U.S. patent application number 12/750329 was filed with the patent office on 2010-08-19 for synthetic anti-candida albicans oligosaccharide based vaccines.
This patent application is currently assigned to THERACARB, INC.. Invention is credited to David R. Bundle, Jim E. Cutler, Mark Nitz.
Application Number | 20100209448 12/750329 |
Document ID | / |
Family ID | 29270748 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100209448 |
Kind Code |
A1 |
Bundle; David R. ; et
al. |
August 19, 2010 |
SYNTHETIC ANTI-CANDIDA ALBICANS OLIGOSACCHARIDE BASED VACCINES
Abstract
The present invention provides methods of making and using
immunogenic oligosaccharide compositions comprising native O-linked
and S-linked oligosaccharides coupled to a protein carrier, wherein
the resultant conjugate elicits a protectively immunogenic
response. These compositions may be useful in vaccines against
pathogenic Candida species.
Inventors: |
Bundle; David R.; (Edmonton,
CA) ; Nitz; Mark; (Toronto, CA) ; Cutler; Jim
E.; (New Orleans, LA) |
Correspondence
Address: |
Swiss Tanner, P.C.;P.O. Box 1749
Four Main Street, Suite 100
Los Altos
CA
94022
US
|
Assignee: |
THERACARB, INC.
|
Family ID: |
29270748 |
Appl. No.: |
12/750329 |
Filed: |
March 30, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10512216 |
Jul 22, 2005 |
7722890 |
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PCT/CA03/00614 |
Apr 25, 2003 |
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12750329 |
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60376016 |
Apr 25, 2002 |
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Current U.S.
Class: |
424/194.1 ;
424/193.1 |
Current CPC
Class: |
A61K 47/65 20170801;
A61P 31/10 20180101; A61K 47/646 20170801 |
Class at
Publication: |
424/194.1 ;
424/193.1 |
International
Class: |
A61K 39/385 20060101
A61K039/385; A61P 31/10 20060101 A61P031/10 |
Claims
1-17. (canceled)
18. A method of preventing or ameliorating infection by Candida
species, which method comprises administering to a mammal an
immunogenic effective amount of an anti-Candida species vaccine
comprising a synthetic method that is an oligosaccharide selected
from the group consisting of
[.beta.-D-mannopyranose-(1.fwdarw.2)-.beta.-D-mannopyranose-(1.fwdarw.2)--
.beta.-D-mannopyranose and
.beta.-D-mannopyranose-(1.fwdarw.2)-.beta.-D-mannopyranose, wherein
each saccharide unit of said oligosaccharide is linked via an
inter-glycosidic atom selected from the group consisting of oxygen
and sulfur; and wherein the oligosaccharide is covalently method to
a protein carrier through a linking group.
19. The method of claim 18, wherein the oligosaccharide is
.beta.-D-mannopyranose-(1.fwdarw.2)-.beta.-D-mannopyranose-(1.fwdarw.2)-.-
beta.-D-mannopyranose.
20. The method of claim 18, wherein the oligosaccharide is
.beta.-D-mannopyranose-(1.fwdarw.2)-.beta.-D-mannopyranose.
21. The method of claim 20, wherein at least one inter-glycosidic
atom is sulfur.
22. The method of claim 21, wherein the inter-glycosidic atom at a
terminal non-reducing end of at least one of the saccharide units
is sulfur.
23. The method of claim 18, wherein the linking group is derived
from diethyl squarate, polyoxyalkylene, succinic anhydride or
maleic anhydride.
24. The method of claim 18, wherein the linking group is derived
from a heterobifunctional or homobifunctional cross coupling
reagent.
25. The method of claim 18, wherein the linking group is derived
from a mammalian lipid mimic or a bacterial lipid mimic wherein the
mammalian lipid mimic or a bacterial lipid mimic is functionalized
to permit covalent attachment to a protein or peptide carrier.
26. The method of claim 25, wherein the mammalian lipid mimic or a
bacterial lipid mimic is selected from the group consisting of
sphingosine, a diacyl glycerol, and a diphytanyl ether of
glycerol.
27. The method of claim 18, wherein the protein carrier is selected
from the group consisting of bovine serum albumin, human serum
albumin, tetanus toxoid, a recombinant outer membrane class 3 porn
(rPorB) from group B Neisseria meningitidis, and peptide carriers
such as PADRE.
28. The method of claim 27, wherein the protein carrier is tetanus
toxoid or bovine serum albumin.
29. The method of claim 18, wherein the Candida species is Candida
albicans.
Description
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] The present invention provides immunogenic oligosaccharide
compositions and methods of making and using them. In particular,
the compositions comprise native O-linked and S-linked
oligosaccharides coupled to a protein carrier, wherein the
resultant conjugate elicits a protectively immunogenic response,
particularly in vaccines against pathogenic Candida species and
more particularly against Candida albicans. Preferably the
pathogenic Candida species are those that possess cell wall
oligosaccharide compositions similar to the .beta.-mannan component
of Candida albicans cell walls.
REFERENCES
[0002] The following publications, patents and patent applications
are cited in this application as superscript numbers. [0003] 1.
Casadevall, A.; Cassone, A.; Bistoni, F.; Cutler, J. E.; Magliani,
W.; Murphy, J. W.; Polonelli, L.; Romani, L. (1998) Med. Mycol. 36,
Suppl. 1, 95-105. [0004] 2. Beck-Sague, C. M.; Jarvis, W. R.,
(1993) J. Infect. Dis. 167, 1247-1251. [0005] 3. Landman, D.;
Saurina, G.; Quale, J. M., (1998) Clin. Infect. Dis. 26:183-184.
[0006] 4. Levitz, S. M., (1992) Clin. Infect. Dis., 14 (Suppl. 1),
S37-42. [0007] 5. Jouault, T.; Delaunoy, C.; Sendid, B.; Ajana, F.;
and Poulain, D., (1997) Clin. Diag. Lab. Immun. 4, 328-333. [0008]
6. Shibata, N., Kobayashi, H., Tojo, M., & Suzuki, S. (1986)
Arch. Biochem. Biophys. 251, 697-708. [0009] 7. Shibata, N.;
Fukasawa, S.; Kobayashi, H.; Tojo, M.; Yonezu, T.; Ambo, A.;
Ohkubo, Y.; Suzuki, S., (1989) Carbohydr. Res. 187, 239-253. [0010]
8. Han, Y.; Cutler, J. E., (1995) Infect. Immun. 63, 2714-2719.
[0011] 9. Han, Y.; Cutler, J. E., (1997) J. Infect. Dis. 175,
1169-1175. [0012] 10. Han, Y., Riesselman, M. H.; Cutler, J. E.,
(2000) Infect. Immun. 68, 1649-1654. [0013] 11. Han, Y.; Kozel, T.
R.; Zhang, M. X.; MacGill, R. S.; Carroll M. C.; Cutler. J. E.,
(2001) J. Immunol. 167, 1550-1557. [0014] 12. Han, Y.; Kanbe, T.;
Cherniak, R.; Cutler, J. E., (1997) Infect. Immun., 65, 4100-4107.
[0015] 13. Trinel, P-A.; Plancke, Y.; Gerold, P.; Jouault, T.;
Delplace, F.; Shwarz, R. T.; Strecker, G.; Poulain, D., (1999) J.
Biol. Chem. 274, 30520-30526. [0016] 14. Trinel, P. A.; Lepage, G.;
Joulault, T.; Strecker, G.; Poulain, D., (1997) FEBS Lett., 416,
203-206. [0017] 15. Han, Y.; Ulrich, M. A.; Cutler, J. E., (1999)
J. Infect. Dis. 179, 1477-1484. [0018] 16. Gridley, J. J.; Osborn
H. M. I., J. Chem. Soc. Perkin Trans 1, 2000, 1471-1491. [0019] 17.
Barresi, F.; Hindsgual, O. Synthesis of .beta.-D-mannose Containing
Oligosaccharides. In Modern Methods in Carbohydrate Synthesis,
Khan, S. H.; O'Neil, R. A. Eds; Harwood Academic Publishers:
Amsterdam, 1996, 251-276. [0020] 18. Lergenmuller, M.; Nukada, T.;
Kuramochi, K.; Akhito, D.; Ogawa, T.; Ito, Y.; Eur. J. Org. Chem.
1999, 1367-1376. [0021] 19. Crich, D.; Sun, S.; J. Am. Chem. Soc.
1998, 120, 435-436. [0022] 20. Nitz, M.; Purse, B. W.; Bundle, D.
R., (2000) Org. Lett., 2, 2939-2943. [0023] 21. Bundle, D. R.;
Nitz, M., (2001) J. Org. Chem., 66, 8411-8423. [0024] 22. Nitz, M.;
Ling, C.-C.; Otter, A.; Cutler, J. E.; Bundle, D. R., (2002) J.
Biol. Chem., 277(5), 3440-3446. [0025] 23. Nitz, M. 2001. Ph.D.
thesis. Synthesis of Trichinella spiralis and Candida albicans
antigens, containing .beta.-mannopyranosyl epitopes, their
conformation and immunochemistry. University of Alberta, Edmonton,
Alberta, May 4. [0026] 24. Cutler, J. E.; Han, Y., U.S. Pat. No.
5,578,309 Issued Nov. 26, 1996. [0027] 25. Bousquet, E.; Spadaro,
A.; Pappalardo, M S.; Bernardini, R.; Romeo, R.; Panza, L.;
Ronisvalle, G., (2000) J. Carbohydr. Chem. 19, 527-541. [0028] 26.
Han, Y.; Morrison, R. P.; Cutler, J. E., (1998) Infect. Immun. 66,
5771-5776. [0029] 27. Kanbe, T.; Cutler, J. E., (1994) Infect.
Immun., 62, 1662-1668. [0030] 28. Kabat, E. A., (1962) Federation
Proc. 21, 694-701. [0031] 29. Kabat, E. A., (1966) J. Immunol. 97,
1-11. [0032] 30. Rodriguez, E. C.; Marcaurelle, L. A.; Bertozzi, C.
R., (1998) J. Org. Chem. 63, 7134-7135.
[0033] All of the above publications, patents and patent
applications are herein incorporated by reference in their entirety
to the same extent as if each individual publication, patent or
patent application was specifically and individually indicated to
be incorporated by reference in its entirety.
STATE OF THE ART
[0034] Candida albicans, the most common etiologic agent in
candidiasis,.sup.1 commonly affects immunocompromised patients and
those undergoing long-term antibiotic treatment..sup.2 The number
of cases of systemic candidiasis has become a major medical problem
in hospitals..sup.2 Treatment of these infections is increasingly
difficult due to drug resistance and the toxicity of known
antifungal compounds..sup.3 Humoral and cell mediated immunity may
both play a major role in host defenses against C. albicans. While
most patients with serious mucosal infections have defects in their
cellular immunity,.sup.4 patients with deep tissue invasion seem to
lack antibodies against the (1.fwdarw.2)-.beta.-mannan oligomer
found in the yeast cell wall..sup.5
[0035] The .beta.-mannan component of Candida albicans cell walls
is a relatively small component of the much larger .alpha.-mannan
to which it is attached via a phosphodiester group..sup.6,7 Not
with standing the importance of the larger mannan in defining some
Candida serogroups, the small .beta.-mannan appears to hold
potential as a protective antigen.
[0036] Monoclonal antibodies that protect mice against the
pathogenic yeast, C. albicans.sup.8,9,10 have been shown to be
specific for the cell wall (1.fwdarw.2)-.beta.-mannan
antigen..sup.6,7 These antibodies raised against C. albicans cell
wall extracts in mice were protective against disseminated
candidiasis and vaginal candidiasis..sup.8-11 Further studies on
these protective monoclonal antibodies indicated the active antigen
to be a (1.fwdarw.2)-.beta.-mannan polymer that is present as a
component of the cell wall phopshomannan,.sup.12 and separately as
a phospholipomannan..sup.13 In both forms, the
(1.fwdarw.2)-.beta.-mannan antigen is relatively small consisting
of between two to fourteen residues..sup.14 The immunochemistry and
solution properties of this antigen are of great interest since
(1.fwdarw.2)-.beta.-mannan oligomers have potential as the key
epitope of conjugate vaccines..sup.15
[0037] The rational synthesis of .beta.-mannosides is a
longstanding problem in glycoside synthesis, that until recently,
lacked a general solution despite several novel
approaches..sup.16-19 In the construction of large homo-oligomers,
the separation of anomeric mixtures posed a major obstacle to
efficient assembly by either block or sequential chain extension
reactions.
[0038] The synthesis of these unique polymers required
modifications of existing procedures for the synthesis of
.beta.-D-mannopyranosides. The modulation of the glycosyl donor's
reactivity, by the introduction of p-chlorobenzyl protecting
groups, was required to stabilize the ulosyl bromide to the
innovative reaction conditions. These conditions employed a
sterically hindered participating solvent and a soluble promoter to
control the stereochemistry of the glycosylation, providing
synthetic access to the interesting
(1.fwdarw.2)-.beta.-D-mannopyranans..sup.20-23 Preparation of these
compounds has facilitated further studies into the interesting
structural properties and activity of the
(1.fwdarw.2)-.beta.-D-mannopyranosides and provided a three
dimensional solution structure that accounts for the immunochemical
properties of this antigen..sup.22
SUMMARY OF THE INVENTION
[0039] In one aspect, this invention provides for conjugates
comprising an oligosaccharide comprising
(1.fwdarw.2)-.beta.-D-mannopyranose or a
(1.fwdarw.2)-.beta.-D-mannopyranose derivative, wherein each
saccharide unit of said oligosaccharide is linked via an
inter-glycosidic atom selected from the group consisting of oxygen
and sulfur, a protein carrier, and a linking group, wherein the
oligosaccharide and the protein carrier are covalently conjugated
to one another through the linking group. In addition the conjugate
produces an immune response in a mammal against Candida species,
wherein this response possesses immunological memory. In a
preferred embodiment the Candida species is Candida albicans.
[0040] In a preferred embodiment the conjugate is an
oligosaccharide selected from the group consisting of disaccharides
through hexasaccharides of (1.fwdarw.2)-.beta.-D-mannopyranose or
(1.fwdarw.2)-.beta.-D-mannopyranose derivatives. In a further
preferred embodiment, the oligosaccharide is the trisaccharide
.beta.-D-mannopyranose-(1.fwdarw.)-.beta.-D-mannopyranose-(1.fwdarw.2)-.b-
eta.-D-mannopyranose or the disaccharide 13-D-mannopyranose-(1
.fwdarw.2)-.beta.-D-mannopyranose.
[0041] In another preferred embodiment, the inter-glycosidic atom
is sulfur.
[0042] Preferably an inter-glycosidic atom at a terminal
non-reducing end of the oligosaccharide is sulphur, a form that is
expected to be more resistant to hydrolysis by mammalian enzymes
and hence a more persistent antigen, yielding superior immune
responses..sup.21,23,25
[0043] In yet another preferred embodiment the linking group is
derived from heterobifunctional or homobifunctional cross coupling
reagents, preferably, diethyl squarate, di-NH.sub.2 or di-CO.sub.2H
capped polyoxyalkylenes (such as Jeffamines), succinic anhydride,
or maleic anhydride. Alternatively the linking group is derived
from a mammalian lipid mimic or a bacterial lipid mimic wherein the
mammalian lipid mimic or a bacterial lipid mimic is functionalized
to permit covalent attachment to a protein or peptide carrier.
Preferably the mammalian lipid mimic or a bacterial lipid mimic is
selected from the group consisting of sphingosine, a diacyl
glycerol, and a diphytanyl ether of glycerol,
{2,3-di-O-[(3R,7R,11R)-3,7,11,15-tetramethylhexadecanyl]-sn-gly-
cerol}.
[0044] In yet another preferred embodiment the protein carrier is
selected from the group consisting of bovine serum albumin, (BSA),
human serum albumin, (HSA), tetanus toxoid, (TT) a recombinant
outer membrane class 3 porin (rPorB) from group B Neisseria
meningitidis, and peptide carriers such as PADRE. In one
embodiment, the protein carrier is tetanus toxoid or bovine serum
albumin.
[0045] In another aspect, this invention drawn to an anti-Candida
species vaccine comprising the conjugate as described above and a
pharmaceutically acceptable adjuvant. In a preferred embodiment the
Candida species is Candida albicans.
[0046] In yet another aspect, this invention is drawn to a method
of preventing or ameliorating infection by a Candida species which
method comprises administering to a mammal an immunogenic effective
amount of an anti-Candida species vaccine comprising a conjugate as
described above and a pharmaceutically acceptable adjuvant. In a
preferred embodiment the Candida species is Candida albicans. In a
particularly preferred embodiment the vaccine is directly
administered to the urogenital track and more preferably with a
topical composition.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention provides immunogenic oligosaccharide
compositions and methods of making and using them. In particular,
the compositions comprise oligosaccharides coupled to a carrier
protein, wherein the resultant conjugate elicits a protectively
immunogenic response, particularly in vaccines against pathogenic
Candida species. In addition and of especial note is the use of
S-linked oligosaccharide as an especially effective hapten for use
in conjugate vaccines where such groups secure an enhanced immune
response toward carbohydrate epitopes. However, prior to describing
this invention in further detail, the following terms will first be
defined:
DEFINITIONS
[0048] The term "oligosaccharide" refers to a carbohydrate
structure having from 2 to about 14 saccharide units wherein each
of the saccharide units is linked via an inter-glycosidic atom
selected from the group consisting of oxygen and sulfur.
[0049] The particular saccharide units employed are not critical
and include, by way of example, all natural and synthetic
derivatives of D-mannose, glucose, galactose, N-acetylglucosamine,
N-acetyl-galactosamine, fucose, sialic acid,
3-deoxy-D,L-octulosonic acid, and the like.
[0050] In addition to being in their pyranose form, all saccharide
units described herein are in their D form except for fucose, which
is in its L form.
[0051] The term "mannopyranose" refers to a 6-carbon (hexose) sugar
having a six-membered ring containing five carbon atoms and one
oxygen atom and of formula (I):
##STR00001##
[0052] The term "mannopyranose derivatives" refers to mannopyranose
as described above with at least one hydrogen of the ring hydroxyl
groups is replaced by another chemical moiety, preferably an acetyl
or C.sub.2-C.sub.6 acyl group or a protecting group such as a
benzyl or a p-chlorobenzyl group. The acetyl and acyl derivatives
are preferably hydrolyzed in vivo to form a mannopyranose
conjugate. The protecting groups, on the other hand, are preferably
removed prior to administration.
[0053] The term "mannan" with respect to Candida and yeasts in
general refer to a complex polymannose polymer that is N-linked via
asparagines to a protein. The structure is shown FIG. 1.
[0054] The polysaccharide is exclusively mannose containing and
very complex with branching side chains. For the most part the main
chain and branches are all alpha linked mannopyranose residues,
however in most Candida there is a short chain linked via a
phosphate ester to the main alpha linked mannan. This short chain
varies in length from 2 beta linked mannopyranose residues up to
10-14 mannopyranose residues.
[0055] The term mannan is most often used to mean the whole
complex. When part of the complex is being referred to it could be
called alpha mannan (also called acid stable mannan) or beta
mannan. The later is also called the acid labile mannan because it
is easily cleaved off.
[0056] The term "protein carrier" or "carrier" refers to a
substance that elicits a thymus dependent immune response that can
be coupled to a hapten or antigen to form a conjugate. In
particular, various protein and/or glycoprotein and/or sub-unit
carriers can be used, including but not limited to, tetanus
toxoid/toxin, diphtheria toxoid/toxin, bacteria outer membrane
proteins, crystalline bacterial cell surface layers, serum albumin,
gamma globulin or keyhole limpet hemocyanin.
[0057] The term "conjugate" refers to oligosaccharides that have
been covalently coupled to a protein or other larger molecule with
a known biological activity through a linker. The oligosaccharide
may be conjugated through the inter-glycosidic oxygen or
sulfur.
[0058] The oligosaccharide is attached though a linker to a protein
carrier using conventional chemical techniques providing for
linkage of the oligosaccharide to the carrier. In one embodiment,
reaction chemistries well known in the art that result in covalent
linkages between the linker and both the protein carrier and the
oligosaccharide and are used. Such chemistries preferably involve
the use of complementary functional groups on the hetero- or
homo-bifunctional cross-coupling reagent. Preferably, the
complementary functional groups are selected relative to the
functional groups available on the oligosaccharide or protein
carrier for bonding or which can be introduced onto the
oligosaccharide or carrier for bonding. Again, such complementary
functional groups are well known in the art. For example, reaction
between a carboxylic acid of either the linker or the protein and a
primary or secondary amine of the protein or the linker in the
presence of suitable, well-known activating agents results in
formation of an amide bond; reaction between an amine group of
either the linker or the protein and a sulfonyl halide of the
protein or the linker results in formation of a sulfonamide bond
covalently; and reaction between an alcohol or phenol group of
either the linker or the protein carrier and an alkyl or aryl
halide of the carrier or the linker results in formation of an
ether bond covalently linking the carrier to the linker. Similarly
these complimentary reactions can occur between the linker and the
oligosaccharide to form a linkage between the oligosaccharide and
the linker. The table below illustrates numerous complementary
reactive groups and the resulting bonds formed by reaction there
between.
TABLE-US-00001 COMPLEMENTARY BINDING CHEMISTRIES FIRST SECOND
REACTIVE REACTIVE GROUP GROUP LINKAGE hydroxyl isocyanate urethane
amine epoxide .beta.-hydroxyamine amine ketone imine amine ketone
secondary amine sulfonyl halide amine sulfonamide carboxyl amine
amide acyl azide amine amide hydroxyl alkyl/aryl halide ether
epoxide alcohol .beta.-hydroxyether epoxide sulfhydryl
.beta.-hydroxythioether maleimide sulfhydryl thioether carbonate
amine carbamate
[0059] The term "heterobifunctional cross coupling reagents" refers
to a reagent that is used to couple two other molecules or species
together by having two different functional groups built into one
reagent. Such cross coupling reagents are well known in the art and
include, for example, X-Q-X', where each of X and X' are preferably
independently cross coupling groups selected, for example, from
--OH, --CO.sub.2H, epoxide, --SH, --N.dbd.C.dbd.S, and the like.
Preferably Q is a group covalently coupling X and X' having from 1
to 20 atoms and preferably from 1 to 20 carbon atoms. Examples of
suitable heterobifunctional cross coupling reagents include
squarate derivatives, as found in the attached appendix, as well as
entities derived from succinic anhydride, maleic anhydride,
polyoxyalkylenes, adepic acid (CO.sub.2H--C.sub.6--CO.sub.2H) and
azelaic acid (CO.sub.2H--C.sub.9--CO.sub.2H). The
heterobifunctional cross coupling reagents may also be a lipid or
lipid mimic, where the carbohydrate hapten may be covalently linked
to the lipid or the lipid is co-administered as an immunological
adjuvant.
[0060] The term "homobifunctional cross coupling reagents" refers
to a reagent that is used to couple two other molecules or species
together by having two of the same functional groups built into one
reagent. Such cross coupling reagents are well known in the art and
include, for example, X-Q-X, where X and Q are as defined above.
1,2-Diaminoethane, a dicarboxylic acid chloride and diethyl
squarate are examples of such homobifunctional cross coupling
reagents. Homobifunctional cross coupling reagents may also be
derived from lipids and lipid mimics.
[0061] The term "linker" or "linking group" refers to the residue
produced after covalent bonding of the homobifunctional or
heterobifunctional cross coupling reagent to the oligosaccharide
and the protein carrier.
[0062] The term "vaccine" refers to a composition used to stimulate
an immune response in a mammal and so confer resistance to the
disease or infection in that mammal, which used herein, infers that
the response has immunologic memory.
[0063] The term "adjuvant" refers to a nonantigenic substance (such
as aluminium hydroxide) that, in combination with an antigen,
enhances antibody production by inducing an inflammatory response,
which leads to a local influx of antibody-forming cells. Adjuvants
are used therapeutically in the preparation of vaccines, since they
increase the production of antibodies against small quantities of
antigen and lengthen the period of antibody production.
[0064] The term "immune response" refers to the reaction of the
body to foreign or potentially dangerous substances (antigens),
particularly disease-producing microorganisms. The response
involves the production by specialized white blood cells
(lymphocytes) of proteins known as antibodies, which react with the
antigens to render them harmless. The antibody-antigen reaction is
highly specific. Vaccines also stimulate immune responses.
[0065] The term "immunologic memory" refers to the ability of the
immune system to remember a previously encountered antigen.
Antibodies are produced as a result of the first exposure to an
antigen and stored in the event of subsequent exposure.
[0066] The term "immunologically effective amount" refers to the
quantity of a immune response inducing substance required to induce
the necessary immunological memory required for an effective
vaccine.
Utility, Formulations and Testing
[0067] The present oligosaccharide-protein conjugates of the
.beta.-mannan component of Candida albicans cell wall antigen are
useful in vaccines against any Candida species possessing the
.beta.-mannan antigen, particularly C. albicans.
[0068] The conjugates of this invention may be used as vaccines, as
immunogens that elicit specific antibody production or stimulate
specific cell mediated immunity responses. They may also be
utilized as therapeutic modalities, for example, to stimulate the
immune system to recognize tumor-associated antigens; as
immunomodulators, for example, to stimulate lymphokine/cytokine
production by activating specific cell receptors; as prophylactic
agents, for example, to block receptors on cell membrane preventing
cell adhesion; as diagnostic agents, for example, to identify
specific cells; and as development and/or research tools, for
example, to stimulate cells for monoclonal antibody production
Pharmaceutical Compositions
[0069] The pharmaceutical compositions of the present invention are
advantageously administered in the form of injectable compositions.
A typical composition for such purpose comprises a pharmaceutically
acceptable carrier. For instance, the composition may contain human
serum albumin in a phosphate buffer containing NaCl. Other
pharmaceutically acceptable carriers include aqueous solutions,
non-toxic excipients, including salts, preservatives, buffers and
the like (REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed., Easton
ed., Mack Publishing Co., pp 1405-1412 and 1461-1487 (1975) and THE
NATIONAL FORMULARY XIV, 14th Ed., American Pharmaceutical
Association, Washington, D.C. (1975), both hereby incorporated by
reference). Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oil and injectable organic esters
such as ethyloleate. Aqueous carriers include water,
alcoholic/aqueous solutions, saline solutions, parenteral vehicles
such as sodium chloride, Ringer's dextrose, etc. Intravenous
vehicles include fluid and nutrient replenishers. The pH and exact
concentration of the various components the pharmaceutical
composition are adjusted according to routine skills in the art.
Goodman and Gilman, THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th
ed.).
[0070] Typically, such vaccines are prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. The preparation also may be emulsified. The active
immunogenic ingredient is often mixed with an excipient that is
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like and combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents,
pH-buffering agents, adjuvants or immunopotentiators that enhance
the effectiveness of the vaccine.
[0071] Adjuvants may increase immunoprotective antibody titers or
cell mediated immunity response. Such adjuvants could include, but
are not limited to, Freunds complete adjuvant, Freunds incomplete
adjuvant, aluminium hydroxide, dimethyldioctadecylammonium bromide,
Adjuvax (Alpha-Beta Technology), Inject Alum (Pierce),
Monophosphoryl Lipid A (Ribi Immunochem Research), MPL+TDM (Ribi
Immunochem Research), Titermax (CytRx), toxins, toxoids,
glycoproteins, lipids, glycolipids, bacterial cell walls, subunits
(bacterial or viral), carbohydrate moieties (mono-, di-, tri-
tetra-, oligo- and polysaccharide) various liposome formulations or
saponins. Combinations of various adjuvants may be used with the
conjugate to prepare the immunogen formulation.
[0072] In another embodiment, the conjugate is formulated for
topical applications including gels, lotions, creams and the like.
Topical formulations are well known in the art.
[0073] A topical formulation may be prepared as follows:
TABLE-US-00002 Ingredient Quantity Active Ingredient 1-10 g
Emulsifying Wax 30 g Liquid Paraffin 20 g White Soft Paraffin 20 to
100 g
[0074] The white soft paraffin is heated until molten. The liquid
paraffin and emulsifying wax are incorporated and stirred until
dissolved. The active ingredient is added and stirring is continued
until dispersed. The mixture is then cooled until solid.
[0075] The vaccines are conventionally administered
intraperitoneally, intramuscularly, intradermally, subcutaneously,
orally, nasally, parenterally or administered directly to the
urogenital tract, preferably topically, to stimulate mucosal
immunity. Additional formulations are suitable for other modes of
administration and include oral formulations. Oral formulations
include such typical excipients as, for example, pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate and the like. The
compositions take the form of solutions, suspensions, tablets,
pills, capsules, sustained release formulations or powders and
contain 10%-95% of active ingredient, preferably 25-70%.
[0076] The term "unit dose" refers to physically discrete units
suitable for use in humans, each unit containing a predetermined
quantity of active material calculated to produce the desired
therapeutic effect in association with the required diluent, i.e.,
carrier or vehicle, and a particular treatment regimen. The
quantity to be administered, both according to number of treatments
and amount, depends on the subject to be treated, capacity of the
subject's immune system to synthesize antibodies, and degree of
protection desired. The precise amounts of active ingredient
required to be administered depend on the judgment of the
practitioner and are peculiar to each individual. However, suitable
dosage ranges are on the order of one to several hundred micrograms
of active ingredient per individual. Suitable regimes for initial
administration and booster shots also vary but are typified by an
initial administration followed in one or two week intervals by one
or more subsequent injections or other administration. Annual
boosters may be used for continued protection.
Methods and Procedures
[0077] The methods and procedures suitable for preparing the
compositions and vaccines of this invention are found in references
20-23.
Synthesis of (1.fwdarw.2)-.beta.-Mannopyranosyl Oligomers.
[0078] Synthesis of .beta.1,2 linked oligomers of mannose have been
realized for the first time.sup.20 by an iterative synthetic
procedure, whereby a suitable glycosyl donor in this case ulosyl
bromide 1 or its related derivative 2 react with selectively
protected mono through pentasaccharide alcohols 3-7 to provide
first the corresponding ulopyranoside (e.g. oligosaccharide bearing
a terminal residue with the 2-keto function), which is reduced to
afford directly a selectively protected oligosaccharide alcohol
(e.g. 4-7) to be used in subsequent chain elongation steps (FIG.
2)..sup.20,21 The donors 1 and 2 are attractive because of their
high diastereoselectivity over the glycosylation and reduction
steps, and the minimization of the number of protecting groups
required. The p-chlorobenzyl protected ulosyl bromide 2 is the
preferred glycosyl donor.
[0079] The synthesis of disaccharide through hexasaccharides was
accomplished as outlined in FIG. 2 with pivaloyl nitrile as a
participating solvent to promote formation of equatorial
glycosides. The steric bulk of the t-butyl group decreases side
reactions at the nitrile carbon. This combination of a
participating solvent and soluble promoter.sup.21 are superior to
the heterogeneous conditions initially employed for the
glycosylation of the complex alcohols..sup.20 Pentasaccharide (7)
and hexasaccharide (8) were synthesized employing these conditions
in 51% and 48% yield respectively (combined yield of glycosylation
and reduction steps)..sup.21 p-Chlorobenzyl protected ulosyl
bromide 2 was found to give higher glycosylation yields than its
benzyl protected counterpart 1 due to the increased stability of
the protecting groups. Formation of the ulosides was accomplished
with good stereoselectivity giving an approximate 4:1
.beta.:.alpha. mixture. Separation of the undesired .alpha.-anomer
was readily accomplished by silica gel chromatography of the
protected oligomer after reduction of the product obtained from the
glycosylation. Excellent diastereoselectivity was observed upon
reduction of the .beta. ulopyranoside to the desired
.beta.-mannoside with L-selectride, since no .beta.-gluco epimer
could be detected by .sup.1H NMR in this reaction. Heteronuclear
one bond coupling constants were used to unambiguously establish
the anomeric configuration of the .beta.-mannopyranosyl
residues..sup.21
[0080] The yields and selectivity achieved in this synthesis
compare well with those reported using 4,6-O-benzylidene protected
mannopyranosyl sulphoxide donors..sup.19 Crich et al. synthesized
.beta.-(1.fwdarw.2) mannopyranosyl pentasaccharides and
hexasaccharides in 68% and 69% respectively but their method
requires an extra deprotection step (achieved in 80-85% yield)
prior to further glycosylation steps, and no large gain in
efficiency is evident (cf reference 19 vs 21).
[0081] The protected oligosaccharides (4-6 and 8) were elaborated
via photo addition of 2-aminoethanethiol to the allyl glycosides to
give the amine functionalized glycosides (9-12)..sup.21 It was
necessary to use long wave (365 nm) UV irradiation to avoid
complications with the aromatic protecting groups (FIG. 3).
Purification of these compounds was difficult, and they were
carried through the subsequent deprotection step with minor
impurities. Compounds (9-12) were deprotected under dissolving
metal conditions to yield the amino functionalized glycosides
(13-16) after purification. Deprotection of compounds (4-8) to
their respective propyl glycosides (17-21) was most efficiently
achieved using a two step procedure. The allyl glycoside was first
reduced to the propyl glycoside via diimide reduction and then the
protecting groups were removed under dissolving metal conditions
(FIG. 3).
Synthesis of Thioglycoside Mimetic
[0082] Installation of a terminal thioglycosidic linkage was
accomplished by reaction of a trisaccharide thiol 22 with the
ulosyl bromide 2 (FIG. 4). Trisaccharide, 22, was synthesized by
glycosylation of disaccharide 4, with the glucosyl donor 23, to
give trisaccharide 24. The trichloracetimidate donor 23 was
prepared from 3-O-benzyl glucose according to standard
conditions..sup.21 Trisaccharide 24 was obtained in high yield from
the imidate donor 23 and the disaccharide acceptor 4.
Transesterification of trisaccharide 24 gave alcohol 25, which was
converted to the 2-O-trifluoromethanesulfonate 26 under standard
conditions. Nucleophilic displacement of the triflate with
thioacetate gave 27 and removal of the acetate proceeded smoothly
to the thiol 22. Reaction of 22 with 2 under basic conditions gave,
after reduction, tetrasaccharide, 28, in 49% yield. The
corresponding 1-thio-.alpha.-gluco epimer was also isolated from
the reaction (.about.12%). The tetrasaccharide 28 was converted to
the corresponding 3-(2-aminoethylthio)-propyl glycoside 29 as
described for compounds 9-12. The tetrasaccharide 28 was reduced
with a solution of hydrazine hydrate, to afford the propyl
glycoside 30. Dissolving metal conditions then efficiently removed
the benzyl and p-chlorobenzyl protecting groups of compounds 29 and
30 to give the desired tetrasaccharides, 31 and 32 (FIG. 5).
Formation of Glycoside Conjugates.
[0083] Reaction of the functionalized mannopyranosyl glycosides
13-16 and 31 with diethyl squarate in a solution of ethanol and
water provided, in high yield, the activated oligosaccharides 33-37
(FIG. 6). Coupling to BSA and tetanus toxoid was performed as
follows: either BSA or tetanus toxoid (.about.20 mg/ml) was
dissolved in borate buffer (Na.sub.2BO.sub.4 0.07M, KHCO.sub.3
0.035M, pH 9.5) the squarate coupled carbohydrate was then added
and the reaction was left for 72 h at room temperature. The
reaction was then diluted with deionized water and dialysed in a
MICROSEP microconcentrator against 5 changes of deionized water (3
ml) where the final volume after concentration was no greater than
500 .mu.L. The solution from the final concentration was
lyophilized to a white solid. MALDI mass spectrometry was preformed
on a Voyager elite system from Applied Biosystems. Conjugation
efficiencies of between 65-70% were achieved with an incorporation
of 13-15 ligands for a 20 fold molar excess of activated
oligosaccharide. Targeted and observed incorporations are tabulated
below (Table 1). The molecular weights of the protein conjugates
determined by MALDI-TOF spectroscopy indicated that all the
conjugates had a degree of hapten incorporation within a narrow
range of .+-.1 haptens.
TABLE-US-00003 TABLE 1 Protein carbohydrate conjugates Target
incorporation (100% Incorporation Incorporation Oligosaccharide
conversion) achieved efficiency 38 M.sub.2-BSA 20 16 80 39
M.sub.3-BSA 20 15 75 40 M.sub.4-BSA 20 13 65 41 M.sub.6-BSA 20 10
50 42 SM.sub.4-BSA 20 15 75 43 SM.sub.4-TT 80 33 41 44 M.sub.4-TT
80 27 34 45 M.sub.6-TT 80 29 36 M.sub.3
(1.fwdarw.2)-.beta.-D-mannopyranotriose,
M.sub.4(1.fwdarw.2)-.beta.-D-mannopyranotetrose, SM.sub.4
(1.fwdarw.2)(1-thio-.beta.-D-mannopyranosyl)(1.fwdarw.2)-.beta.-D-
-mannopyranotriose, M.sub.6 (1-2)-.beta.-D-mannopyranohexose
[0084] Preferably the conjugates of the present invention exclude
conjugates that originate from the use of a reducing sugar residue
to make the linkage to the protein carrier either directly or via a
tether using reactions such as the conversion of a terminal alditol
to an aldehyde and hence coupling by reductive amination to an
amine or lysine, its conversion to a carboxylic acid derivative or
the direct coupling of a terminal aldose by reductive amination or
by creation of a glycosylamine and then coupling via its amino
group.
Binding of Propyl (1.fwdarw.2)-.beta.-Mannosides to Monoclonal
Antibodies
[0085] Two antibodies produced in response to a C. albicans native
antigen liposomal vaccine.sup.15 and which protect mice against
live challenge with C. albicans.sup.10,26 were used to evaluate the
biological activity of the synthetic oligomanns 17-21 and 32. These
monoclonal antibodies are IgM B6.1 and IgG C3.1. The affinity of
these monoclonal antibodies for the synthesized propyl glycosides
was determined in a competitive inhibition ELISA. The monoclonal
antibodies, MAb B6.1 (IgM) and MAb C3.1 (IgG3) were produced as
concentrated tissue culture supernatants and diluted
.about.1:40,000 (B6.1) and .about.1:2000 (C3.1). C. albicans mannan
obtained by 2-mercaptoethanol extraction of whole cells without
subsequent affinity fractionation.sup.27 was dissolved in PBS (10
.mu.g/mL) and the solution was used to coat 96 well ELISA plates
(100 .mu.L, 18 h at 4.degree. C.). The plate was washed five times
with PBST containing Tween 20, 0.05% v/v) and blocked for 1 h at
room temperature (2% BSA PBS, 100 .mu.L). The monoclonal antibodies
were mixed with inhibitor dissolved in PBST at concentrations in
the range between 0.1 .mu.M to 1 mM, and the resulting solutions
were added to the coated microtitre plate in triplicate and
incubated at room temperature for 18 h. The plate was washed with
PBST (5.times.), and goat anti-mouse (IgG or IgM) antibody
conjugated to horseradish peroxidase (diluted 1:2000, Kirkegaard
and Perry Lab) in PBST (100 .mu.L) was added and incubated for 1 h
at room temperature. The plate was washed with PBST (5.times.),
3,3',5,5'-tetramethylbenzidine (TMB, 100 .mu.L, Kirkegaard and
Perry Lab) was added, and after 2 minutes the colour reaction was
stopped by the addition of 1M phosphoric acid (100 .mu.L).
Absorbance was read at 450 nm and percent inhibition was calculated
relative to wells containing antibody without inhibitor, and the
concentration of ligand that gives 50% inhibition is quoted as an
IC.sub.50 value.
[0086] The IgM antibody B6.1 shows a surprisingly high affinity for
the di and trisaccharides when compared to the tetra and
hexasaccharides. The propyl
(1-thio-.beta.-D-mannopyranosyl)-(1.fwdarw.2)-.beta.-D-mannopyrano-
trioside falls between the tri and tetrasaccharide in affinity
(FIG. 7).
[0087] The IgG antibody showed a similar trend of affinities, to
that of the IgM antibody, for the same panel of oligo-mannans. Di-
and trisaccharides had higher affinities, five and two fold
respectively, for the IgG antibody than for the IgM MAb and the
remaining antigens had a similar affinity (FIG. 8). The observation
of higher affinity for smaller oligomers is unprecedented for
antibodies generated to polysaccharide antigens. As a rule the
larger oligomers have higher affinity for the antibodies..sup.28,29
The expected trend is to observe oligomer inhibition that increases
with oligosaccharide length to a maximum of 5-8 hexose
residues..sup.28,29
[0088] Furthermore, it is very surprising that the IgG and IgM
antibodies have similar specificity and affinity. A polydisperse
antigen such as the extract used to generate antibodies B6.1 and
C3.1.sup.15 would be expected to generate antibodies to differing
epitopes. The finding that both monoclonal antibodies recognize
similar epitopes suggests a single immunodominant epitope in the
extract of the C. albicans cell wall used for immunization. As
these antibodies are known to be protective, the inhibition results
indicate a near ideal situation for the generation of synthetic
vaccines..sup.22 This unexpected result has been rationalized with
the compact helical nature of the (1.fwdarw.2)-.beta.-mannan
polymer and potential steric interactions with the binding site.
This finding suggests a synthetically simple disaccharide or
trisaccharide conjugate may be all that is necessary to generate a
protective immune response.
Immunization of Experimental Rabbits with Tetrasaccharide-TT
Conjugate 44 and Hexasaccharide-TT Conjugate 45
[0089] 2 New Zealand white rabbits where immunized with a tetanus
toxoid protein conjugate (50 .mu.g in 500 .mu.L PBS and 500 .mu.L
Freunds complete adjuvant, homogenised) in three injections one
intramuscularly in the rear thigh and two subcutaneously. On day 36
the rabbits were boosted with (50 .mu.g in 500 .mu.L PBS and 500
.mu.L Freunds incomplete adjuvant, homogenised). On day 59 a third
boost with (50 .mu.g in 500 .mu.L PBS and 500 .mu.L Freunds
incomplete adjuvant, homogenised) was carried out and on day 70 the
rabbits were sacrificed. The titers of antibodies produced in
rabbits were analyzed by ELISA.
[0090] 96 well ELISA plates were coated with .beta.-mannan
preparation extracted from Candida albicans and the plate was
washed. Serial dilutions of the sera from the immunized animal were
added to the plate in triplicate. Goat anti-rabbit horse radish
peroxidase conjugated antibody was used to quantify specific
antibody..sup.23
[0091] Rabbits immunized with the tetanus toxoid conjugates
exhibited a strong IgG response after three injections. A high
titers were observed for the immunizing antigen and in addition
these antibodies showed extremely strong specificity for C.
albicans native antigen with a litre between 10.sup.5-10.sup.6
(FIG. 9). Both the hexa and tetrasaccharide TT conjugates raised
similar antibody titers.
Antibody Levels in Mice
[0092] Mice developed a strong IgM response against the
hexasaccharide tetanus toxoid conjugate after two immunizations, as
judged by ELISA assay, using the BSA conjugated hexasaccharide.
Modest IgG antibodies were also present after this time. Groups of
5 mice were given monthly injections of 5-10 ug of the SM.sub.4-TT
conjugate. Two mice were sacrificed after the sixth injection and
using standard hybridoma protocols monoclonal antibody were
produced. Three IgG and IgM monoclonal antibodies that bound the C.
albicans native .beta.-mannan antigen were selected and these
showed activity in in-vivo mouse protection studies..sup.15
* * * * *