ-聚谷氨酸发酵液提取纯化γ-聚谷氨酸的方法

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WO2005007804A2 (fr) * 2003-04-10 2005-01-27 President And Fellows Of Harvard College Vaccin conjugue contre l'anthrax et anticorps diriges contre des bacilles et des toxines de l'anthrax
EP1633778B1 (fr) * 2003-06-05 2013-09-11 GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Conjugues d'acide poly-gamma-glutamique pour solliciter des reponses immunitaires dirigees contre des bacilles
US20080206276A1 (en) * 2005-07-08 2008-08-28 Michael Otto Targeting Poly-Gamma-Glutamic Acid to Treat Staphylococcus Epidermidis and Related Infections

Cited By (1)

* Cited by examiner, † Cited by third party
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CN118240201A (zh) * 2024-05-22 2024-06-25 成都金开生物工程有限公司 利用γ-聚谷氨酸发酵液提取纯化γ-聚谷氨酸的方法

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