CA2346061A1 - Carbohydrate vaccines for viral diseases - Google Patents

Abstract

This invention provides a method for treating or preventing a viral disease in a subject comprising administering to the subject an effective amount of 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. This invention also provides a vaccine for treating or preventing a viral disease comprising 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.

Description

CARBOHYDRATE VACCINES FGR VIRAL DISEASES
This application claims the benefit of U..S. Provisional Application No. 60/102,657, filed October 1, 1998, the contents of which are hereby incorporated by reference.
5 Throughout this application; various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art _ ,to which this invention pertains.
to Backaround of the Invention Dramatic changes in carbohydrate metabolism and cell surface expression have been observed in a:~sociation with infectious diseases, such as viral infection's. For example, in vivo and 15 in vitro infection of human lymphocytes with retroviruses such as the human immunodeficiency virus (HIV) or human T
lymphotropic virus type-1 (HTLV-1) leads to increased cell ~~
surface expression of the Lewis y antigen (Adachi et al., J.
Exp. Med, 167:323, 1988) and of t:he gangliosides GM2, GD2, 20 GM1, GMIa, GDla, and GM3 (Sorice, et al., JAIDS, 12:112, 1996; Misasi et al., Clin. Immunol. Immunopathol., 67:216, 1993; Furukawa et al., PNAS, 90:1972, 1993; Matsuda et al., Biochem. Biophys. Acta, 1168:12?'., 1993; Auci et: al., J.
Leukoc. Biol. 52:282, 1992). Increased expression of GM2, 25 GD2 and GM3 was observed in cell lines transfected with human adenovirus genes or infected with a hybrid adenovirus (Sanai et al., J. Biochem. [Tokyo] 107:740, 1990, Jambrosic et al., Int. J. Cancer, 44:1117, :1989).
30 GM2, GD2 and other gangliosides acre 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 35 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, 1.995). The Lewes 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.
As with virally infected cells, a variety of human tumors are characterized by altered expression of carbohydrate structures, including GM2, GiD2, and the Lewis y antigen(reviewed in Ragupathi, Cancer Immunol. Immunother.
43:152, 1996). Melanoma patients with naturally occurring antibodies to GM2 have prolonged! disease-free and overall survival periods. Cell surface carbohydrate antigens have thus been identified as target:a for active and passive immunotherapy of cancers, and different approaches have been adopted to induce immune responses against these structures.
These 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.
While each vaccine approach has shown promise in initial experimentation, adsorption or covalent attachment of purified carbohydrate antigens to immunogenic T-dependent protein carriers is the concept that has been pursued most vigorously, resulting in vaccines that have ~ in same instances been shown to be effective in clinical trials.
3a In studies aimed at inducing a humoral immune response against gangliosides in melanoma patients by active immunization, 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). In a randomized study with 122 melanoma patients, who were disease-free after surgery, that it was shown that, out of 64 patients treated with BCG alone and 58 patients with GM2/BCG, the majority of patients (860) receiving the GM2 vaccine produced. antibodies. Patients who produced anti-GM2 antibodies had a significantly longer disease free interval and overall survival than antibody 5 negative patients. (Livingston et al., J. Clin. Oncol.
12:1036, 1994).
Improved humoral immune responses were elicited in humans by a vaccine containing GM2 covalently linked to the carrier 10 protein Keyhole Limpet ~Hem~cyanin (KLH) and administered with the adjuvant QS-21. 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 ~~ series of these saponins 15 have been described (Kensil, Crit. Rev. Ther. Drug Carrier Syst. 13:1, 1996). Studies have s_dentified the 100 ug dose of QS-2l as the optimal well tolerated dose for induction of antibodies against GM2 and KLH in humans (Livingston et al., Vaccine 22:1275, 1994).

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). Tn the 25 majority of treated patients, the 'vaccine also induced anti-GM2 IgG antibodies,. which have the ability to mediate antibody-dependent oe~Il-mediated cytotoxicity (ADCC). The elicited antibodies were shown to specifically bind and kill cancer cells via complement mediated lysis and ADCC.

In addition to gangliosides, other carbohydrate antigens have been made immunogenic by conjugation to appropriate carrier proteins. Coupling of capsular polysaccharides from the bacterium Haemophilus influenzae type b to diphtheria 35 toxiod resulted in a significant increase in immune response and protection (Eskola et al., New England J. Med. 323:1381, 1990). This product has been licensed by the U.S. Food and Drug Administration for use in children and in adults with _4 splenic dysfunction. Similarly, robust humoral immune responses were elicited in children by pneumococcal polysaccharides conjugated to either tetanus toxoid or the outer membrane protein complex of Neisseria meningitides (Kayhty et al., J. Infect. Dis. L?2:1273, 1995; Anttila et al., J. Infect. Dis., 177:1614, 1998). In addition, vaccination of ovarian cancer patients with synthetic Thompson Friedenreich tumor antic3en conjugated to. keyhole limpet.hemocyanin elicited humoral IgM and IgG responses (MacLean et al., J. Immunother<~py 11:292, 1992}. The important finding common in theses studies a as the isotype switch from a IgM response of short duration to a long lasting, high affinity IgG ~ response indicating that activation of T-cell dependent pathways against carbohydrates is likely to occur.
This approach is now applied to the use of ganglioside and other carbohydrate-based vaccines for the treatment or prevention of viral infections wherein carbohydrate metabolism and expression are altered. The goal is to similarly induce immune responses to carbohydrate antigens associated with diseased cells. Through well known mechanisms, the immune system can destroy the infected cells or viruses that display the carbohydrate antigens and thereby prevent or favorably alter the course of the viral infection.
Antibodies are known to be an important component of the host defense against viruses (Klein; Immunology, Blackwell Scientific Publications, Boston, 1HA, 1990). Antibodies can bind to viruses and eliminate their infectivity by direct neutralization, complement-mediated virolysis, or Fc receptor-mediated phagocytosis. IrA addition, antibodies can eliminate virally infected cells via either complement-mediated cytolysis or antibody-dependent cell-mediated cytotoxicity. Incorporation of host cell antigens into virus particles is a well-established phenomenon, and it has been further demonstrated that protection from viral infections can be afforded by the immunological response to host cell antigens as well as virally encoded antigens iSchultz and Stott, AIDS, 8:[suppl 1]:5203, 1994).
5 Similarly, cell-mediated immunity is known to play a major role in controlling viral infections. Importantly, cytotoxic T lymphocytes can eliminate virally infected cells wherein antigen expression is altered. As described above, cytotoxic T lymphocytes can recognize carbohydrate antigens to that are either lipid-linked and. presented in association with CD1 molecules or peptide-linked and 'presented in association with major histocompatibility molecules.

Summnarv of the Invention 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 5 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. In an embodiment, the viral disease is caused by the human immunodeficiency virus type-1.
to In an embodiment, the carbohydrate is the Le~niis 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. In a Z5 preferred embodiment, the gangl:ioside is GM2, ~GD2 or a combination thereof.
In an embodiment, the molecular mimic is an anti-idiotype antibody. In another embodiment, the molecular mimic is a 20 peptide mimotope..
In an embodiment, the cellular carbohydrate antigen or.a molecular mimic thereof is administered to the subject together with a suitable adjuvant. In a preferred 25 embodiment, the adjuvant is QS-21.
In a further embodiment, the cellular carbohydrate antigen or a molecular mimic thereof is adsorbed, covalently linked or otherwise conjugated to an imrriunogenic carrier protein.
30 In a preferred embodiment, the carrier protein is Keyhole Limpet Hemocyanin.
This invention also provides a vaccine for treating or preventing a viral disease in a subject comprising 35 administering to the subject a cellular carbohydrate antigen that is overe~cpressed 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.

WO 00/1$432 PCTIUS99/23013 _g.
Detailed Desariytion of the Invention This invention provides a method for treating or preventing a viral disease in a subject compr:i.sing administering to the subject a cellular carbohydrate antigen that is 5 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.
As used herein, the molecular mimics is defined as a compound which when administered to an appropriate host, stimulates or enhances an immune respon~~ that recognizes the cellular carbohydrate. Such mimics include, but are not limited to, mimotopes and anti-idiotype antibodies. Mimotopes are peptides which mimic the zmmunogenicity of the cellular 15 carbohydrate. It is known that anti-idiotype antibodies can mimic carbohydrate antigens for the purposes of inducing anti- carbohydrate immune responses. For example, U.S.
Patent No. 5,792,455, entitled, "Anti-idiotype antibody vaccine," describes an anti-idiotype antibody which mimics 20 the ganglioside GD3; and U.S. Patent No. 5,653,977, entitled, "Anti-idiotypic antibady that mimics the GD2 antigen," describes an anti-idiotypic monoclonal antibody which elicits an immune response reactive against the ganglioside GD2 antigen. The content of these patents is 25 incorporated into this application by reference.
In an embodiment, the cellular carbohydrate antigen or a molecular mimic thereof is administered to the subject in the presence of a suitable adjuvant. Suitable adjuvants 30 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. In a preferred embodiment, the 35 adjuvant is QS-21.
Im an embodiment, the viral disease is caused by human immunodeficiency virus type-1 WO 00/18432 PCTIUS99/23fl3 _g_ In an embodiment, the cellular carbohydrate antigen is the Lewis y antigen, a ganglioside, or the oligosaccharide portion of a ganglioside. Thn gangliosides which are applicable to this invention include, but are not limited 5 to, GM1, GMla, GM2, GM3, GDla, and GD2. In a preferred embodiment, the ganglioside is GM2, GD2 or a combination thereof .
In an embodiment, 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 I5 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.
20 In a preferred embodiment of this invention, 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 25 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 ar conjugated oligosaccharide 30 portion thereof to the subject with or without a suitable adjuvant, and then examine the immune response.
In an embodiment, the effective amount of conjugated ganglioside or conjugated oligosaccharide portion thereof is 35 an amount between about 1 ~g and about 500 ,ug.
In another embodiment, the effective amount of conjugated ganglioside or conjugated oligrisaccharide portion thereof is an amount between about 50 ~.g and about 90 fig. In an embodiment, the effective amount of conjugated ganglioside or conjugated oligosaccharide portion thereof is about 70 ~g-In another embodiment, the effeci~ive amount of conjugated ganglioside or conjugated oligosaccharide portion thereof is between about 1 ~g and about 10 ~.g. In amore specific embodiment, the effective amount of conjugated ganglioside to or conjugated oligosaccharide portion thereof is between about 7 ~.g and about 10 fig. In an embodiment, the effective amount of conjugated ganglioside or conjugated oligosaccharide portion thereof is about 7 fig.
In addition, the effective amount of the adjuvant may also be similarly determined, i.e. by administering different amounts of the adjuvant with t:he cellular carbohydrate antigen or a molecular mimic thereof and examining the immune response so as to determine which amount is effective. When using QS-21 as adjuvant, the effective amount of QS-21 may also to be similarly determined.
In a preferred embodiment, the effective amount of.QS-21 is an amount between about lO~Cg anal about 200 ~,g. In an 25.embodiment, the effective amount of QS-21 is about 100 ~.g.
In another embodiment, the effecaive amount of QS-21 is about 2 0 0 ~.g .
In a preferred embodiment, the cellular carbohydrate antigen or a molecular mimic thereof is conjugated to an immunogenic protein. As used herein, 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. In a further embodiment, 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 Hemc>cyanin may be generated by direct linkage of at least one immunological adjuvant such as monophospholipid A or non-ionic block copolymers or cytokines to Keyhole Limpet Hemocyanin. Cytokines are well known to an ordinary skilled practitioner. Example cytokines 10 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.
15 In an embodiment, a ganglioside is conjugated to the immunogenic protein by the proceas of reductive amination following oxidation of a ceramide alkene structure to an aldehyde. In another embodiment, the conjugation of the ganglioside occurs through an aminolysyl group of the 20 Keyhole Limpet Hemocyanin.
In addition to reductive amination, various other conjugation techniques may to be: used for this invention.
The conjugation techniques may include chemical linker 25 groups and should not adversely affect the immunogenicity of the carbohydrate.
As used herein, a suitable adjuvant is an adjuvant which when administered together with the carbohydrate or a 30 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. In an embodiment, the adjuvant is QS-:Z1. There are other known adjuvants which may to be applicable to this invention.
35 There may be classes of QS-21 or QS-21 like chemicals which may be similarly used in accordance with this invention.
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 5 prevent the viral disease, and a pharmaceutically acceptable carrier.
In an embodiment of this invention, the subject is a human.
10 In an embodiment, 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 15 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-20 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 they vaccine is administered, an immune response which recognizes a cellular. carbohydrate antigen that is overexpressed during a viral disease 30 comprising an amount of such carbohydrate or molecular mimic thereof effective to stimulate: or enhance an anti-carbohydrate immune response a.rl the subjects and a pharmaceutically acceptable vehicle, wherein the subject is susceptible to the viral disease and the immune response 35 produced in the subject upon administration of the vaccine effectively. prevents the viral disease.
This invention further provides a vaccine for a viral WO 00!18432 PCT/US99I23013 _13_ _ 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 effective 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 derivat-ive of Keyhole Limpet Hemocyanin.. This invention further provides a method of using the above-described vaccine wherein the adjuvant is QS-21.

WO 00/18432 PCTlUS99/23013 _14_ 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.
For the purposes of this invention "pharmaceutically acceptable vehicles" means any of the standard pharmaceutical vehicles. Examples of 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:~hate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion, and various t.~rpes 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.
In a preferred embodiment 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 adminie~tering 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. In a specific embodiment,. the administering comprises administering at three sites.

This invention will to be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate i~hat the specific methods and results discussed are merely illustrative of the invention as described more fully i.n the claims which follow thereafter.
Experimental Details Flow cYtometric analysis of an~ibodv bindincr to HIV-1 infected cells Approximately 106 HIV-infected or control cells are stained first with approximately 50,1 of anti-carbohydrate antibody (~5 ~.g/mL) for approximately 20 mini at 4 °C in PBS/1%FBS/O.I%
sodium azide (assay buffer) washed with assay buffer and then stained with a phycoerythri.n-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 Complement-mediated cytotoxicity assays axe performed by a 4h S~Cr release assay. 2xlC~ cells are labelled with 100~.Ci Na25~Cr04 (New England Nuclear; Boston, MA) in 10% FCS RPMI
for lh at 37°C in a C02 incubator. The cells are viashed twice, and 104 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 C02 incubator. The cells are washed. and incubated with human complement (Sigma) at a dilution of 1:4 for 4h at 37°C. The 5 plates are spun at 5008 for 5 min, and a 125.1 aliquot of supernatant of each well i.s harvested for determination of released S~Cr. All assays axe performed in triplicate and include control wells for maximum release in 1% NP-40 (Sigma) and for spontaneous release in the absence of 10 complement. The GD2- and GM2-po;aitive melanoma cell line SK-MEL-31 can serve as positive control target cells for assays using anti-GM2 and anti-GD2 antibodies.
The percentage of specific lysis is calculated as follows:

Experimental release - spontaneous release o - x 100 cytatoxicity Maximum release - spontaneous release The antibodies may to be monoclonal antibodies, purified hyperimmune immunoglobulin, or human or animal sera known to contazn high levels of carbohydrate-reactive antibodies ,produced naturally or elicited by active immunization. The 25 HIV-1 infected cells may be either cell lines competent for HIV-1 infection or PBMC. The HIV-I viruses may include .labaratory adapted X4 viruses and primary R5, X4 arid R5X4 isolates.
30 Anti-carbohydrate antibody-dependent cell-mediated cytotoxicitv (ADCC) assay PBMC are suspended in RPMI media supplemented with loo heat-inactivated FBS (assay media) at a density of 4x106 cells/ml.
These cells are then incubated overnight at 37°C prior to use 35 as effectors in the ADCC assay. The following day, target cells are labeled with Na25~Cr04 (100~.Ci per 2x106 cells) for approximately 3 h in RPMI 1640 assay media prior to use in the assay. ' In each well, 104 S~Cr_-labeled target cells are preincubated with various dilutions of sera or 40 concentrations of antibodies in 150.1 total assay volume for WO 00/18432 PCT/(IS99/23013 30 min at 37°C. Then 106 PBMC are added in 50,1 assay media, and the plates are centrifuged at 3008 for 5 minutes prior to incubation at 37°C for 4h. Plates are then centrifuged at 300g, and 1001 aliquots of cell supernatants are collected.
S~Cr release is detected using a Wallac 1470 gamma counter.
A11 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 percenta~ ;e of specific lysis :is calculated as follows Experimental release - :spontaneous release - x 100 cytotoxicity Maximum release - spontaneous release The antibodies rnay 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 HTV-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.
HIV-1 neutralization assays Virus neutralization is assessed using phytohemagglutinin-stimuiated 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-food serial dilutions with virus and/or cells for 1 h at 3'7 °C. The virus is then added to the PBMC at a density of 4 x 106 /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 immunoglobuli.n, or human or animal sera known to contain high levels of carbohydrate-reactive antibodies produced naturally or elicited by active immunization. The HIV-1 viruses may include laboratory adapted X4 viruses and primary R5, X4 and R5X4 isolates.

Preparation of Clinical Grape Ganqlioside Con~uqate Vaccine CHEMISTRY AND MANUFACTURING
DRUG SUBSTANCE
NAME AND SOURCE
Proper name:

Chemical name:
113NeuAc-GgOse3Cer-keyhole limpet hemocyanin (KLH) 20 Manufacturer: Progenies Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591, United States of America.

MATERIAL SUPPLIER GRADE

Acetone BDH ACS

Ammonia Solution BDH ACS

Chloroform BDH ACS

Ethanol Commercial Alcohol Ltd.

Ethyl Ether BDH ACS

Methanol BDH ACS

2-Propanol Fisher UJ.V1219 ACS

Water Travanol sterile water for irrigation.

Calcium Chloride Fisher Certified (anhydrous - 20 mesh granular) Dimethyl Sulfide Aldrich 99% +
GM2 Fidia ---Oxygen Linde UN1072 ' USP
Silica Gel E Merck Kieselgel 50H Art 7736 Sodium Aldrich 95% Pure Cyanoborohydride TLC Plates E Merck K i a s a 1 g a 1 MATERIALS USED IN THE CONJUGATION PROCEDURE
MATERIAL SUPPLIER GRADE

Keyhole limpet hemocyanin Perimmune, Inc. USP

(KLH) Rockville, MD

Deoxycholic acid, sodium salt Aldrich Analytical (DOC) (monohydrate) 98%

Ethylenediamine tetraacetic Aldrich ACS

acid di-sodium hydrogen orthophos- BDH Analytical . 20 phate (anhydrous) (NaZHP04) Sodium chloride BDH Analytical (NaCl) Potassium dihydrogen BDH Analytical orthophosphate (KHZP04) 25Sodium hydroxide BDH Analytical (NaOH) Tris (hydroxymethyl) Sigman aminomethane hydrochloride Sodium cyanoborohydride Aldrich 3 0 ( NaBH3CN ) Sepharose CL-4B Pharmacia Nitrogen gas (filtered) Medigas GM2 aldehyde Progenics Data for the GM2 and GM2 Aldehyde:
The structures of GM2 and GM2 aldehyde were characterized by ;H NMR spectroscopy, thin layer chromatography (TLC), FAB-MS

and FT-IR.

STRUCTURAL MOLECULAR MOLECULAR.

FORMULA FORMULA WEIGHT

GM2 - ganglioside {compound #1) GaINAcBI-4GaLB1-4 GlcB1-lCer C6~'~721026N3 M-1--1382 Solid Neu5Aca2 I3NeuAc-GgOse3Cer C69~~125026N3 M-1=1410 (acid) IO TLC: Rf=0.21 (65:35:8 CHCL3-CH30H-~H20) Rf=0.60 (5:4:1 CHC13-CH30H-0.2% aqueous CaCl2) Rf=0.2 {7:1:1 {CH~)ZCHOH-NH~OH-H20) STRUCTURAL MOLECULAR MOL. PHYSICO-CHEMICAL

FORMULA FORMULA WT. CHARACTERISTICS

GM2-aldehyde C53H9302~3 1204.29 Cream White, (compound #2) Odorless, Amphorous Solid STRUCTURAL DATA
~H (DMSO-d6:D2) b : 9 . 48 (d,1H, J=2, OHz) , 4 . 79 (d, 1H, J=8 . 5Hz, III-1), 4.26(d,lH,J=8.OHz,II-1), 4.19 (d,IH,J=8,OHZ,I-1),2.54(dd,lH, A-3e)', 1.88{s,3H,Ac), 1.78(s,3H,Ac),0.85(t,3H,J=6.6Hz,CH3).
25 FT-IR (KBr Cast, CM-~}: 3439, 3420, 2952, 2923, 2851, 1634, 1070 (possibly the gem diol).
TLC Rf=0.5 {5:4:1 CHC13-CH30H-0.2% aquecous CaCl2) Data for KLH and GM2-KLH:
30 Keyhole limpet hemocyanin (KLH) is a large, complex protein composed of a number of smaller molecular weight subunits.
KLH is extracted and purified from the keyhole limpet mollusk (Megathura crenulata).
3 S COMPOUND SEPHAROSE Ch-4B ISOET.ECTRIC RESORCIPIOL-GEL
CHROMATOGRAPHY FOCUSIHIG HCl Molecular Weight (Isoelec. pts.) moles of GM2/
. (daltons) moles of protein 4 0 KZH Whole mol.(2):>2x106 Mult. bands Subunits: 2-7 x 105 between pH 4.65 iii WO 00/18432 PCTlUS99/23013 -21- _ and pH 6 GM2-ItI~H Whole mol. t2)>2x106 Multiple bands Subunits: 2-7 x105 between pH 4.65 200-and pH 6 1900 STEP 1 - PURIFICATION OF GM2:
10 GM2 (FIDIA) y Silica Gel Column Chromatography 1. 65:35 Chloroform - methanol 2. 65:35:4 chloroform - methanol - water y In-process QC:
1. TLC
y STEP 2 - SYNTHESIS OF GM2 ALDEH'YDE (COMPOUND #2):
2 0 GM2 ( Compound #1 ) y ( 1 ) 03, MeoH
y ( 2 ) CH3SCH:~
y.
GM2 Aldehyde (Compound #2, may be the gem diol) y In-process tests done in-house:
1. TLC
w y STEP 3 - CONJUGATION OF THE GM2 ADEHYDE TO KLH:
Sterile pyrogen free KLH
y KLH added to GM2 aldehyde in 4:1 ratio (w/w).
y Incubated at room temperature with shaking for 3 minutes y NaBH3CN is added to GM2 Adehyde/KLH mixture in 1:1 ratio (w/w) 40 y Reaction mixture is gently stirred at room temperature WO 00118432 PCTlUS99123013 overnight then at 40°C for 4 days y STEP 4 - DIAFILTRATION OF THE CON~TUGATE:
Conj ugate is diaf i7.tered vs .
-PBS pH 7.5 -TRIS/EDTA pH 7.75 -TRIS/EDTA/0.05~ DOC pH 7.75 -TRIS/EDTA pH 7.5 -PBS pH 7.5 Conjugate aseptically removed from the Amicon filtration unit y Centrifuged y Conjugate sterile filtered In-process QC tests:
1. BioRad Protein Assay 2. Sepharose gel filtration 3. Isoelectric focusing (IEF) y Concentration of conjugate aseptically adjusted to 1 mg/mL
Conjugate dispensed into 1 mL sterile, pyrogen free vials and stored at 2-8°C
y Final QC testing 1. Enzyme immunoassay (EIA) 5. Rabbit pyrogen test 2. LAL pyrogen test 6. - General safety test 3. BioRad protein assay 7. Sterility test 4. Resorcinol-HCl assay 8. Impurity test for cyanide METHOD OF MANUFACTURE OF GM2-KLH (~ONJUGATE
The manufacturing of the GM2-KLH conjugate is carried out in 4 steps:
1. Purification of incoming GM2 (bovine source) (compound ## 1 ) .

WO 00/1$432 PCT/US99/23Q13 2. Synthesis of GM2 aldehyde (compound #2).
3. Conjugation of the GM2 aldehyde to KLH.
4. Diafiltration of the conjugate.
Step 1: Purification to GM2 (Compound #1}:
Name: GM2 ganglioside Abbreviated Name: II3NeuAc-GgOse3Cer GM2 ganglioside (bovine source) starting material is supplied by FIDIA. All glass wares is washed with distilled acetone followed by distiller ethanol arid then'dried (130°C) for 18 hours prior to use. A column (Michel-Miller S 795-10) of silica gel (30.58, 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 chlorofoz-m-methanol solution and elution is performed with this solvent, followed by 65:35:4 chloroform-methanol-water. The fractions are analyzed by TLC (Rf 0.6, 5x4:1 chloroform-methanol-0.2~ aqueous CaCl2).
The GM2 containing fractions are pooled and evaporated to give a creamy white amorphous solid.
In-process testing for this material (compound #1).includes ~H NMR and thin layer chromatography (TLC) to confirm the identity and purity of this ganc3lioside. The in-process test results must meet the specifications listed under developmental chemistry. If this material is found to be impure, the above purification is repeated.
Step 2: Synthesis of GM2 Aldehyde (Compound #2):
All glassware is rinsed with distilled methanol and dried (130°C) for 18 hours prior to use. A solution of the purified GM2 ganglioside (compound #1) (40 mg) in distilled methanol (10 mL) is stirred at ~7.5°C (dry ice-ethanol) and ozone gas .(Orec 03V10-0 ozonator) is passed through the solution for 7 minutes. A stream of argon is then passed through the solution while the reaction is checked by TLC

WO 00/i8432 PCTIUS99I23013 (5:4:1 chloroform-methanol- 0.2% aqueous CaCl2). The solvents are then removed under reduced pressure and the resulting material is dissolved i.n distilled methanol. To this solution is added methylsulfide (200 m~) and the 5 reaction mixture is stirred at room temperature for one hour. The solvents are then removed and the residue is washed with ethyl ether(4 x 25 nnL). The resulting white solid (compound #2) is dried in vacuo for 15 minutes to remove~any remaining solvent and is then used directly in the subsequent canjugation step.
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 15 the presence of a small amount of sphinganine or phytosphingosine analog (same Rf a;s compound #1) and a small amount of reducing sugar (Rf 0.32).
Step 3: Conjugation of GM2 Alde:hyde to KLH:
All manipulations are done in a Class 100 biological safety cabinet.
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 ag~.tated at room temperature for 3 minutes until all of the GM2 Aldehyde has gone into solution.
30 The sodium cyanoborohyd~ide (NaBH~CN) (40 mg) is added to the GM2 Aldehyde/KLH solution then tile 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
3 5 f or 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 5 Amicon unit. The conjugate is thE~n diafiltered against the following sterile, pyrogen-free ar. low pyrogen content buffers successively:
1. 2 complete changes of PBS pH 7.5 (sterile, pyrogen-free) 10 2. 2 complete changes of TRIS-HCI, EDTA pH 7.75 (sterile, low pyrogen content) 3. 2 complete changes of TRIS-HCI pH 7.75 with 0.50 Deoxycholic acid (DOC) (sterile, low pyrogen content) 4. 4 complete changes of TRIS-:EiCI pH 7.75 (sterile, low 15 pyrogen content) 5. 3 complete changes of PBS pH 7.5 (sterile, pyrogen-free) The glycoconjugate is then aseptically removed from the 20 filtration unit and spun at 2000 rpm for 30 minutes. The supernatant is thenwsterile filtered with a 0.22 mm low protein binding filter.
.A sample of the glycoconjugate is obtained and the following 25 'in-process QC tests are done:
1. Sepharose gel filtration 2. Isoelectric focusing (IEF) 3. BioRad protein assay 30 Based on the results of the protean assay, the final volume of the glycoconjugate is adjusted with sterile, pyrogen-free pH 7.5 PBS buffer to yield a protein concentration of lmg/mL.
35 Inside of a Class 100 biological safety cabinet, 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. During the-f~.lling procedure, 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 5 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 verifiedyby the Quality Control department.
The product is then stored at 2-8°C.
Each lot of GM2 - KLH goes through the following Final Quality Control tests:
15 1. Enzyme Immunoassay (EIA) 2. T;AT, pyrogen test 3. BioRad protein assay 4. Resorcinol-HC1 carbohydrate assay 5. Rabbit pyrogen test 20 6. General safety test 7. Sterility test 8. Impurity testing for cyanide

Claims (17)

What is claimed is:

1. 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.

2. The method of claim 1, wherein the subject is a human.

3. The method of claim 1, wherein the viral disease is caused by the human immunodeficiency virus type-1.

4. The method of claim 1, wherein the carbohydrate is the Lewis y antigen, a ganglioside or the oligosaccharide portion of a ganglioside.

5. The method of claim 4, wherein the ganglioside is GM1, GM1a, GM2, GM3, GD1a, or GD2 or a combination thereof.

6. The method of claim 4, wherein the ganglioside is GM2 or GD2 or a combination thereof.

7. The method of claim 1, wherein the cellular carbohydrate antigen or a molecular mimic thereof is conjugated to a carrier protein.

8. The method of claim 7, wherein the carrier protein is Keyhole Limpet Hemocyanin or a derivative thereof.

9. The method of claim 1, wherein the cellular carbohydrate antigen or a molecular mimic thereof is administered to the subject together with a suitable adjuvant.

10. The method of claim 9, wherein the adjuvant is QS-21.

11. The method of claim 6, wherein the viral disease is caused by the human immunodeficiency virus type-1.

12. The method of claim 11, wherein the ganglioside or oligosaccharide portion thereof is conjugated to Keyhole Limpet Hemocyanin or a derivative thereof.

13. The method of claim 12, wherein the conjugated ganglioside or oligosaccharide portion thereof is administered together with the adjuvant QS-21.

14. The method of claim 1, wherein the molecular mimic is an anti-idiotype antibody.

15. The method of claim 1, wherein the molecular mimic is a peptide mimotope.

16. A vaccine for treating or preventing a viral disease comprising 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.

17. The vaccine of claim 16, wherein the carbohydrate is the Lewis y antigen, a ganglioside or the oligosaccharide portion of a ganglioside.