EP1117432A1 - Vaccins aux hydrates de carbone contre les maladies virales - Google Patents

Vaccins aux hydrates de carbone contre les maladies virales

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Publication number
EP1117432A1
EP1117432A1 EP99950134A EP99950134A EP1117432A1 EP 1117432 A1 EP1117432 A1 EP 1117432A1 EP 99950134 A EP99950134 A EP 99950134A EP 99950134 A EP99950134 A EP 99950134A EP 1117432 A1 EP1117432 A1 EP 1117432A1
Authority
EP
European Patent Office
Prior art keywords
carbohydrate
ganglioside
viral disease
subject
mimic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99950134A
Other languages
German (de)
English (en)
Inventor
Paul J. Maddon
William C. Olson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Progenics Pharmaceuticals Inc
Original Assignee
Progenics Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Progenics Pharmaceuticals Inc filed Critical Progenics Pharmaceuticals Inc
Publication of EP1117432A1 publication Critical patent/EP1117432A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • GM2 , GD2 and other gangliosides are sialic acid containing glycosphingolipids composed of a complex carbohydrate moiety linked to a hydrophobic ceramide portion. Embedded within the outer leaflet of the cell membrane, the carbohydrate chain is exposed to the extracellular matrix.
  • the oligosaccharide portion of gangliosides such as GD2 may also be linked to peptide moieties and activate cytotoxic T lymphocytes when presented on the cell surface in association with major histocompatibility molecules (Zhao and Cheung, J. Exp. Med. 182:67, 1995).
  • the Lewis y antigen is a neutral oligosaccharide found on glycolipids and glycoproteins . A growing number of glycolipids are known to elicit cytotoxic T lymphocyte responses when presented on the cell surface in association with members of the CD1 family of antigen presenting molecules.
  • purified carbohydrates include whole or lysed tumor cells, purified carbohydrates, mimotopes and anti-idiotype antibodies.
  • the immunogenicity of purified carbohydrates can be improved via their conjugation to immunogenic carrier proteins.
  • GM2/Bacillus Calmette-Guerin (BCG) vaccines were shown to be effective (Livingston et al . , Proc. Natl . Acad. Sci . USA 84:2911, 1987; Livingston et al . , Cancer Res. 49:4045, 1989).
  • BCG Calmette-Guerin
  • QS- 21 is a carbohydrate extracted from the bark of the South American tree Quillaja saponaria Molina. The monosaccharide composition, molecular weight, adjuvant effect and toxicity for a series of these saponins have been described (Kensil, Crit. Rev. Ther. Drug Carrier Syst. 13:1, 1996). Studies have identified the 100 ug dose of QS-21 as the optimal well tolerated dose for induction of antibodies against GM2 and KLH in humans (Livingston et al . , Vaccine 12:1275, 1994).
  • the GM2-KLH conjugate vaccine In the presence of QS-21 adjuvant, the GM2-KLH conjugate vaccine consistently induced high-titer, long-lived IgM responses against GM2 in melanoma patients (Livingston et al . , Cancer Immunol. Immunother. 43:324, 1997). In the majority of treated patients, the vaccine also induced anti- GM2 IgG antibodies, which have the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) . The elicited antibodies were shown to specifically bind and kill cancer cells via complement mediated lysis and ADCC.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Antibodies are known to be an important component of the host defense against viruses (Klein, Immunology, Blackwell Scientific Publications, Boston, MA, 1990) . Antibodies can bind to viruses and eliminate their infectivity by direct neutralization, complement-mediated virolysis, or Fc receptor-mediated phagocytosis. In addition, antibodies can eliminate virally infected cells via either complement- mediated cytolysis or antibody-dependent cell -mediated cytotoxicity.
  • cytotoxic T lymphocytes can eliminate virally infected cells wherein antigen expression is altered.
  • cytotoxic T lymphocytes can recognize carbohydrate antigens that are either lipid-linked and presented in association with CD1 molecules or peptide-linked and presented in association with major histocompatibility molecules.
  • This invention provides a method for treating or preventing a viral disease in a subject comprising administering to the subject a cellular carbohydrate antigen that is overexpressed during the viral disease, or a molecular mimic thereof, the amount of such carbohydrate or such mimic being effective to treat or prevent the viral disease.
  • the viral disease is caused by the human immunodeficiency virus type-1.
  • the carbohydrate is the Lewis y antigen, a ganglioside, or the oligosaccharide portion of a ganglioside.
  • the gangliosides may include, but not to be limited to, GM1 , GMla, GM2 , GM3 , GDla, and GD2.
  • the ganglioside is GM2 , GD2 or a combination thereof.
  • the molecular mimic is an anti-idiotype antibody. In another embodiment, the molecular mimic is a peptide mimotope .
  • the cellular carbohydrate antigen or . a molecular mimic thereof is administered to the subject together with a suitable adjuvant.
  • the adjuvant is QS-21.
  • the cellular carbohydrate antigen or a molecular mimic thereof is adsorbed, covalently linked or otherwise conjugated to an immunogenic carrier protein.
  • the carrier protein is Keyhole
  • This invention also provides a vaccine for treating or preventing a viral disease in a subject comprising administering to the subject a cellular carbohydrate antigen that is overexpressed during the viral disease, or a molecular mimic thereof, the amount of such carbohydrate or such mimic being effective to treat or- prevent the viral disease, and a pharmaceutically acceptable carrier.
  • This invention provides a method for treating or preventing a viral disease in a subject comprising administering to the subject a cellular carbohydrate antigen that is overexpressed during the viral disease, or a molecular mimic thereof, the amount of such carbohydrate or such mimic being effective to treat or prevent the viral disease.
  • the molecular mimic is defined as a compound which when administered to an appropriate host, stimulates or enhances an immune respons 3 that recognizes ' the cellular carbohydrate.
  • Such mimics include, but are not limited to, mimotopes and anti-idiotype antibodies.
  • Mimotopes are peptides which mimic the immunogenicity of the cellular carbohydrate.
  • anti-idiotype antibodies can mimic carbohydrate antigens for the purposes of inducing anti- carbohydrate immune responses.
  • U.S. Patent No. 5,792,455 entitled, "Anti-idiotype antibody vaccine," describes an anti-idiotype antibody which mimics the ganglioside GD3 ; and U.S. Patent No.
  • the cellular carbohydrate antigen or a molecular mimic thereof is administered to the subject in the presence of a suitable adjuvant.
  • suitable adjuvants include the precipitated aluminum salts collectively known as alum, cytokines such as IL-2 and interferon-gamma, block copolymer-based adjuvants such as titermax, Ribi Detox and other monophosphoryl lipid A containing adjuvants, and saponins such as QS-21.
  • the adjuvant is QS-21.
  • the viral disease is caused by human immunodeficiency virus type-1
  • the cellular carbohydrate antigen is the Lewis y antigen, a ganglioside, or the oligosaccharide portion of a ganglioside.
  • the gangliosides which are applicable to this invention include, but are not limited to, GM1, GMla, GM2 , GM3 , GDla, and GD2.
  • the ganglioside is GM2 , GD2 or a combination thereof .
  • the cellular carbohydrate antigen or a molecular mimic thereof is adsorbed, covalently linked or otherwise conjugated to an immunogenic carri .r protein.
  • Ganglioside conjugate vaccines have been described. See e.g. Livingston and Helling, "Ganglioside-KLH Conjugate Vaccines with QS-21" Patent Cooperation Treaty (PCT) Application No: PCT/US94/00757 , International Publication Number: WO/94/16731, the content of which is incorporated into this application by reference.
  • PCT Patent Cooperation Treaty
  • the conjugated ganglioside is GM2 , GD2 or a combination thereof.
  • Different effective amounts of the conjugated ganglioside or oligosaccharide portion thereof may be used according to this invention.
  • a person of ordinary skill in the art can perform simple titration experiments to determine what amount is required for effective immunization.
  • An example of such titration experiment is to inject different amounts of the conjugated ganglioside or conjugated oligosaccharide portion thereof to the subject with or without a suitable adjuvant, and then examine the immune response.
  • the effective amount of conjugated ganglioside or conjugated oligosaccharide portion thereof is an amount between about 1 ⁇ g and about 500 ⁇ g .
  • the effective amount of conjugated ganglioside or conjugated oligosaccharide portion thereof is -loan amount between about 50 ⁇ g and about 90 ⁇ g. In an embodiment, the effective amount of conjugated ganglioside or conjugated oligosaccharide portion thereof is about 70 ⁇ g.
  • the effective amount of conjugated ganglioside or conjugated oligosaccharide portion thereof is between about 1 ⁇ g and about 10 ⁇ g. In a more specific embodiment, the effective amount of conjugated ganglioside or conjugated oligosaccharide portion thereof is between about 7 ⁇ g and about 10 ⁇ g. In an embodiment, the effective amount of conjugated ganglioside or conjugated oligosaccharide portion thereof is about 7 ⁇ g.
  • the effective amount of the adjuvant may also be similarly determined, i.e. by administering different amounts of the adjuvant with the cellular carbohydrate antigen or a molecular mimic thereof and examining the immune response so as to determine which amount is effective.
  • the effective amount of QS-21 may also to be similarly determined.
  • the effective amount of QS-21 is an amount between about lO ⁇ g and about 200 ⁇ g. In an embodiment, the effective amount of QS-21 is about 100 ⁇ g. In another embodiment, the effective amount of QS-21 is about 200 ⁇ g.
  • the cellular carbohydrate antigen or a molecular mimic thereof is conjugated to an immunogenic protein.
  • an immunogenic protein is a polypeptide that, when conjugated to a carbohydrate or a molecular mimic thereof stimulates or enhances an immune response to the carbohydrate in the subject.
  • the immunogenic protein is Keyhole Limpet Hemocyanin or a derivative thereof.
  • This invention also provides the above-described vaccine wherein the cellular carbohydrate antigen or a molecular mimic thereof is conjugated to Keyhole Limpet Hemocyanin or a derivative of Keyhole Limpet Hemocyanin.
  • Keyhole Limpet Hemocyanin is a well-known protein.
  • a derivative of Keyhole Limpet Hemocyanin may be generated by direct linkage of at least one immunological adjuvant such as monophospholipid A or non-ionic block copoly ⁇ ners or cytokines to Keyhole Limpet Hemocyanin.
  • Cytokines are well known to an ordinary skilled practitioner.
  • Example cytokines with adjuvant properties include interleukin 2 and interferon-gamma.
  • There are other known cytokines in the art which may to be linked to Keyhole Limpet Hemocyanin, forming a derivative of Keyhole Limpet Hemocyanin.
  • a ganglioside is conjugated to the immunogenic protein by the process of reductive amination following oxidation of a ceramide alkene structure to an aldehyde.
  • the conjugation of the ganglioside occurs through an aminolysyl group of the Keyhole Limpet Hemocyanin.
  • conjugation techniques may include chemical linker groups and should not adversely affect the immunogenicity of the carbohydrate .
  • a suitable adjuvant is an adjuvant which when administered together with the carbohydrate or a molecular mimic thereof or a conjugated carbohydrate or a conjugated molecular mimic thereof stimulates or enhances an immune response to the carbohydrate in the subject.
  • the adjuvant is QS-21.
  • This invention also provides a vaccine for treating or preventing a viral disease comprising a cellular carbohydrate antigen that is overexpressed during the viral infection, or a molecular mimic thereof, the amount of such carbohydrate or such mimic being effective to treat or prevent the viral disease, and a pharmaceutically acceptable carrier .
  • the subject is a human.
  • the cellular carbohydrate antigen is the Lewis y ar ;igen, a ganglioside or the oligosaccharide portion of a ganglioside.
  • This invention further provides a vaccine for stimulating or enhancing in a subject to which the vaccine is administered, an immune response which recognizes a cellular carbohydrate antigen that is overexpressed during a viral disease comprising an amount of such carbohydrate or a molecular mimic thereof effective to stimulate or enhance an anti- carbohydrate immune response in the subject, and a pharmaceutically acceptable vehicle, wherein the subject is afflicted with the viral disease and the immune response produced in the subject upon administration of the vaccine effectively treats the viral disease.
  • This invention also provides a vaccine for stimulating or enhancing in a subject to which the vaccine is administered, an immune response which recognizes a cellular carbohydrate antigen that is overexpressed during a viral disease comprising an amount of such carbohydrate or molecular mimic thereof effective to stimulate or enhance an anti- carbohydrate immune response in the subject, and a pharmaceutically acceptable vehicle, wherein the subject is susceptible to the viral disease and the immune response produced in the subject upon administration of the vaccine effectively prevents the viral disease.
  • This invention further provides a vaccine for a viral disease, wherein the virus or the virus-infected cells have gangliosides on their surface.
  • This invention further provides a method for treating viral diseases in a subject afflicted with a viral disease comprising administering to the subject an effective dose of a vaccine for stimulating or enhancing in a subject to which the vaccine is administered, production of an antibody which recognizes a ganglioside, comprising an amount of ganglioside or oligosaccharide portion thereof conjugated to an immunogenic protein eff ⁇ tive to stimulate or enhance antibody production in the subject, an effective amount of adjuvant and a pharmaceutically acceptable vehicle, wherein the subject is afflicted with a viral disease and the antibody produced in the subject upon administration of the vaccine effectively treats the viral disease.
  • This invention further provides a method for preventing a viral disease in a subject susceptible to a viral disease comprising administering to the subject an effective dose of a vaccine for stimulating or enhancing in a subject to which the vaccine is administered, production of an antibody which recognizes a ganglioside, comprising an amount of ganglioside or oligosaccharide portion thereof conjugated to an immunogenic protein effective to stimulate or enhance antibody production in the subject, an effective amount of adjuvant and a pharmaceutically acceptable vehicle, wherein the subject is susceptible to a viral disease and the antibody produced in the subject upon administration of the vaccine effectively prevents the viral disease.
  • This invention also provides a method of using the above- described vaccine, wherein the ganglioside or oligosaccharide portion thereof is conjugated to Keyhole Limpet Hemocyanin or a derivative of Keyhole Limpet Hemocyanin.
  • This invention further provides a method of using the above-described vaccine wherein the adjuvant is QS-21.
  • This invention further provides a method of using the above- described vaccine for treating or preventing viral disease, wherein the virus or virus-infected cells have gangliosides on their surface.
  • pharmaceutically acceptable vehicles means any of the standard pharmaceutical vehicles.
  • suitable vehicles are well known in the art and may include, but not limited to, any of the standard pharmaceutical vehicles such as a phosphate buffered saline solutions, pho? Dhate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion, and various types of wetting agents.
  • the vaccine of this invention may be administered intradermally, subcutaneously and intramuscularly. Other methods well known by a person of ordinary skill in the art may also be used.
  • this invention provides a method for stimulating or enhancing in a subject production of antibodies which recognize a ganglioside comprising administering to the subject an effective dose of a vaccine for stimulating or enhancing in a subject to which the vaccine is administered, production of an antibody which recognizes a ganglioside, comprising an amount of ganglioside or oligosaccharide portion thereof conjugated to an immunogenic protein effective to stimulate or enhance antibody production in the subject, an effective amount of adjuvant and a pharmaceutically acceptable vehicle, wherein the administering comprises administering the effective dose at two or more sites.
  • administering the effective dose at two or more sites means that the effective dose is divided into two or more portions and each portion is administered at a different site of the subject.
  • the administering comprises administering at three sites .
  • Approximately 10° HIV-infected or control cells are stained first with approximately 50 ⁇ l of anti-carbohydrate antibody (-5 ⁇ g/mL) for approximately 20 min at 4 °C in PBS/l%FBS/0.1% sodium azide (assay buffer) washed with assay buffer and then stained with a phycoerythrin-conjugated reporter antibody for 20 minutes at 4 °C.
  • the cells are washed with assay buffer, fixed overnight at 4°C with PBS/1% FBS/1% formaldehyde, and analyzed on a flow cytometer.
  • the GD2- and GM2 -positive melanoma cell line SK-MEL-31 can serve as a positive control in assays using anti-GM2 and anti-GD2 antibodies .
  • the antibodies may to be monoclonal antibodies, purified hyperimmune immunoglobulin, or human or animal sera known to contain high levels of carbohydrate-reactive antibodies produced naturally or elicited by active immunization.
  • the HIV-1 infected cells may be either cell lines competent for HIV-1 infection or phytohemagglutinin-stimulated human peripheral blood mononuclear cells (PBMC) .
  • the HIV-1 viruses may include laboratory adapted X4 viruses and primary R5 , X4 and R5X4 isolates.
  • Complement-mediated lysis of HIV-1 infected cells are performed by a 4h 51 Cr release assay.
  • 2xl( cells are labelled with lOO ⁇ Ci Na 2 51 Cr0 4 (New England Nuclear, Boston, MA) in 10% FCS RPMI for lh at 37°C in a C0 2 incubator.
  • the cells are washed twice, and 10 4 cells/well in 96-well round-bottom plates (Corning, New York, NY) are labelled and incubated with sera, antibodies or with medium alone for lh at 37°C in a C0 2 incubator.
  • the cells are washed and incubated with human complement (Sigma) at a dilution of 1:4 for 4h at 37°C.
  • the plates are spun at 500g for 5 min, and a 125 ⁇ l aliquot of supernatant of each well is harvested for determination of released 5 Cr. All assays are performed in triplicate and include control wells for maximum release in 1% NP-40 (Sigma) and for spontaneous release in the absence of complement.
  • the GD2- and GM2-positive melanoma cell line SK-MEL-31 can serve as positive control target cells for assays using anti-GM2 and anti-GD2 antibodies.
  • the antibodies may to be monoclonal antibodies, purified hyperimmune immunoglobulin, or human or animal sera known to contain high levels of carbohydrate-reactive antibodies produced naturally or elicited by active immunization.
  • the HIV-1 infected cells may be either cell lines competent for HIV-1 infection or PBMC .
  • the HIV-1 viruses may include ⁇ laboratory adapted X4 viruses and primary R5 , X4 and R5X4 isolates .
  • ADCC Anti -carbohydrate antibody-dependent cell-mediated cytotoxicity
  • PBMC peripheral blood mononuclear cells
  • RPMI media supplemented with 10% heat- inactivated FBS (assay media) at a density of 4xl0 6 cells/ml. These cells are then incubated overnight at 37°C prior to use as effectors in the ADCC assay.
  • target cells are labeled with Na 2 51 Cr0 4 (lOO ⁇ Ci per 2xl0 6 cells) for approximately 3 h in RPMI 1640 assay media prior to use in the assay.
  • 10 4 51 Cr-labeled target cells are preincubated with various dilutions of sera or concentrations of antibodies in 150 ⁇ l total assay volume for 30 min at 37°C.
  • the antibodies may to be monoclonal antibodies, or purified hyperimmune immunoglobulin, or human or animal sera known to contain high levels of carbohydrate-reactive antibodies produced naturally or elicited by active immunization.
  • the HIV-1 infected cells may be either cell lines competent for HIV-1 infection or PBMC.
  • the HIV-1 viruses may include laboratory adapted X4 viruses and primary R5 , X4 and R5X4 isolates.
  • the GD2- and GM2 -positive melanoma cell line SK- MEL-31 can serve as positive control target cells for assays using anti-GM2 and anti-GD2 antibodies.
  • Virus neutralization is assessed using phytohemagglutinin- stimulated PBMC as indicator cells, with determination of p24 antigen production as the endpoint .
  • PBMC are stimulated with PHA for 48 h before removal of the mitogen by washing.
  • Antibodies are combined in 2-fold serial dilutions with virus and/or cells for 1 h at 37 °C.
  • the virus is then added to the PBMC at a density of 4 x 10 6 /ml and the cultures incubated for 7 days.
  • the culture supernatants are harvested, treated with 1% Empigen detergent before determination of the p24 concentration by ELISA.
  • the Leu 3a HIV-1 inhibitory monoclonal antibody (Becton Dickinson) may to be used as a positive control.
  • the antibodies may to be monoclonal antibodies, purified hyperimmune immunoglobulin, or human or animal sera known to contain high levels of carbohydrate-reactive antibodies produced naturally or elicited by active immunization.
  • HIV-1 viruses may include laboratory adapted X4 viruses and primary R5 , X4 and R5X4 isolates.
  • TLC Rf 0.5 (5:4:1 CHCl 3 -CH 3 OH-0.2% aquecous CaCl.
  • KLH Keyhole limpet hemocyanin
  • GM2 Aldehyde (Compound #2, may be the gem diol) ⁇
  • Conjugate is diafiltered vs.: -PBS pH 7.5
  • EIA Enzyme immunoassay
  • Step 1 Purification to GM2 (Compound #1) : Name: GM2 ganglioside
  • GM2 ganglioside (bovine source) starting material is supplied by FIDIA. All glass ware is washed with distilled acetone followed by distillec' ethanol and then dried (130°C) for 18 hours prior to use. A column (Michel-Miller S 795- 10) of silica gel (30.5g, Kieselgel 60H, Art 7736, E. Merck) is packed at 75 psi (SSI Model 300 Lo pump) using 65:35 chloroform: methanol as solvent. GM2 (200 mg) is applied as a concentrated 65:35 chloroform-methanol solution and elution is performed with this solvent, followed by 65:35:4 chloroform-methanol-water.
  • silica gel 30.5g, Kieselgel 60H, Art 7736, E. Merck
  • the fractions are analyzed by TLC (Rf 0.6, 5:4:1 chloroform-methanol-0.2% aqueous CaCl 2 ) .
  • the GM2 containing fractions are pooled and evaporated to give a creamy white amorphous solid.
  • In-process testing for this material includes 1 H NMR and thin layer chromatography (TLC) to confirm the identity and purity of this ganglioside.
  • TLC thin layer chromatography
  • compound #2 Due to the unstable nature of the resulting aldehyde (B- elimination) , compound #2 is identified on a routine basis only by TLC.
  • the TLC of a typical run generally indicates the presence of a small amount of sphinganine or phytosphingosine analog (same Rf as compound #1) and a small amount of reducing sugar (Rf 0.32).
  • Step 3 Conjugation of GM2 Aldehyde to KLH:
  • the KLH protein (160 mg) is aseptically measured and added to the flask containing the lyophilized GM2 Aldehyde and a magnetic stir bar. The solution is gently agitated at room temperature for 3 minutes until all of the GM2 Aldehyde has gone into solution.
  • the sodium cyanoborohydride (NaBH 3 CN) (40 mg) is added to the GM2 Aldehyde/KLH solution then the flask is sealed with a stopper equipped with a sterile filter needle. The solution is gently shaken then incubated overnight at room temperature. The solution is then further incubated at 40 °C for 4 days.
  • Step 4 Diafiltration of the Glycoconjugates (GM2-KLH):
  • the contents of the GM2/KLH reaction vial are aseptically transferred to a sterile, pyrogen-free Amicon ultrafiltration unit with a YM-30 filter. Filtered nitrogen is used to provide an operating pressure of 16 psi for the Amicon unit.
  • the conjugate is then diafiltered against the following sterile, pyrogen-free or low pyrogen content buffers successively: 1. 2 complete changes of PBS pH 7.5 (sterile, pyrogen- free) 2. 2 complete changes of TRIS-HCI, EDTA pH 7.75 (sterile, low pyrogen content)
  • glycoconjugate is then aseptically removed from the filtration unit and spun at 2000 rpm for 30 minutes. The supernatant is then sterile filtered with a 0.22 mm low protein binding filter.
  • the final volume of the glycoconjugate is adjusted with sterile, pyrogen-free pH 7.5 PBS buffer to yield a protein concentration of l g/mL .
  • the final glycoconjugate is then dispensed in 1.0 mL aliquots with an overfill volume of 0.1 mL into 2 mL sterile, pyrogen-free, clear, borosilicate serum vials with rubber stoppers and stored at 2-8°C.
  • the air inside the filing area is monitored by exposing two blood agar plates to the air near the work area inside of the hood for a minimum of thirty minutes. These plates are then transferred to a 37°C incubator and incubated for 1-2 days. The plates are then examined for any bacterial or fungal colonies .
  • the product is labeled by the manufacturing personnel and the labeling is verified by the Quality Control department. The product is then stored at 2-8°C.

Abstract

Un procédé permettant de traiter ou de prévenir une maladie virale chez une personne consiste à administrer à cette personne une quantité efficace d'un antigène glucidique cellulaire qui est surexprimé pendant la maladie virale, ou un analogue moléculaire de ce dernier, suivant une quantité d'antigène glucidique cellulaire ou d'analogue de ce dernier efficace pour traiter ou prévenir la maladie virale. Cette invention concerne également un vaccin permettant de traiter ou de prévenir une maladie virale, ce vaccin contenant un antigène glucidique cellulaire qui est surexprimé pendant la maladie virale, ou un analogue de ce dernier, suivant une quantité d'antigène glucidique cellulaire ou d'analogue de ce dernier efficace pour traiter ou prévenir la maladie virale, et un support pharmaceutiquement acceptable.
EP99950134A 1998-10-01 1999-10-01 Vaccins aux hydrates de carbone contre les maladies virales Withdrawn EP1117432A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US10265798P 1998-10-01 1998-10-01
US102657P 1998-10-01
PCT/US1999/023013 WO2000018432A1 (fr) 1998-10-01 1999-10-01 Vaccins aux hydrates de carbone contre les maladies virales

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EP1117432A1 true EP1117432A1 (fr) 2001-07-25

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EP (1) EP1117432A1 (fr)
JP (1) JP2002525339A (fr)
AU (1) AU6285899A (fr)
CA (1) CA2346061A1 (fr)
MX (1) MXPA01003195A (fr)
WO (1) WO2000018432A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
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EP1655623A1 (fr) 2004-11-04 2006-05-10 Leuze electronic GmbH + Co. KG Capteur optique

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US5102663A (en) * 1988-10-18 1992-04-07 Sloan-Kettering Instutute For Cancer Research Vaccine for stimulating or enhancing production of antibodies against 9-O-acetyl GD3

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AU6285899A (en) 2000-04-17
WO2000018432A1 (fr) 2000-04-06
MXPA01003195A (es) 2003-07-14
JP2002525339A (ja) 2002-08-13

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