U.S. patent application number 11/469270 was filed with the patent office on 2007-03-29 for methods and reagents for the analysis and purification of polysaccharides.
This patent application is currently assigned to Academia Sinica. Invention is credited to Szu-Ting Chen, Shih-Chin Cheng, Shie-Liang Hsieh, Tsui-Ling Hsu, Chie-Huey Wong.
Application Number | 20070072247 11/469270 |
Document ID | / |
Family ID | 44209631 |
Filed Date | 2007-03-29 |
United States Patent
Application |
20070072247 |
Kind Code |
A1 |
Wong; Chie-Huey ; et
al. |
March 29, 2007 |
METHODS AND REAGENTS FOR THE ANALYSIS AND PURIFICATION OF
POLYSACCHARIDES
Abstract
The disclosure provides fusion proteins comprising a
carbohydrate recognition domain of an innate immunity receptor and
a heterologous polypeptide. The fusion proteins of the disclosure
may be used, for example, to fingerprint polysaccharide
compositions and to purify polysaccharide compositions.
Polysaccharide compositions include those isolated from Ganoderma
lucidum (Reishi). The methods and reagents of the disclosure may
also be used to identify innate immunity receptors and cell types
that bind to polysaccharide compositions (including polysaccharide
compositions associated with pathogens), whereupon modulators of
the identified receptors can then be obtained. The fusion proteins
also may be used to inhibit the interaction between a
polysaccharide composition and an innate immunity receptor on a
cell surface. The methods and reagents of the disclosure are used
in one example to determine that the DLVR1 innate immunity receptor
on macrophages interacts with Dengue virus (DV), and that DLVR1 is
responsible for DV-mediated secretion of proinflammatory cytokines
from macrophages. The disclosure also provides DVLR1 antibodies
that prevent the secretion of proinflammatory cytokines by
DV-infected macrophages.
Inventors: |
Wong; Chie-Huey; (Rancho
Santa Fe, CA) ; Hsieh; Shie-Liang; (Taipei, TW)
; Hsu; Tsui-Ling; (Taipei, TW) ; Cheng;
Shih-Chin; (Nantou City, TW) ; Chen; Szu-Ting;
(Taipei, TW) |
Correspondence
Address: |
GREENBERG TRAURIG LLP
2450 COLORADO AVENUE, SUITE 400E
SANTA MONICA
CA
90404
US
|
Assignee: |
Academia Sinica
Taipei
TW
|
Family ID: |
44209631 |
Appl. No.: |
11/469270 |
Filed: |
August 31, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60713463 |
Aug 31, 2005 |
|
|
|
Current U.S.
Class: |
435/7.2 ;
435/320.1; 435/326; 435/69.7; 530/388.22; 530/391.1; 536/23.5 |
Current CPC
Class: |
Y02A 50/30 20180101;
G01N 33/56983 20130101; C07K 2319/30 20130101; C07K 16/2851
20130101; Y02A 50/53 20180101; C07K 2317/76 20130101; G01N 33/56972
20130101; G01N 2333/18 20130101; G01N 2500/02 20130101 |
Class at
Publication: |
435/007.2 ;
435/069.7; 435/326; 435/320.1; 530/388.22; 530/391.1;
536/023.5 |
International
Class: |
G01N 33/567 20060101
G01N033/567; C07H 21/04 20060101 C07H021/04; C12P 21/04 20060101
C12P021/04; C07K 16/46 20060101 C07K016/46 |
Claims
1. A fusion protein comprising: (a) a carbohydrate recognition
domain of an innate immunity receptor; and (b) a heterologous
polypeptide.
2. The fusion protein of claim 1 wherein said heterologous
polypeptide comprises an immunoglobulin or a fragment of an
immunoglobulin.
3. The fusion protein of claim 2 wherein said immunoglobulin is
IgG.
4. The fusion protein of claim 1 wherein said heterologous
polypeptide is IgG Fe.
5. The fusion protein of claim 1 wherein said heterologous
polypeptide is human IgG1 Fe.
6. The fusion protein of claim 5 wherein said human IgG1 Fe is a
variant that does not bind to Fe receptors.
7. A method for determining whether a specific carbohydrate
component is present in a composition comprising a polysaccharide,
the method comprising: (a) contacting said polysaccharide with a
fusion protein comprising: (i) a carbohydrate recognition domain of
an innate immunity receptor, wherein said carbohydrate recognition
domain is capable of binding to said specific carbohydrate
component; and (ii) a heterologous polypeptide; and (b) determining
whether said fusion protein has bound to said polysaccharide.
8. The method of claim 7 wherein said composition comprising said
polysaccharide is immobilized on a solid support, and wherein step
(b) is accomplished by determining whether said heterologous
polypeptide is present on said solid suppport, wherein the presence
of said heterologous polypeptide is indicative of the presence of
said specific carbohydrate in said composition.
9. The method of claim 8 wherein said heterologous polypeptide is
an immunoglobulin or a fragment of an immunoglobulin.
10. A kit comprising: (a) a fusion protein comprising: (i) a
carbohydrate recognition domain of an innate immunity receptor; and
(ii) a heterologous polypeptide; and (b) reagents for detecting the
presence of said heterologous polypeptide on said solid
support.
11. A method for determining whether an innate immunity receptor
binds to a pathogen, the method comprising: (a) contacting said
pathogen with a fusion protein comprising: (i) a carbohydrate
recognition domain of said innate immunity receptor, wherein said
carbohydrate recognition domain is capable of binding to a specific
carbohydrate component; and (ii) a heterologous polypeptide; (b)
determining whether said fusion protein has bound to said
pathogen.
12. The method of claim 11 wherein said fusion protein is
immobilized on a solid support, and wherein step (b) is
accomplished by using an antibody specific for said pathogen.
13. A method of inhibiting the interaction between an innate
immunity receptor on the surface of a cell and a polysaccharide
that binds to a carbohydrate recognition domain of said innate
immunity receptor, the method comprising contacting said cell with
a fusion protein comprising: (a) said carbohydrate recognition
domain of said innate immunity receptor; and (b) a heterologous
polypeptide.
14. A method of inhibiting the interaction between an innate
immunity receptor on the surface of a cell and a
pathogen-associated polysaccharide that binds to a carbohydrate
recognition domain of said innate immunity receptor, the method
comprising contacting said cell with an antibody antagonist of said
innate immunity receptor.
15. The method of claim 14 wherein said pathogen is selected from
the group consisting of an enveloped virus, a fungal cell, and a
bacterial cell.
16. The method of claim 14 wherein said interaction is inhibited in
vivo by administering a pharmaceutical composition comprising said
antibody to an organism suspected of coming into contact with said
pathogen.
17. The method of claim 14 wherein said antibody is a monoclonal
antibody.
18. A method of treating an organism infected with an enveloped
virus, the method comprising administering to said organism an
agent that inhibits the activity of DVLR1.
19. The method of claim 18 wherein said organism is a human.
20. The method of claim 19 wherein said virus is influenza
virus.
21. The method of claim 19 wherein said virus is a member of the
Flaviviridae family.
22. The method of claim 21 wherein said virus is a member of the
Flavivirus genus or the Hepacivirus genus.
23. The method of claim 22 wherein said virus is a member of the
Flavivirus genus selected from the group consisting of West Nile
Virus, Japanese encephamyelitis virus, yellow fever virus,
tick-borne encephamyelitis virus, and Dengue virus.
24. The method of claim 23 wherein said virus is Dengue virus.
25. The method of claim 24 wherein said agent is an antibody
against DVLR1.
26. The method of claim 24 wherein said agent is a monoclonal
antibody against DVLR1.
27. The method of claim 24 wherein said agent is a humanized
antibody against DVLR1.
28. The method of claim 24 wherein said agent is a fragment of an
antibody against DVLR1.
29. The method of claim 24 wherein said agent is a mediator of RNA
inteference.
30. The method of claim 29 wherein said agent is an siRNA
comprising sequence from DVLR1.
31. An isolated antibody, or epitope-binding fragment thereof, that
binds to DVLR1 on CD14+ macrophages and which inhibits Dengue
virus-mediated TNF-.alpha. secretion from CD14+ macrophages.
32. A purified monoclonal antibody, or epitope-binding fragment
thereof, which binds to a DVLR1 epitope bound by a monoclonal
antibody selected from the group consisting of monoclonal
antibodies 9B12, 3E12A2, 3E12C1, 3E12G9, and 8H8F5, wherein said
antibody or fragment thereof inhibits TNF-.alpha. secetion by
macrophages after infection with Dengue virus.
33. A pharmaceutical composition comprising the monoclonal
antibody, or epitope-binding fragment thereof, of claim 32 and a
pharmaceutically acceptable excipient.
34. A monoclonal antibody, or epitope-binding fragment thereof, the
monoclonal antibody selected from the group consisting of
monoclonal antibodies 9B12, 3E12A2, 3E12C1, 3E12G9, and 8H8F5.
35. A pharmaceutical composition comprising the monoclonal
antibody, or epitope-binding fragment thereof, of claim 34 and a
pharmaceutically acceptable excipient.
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/713,463, filed Aug. 31, 2005, the
disclosure of which is incorporated herein by reference in its
entirety.
[0002] This work was supported by grant 94F008-5, NSC
95-2320-B-010-010 and NSC 95-3112-B-010-017 from the National
Sciences Council, Taiwan. This work was also supported by grant
94M002-1 from the Academia Sinica, Taiwan, and by grant 95A-CT8G02
from the National Yang-Ming University.
BACKGROUND
[0003] Citation to any reference in this specification is not, and
should not be taken as, an acknowledgment or any form of suggestion
that this reference forms part of the common general knowledge or
of the prior art in any country. All references cited herein are
specifically incorporated herein by reference in their
entirety.
[0004] The immune system enables a host organism to discriminate
self from non-self antigens, as well as to recognize and eradicate
invasive pathogens. The adaptive immunity system relies on highly
polymorphic molecules, such as class I and class II antigens of the
major histocompatibility complex (MHC), T cell receptors, and B
cell receptors, to present antigens to T cells and B cells, thus
leading to the activation of immune system. The mechanism by which
the innate immunity system can recognize these diverse antigens
remained unsolved until the emergence of the concept of `pattern
recognition receptors (PPRs)` proposed by Janeway (Janeway, 1989,
Cold Spring Harb Symp Quant Biol 54 Pt 1, 1-13). This hypothesis
was later proved correct by the identification of
pathogen-associated molecular patterns (PAMPs) which are recognized
by TOLL-like receptors (Aderem and Ulevitch, 2000 Nature 406,
782-7; Akira and Takeda, 2004, Nat Rev Immunol 4, 499-511; Athman
and Philpott, 2004, Curr Opin Microbiol 7, 25-32), lectin receptors
(Cambi and Figdor, 2003, Curr Opin Cell Biol 15, 539-46),
immunoglobulin-like (Ig-like) receptors (Daws et al., 2003, J
Immunol 171, 594-9), and NOD proteins (Athman and Philpott, 2004,
Curr Opin Microbiol 7, 25-32), and others (Liu et al., 2001, J Biol
Chem 276, 34686-94; McDonald et al., 2005, J Biol Chem 280,
20177-80).
[0005] In addition to the well characterized PAMPs recognized by
TOLL-like receptors (Akira and Takeda, 2004, Nat Rev Immunol 4,
499-511), recent study indicates that the host immune system can
recognize invasive pathogens through specific carbohydrate
antigens. For example, mannose receptors can recognize the high
mannose sugar moiety expressed on the surface of pathogens (Stahl
and Ezekowitz, 1998, Curr Opin Immunol 10, 50-5), while the
Dectin-1 receptor can bind specifically to .beta.-glucan, the major
backbone of polysaccharides on fungus walls (Brown and Gordon,
2001, Nature 413, 36-7; Herre et al., 2004, Mol Immunol 40,
869-76). This suggests that the carbohydrate structures associated
with pathogens are one of the targets recognized by the innate
immunity receptors of immune cells.
[0006] Ganoderma species and Cordyceps species are groups of
medical fungus which are the most popular herbal drugs taken in
China. Polysaccharides extracted from Ganoderma lucidum (also known
as Ling zhi, Reishi) have been used in traditional Chinese medicine
(TCM) as anti-tumor agents and as immuno-modulating agents (Lien,
1990, Prog Drug Res 34, 395-420; Wang et al., 2002, Bioorg Med Chem
10, 1057-62; Shiao, 2003, Chem Rec 3, 172-80), while those
extracted from Cordyceps sinensis (Cordyceps, Caterpillar fungus)
have been shown to alter apoptotic homeostasis, and to improve
respiratory, renal and cardiovascular functions (Buenz et al.,
2005, J Ethnopharmacol 96, 19-29; Zhu et al., 1998, J Altern
Complement Med 4, 289-303; Zhu et al., 1998, J Altern Complement
Med 4, 429-57), as well as to increase whole body sensitivity to
insulin (Balon et al., 2002, J. Altern Complement Med 8, 315-23).
However, the polysaccharide composition of the extracts vary when
they the polysaccharides are extracted from different sources, from
different strains, and under different growing conditions.
[0007] Analytical methods relying on high-performance liquid
chromatography (HPLC) and proton-nuclear magnetic resonance have
been applied to investigate the components of polysaccharides
isolated from Ganoderma lucidum and Cordyceps sinensis (He and
Seleen, 2004, Int. J. Med. Mushrooms 6, 253). However, the HPLC
chromatogram is based on the comparison with ganoderic acid A and C
(two major triterpenes of Ganoderma lucidum) or adenosine. It is
still difficult to know whether the extracts contain the active
components of polysaccharides based on the mass spectrum.
SUMMARY
[0008] In one series of embodiments, the disclosure provides a
fusion protein comprising a carbohydrate recognition domain of an
innate immunity receptor; and a heterologous polypeptide. In one
embodiment, the heterologous polypeptide comprises an
immunoglobulin, such as human IgG, or a fragment of an
immunoglobulin, such as human IgG Fc, or a variant of human IgG Fc
that does not bind to Fc receptors. The fusion protein may further
comprise a linker peptide between the carbohydrate recognition
domain and the heterologous polypeptide.
[0009] In some embodiments, the innate immunity receptor from which
the carbohydrate recognition domain is derived may be a C-type
lectin, such as a C-type lectin selected from the group consisting
of ASGR1, ASGR2, CD207 (CLEC4K), CD209, CD302, CLEC1A, CLEC1B,
CLEC2A, CLEC2B, CD69, CLEC2D, CLEC2L, CLEC3A, CLEC3B, CLEC30,
CLEC3Q, CLEC4A, CLEC4C, CLEC4D, CLEC4E, CLEC4F, CLEC4G, CLEC4M,
CD209, CLEC5A, CLEC6A, CLEC7A, CLEC9A, CLEC10A, CLEC11A, CLEC12A,
CLEC14A, FCER2, KLRB1, KLRF1, LY75, MRC1, MRC1L1, MRC2, OLR1,
PLA2R1, mKCR, and COLEC10.
[0010] In some embodiments, the innate immunity receptor from which
the carbohydrate recognition domain is derived may be an
immunoglobulin-like receptor.
[0011] In some embodiments the innate immunity receptor from which
the carbohydrate recognition domain is derived is selected from the
group consisting of CD300 Antigen Like Family Member B (CD300LB),
CD300 Antigen Like Family Member G (CD300LG), TREM1, TREM2, TREML1,
TREML2, TREML3, and TREML4.
[0012] In some embodiments the innate immunity receptor from which
the carbohydrate recognition domain is derived is selected from the
group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,
TLR9, TLR10, TLR11, TLR12, TLR13, TICAM1, and TICAM2.
[0013] In some embodiments the innate immunity receptor from which
the carbohydrate recognition domain is derived is selected from the
group consisting of CD22, CD33, Myelin Associated Glycoprotein
(MAG), SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9, SIGLEC10,
SIGLEC11, SIGLEC12, SIGLEC13, and Sialoadhesin (SN).
[0014] In another aspect, the disclosure provides a method for
determining whether a specific carbohydrate component is present in
a composition comprising a polysaccharide. The method comprises
contacting the polysaccharide with a fusion protein comprising:
[0015] (i) a carbohydrate recognition domain of an innate immunity
receptor, wherein the carbohydrate recognition domain is capable of
binding to the specific carbohydrate component; and [0016] (ii) a
heterologous polypeptide; and and then determining whether the
fusion protein has bound to the polysaccharide.
[0017] In some embodiments, the aforementioned method may be
performed wherein the composition comprising the polysaccharide is
immobilized on a solid support. The determination of binding
between the fusion protein and the polysaccharide is accomplished
by determining whether the heterologous polypeptide is present on
said solid suppport, wherein the presence of the heterologous
polypeptide is indicative of the presence of the specific
carbohydrate in the composition.
[0018] The heterologous polypeptide may be, for example, an
immunoglobulin or a fragment of an immunoglobulin. In some
embodiments, the heterologous polypeptide is conjugated to at least
one biotin, which allows detection of the heterologous polypeptide
using, for example, a streptavidin-conjugated enzyme. In other
embodiments, the heterologous polypeptide is detected using an
antibody that binds to the polypeptide. In further embodiments, the
heterologous polypeptide is labelled with a detectable moiety, such
as an enzyme or a fluorophore.
[0019] In another aspect, the disclosure provides a kit comprising
one of the aforementioned fusion proteins and further comprising
reagents for detecting the presence of said heterologous
polypeptide on said solid support.
[0020] In another aspect, the disclosure provides a method for
isolating a composition comprising a polysaccharide from a mixture,
wherein the polysaccharide comprises a specific carbohydrate
component, the method comprising providing a solid support
comprising an immobilized fusion protein (comprising a carbohydrate
recognition domain of an innate immunity receptor, wherein the
carbohydrate recognition domain is capable of binding to the
specific carbohydrate component; and a heterologous polypeptide);
contacting the solid support with the mixture wherein the specific
carbohydrate component binds to said carbohydrate recognition
domain; washing the solid support; and dissociating the specific
carbohydrate component from the fusion protein, whereby the
composition comprising the polysaccharide may be isolated. In some
embodiments, the composition comprising a polysaccharide is a
peptidoglycan. In other embodiments, the composition comprising a
polysaccharide is a fungal cell. In still further embodiments, the
composition comprising a polysaccharide is a glycoprotein.
[0021] In another aspect, the disclosure provides a method for
determining whether an innate immunity receptor binds to a
pathogen, the method comprising: contacting the pathogen with a
fusion protein comprising: [0022] (i) a carbohydrate recognition
domain of the innate immunity receptor, wherein the carbohydrate
recognition domain is capable of binding to a specific carbohydrate
component; and [0023] (ii) a heterologous polypeptide; and
determining whether the fusion protein has bound to the
pathogen.
[0024] In some embodiments, the pathogen is immobilized on a solid
support, such as a microtiter plate, and the presence of the
heterologous polypeptide on the solid support is indicative of the
binding of the fusion protein to the pathogen. The pathogen may be
immobilized on the solid support using an antibody specific for the
pathogen.
[0025] In another embodiment, the fusion protein is immobilized on
a solid support, and determining whether the fusion protein has
bound to the pathogen is accomplished by using an antibody specific
for the pathogen.
[0026] The pathogen can be, without limitation, a virus, a fungal
cell, or a bacterial cell. The virus can be, without limitation, an
enveloped virus such as a flu virus. The virus may be a virus from
the Flaviviridae family, more specifically a member of the
Flavivirus genus, such as, but not limited to, Dengue virus.
[0027] In another aspect, the disclosure provides a method of
inhibiting the interaction between an innate immunity receptor on
the surface of a cell and a polysaccharide that binds to a
carbohydrate recognition domain of the innate immunity receptor.
The method comprises contacting the cell with a fusion protein
comprising: [0028] (a) the carbohydrate recognition domain of the
innate immunity receptor; and [0029] (b) a heterologous
polypeptide.
[0030] In some embodiments of this method, the heterologous
polypeptide is a variant of human IgG Fc that does not bind to Fe
receptors. The method may be performed by administering the fusion
protein to an organism as a pharmaceutical composition. The
pharmaceutical may comprise, in addition to the fusion protein, one
or more pharmaceutically acceptable excipients. Accordingly, the
disclosure also provides pharmaceutical compositions comprising any
of the aforementioned fusion proteins and one or more
pharmaceutically acceptable excipients.
[0031] In another aspect, the disclosure provides a method of
inhibiting the interaction between an innate immunity receptor on
the surface of a cell and a pathogen-associated polysaccharide that
binds to a carbohydrate recognition domain of the innate immunity
receptor. The method involves contacting the cell with an antibody
antagonist of the innate immunity receptor. The antibody may be a
monoclonal antibody, or fragment thereof. The antibody may also be
a humanized antibody. The pathogen may be, without limitation, a
virus (including enveloped viruses), a bacterial cell, or a fungal
cell.
[0032] In another aspect, the disclosure provides a method of
treating an organism (such as a human) infected with an enveloped
virus, the method comprising administering to the organism an agent
that inhibits the activity of DVLR1. The virus in this method may
be, for example, an influenza virus. In some embodiments, the virus
is a member of the Flaviridae family, such as a Flavivirus genus
member (including, but not limited to, West Nile Virus, Japanese
encephamyelitis virus, yellow fever virus, tick-borne
encephamyelitis virus, and Dengue virus) or a Hepacivirus genus
member (including, but not limited to, Hepatitis C).
[0033] In some embodiments, the agent that inhibits the activity of
DVLR1 is an antibody against DVLR1. The antibody may be a
monoclonal antibody, including a humanized mononclonal antibody.
The antibody may be a fragment of an antibody against DVLR1.
[0034] In further embodiments, the agent that inhibits the activity
of DVLR1 is a mediator of RNA inteference, such as an siRNA
comprising sequence from DVLR1. The siRNA may be administered to
the organism, or a construct may be administered to the organism
that is transcribed in vivo to yield an siRNA (for example, a
shRNA).
[0035] In another aspect, the disclosure provides an isolated
antibody that binds to DVLR1 on CD14+ macrophages and which
inhibits Dengue virus-mediated TNF-.alpha. secretion from CD 14+
macrophages.
[0036] In a further aspect, the disclosure provides a purified
monoclonal antibody, or epitope-binding fragment thereof, which
binds to a DVLR1 epitope bound by a monoclonal antibody selected
from the group consisting of 3E12A2, 3E12C1, 3E12G9, 9B12, and
8H8F5, wherein the antibody or fragment thereof inhibits
TNF-.alpha. secetion by macrophages after infection with Dengue
virus.
[0037] In a further aspect, the disclosure provides a monoclonal
antibody, or epitope-binding fragment thereof, the monoclonal
antibody selected from the group consisting of monoclonal
antibodies 3E12A2, 3E12C1, 9B12, 3E12G9, and 8H8F5. The disclosure
also provides hybridoma cells that secrete these monoclonal
antibodies, which are deposited with ______ and have the following
accession numbers ______.
[0038] The disclosure also provides pharmaceutical compositions
comprising any of the aforementioned DVLR1 monoclonal antibodies
and a pharmaceutically acceptable excipient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1A shows DNA fragments of innate immunity receptors
amplified by RT-PCR, then fractionated on 0.8% agarose and
visualized by ethidium bromide staining. FIG. 1B shows the
expressed recombinant receptor.Fc fusion proteins following
electrophoresis on a12% SDS-PAGE gel.
[0040] FIG. 2A shows a dot blot of membrane-immobilized
biotinylated GLPS F3 contacted with streptavidin-conjugated
horseradish peroxidase (HRP). FIG. 2B shows a dot blot of
membrane-immobilized non-biotinylated GLPS F3 contacted with a
Dectin-1.Fc fusion protein, followed by incubation with goat
HRP-conjugated anti IgG1 antibody. FIG. 2C shows a dot density
analysis of the blot of FIG. 2B. FIG. 2D shows the effects on dot
density of competitor .beta.-glucan on the binding of Dectin-1.Fc
to membrane-immobilized GLPS F3. FIG. 2E shows a dot blot of
immobilized GLPS F3 contacted with Dectin-1.Fc fusion protein
followed by incubation with goat HRP-conjugated anti IgG1 antibody
in the presence varying amounts of competitor polysaccharides
(.beta.-glucan, D-glucose, and D-galactose).
[0041] FIG. 3 shows a semi-quantitative analysis of dot blots of
membrane-immobilized GLPS F3 and GLPS F3C contacted with 27
different fusion proteins, each comprising the extracellular domain
of the listed innate immunity receptor coupled to IgG1 Fe.
[0042] FIG. 4A shows a dot blot of membrane-immobilized GLPS F3 and
GLPS F3C probed with the 27 fusion proteins listed in FIG. 3. FIG.
4B shows the effect of EDTA on the binding of Dectin-1.Fc,
DC-SIGNR.Fc, KCR.Fc, and TLT-2.Fc to membrane immobilized GLPS F3.
FIG. 4C shows a dot blot of membrane-immobilized .beta.-glucan
probed with Dectin-1.Fc, DC-SIGNR.Fc,.KCR.Fc, and TLT-2.Fc fusion
proteins.
[0043] FIG. 5A shows dot blots of polysaccharide samples probed
with Dectin-1.Fc, DC-SIGNR.Fc, KCR.Fc, and TLT-2.Fc fusion
proteins. FIG. 5B shows the identity of the sample numbers and
provides the dot densities of FIG. 5A in semi-quantitative
form.
[0044] FIG. 6A shows the amount of biotinylated GLPS-F3 coated onto
a microtiter plate as measured using a peroxidase-conjugated avidin
assay and reading at OD450 nm to detect the yellow-colored reaction
product. FIG. 6B depicts in graphical form the affinity of various
receptor.Fc fusion proteins for GLPS-F3 immobilized on a microtiter
plate. The absolute binding of each receptor.Fc fusion protein is
depicted on the left Y axis (as an OD450nm reading) and the right Y
axis depicts the relative binding in comparison to the binding of
Dectin-1.Fc.
[0045] FIG. 7 illustrates graphically the percentage binding of
various receptor.Fc fusion proteins to GLPS-F3 in a competition
assay with the polysaccharides mannan and .beta.-glucan, and with
the monosaccharides D-mannose (Man), D-glucose (Glc),
N-acetyl-glucosamine (GlcNAc), D-galactose (Gal),
N-acetyl-galactosamine (GalNAc), L-fucose (Fuc) and sialic
acid.
[0046] FIG. 8A shows graphically the binding of receptor.Fc fusion
proteins to Dengue Virus, in comparison to a human IgG negative
control. FIG. 8B shows a Western blot of immunocomplexes of Dengue
Virus with three receptor.Fc fusion proteins and a human IgG
negative control, probed with an antibody against the Dengue Virus
E protein. FIG. 8C shows graphically that EDTA inhibits the binding
of Dengue Virus to DC-SIGN.Fc fusion protein, but not the binding
to DVLR1.Fc fusion protein. FIG. 8D shows the binding of a DVLR1.Fc
fusion protein to Dengue Virus treated with PNGaseF, dithiothreitol
(DTT), heat, or UV irradiation, and to untreated Dengue Virus
(non).
[0047] FIG. 9A shows the expression of DVLR1 in various immune cell
types by flow cytometry using an anti-DVLR1 antibody. Expression of
DVLR1 is indicated where the DVLR1 profile (dotted line trace) does
not match the antibody isotype control (shaded area). FIG. 9B shows
the expression of DC-SIGN in various immune cell types by flow
cytometry using an anti-DC-SIGN antibody. Expression of DC-SIGN is
indicated where the DC-SIGN profile (dotted line trace) does not
match the antibody isotype control (shaded area).
[0048] FIG. 10A shows flow cytometry analysis of the expression of
NS3 protein using an anti-NS3 antibody in CD14+ macrophages
contacted with live or UV irradiated (UV-DV) Dengue Virus, in
comparison to a matched antibody isotype control (shaded area).
FIG. 10B shows graphically extracellular Dengue virus titers over
time for CD 14+ macrophages infected with Dengue Virus at different
multiplicities of infection (MOI) or with UV irradiated Dengue
Virus. FIG. 10C shows an immunoblot illustrating total DAP12 and
phosphorylated DAP12 in CD14+ macrophages infected with Dengue
virus at different MOIs. FIG. 10D shows an immunoblot illustrating
total DAP12 and phosphorylated DAP12 in CD14+ macrophages infected
with Dengue virus at different times following infection with live
Dengue virus or UV irradiated Dengue virus at MOI=5.
[0049] FIG. 11 shows an immunoblot illustrating total DAP12 and
phosphorylated DAP12 in CD14+ macrophages electroporated with
pLL3.7 vector (control) or with DVLR1-shRNA prior to infection with
Dengue virus.
[0050] FIG. 12A shows the secretion of TNF-.alpha. at 6 hours after
infection of CD14+ macrophages with live or UV-irradiated Dengue
Virus at the specified MOIs. FIG. 12B shows the secretion of
TNF-.alpha. at 12 hours after infection of CD14+ macrophages with
live or UV-irradiated Dengue Virus at the specified MOIs. FIG. 12C
shows time course measurements of the secretion of TNF-.alpha.
following infection of CD14+ macrophages.
[0051] FIG. 13A shows the expression of DC-SIGN and DVLR1 by
Western blot in CD14+ macrophages transfected with DC-SIGN-shRNA or
DVLR1-shRNA, or with vector controls (pWTSI and pLL3.7). FIG. 13B
shows flow cytometry analysis of NS3 expression (using anti-NS3
antibody) in CD14+ macrophages electroporated with DC-SIGN-shRNA,
DVLR1-shRNA, or pLL3.7 vector control prior to infection with
Dengue virus. The shaded area is an isotype control for the NS3
antibody. FIG. 13C illustrates a time course analysis of virus
titer in the supernatant of CD14+ macrophages electroporated with
DC-SIGN-shRNA, DVLR1-shRNA, or vector controls, prior to infection
with Dengue virus at t=0.
[0052] FIG. 14A shows a time course analysis of the secretion of
various cytokines by CD14+ macrophages that were electroporated
with DC-SIGN-shRNA, DVLR1-shRNA, or vector controls prior to
infection with Dengue virus at t=0. FIG. 14B shows a time course
analysis for the cytokine IFN-.alpha. under the same
conditions.
[0053] FIG. 15 shows ELISA measurements of TNF-.alpha. secreted
into culture supernatants by CD14+ macrophages infected with Dengue
virus and treated with the specified monoclonal antibody against
DVLR1 at the specified concentrations.
DETAILED DESCRIPTION
[0054] In one implementation, the disclosure provides fusion
proteins comprising a carbohydrate recognition domain of an innate
immunity receptor and a heterologous polypeptide. By innate
immunity receptor is meant: [0055] 1) receptors encoded by genes
within the leukocyte receptor complex (LRC) and LRC-related genes
on human chromosome 19, including, but not limited to, the CD66
family (CEACAM1 and PSG1), the SIGLEC family, NGK7, FCGRT, the
ILT/LILRA/LILRB (CD85) family, the LAIR family, the KIR (CD158)
family (including the KIR2DL subfamily, KIR2DS subfamily, and
KIR3DL subfamily), FCAR (CD89), NKp46 (NCR1), and GPVI (GP6); and
[0056] 2) receptors encoded by genes within the natural killer
receptor complex (NKC) on human chromosome 12, including but not
limited to MAFA-L (KLRG1), A2M, NKR-P1A (KLRB1), LLt1 (CLEC2D),
CD69 (CLEC2C), KLRF1, AICL (CLEC2B), CLEC-2 (CLECFS2), Lox-1
(OLR1), CD94 (KLRD1), NKG2-D (KLRK1), NKG2-F (KLRC4), NKG2-E
(KLRC3), NKG2-C (KLRC2), NKG2-A (KLRC1), Ly49L (KLRA1) and PRB3;
and [0057] 3) all human C-type lectin (CLEC) family genes, all
human Sialic Acid Binding Ig-Like (SIGLEC) genes, all human
Triggering Receptor Expressed on Myeloid Cells (TREM) genes, all
human TREM-like (TREML/TLT) genes, all human Toll-Like Receptor
(TLR) genes, and all human Fc Receptor-like (including FCRL1
through FCLR6, and also FCLRM1 and FCLRM2) genes found on human
chromosomes.
[0058] Additional genes within these groupings that may be used in
the methods of the disclosure may be found using the Human Genome
Organization (HUGO) search engine website. See also the locus
descriptions in Immunological Reviews 2001 Vol. 181: 20-38,
incorporated herein by reference in its entirety.
[0059] Orthologues of any of the aforementioned genes from
non-human species may be also be used in the methods of the
disclosure.
[0060] C-type lectin genes that are contemplated for use in the
present disclosure include, but are not limited to the following
human genes,: ASGR1, ASGR2 (CLEC4H2), CD207 (CLEC4K/Langerin),
CD209 (DC-SIGN/CLEC4L), CD302 (CLEC13A), CLEC1A, CLEC1B (CLEC-2),
CLEC2A, CLEC2B, CD69, CLEC2D, CLEC2L, CLEC3A, CLEC3B, CLEC3O,
CLEC3Q, CLEC4A, CLEC4C, CLEC4D (CLEC-6), CLEC4E, CLEC4F (KCLR),
CLEC4G, CLEC4M (DC-SIGNR), CD209, CLEC5A, CLEC6A (Dectin-2), CLEC7A
(Dectin-1), CLEC9A, CLEC10A, CLEC11A, CLEC12A, CLEC14A, FCER2,
KLRB1, KLRF1, LY75 (DEC205), MRC1, MRC1L1, MRC2 (Endo180), OLR1,
PLA2R1, DCAL1, and COLEC10. Homologues of any of these genes are
also contemplated, as are orthologues from other animal species
such as mice and rats. Homologues and orthologues may be 50%, 70%,
80%, 80.6%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or
99.9% identical to any of the enumerated C-type lectin genes. A
specifically contemplated orthologue is the Kupffer Cell Receptor
(mKCR) gene in mice (homologous to human CLEC4F).
[0061] TREM genes and TREML genes that are contemplated for use in
the present disclosure include, but are not limited to the
following human genes: CD300 Antigen Like Family Member B
(CD300LB), CD300 Antigen Like Family Member G (CD300LG), TREM1,
TREM2, TREML1 (TLT1), TREML2 (TLT2), TREML3 (TLT3), and TREML4
(TLT4). Homologues of any of these genes are also contemplated, as
are orthologues from other animal species such as mice and rats.
Homologues and orthologues may be 50%, 70%, 80%, 80.6%, 83%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,
99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any
of these enumerated genes. Specifically contemplated orthologues
include mTREM1, mTREM2, mTLT1, and mTLT4 from mouse.
[0062] TLR genes that are contemplated for use in the present
disclosure include, but are not limited to, the following human
genes: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10,
TLR11, TLR12, and TLR13. Homologues of any of these genes are also
contemplated, as are orthologues from other animal species such as
mice and rats. Homologues and orthologues may be 50%, 70%, 80%,
80.6%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%
identical to any of the enumerated TLR genes.
[0063] SIGLEC genes that are contemplated for use in the present
disclosure include, but are not limited to, the following human
genes: CD22, CD33, Myelin Associated Glycoprotein (MAG), SIGLEC5,
SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9, SIGLEC10, SIGLEC11, SIGLEC12,
SIGLEC13, and Sialoadhesin (SN). Homologues of any of these genes
are also contemplated, as are orthologues from other animal species
such as mice and rats. Homologues and orthologues may be 50%, 70%,
80%, 80.6%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or
99.9% identical to any of the enumerated SIGLEC genes.
[0064] Other innate immunity receptors suitable for use in the
instant disclosure include those recited in the Examples below.
[0065] The fusion protein may comprise the entire extracellular
domain of the innate immunity receptor, including a carbohydrate
recognition domain, or it may comprise a portion of the
extracellular domain, including a carbohydrate recognition domain,
or it may comprise only a carbohydrate recognition domain.
[0066] The heterologous polypeptide may comprise any polypeptide to
which a carbohydrate recognition domain of an innate immunity
receptor may be fused such that the heterologous polypeptide does
not interfere with the binding of a carbohydrate domain to its
cognate specific carbohydrate, either in vivo or in vitro.
Preferably, the heterologous polypeptide is an immunoglobulin, such
as human IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgE, IgD, IgAa, and
IgA2, or an immunoglobulin from other animal species. Preferably, a
fragment of an immunoglobulin is used as the heterologous
polypeptide, for example an Fc fragment of an IgG. In preferred
embodiments, the heterologous polypeptide is an immunoglobulin
variant that does not bind to human Fc receptors. Such variants are
well known in the art. For example, a human IgG1 Fc variant
comprising the following mutations may be used: L234A, L235E,
G237A, and P331S.
[0067] The heterologous polypeptides may further comprise one or
more finctional domains that permit the fusion polypeptide to be
immobilized on a solid support, or purified from a complex mixture.
By way of example, the heterologous polypeptide may comprise a His6
tag to permit attachment of the fusion protein to a Ni-NTA solid
support according to methods well known in the art. Also by way of
example, the heterologous polypeptide may comprise a
glutathione-S-transferase domain so that the resulting fusion
protein can be adsorbed onto, for example, glutathione beads or
glutathione derivatized microtiter plates.
[0068] The heterologous polypeptide may also comprise one or more
biotins, or biotin derivatives. In this way, fusion proteins may be
immobilized to streptavidin-conjugated solid supports, or a
streptavidin-conjugated enzyme may be bound to the fusion
protein.
[0069] The fusion protein may optionally further comprise a linker
between the heterologous polypeptide and a carbohydrate recognition
domain of the innate immunity receptor. The linker may be a peptide
linker, or it may be a non-peptidic linker, such as a polyethylene
glycol.
[0070] The carbohydrate recognition domain may be C-terminal
relative to the heterologous polypeptide or it may be N-terminal
relative to the heterologous polypeptide in the fusion protein.
[0071] The fusion proteins of the disclosure may be prepared by any
method known in the art for the production of proteins. Preferably,
the fusion proteins are prepared using recombinant DNA technology
and protein expression technology well known in the art. For
example, DNA encoding the carbohydrate recognition domain of an
innate immunity receptor may be obtained by reverse-transcriptase
PCR (RT-PCR) of mRNA using primers specific for the carbohydrate
recognition domain of the particular innate immunity receptor of
interest. The resulting DNA may then be cloned into an expression
vector in frame with DNA encoding the heterologous polypeptide
sequence. Expression vectors useful in the present disclosure
typically contain an origin of replication, a promoter located 5'
to (i.e., upstream of) and followed by the DNA sequence coding for
the fusion protein, transcription termination sequence, and the
remaining vector. The expression vectors may also include other DNA
sequence known in the art, for example, stability leader sequences
that provide for stability of the expression product, secretory
leader sequences which provide for secretion of the expression
product, and sequences which allow expression of the fusion protein
to be modulated or induced. The expression vector may also contain
viral sequences that allow the fusion protein to be expressed using
a viral expression system, such as the baculovirus expression
system well known in the art. The expression vector may be
introduced into host cells, such as microbial cells, yeast cells,
mammalian cells, or insect cells. The expression vector may be
introduced into cells as naked DNA, or it may be encapsulated
within a virus (such as a baculovirus). The expression vector may
be maintained within the host cell, or it may integrate into the
host cell genome.
[0072] Preferably, the expression vector comprises DNA sequence
that lead to the addition of a secretory leader sequence on the
fusion protein, thereby causing the fusion protein to be secreted
into the medium surrounding the host cells. The fusion protein can
then be purified from the medium using techniques known in the art.
By way of example, if the fusion protein comprises IgG as the
heterologous polypeptide, then a Protein A column may be used to
bind to the fusion protein to permit the fusion protein to be
partitioned from other proteins in the surrounding medium.
[0073] Fusion proteins may also be produced by in vitro translation
of a mRNA encoding the fusion protein using an in vitro expression
system, such as a Xenopus oocyte expression system.
[0074] In an embodiment, the fusion proteins are produced
separately and then coupled to one another using chemical
techniques known in the art. For example, the carbohydrate
recognition domain and the heterologous polypeptide may be produced
separately and then coupled to one another using
glutaraldehyde.
[0075] Following production of the fusion protein, the fusion
protein may be labeled with a detectable label, such as a
fluorophore, radiolabel, an enzyme, an enzyme substrate, a dye, a
chemiluminescent agent, a magnetic particle, a quantum dot, or any
other moiety that produces, directly or indirectly, a detectable
signal. Many methods for the conjugation of such detectable labels
to proteins are known in the art. By way of example only, an
N-hydroxysuccinimide-activated dye, most preferably an
N-hydroxysuccinimide-activated fluorophore, may be conjugated to
the fusion protein by reaction with primary amines on the fusion
protein.
[0076] In some embodiments, the fusion protein is biotinylated
using methods known in the art such that the fusion protein
comprises one or more biotins, or one or more biotin derivatives.
In this way, the fusion protein may be attached to a
streptavidin-detectable moiety conjugate, such as an
enzyme-streptavidin conjugate.
[0077] In one series of embodiments, the fusion proteins of the
disclosure are used to determine whether a specific carbohydrate
component is present in a composition that comprises a
polysaccharide. The methods involve contacting the polysaccharide
with a fusion protein that binds to a specific carbohydrate
component of a polysaccharide, and then determining whether the
fusion protein has bound to polysaccharide in the composition. For
example, it is known that the carbohydrate recognition domain of
CLEC7A (also known as Dectin-1), can interact with
.beta.-1,3-D-glycans (see Brown, G. D. and Gordon, S., 2001, Nature
413, 36-7, incorporated herein by reference in its entirety).
Binding of a fusion protein comprising the carbohydrate recognition
domain of CLEC7A to a polysaccharide composition therefore
indicates that the polysaccharide composition comprises .beta.-1,3
glucan. Similarly, since the rodent Kupffer cell receptor (KCR;
homologous to human CLEC4F) has high affinity to D-galactose and
N-acetylgalactosamine, and is able to clear D-galactose and
D-fucose terminated glycoproteins from serum (see Fadden, A. J.,
Holt, O. J. and Drickamer, K. (2003), Glycobiology 13, 529-37,
incorporated herein by reference in its entirety), binding of a
fusion protein comprising the carbohydrate recognition domain of
KCR to a polysaccharide composition therefore indicates that the
polysaccharide composition comprises D-galactose and/or
N-acetylgalactosamine and/or D-galactose terminated glycoproteins
and/or D-fucose terminated glycoproteins. In addition, CD209 (also
known as DC-SIGN and CLEC4L) and CLEC4M (also known as DC-SIGNR and
L-SIGN) can both bind to Man.sub.9GlcNAc.sub.2Asn glycopeptide, but
only CD209 and not CLEC4M can bind to glycans with a terminal
fucose residue (see Guo et al (2004) Nat Struct Mol Biol 11,
591-8); therefore, fusion proteins of CD209 and CLEC4M can
discriminate between polysaccharide compositions comprising these
carbohydrate components. The methods and reagents of the disclosure
may therefore be used to determine the identity of the carbohydrate
components of a polysaccharide composition and to determine the
relative amounts of those carbohydrate components e.g. to
"fingerprint" a polysaccharide composition. For example, the
methods and reagents of the disclosure may be used to determine the
carbohydrate components of a polysaccharide composition that has
immunomodulatory activity.
[0078] In addition, if one knows the identity of the cells that
express the innate immunity receptors from which the carbohydrate
recognition domain of the fusion protein is derived, then the
assays disclosed herein reveal the identity of the cells in the
body that bind to the polysaccharide under investigation. Such
knowledge, for example, can help reveal the mechanism by which a
particular polysaccharide composition (such as polysaccharides
isolated from Ganoderma lucidum) exerts beneficial or deleterious
effects on an organism which comes into contact with the
polysaccharide. It is not necessary to know the identity of the
carbohydrate component bound by the carbohydrate recognition domain
in this embodiment.
[0079] The binding of the fusion proteins of the disclosure to
their cognate carbohydrate component can be performed by
immobilizing the composition comprising the polysaccharide to a
solid support, and then contacting the solid support with a fusion
protein. Binding of the fusion protein may be detected by detecting
the presence of the fusion protein on the surface of the solid
support, for example, by detecting the presence of the heterologous
polypeptide on the surface of the solid support or by detecting the
presence of the carbohydrate recognition domain on the surface of
the solid support. For example, if the heterologous polypeptide is
conjugated to a fluorophore, then the presence of the fluorophore,
following washing, on the surface of the solid support is
indicative of the presence of the fusion protein, which in turn is
indicative of the presence of a polysaccharide comprising the
specific carbohydrate component recognized by the carbohydrate
recognition domain of the fusion protein.
[0080] As used herein, "solid support" is defined as any surface to
which molecules may be attached through either covalent or
non-covalent bonds. This includes, but is not limited to, membranes
(for example, polyvinylidene fluoride (PVDF) membranes), plastics
(for example, microtiter plates), paramagnetic beads, charged
paper, nylon, Langmuir-Bodgett films, functionalized glass,
germanium, silicon, PTFE, polystyrene, gallium arsenide, gold and
silver. Any other material known in the art that is capable of
having functional groups such as amino, carboxyl, thiol or hydroxyl
incorporated on its surface, is also contemplated. This includes
surfaces with any topology, including, but not limited to,
spherical surfaces, grooved surfaces, and cylindrical surfaces
e.g., columns.
[0081] The composition comprising a polysaccharide (also referred
to herein as a "polysaccharide composition") can be, without
limitation, any composition that includes a polysaccharide
including, for example, a glycoprotein (including a proteoglycan),
a glycolipid, peptidoglycan, a microbial cell wall, a viral
particle, and a fungal cell wall. In other embodiments, the
composition comprising a polysaccharide is a polysaccharide free in
solution e.g. a polysaccharide that is not attached to a protein or
lipid. As used herein, a "polysaccharide" means a carbohydrate
molecule that comprises two or more monosaccharides.
[0082] Immobilization of the composition comprising a
polysaccharide on a solid support may be achieved, for example, by
biotinylating the polysaccharides in the composition, and then
immobilizing on a streptavidin-conjugated solid support. In
addition, polysaccharides may be immobilized on, for example,
methanol-activated PVDF membranes. It is specifically contemplated
that the methods of the disclosure can be performed in a "dot blot"
format using dots of polysaccharide immobilized on a PVDF
membrane.
[0083] In some embodiments, binding of the fusion protein to an
immobilized polysaccharide is detected by binding a secondary
reagent to the fusion protein, preferably to the heterologous
polypeptide, and then detecting the presence of the secondary
reagent. For example, a biotinylated fusion protein may be attached
to a streptavidin-conjugated enzyme, and the presence of the enzyme
detected by adding a substrate that yields a detectable product. A
non-biotinylated fusion protein may be detected using, for example,
an antibody that binds to the heterologous polypeptide (such as an
anti-IgG antibody if the heterologous polypeptide is IgG, or a IgG
Fc), which secondary antibody is conjugated to an enzyme. For
example, if the enzyme is horseradish peroxidase (HRP), then
detection of fusion protein binding may be performed using the
Enhanced Chemiluminescence (ECL) technique well known in the art.
The secondary reagent may also, or alternatively, be conjugated to
a detectable label such as a fluorophore or a radionuclide. Many
other techniques are known in the art which may be used to detect
the binding of the disclosed fusion proteins to a solid
support.
[0084] It is specifically contemplated that the aforementioned
assays may be carried out in a multiplexed array format. For
example, a solid support may be partitioned into a plurality of
spatially discrete addresses onto which a plurality of different
compositions may be bound. Then the solid support may be contacted
with a fusion protein, and the binding of the fusion protein
detected. In this way, it can be determined which, if any, of the
immobilized polysaccharide compositions comprises the particular
carbohydrate component bound by the carbohydrate recognition domain
of the fusion protein.
[0085] In another embodiment, a single composition is immobilized
on a solid support which is partitioned into a plurality of
spatially discrete addresses. Each address is then contacted with a
different fusion protein, each different fusion protein comprising
a different carbohydrate recognition domain. Following washing to
remove non-specifically bound material, binding of the fusion
proteins may then be detected as described above; the spatial
address of each binding reaction detected reveals the identity of
the fusion protein that has bound. In this way, the composition can
be probed with a number of different fusion proteins in parallel.
In this embodiment, each fusion protein may comprise the same
heterologous polypeptide, thereby allowing a single secondary
reagent to simultaneously detect binding at each address. For
example, if each fusion protein comprises IgG Fe as the
heterologous polypeptide, then either an anti-IgG antibody, or
Protein A, or Protein G, may be used to detect binding of the
fusion protein.
[0086] The fusion proteins and methods of the disclosure may be
used to "fingerprint" any composition which comprises
polysaccharides, including, but not limited to, polysaccharide
compositions obtained from herbal preparations, such as
polysaccharide-containing fractions isolated from the fungi Reishi
(Ganoderma lucidim), Cordyceps sinensis, and Lentinus edodes; and
from the plant Dendrobium huoshanense. In particular, it is
specifically contemplated that the methods used herein are used to
determine the carbohydrate components of the F3 polysaccharide
fraction of Reishi polysaccharide (see Wang, et al (2002) Bioorg
Med Chem 10, 1057-62; Chen, et al (2004) Bioorg Med Chem 12,
5595-601; Chien,et al (2004) Bioorg Med Chem 12, 5603-9.; and Hsu
et al (2004) J Immunol 173, 5989-99, each of which is specifically
incorporated herein by reference in its entirety).
[0087] The methods provided herein can be used to "fingerprint"
complex mixtures that include a number of different polysaccharide
compositions, or they can be used on preparations that contain only
a single polysaccharide species e.g. a single glycoprotein or a
single polysaccharide.
[0088] If one knows the identity of the cells that express the
innate immunity receptor from which the carbohydrate recognition
domain is derived, then the aforementioned assays reveal which
cells in the body bind to the polysaccharide upon introduction of
the polysaccharide composition into the body. It is then possible
to obtain agents that modulate the activity of the identified
innate immunity receptor. For example, agents that mimic the
structure of the polysaccharide or that potentiate the interaction
of the polysaccharide with the innate immunity receptor may be
generated if interaction of the innate immunity receptor with the
polysaccharide leads to beneficial effects in the body. See the
section below entitled "Modulators."
[0089] In another series of embodiments, the methods and fusion
proteins of the disclosure are used to determine the identity of
polysaccharides displayed on the surface of a pathogen, such as a
fungal cell, a bacterial cell, or a virus, such as an enveloped
virus, including but not limited to influenza virus, and also
including but not limited to viruses from the Flaviviridae family.
Flaviviridae viruses suitable for use in the methods disclosed
herein include, but are not limited to, members of the genus
Flavivirus (such as, for example, Dengue virus, West Nile Virus,
Japanese encephamyelitis virus (JEV), yellow fever virus, and
tick-borne encephamyelitis virus) and members of the genus
Hepacivirus (such as, for example, Hepatitis C virus). In one such
embodiment, a fusion protein is immobilized on a solid support (for
example, using a Protein A derivatized solid support if the
heterologous polypeptide is IgG or a fragment thereof), and the
solid support is this contacted with a composition comprising the
pathogen of interest. Following washing, the binding of the
pathogen is then detected using, for example, a secondary reagent
that binds specifically to the pathogen in a manner that does not
compete with the binding of the fusion protein. For example, a
secondary antibody that is specific for the pathogen may be used.
Binding of the secondary reagent is then detected as described
above (for example using HRP-conjugated secondary antibody), or it
may be detected using a tertiary reagent that binds to the
secondary reagent (for example, using an anti-IgG antibody
conjugated to HRP if the secondary reagent is an anti-pathogen
IgG). If binding of the secondary reagent is detected, then this
reveals that the pathogen comprises a polysaccharide that comprises
the specific carbohydrate component recognized by the carbohydrate
recognition domain of the fusion protein.
[0090] Alternatively, the assay may be performed by immobilizing a
reagent that binds specifically to the pathogen on a solid support.
For example, an antibody which binds to the pathogen can be
immobilized on a solid support, then contacted with a composition
comprising the pathogen. The solid support is then contacted with
the fusion protein(s), and the binding of the fusion proteins is
then detected as described above (preferably, the fusion protein
does not compete for pathogen binding with the immobilized
reagent). For example, if the heterologous polypeptide of the
fusion protein is IgG Fc, then an anti-IgG antibody can detect
binding of the fusion protein to the pathogen; alternatively, if
the fusion protein is conjugated to a detectable label, then
detection of the label is used to detect binding.
[0091] It is expressly contemplated that the aforementioned
pathogen assays can be carried out in a multiplexed format using,
for example, a plurality of different fusion proteins
simultaneously. For example, an antibody that binds to the pathogen
may be immobilized at a plurality of discrete addresses on a solid
support; then the solid support is contacted with a composition
comprising the pathogen; and then each specific address is
contacted with a different fusion protein, each different fusion
protein comprising a different carbohydrate recognition domain. If
each fusion protein comprises the same heterologous polypeptide,
then binding of the fusion protein may be detected using a single
reagent that binds to the heterologous polypeptide. For example, if
the heterologous polypeptide is IgG Fc, then an anti-IgG antibody
can be used to detect binding of the fusion protein(s). The spatial
address of each binding reaction then reveals the identity of the
fusion protein. Alternatively, a multiplexed assay may be carried
out using a plurality of different fusion proteins immobilized on
the solid support at spatially discrete addresses, by contacting
the solid support with the composition comprising the pathogen,
followed by contacting the solid support with a secondary reagent
that binds specifically to the pathogen. For example, if the
pathogen is Dengue virus, then the secondary reagent may be an
antibody against the E envelope protein. As in all the preceding
assays, washing may be used to remove non-specifically bound
material from the solid support.
[0092] Using the methods disclosed herein, it has been discovered
that Dengue virus binds to DVLR1/MDL-1 on the surface of CD14+
macrophages. See Example 11. It has further been shown that
DVLR1/MDL-1 binding to Dengue virus results in the activation of
DAP12, which in turn leads to the release of the proinflammatory
cytokines TNF-.alpha., MIP-1.alpha., IFN-.alpha., and IL-8 from
macrophages. See Example 12. The release of these cytokines is
implicated in the development of Dengue hemorrhagic fever (DHF) and
Dengue shock syndrome (DSS).
[0093] Knowledge of the identity of the innate immunity receptor(s)
that interact with a pathogen may then be used to develop agents
that modulate the activity of the innate immunity receptor. For
example, modulators that activate an identified innate immunity
receptors may be obtained in order to augment the immune response
to a particular pathogen. In cases where interaction of an innate
immunity receptor to a particular polysaccharide composition is
detrimental to the body (for example, when a pathogen causes
excessive inflammation), modulators may be obtained that reduce the
activity of the innate immunity receptors. For example, agents
(such as antibodies) that block the binding of a pathogen to an
innate immunity receptor may be used to prevent the occurrence of
an undesirable proinflammatory reaction to infection with said
pathogen. Similarly, if the screening methods disclosed herein
reveal that a particular pathogen (such as a virus) uses an innate
immunity receptor to gain entry into a cell, then an agent that
blocks the binding of the pathogen to the innate immunity receptor
will prevent entry of the pathogen into the cell.
[0094] In another series of embodiments, the fusion proteins of the
disclosure are used to disrupt or prevent the interaction between a
polysaccharide and an innate immunity receptor on a cell surface.
In this series of embodiments, the fusion protein comprises the
carbohydrate recognition domain of the innate immunity receptor
that is expressed on the cell surface. The cell expressing the
innate immunity receptor is then contacted with the fusion protein,
either in vivo or in vitro, whereby the fusion protein competes
with the polysaccharide for binding to the innate immunity
receptor.
[0095] If interaction of the polysaccharide with the innate
immunity receptor on the cell surface leads to deleterious effects
in an organism, then a therapeutically effective amount of the
fusion protein may be administered to the organism in a
pharmaceutical composition to prevent or diminish the interaction.
Preferably, the heterologous polypeptide of the administered fusion
protein does not bind to an any cell surface receptor. For example,
the heterologous polypeptide may be comprised of a mutated variant
of IgG Fc that does not bind to Fc receptors on cell surfaces.
Purification
[0096] In another series of embodiments, the fusion proteins are
used to at least partially purify or isolate polysaccharides that
comprise the specific carbohydrate component recognized by the
carbohydrate recognition domain of the fusion protein. For example,
the fusion protein may be immobilized on a solid support, and a
composition suspected of containing, or known to contain, a
polysaccharide composition is contacted with the solid support. If
the composition comprises a polysaccharide that can bind to the
carbohydrate recognition domain of the fusion protein, then that
polysaccharide will bind to the fusion protein. The solid support
can then be washed to remove non-specifically bound components of
the composition, and the bound polysaccharide may then be eluted by
dissociating the interaction with the fusion protein, and
collected. For example, if the fusion protein comprises the
carbohydrate recognition domain of a lectin receptor, then the
interaction may be dissociated using EDTA to chelate Ca.sup.2+. In
this way, it is possible to purify specific polysaccharide
compositions from complex mixtures. In preferred embodiments, this
method is used to purify polysaccharides isolated from Ganoderma
lucidum (Reishi).
[0097] For the aforementioned purification method, the solid
support may comprise, for example, a column to which the fusion
protein is bound. Suitable columns include Sepharose Protein A
columns, to which fusion proteins comprising IgG as the
heterologous polypeptide may be bound via interaction with of the
IgG domain of the fusion protein with Protein A. Alternatively,
CNBr activated column media may be bound to fusion proteins.
[0098] The present disclosure also provides kits that can be used
in any of the above methods. In one embodiment, a kit comprises a
fusion protein according to the disclosure, in one or more
containers. The kit may also comprise a secondary reagent, such as
an antibody that specifically binds to the heterologous polypeptide
domain of the fusion protein e.g. an anti-IgG antibody if the
heterologous polypeptide is IgG, or a fragment thereof. The kit may
also comprise reagents and buffers for detecting the binding of a
fusion protein to a polysaccharide. For example, in embodiments
where a HRP-conjugated secondary antibody is used to detect the
binding of a fusion protein to a polysaccharide, the kit may
comprise the reagents necessary to establish an enhanced
chemiluminescence reaction e.g. one or more containers comprising
luminol, p-coumaric acid, Tris buffer, and hydrogen peroxide. The
kit may also comprise one or more positive control polysaccharides.
The kit may also comprise one or more solid supports for use in the
aforementioned methods, for example, one or more PVDF membranes or
one or more multiwell microtiter plates.
Modulators
[0099] As described above, the methods of the disclosure identify
innate immunity receptor(s) that interact with a particular
polysaccharide. This information then allows one to obtain
modulators of the identified innate immunity receptor. A modulator
can be an agonist, an antagonist (including competitive and
non-competitive antagonists), or an inverse agonist of an innate
immunity receptor. A modulator may, without limitation: inhibit the
binding of a polysaccharide to an innate immunity receptor;
potentiate the binding of a polysaccharide to an innate immunity
receptor; or function as a mimetic of a polysaccharide that binds
to an innate immunity receptor, thereby activating the innate
immunity receptor even in the absence of the polysaccharide.
[0100] Modulators of innate immunity receptors include antibodies.
For example, an antagonistic antibody against an innate immunity
receptor can prevent binding of a pathogen to the innate immunity
receptor. In some cases, such an antibody is a neutralizing
antibody as it prevents the entry of the pathogen into the cell
that expresses the innate immunity receptor. Alternatively, an
agonistic antibody may function as a mimetic of a polysaccharide
composition that exerts a beneficial effect on a cell. An
antagonistic antibody may also bind to an innate immunity receptor
in such a way as to block the downstream signaling by the receptor
upon pathogen binding. Antibodies may be, without limitation,
polyclonal, monoclonal, monovalent, bispecific, heteroconjugate,
multispecific, human, humanized or chimeric antibodies, single
chain antibodies, Fab fragments, F(ab') fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies, and epitope-binding fragments of any of the above. The
term "antibody," as used herein, refers to immunoglobulin molecules
and immunologically active portions of immunoglobulin molecules,
i.e., molecules that contain an antigen binding site that
immunospecifically binds an antigen. The immunoglobulin molecules
can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of
immunoglobulin molecule. Moreover, the term "antibody" (Ab) or
"monoclonal antibody" (Mab) is meant to include intact molecules,
as well as, antibody fragments (such as, for example, Fab and
F(ab')2 fragments) which are capable of specifically binding to
protein. Fab and F(ab')2 fragments lack the Fe fragment of intact
antibody, clear more rapidly from the circulation of the animal or
plant, and may have less non-specific tissue binding than an intact
antibody (Wahl et al., J. Nucl. Med. 24: 316-325 (1983)). Methods
for producing antibody agonists are described in, for example, PCT
publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al.,
Blood 92 (6): 1981-1988 (1998); Chen et al., Cancer Res. 58 (16):
3668-3678 (1998); Harrop et al., J. Immunol. 161 (4): 1786-1794
(1998); Zhu et al., Cancer Res. 58 (15): 3209-3214 (1998); Yoon et
al., J. Immunol. 160 (7): 3170-3179 (1998); Prat et al., J. Cell.
Sci. 111 (Pt2): 237-247 (1998); Pitard et al., J. Immunol. Methods
205 (2): 177-190 (1997); Liautard et al., Cytokine 9 (4): 233-241
(1997); Carlson et al., J. Biol. Chem. 272 (17): 11295-11301
(1997); Taryman et al., Neuron 14 (4): 755-762 (1995); Muller et
al., Structure 6 (9): 1153-1167 (1998); Bartunek et al., Cytokine 8
(1): 14-20 (1996); Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (which are all incorporated by reference
herein in their entireties).
[0101] The disclosure provides non-limiting specific examples of
anti-DVLR1 monoclonal antibodies that prevent TNF-.alpha. release
from macrophages following DV infection. See Example 15. These
antibodies can be used in the pharmaceutical compositions and the
methods of treatment specified herein, particularly in compositions
and methods for the treatment or prophylaxis of DV infection in
humans.
[0102] Modulators of innate immunity receptors also include small
molecules identified by high throughput screening methods. Such
high throughput screening methods typically involve providing a
combinatorial chemical or peptide library containing a large number
of potential therapeutic compounds (e.g., ligand or modulator
compounds). Such combinatorial chemical libraries or ligand
libraries are then screened in one or more assays to identify those
library members (e.g., particular chemical species or subclasses)
that bind to the innate immunity receptor of interest. The
compounds so identified can serve as conventional lead compounds,
or can themselves be used as potential or actual therapeutics.
[0103] A combinatorial chemical library is a collection of diverse
chemical compounds generated either by chemical synthesis or
biological synthesis, by combining a number of chemical building
blocks (i.e., reagents such as amino acids). As an example, a
linear combinatorial library, e.g., a polypeptide or peptide
library, is formed by combining a set of chemical building blocks
in every possible way for a given compound length (i.e., the number
of amino acids in a polypeptide or peptide compound). Millions of
chemical compounds can be synthesized through such combinatorial
mixing of chemical building blocks.
[0104] The preparation and screening of combinatorial chemical
libraries is well known to those having skill in the pertinent art.
Combinatorial libraries include, without limitation, peptide
libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991, Int. J. Pept.
Prot. Res., 37: 487-493; and Houghton et al., 1991, Nature, 354:
84-88). Other chemistries for generating chemical diversity
libraries can also be used. Nonlimiting examples of chemical
diversity library chemistries include, peptides (PCT Publication
No. WO 91/019735), encoded peptides (PCT Publication No. WO
93/20242), random bio-oligomers (PCT Publication No. WO 92/00091),
benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as
hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993,
Proc. Natl. Acad. Sci. USA, 90: 6909-6913), vinylogous polypeptides
(Hagihara et al., 1992, J. Amer. Chem. Soc., 114: 6568),
nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et
al., 1992, J. Amer. Chem. Soc., 114: 9217-9218), analogous organic
synthesis of small compound libraries (Chen et al., 1994, J. Amer.
Chem. Soc., 116: 2661), oligocarbamates (Cho et al., 1993, Science,
261: 1303), and/or peptidyl phosphonates (Campbell et al., 1994, J.
Org. Chem., 59: 658), nucleic acid libraries (for example, see U.S.
Pat. No. 5,270,163 describing the generation of nucleic acid
ligands, also known as "aptamers"), peptide nucleic acid libraries
(U.S. Pat. No. 5,539,083), antibody libraries (e.g., Vaughn et al.,
1996, Nature Biotechnology, 14 (3): 309-314) and PCT1JS96/10287),
carbohydrate libraries (e.g., Liang et al., 1996, Science,
274-1520-1522) and U.S. Pat. No. 5,593,853), small organic molecule
libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,
page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.
5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506,337; and the like).
[0105] Devices for the preparation of combinatorial libraries are
commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech,
Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied
Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford,
Mass.). In addition, a large number of combinatorial libraries are
commercially available (e.g., ComGenex, Princeton, N.J.; Asinex,
Moscow, Russia; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd.,
Moscow, Russia; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences,
Columbia, Md., and the like).
Pharmaceutical Compositions
[0106] The instant disclosure also provides pharmaceutical
compositions. In some embodiments, the pharmaceutical compositions
comprise the fusion proteins of the disclosure. In other
embodiments, the pharmaceutical compositions comprise a modulator
of an innate immunity receptor (for example antibodies against an
innate immunity receptor such as DVLR1, including the antibodies
exemplified in Example 15). In such pharmaceutical compositions,
the fusion protein or the innate immunity receptor modulator form
the "active compound." In some embodiment, the pharmaceutical
compositions are administered to a subject in order to treat or
prevent diseases or disorders characterized by the binding of a
polysaccharide to an innate immunity receptor on the surface of a
cell in that subject. In other embodiments, the pharmaceutical
compositions are administered to a subject to activate an innate
immunity receptor in circumstances where increasing the activity of
that receptor is beneficial to the subject. In still other
embodiments, the pharmaceutical compositions are administered to a
subject to potentiate the binding of a polysaccharide composition
to an innate immunity receptor.
[0107] In addition to active compound, the pharmaceutical
compositions preferably comprise at least one pharmaceutically
acceptable carrier. As used herein the language "pharmaceutically
acceptable carrier" includes solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical
administration. Supplementary active compounds can also be
incorporated into the compositions. A pharmaceutical composition is
formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral
(e.g., inhalation), transdermal (topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0108] Subject as used herein refers to humans and non-human
primates (e.g. guerilla, macaque, marmoset), livestock animals
(e.g. sheep, cow, horse, donkey, pig), companion animals (e.g. dog,
cat), laboratory test animals (e.g. mouse, rabbit, rat, guinea pig,
hamster), captive wild animals (e.g. fox, deer) and any other
organisms who can benefit from the agents of the present
disclosure. There is no limitation on the type of animal that could
benefit from the presently described agents. The most preferred
subject of the present disclosure is a human. A subject regardless
of whether it is a human or non-human organism may be referred to
as a patient, individual, animal, host or recipient.
[0109] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0110] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0111] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0112] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0113] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmueosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0114] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0115] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to
cell-specific antigens) can also be used as pharmaceutically
acceptable carriers. These can be prepared according to methods
known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811.
[0116] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
[0117] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
high therapeutic indices are preferred. While compounds that
exhibit toxic side effects can be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0118] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
subjects. The dosage of such compounds lies preferably within a
range of circulating concentrations that include the ED50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
disclosure, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in subjects. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
[0119] As defined herein, a therapeutically effective amount of an
active compound of the disclosure may range from about 0.001 to 30
mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight,
more preferably about 0.1 to 20 mg/kg body weight, and even more
preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7
mg/kg, or 5 to 6 mg/kg body weight. Without limitation, the active
compound can be administered between one time per week and three or
more times per day, for between about 1 to 10 weeks, preferably
between 2 to 8 weeks, more preferably between about 3 to 7 weeks,
and even more preferably for about 4, 5, or 6 weeks. The skilled
artisan will appreciate that certain factors can influence the
dosage and timing required to effectively treat a subject,
including but not limited to the severity of the disease or
disorder, previous treatments, the general health and/or age of the
subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a pharmaceutical
composition of the disclosure can include a single treatment or,
preferably, can include a series of treatments.
Gene Therapy and RNAi
[0120] Constructs encoding the fusion proteins of the disclosure
can be used as a part of a gene therapy protocol to deliver
therapeutically effective doses of a receptor fusion protein to a
subject. A preferred approach for in vivo introduction of nucleic
acid into a cell is by use of a viral vector containing nucleic
acid, encoding a fusion protein of the disclosure. Infection of
cells with a viral vector has the advantage that a large proportion
of the targeted cells can receive the nucleic acid. Additionally,
molecules encoded within the viral vector, e.g., by a cDNA
contained in the viral vector, are expressed efficiently in cells
which have taken up viral vector nucleic acid.
[0121] Retrovirus vectors and adeno-associated virus vectors can be
used as a recombinant gene delivery system for the transfer of
exogenous nucleic acid molecules encoding fusion proteins in vivo.
These vectors provide efficient delivery of nucleic acids into
cells, and the transferred nucleic acids are stably integrated into
the chromosomal DNA of the host. The development of specialized
cell lines (termed "packaging cells") which produce only
replication-defective retroviruses has increased the utility of
retroviruses for gene therapy, and defective retroviruses are
characterized for use in gene transfer for gene therapy purposes
(for a review see Miller, A. D. (1990) Blood 76:27 1). A
replication defective retrovirus can be packaged into virions which
can be used to infect a target cell through the use of a helper
virus by standard techniques. Protocols for producing recombinant
retroviruses and for infecting cells in vitro or in vivo with such
viruses can be found in Current Protocols in Molecular Biology,
Ausubel, F. M. et al., (eds.) Greene Publishing Associates, (1989),
Sections 9.10-9.14 and other standard laboratory manuals.
[0122] Another useful viral gene delivery system uses
adenovirus-derived vectors. The genome of an adenovirus can be
manipulated such that it encodes and expresses a gene product of
interest but is inactivated in terms of its ability to replicate in
a normal lytic viral life cycle. See, for example, Berkner et al.,
BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434
(1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitable
adenoviral vectors derived from the adenovirus strain Ad type 5
d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are
known to those skilled in the art. Recombinant adenoviruses can be
advantageous in certain circumstances in that they are not capable
of infecting nondividing cells and can be used to infect a wide
variety of cell types, including epithelial cells (Rosenfeld et
al., (1992) cited supra). Furthermore, the virus particle is
relatively stable and amenable to purification and concentration,
and as above, can be modified so as to affect the spectrum of
infectivity. Additionally, introduced adenoviral DNA (and foreign
DNA contained therein) is not integrated, into the genome of a host
cell but remains episomal, thereby avoiding potential problems that
can occur as a result of insertional mutagenesis in situations
where introduced DNA becomes integrated into the host genome (e.g.,
retroviral DNA). Moreover, the carrying capacity of the adenoviral
genome for foreign DNA is large (up to 8 kilobases) relative to
other gene delivery vectors (Berkner et al., cited supra;
Haj-Ahmand et al., J. Virol. 57:267 (1986)).
[0123] In another embodiment, non-viral gene delivery systems of
the present disclosure rely on endocytic pathways for the uptake of
the subject nucleotide molecule by the targeted cell. Exemplary
gene delivery systems of this type include liposomal derived
systems, poly-lysine conjugates, and artificial viral envelopes. In
a representative embodiment, a nucleic acid molecule encoding a
fusion protein of the disclosure can be entrapped in liposomes
bearing positive charges on their surface (e.g., lipofectins) and
(optionally) which are tagged with antibodies against cell surface
antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka
20:547-551; PCT publication WO91/06309; Japanese patent application
1047381; and European patent publication EP-A43075).
[0124] Gene delivery systems for a gene encoding a fusion protein
of the disclosure can be introduced into a subject by any of a
number of methods. For instance, a pharmaceutical preparation of
the gene delivery system can be introduced systemically, e.g. by
intravenous injection, and specific transduction of the protein in
the target cells occurs predominantly from specificity of
transfection provided by the gene delivery vehicle, cell-type or
tissue-type expression due to the transcriptional regulatory
sequences controlling expression of the receptor gene, or a
combination thereof. In other embodiments, initial delivery of the
recombinant gene is more limited with introduction into the animal
being quite localized. For example, the gene delivery vehicle can
be introduced by catheter (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3
054-3057). The pharmaceutical preparation of the gene therapy
construct can consist essentially of the gene delivery system in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Where the fusion protein can
be produced intact from recombinant cells, e.g. retroviral vectors,
the pharmaceutical preparation can comprise one or more cells which
produce the fusion protein.
[0125] In another embodiment, the expression of an innate immunity
receptor that is identified according to the methods disclosed
herein as being involved in the pathogenesis is reduced or
completely inhibited using RNA interference (RNAi). RNAi is well
known in the art and may be accomplished using small interfering
RNA (siRNA). siRNAs according to the invention could have up to 29
bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any
integer thereabout or therebetween. Such siRNAs can be
administered, e.g., in a form encoded by a vector (for example, a
vector encoding a small hairpin RNA (shRNA)) or as a liposome
nucleic acid complex. The preparation of lipid:nucleic acid
complexes, including targeted liposomes such as immunolipid
complexes, is well known to one of skill in the art (see, e.g.,
Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene
Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389
(1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et
al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.
52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344,
4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028,
and 4,946,787). Accordingly, the present disclosure also provides
pharmaceutical compositions comprising RNA molecules that are
capable of mediating RNA interference of an innate immunity
receptor when administered to a subject.
[0126] The present disclosure provides a non-limiting example of
the RNAi-mediated "knock down" of the DVLR1 gene in macrophages.
The attenuation of DVLR1 in this manner significantly reduces the
secretion of proinflammatory cytokines in DV-infected macrophages,
thereby indicating that RNAi-mediated attenuation of DVLR1 will be
useful for the treatment of DV.
[0127] It is specifically contemplated that siRNA or shRNA that
attenuates expression of DVLR1 is used for the RNAi-mediated
treatment of subjects infected with Dengue virus. Methods for
designing, synthesizing, and administering shRNA and siRNA in order
to attenuate the expression of a specific gene are well known in
the art and are described in, for example, U.S. Pat. No. 7,022,828.
Non-limiting examples of agents suitable for formulation with the
shRNA constructs and siRNA molecules of the disclosure include: PEG
conjugated nucleic acids, phospholipid conjugated nucleic acids,
nucleic acids containing lipophilic moieties, phosphorothioates,
P-glycoprotein inhibitors (such as Pluronic P85) which can enhance
entry of drugs into various tissues, for example the CNS
(Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13,
16 26); biodegradable polymers, such as poly
(DL-lactide-coglycolide) microspheres for sustained release
delivery after implantation (Emerich, DF et al, 1999, Cell
Transplant, 8, 47 58) Alkermes, Inc. Cambridge, Mass.; and loaded
nanoparticles, such as those made of polybutylcyanoacrylate, which
can deliver drugs across the blood brain barrier and can alter
neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol
Psychiatry, 23, 941 949, 1999). Other non-limiting examples of
delivery strategies, including CNS delivery of the nucleic acid
molecules of the instant disclosure include material described in
Boado et al., 1998, J. Pharm. Sci., 87, 1308 1315; Tyler et al,
1999, FEBS Lett., 421, 280 284; Pardridge et al., 1995, PNAS USA.,
92, 5592 5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73 107;
Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910 4916;
and Tyler et al., 1999, PNAS USA., 96, 7053 7058. All these
references are hereby incorporated herein by reference. In
addition, compositions comprising surface-modified liposomes
containing poly (ethylene glycol) lipids (PEG-modified, or
long-circulating liposomes or stealth liposomes) may also be used
with the nucleic acids of the disclosure. Nucleic acid molecules of
the disclosure can also comprise covalently attached PEG molecules
of various molecular weights. These formulations offer a method for
increasing the accumulation of drugs in target tissues. This class
of drug carriers resists opsonization and elimination by the
mononuclear phagocytic system (MPS or RES), thereby enabling longer
blood circulation times and enhanced tissue exposure for the
encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601 2627;
Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005 1011). Such
liposomes have been shown to accumulate selectively in tumors,
presumably by extravasation and capture in the neovascularized
target tissues (Lasic et al., Science 1995, 267, 1275 1276; Oku et
al., 1995, Biochim. Biophys. Acta, 1238, 86 90). The
long-circulating liposomes enhance the pharmacokinetics and
pharmacodynamics of DNA and RNA, particularly compared to
conventional cationic liposomes which are known to accumulate in
tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864
24870; Choi et al., International PCT Publication No. WO 96/10391;
Ansell et al., International PCT Publication No. WO 96/10390;
Holland et al., International PCT Publication No. WO 96/10392; all
of which are incorporated by reference herein). Long-circulating
liposomes are also likely to protect drugs from nuclease
degradation to a greater extent compared to cationic liposomes,
based on their ability to avoid accumulation in metabolically
aggressive MPS tissues such as the liver and spleen.
EXAMPLES
[0128] The present disclosure is further described by the following
non-limiting examples:
Example 1
Preparation of Innate Immunity Receptor:Fc Fusion Protein
Cell culture
[0129] 293F cells (Invitrogen, R790-07) were cultured in serum-free
293 FREESTYLE 203 expression medium (Invitrogen, 12338-018) in a
125 mL flask on an orbital shaker (125 rpm) at 37.degree. C. in a
CO.sub.2 incubator.
Construction of Receptor Fcfusion Genes
[0130] The extracellular domains of lectin receptors, TREMs and
TLTs were cloned by the reverse-transcriptase polymerase chain
reaction (RT-PCR), followed by subcloning into a yT&A vector
and then into a pcDNA3.1 (+)hIgG 1.Fc expression vector. The
resulting receptor.Fc construct encodes recombinant proteins that
are fused with a mutated human IgG1Fc portion, which does not bind
to human Fc receptors. The mutations in the IgG1 Fc portion are
L234A, L235E, G237A, and P331S. The sequences of the primers used
to RT-PCR amplify the extracellular domains are: TABLE-US-00001
CLEC1A/CLEC-1 sense primer 5'-GAATCCTTTCAGTACTACCAGCTCTCC-3' SEQ ID
NO:1 antisense primer 5'-GAATTCTCAGTCACCTTCGCCTAATGT-3' SEQ ID NO:2
CLEC1B/CLEC-2 sense primer 5'-GGATCCCTGGGGATTTGGTCTGTC-3' SEQ ID
NO:3 antisense primer 5'-GAATTCTTAAGGTAGTTGGTCCAC-3' SEQ ID NO:4
CLEC2B/AICL sense primer 5'-GGATCCTCTCAGAGTTTATGCCCC-3' SEQ ID NO:5
antisense primer 5'-GGATCCCCCCATTATCTTAGACAT-3' SEQ ID NO:6
CLEC4A/DCIR sense primer 5'-GGATCCTTTCAAAAATATTCTCAGCTTCTT-3' SEQ
ID NO:7 antisense primer 5'-GAATTCTCATAAGTGGATCTTCATCATC-3' SEQ ID
NO:8 CLEC4C/BDCA-2 sense primer
5'-GGATCCTTTATGTATAGCAAAACTGTCAAG-3' SEQ ID NO:9 antisense primer
5'-GAATTCTTATATGTAGATCTTCTTCATCTT-3' SEQ ID NO:10 CLEC4D/CLEC-6
sense primer 5'-GAATCCCATCACAACTTTTCACGCTGT-3' SEQ ID NO:11
antisense primer 5'-GAATTCCTAGTTCAATGTTGTTCCAGG-3' SEQ ID NO:12
CLEC4E/MINCLE sense primer 5'-GAAGATCTACATTTCGCATCTTTCAAACC-3' SEQ
ID NO:13 antisense primer 5'-GCGGTTAAAGAGATTTTCCTTTGTTCA-3' SEQ ID
NO:14 CLEC4K/Langerin sense primer 5'-GGATCCCGGTTTATGGGCACCATA-3'
SEQ ID NO:15 antisense primer 5'-GGATCCTCACGGTTCTGATGGGAC-3' SEQ ID
NO:16 CLEC4L/DC-SIGN sense primer 5'-GGATCCAAGGTCCCCAGCTCCATAAG-3'
SEQ ID NO:17 antisense primer 5'-GAATTCCTACGCAGGAGGGGGGT-3' SEQ ID
NO:18 CLEC4M/DC-SIGNR/L-SIGN sense primer
5'-GGATCCTCCAAGGTCCCCAGCTCC-3' SEQ ID NO:19 antisense primer
5'-GAATTCCTATTCGTCTCTGAAGCAGG-3' SEQ ID NO:20 CLEC5A/MDL-1 sense
primer 5'-AGATCTAGTAACGATGGTTTCACCAC-3' SEQ ID NO:21 antisense
primer 5'-GAATTCCTGTGATCATTTGGCATTCTT-3' SEQ ID NO:22
CLEC6A/Dectin-2 sense primer 5'-GGATCCACATATGGTGAAACTGGC-3' SEQ ID
NO:23 antisense primer 5'-GAATTCCATCAGTCGATGGGC-3' SEQ ID NO:24
CLEC7A/Dectin-1 sense primer 5'-GGATCCACCATGGCTATTTGGAGATCC-3' SEQ
ID NO:25 antisense primer 5'-GAATTCTTACATTGAAAACTTCTTCTCACA-3' SEQ
ID NO:26 CLEC10A/ML2 sense primer 5'-GGATCCTCCAAATTTCAGAGGGACCTG-3'
SEQ ID NO:27 antisense primer 5'-GAATTCTCAGTGACTCTCCTGGCTG-3' SEQ
ID NO:28 CLEC12A/CLL-1 sense primer
5'-GGATCCGTAACTTTGAAGATAGAAATGAAA-3' SEQ ID NO:29 antisense primer
5'-GAATCCTCATGCCTCCCTAAAATATGTA-3' SEQ ID NO:30 CLEC13A/BIMLEC
sense primer 5'-GGATCCTCATGCTCCGGGCCGCG-3' SEQ ID NO:31 antisense
primer 5'-GAATTCGCTAGCAATCACCAATGCTGA-3' SEQ ID NO:32 COLEC12/CL-P1
sense primer 5'-AGAGGTGACAGAGGATCCCA-3' SEQ ID NO:33 antisense
primer 5'-GAATTCGTGATCCCATCACAGTCC-3' SEQ ID NO:34 MAFA-L/KLRG-1
sense primer 5'-GGATCCTGCCAGGGCTCCAACT-3' SEQ ID NO:35 antisense
primer 5'-ATGACAGATCTGAGGGTCA-3' SEQ ID NO:36
Expression and Purification of Recombinant Receptor.Fc Fusion
Proteins
[0131] The receptor.Fc proteins were over-expressed using the
FREESTYLE 293 Expression System (Invitrogen, Carlsbad, Calif.) and
purified on protein A columns. Briefly, 3.times.10.sup.7 293-F
cells were spun down at 1,500 rpm, then resuspended in 28 ml
FREESTYLE 293 expression medium. Then, 40 .mu.l of 293FECTIN was
mixed with 1 ml OPTI-MEM (Invitrogen, 31985-062) for 5 min at room
temperature, then incubated with 30 .mu.g plasmid DNA in 1 ml
OPTI-MEM (Invitrogen, 31985-062) for another 20 min, before
addition to the 293-F cells. After 48 h, the supernatant was
harvested and the recombinant fusion proteins were purified by
protein A columns.
Example 2
Preparation of Polysaccharide Extracts
Crude Extracts of Reishi
[0132] Crude Reishi extract (prepared via alkaline extraction,
neutralization and ethanol precipitation) was obtained from
Pharmanex Co. (CA, USA). Spectrapor.RTM. dialysis membrane tubing
with molecular weight cut off (MWCO) 6000-8000 dalton, Thermo
bio-basic SEC-1000 columns, Tosoh TSK G5000PWx1 SEC columns, and
all chemicals and reagents were from Sigma, or Aldrich Co., unless
indicated.
Purification of Reishi extract
[0133] Crude Reishi powder (6 g) (obtained from Pharmanex Co.) was
dissolved in 120 mL of ddH.sub.2O, stirred at boiling water
(100.degree. C.) for 2h, and centrifuged (1000 rpm) for 1 h to
remove insoluble material. The resulting solution was concentrated
at between about 40.degree. C. and about 50.degree. C. to give a
small volume, and then lyophilized to generate 5 g (83%) powder of
dark-brown color (G. lucidum polysaccharides; GLPS). This water
soluble residue was stored at -20.degree. C. until further
purification.
Standardization-Isolation of the F3 Fraction of Reishi
Polysaccharide
[0134] G. lucidum polysaccharide fraction 3 (hereinafter referred
to as "GLPS F3" and "F3") was isolated from the dark powder of
water soluble residue of Reishi polysaccharide. All chromatography
steps were performed at 4.degree. C. in a cold room. The sample
(2.1 g) was dissolved in a small volume of Tris buffer (pH 7.0, 0.1
N) containing 0.1 N sodium azide, and purified by gel filtration
chromatography using a Sephacryl S-500 column (95.times.2.6 cm)
with 0.1 N Tris buffer (pH 7.0) as the eluent. The flow rate was
set at 0.6 mL/min, and 6.0 mL per tube was collected. After
chromatography, each fraction was subjected to the
phenol-H.sub.2SO.sub.4 method to detect the content of sugar in
each tube. Five fractions were collected (fraction 1-5). Fraction 3
(F3) was concentrated at about 40-50.degree. C. in a rotary
vaporizer to give a small volume which was then dialyzed using a
6000-8000 dalton MWCO membrane to remove excessive salt and sodium
azide. Following dialysis, F3 was then lyophilized to give 520mg of
solid.
Preparation of Polysaccharides from Cordyceps sinensis
[0135] To purify the polysaccharides from Cordyceps sinensis,
samples were chopped into 0.2 cm.sup.3 pieces then incubated in
deionized boiling water (100.degree. C.) for 60 min, then cooled
down to room temperature before passing through the 0.2 .mu.m
filter, followed by addition of an equal volume of ethanol to
precipitate the polysaccharides. The precipitates were dried using
a lyophilizer and stored at 4.degree. C. Total sugar analysis of
the polysaccharides was determined by the Phenol-H.sub.2SO.sub.4
method, by measuring OD at 485 nm, while the purity of the
polysaccharides was determined by HPLC using a Thermo Bio-Basic
SEC-1 000 column with UV detection at 280 nm and using a RI
detector.
Preparation of Polysaccharides from Dendrobium huoshanense
[0136] Air-dried D. huoshanense was crushed and ground to a powder,
homogenized in distilled water, and stirred at 4.degree. C.
overnight. The insoluble material was collected by centrifugation.
The supernatant was concentrated to a small volume, and then added
to 1 volume of ethanol to yield a precipitate (O) and supernatant
(N). A TSK G-5000 PW size exclusion column was used in high
performance liquid chromatography (HPLC) for polysaccharides
analysis with standard pullulan fractions having defined molecular
weights. The molecular weight of polysaccharides in N was estimated
as between 1.2.times.10.sup.5-4.1.times.10.sup.5 daltons, and the
molecular weight of polysaccharides in O was estimated as between
1.0.times.10.sup.6-2.2.times.10.sup.5 daltons. The total
carbohydrate content was measured by the phenol-sulfuric acid
method. Polysaccharides in O were 83%, and polysaccharides in N
were 77%. Both O and N test positive with an iodine reaction
(.lamda.max 440 nm, deep blue color) suggesting that the
polysaccharides in these fractions are primarily
.alpha.-D-glucan.
Preparation of Polysaccharides from Mushroom
[0137] Air-dried Lentinus edodes was crushed and ground to a
powder, homogenized in distilled water, and stirred at 4.degree. C.
overnight. Residues were removed by centrifugation and supernatant
was concentrated to a small volume, then lyophilized to give crude
polysaccharide L. Then, 0.25N NaOH solution was added to the water
insoluble residue (which was isolated by centrifugation), and the
mixture was then stirred at room temperature overnight before
adding 2 volume of ethanol to precipitate the polysaccharides.
Distilled water was then added to the precipitated polysaccharide,
followed by acetic acid to neutralize pH. The resulting solution
was centrifuged and lyophilized to give polysaccharide M. HPLC
using a TSK G-5000 PW size exclusion column was then performed in
order to analyze the polysaccharides. The total carbohydrates
content was measured by the phenol-sulfuric acid method with L
comprising 79% carbohydrates, and M comprising 90% carbohydrates. A
comparison with data of the fractions of polysaccharides from
Lentinus edodes suggested that the polysaccharides L and M are
primarily .beta.-1,3-D-glucan.
Preparation of .beta.-1,3-glucan, D-glucose and D-galactose
[0138] To prepare samples for a competition assay, 100 mg of
.beta.-1,3-glucan (Fluka, Japan) was suspended in 7.5 ml of water,
and 50 .mu.l of a 40% (w/w) aqueous solution of sodium hydroxide
was added. The mixture was heated under reflux for 1.5 hours, and
cooled. Then, methanol was added to precipitate .beta.-1,3-glucan.
The .beta.-1,3-glucan precipitate was dissolved in water, dialyzed
with 4 L dd-H2O four times, and concentrated at reduced pressure to
obtain the water-soluble .beta.-1,3-glucan. D-Glucose (Sigma) and
D-galactose (Sigma) were dissolved in dd-H2O (100 mg/ml) and stored
at 4.degree. C.
Preparation of biotinyl-F3
[0139] Reishi polysaccharides-F3 were labeled with biotin using a
"one pot" reaction. Specifically, Reishi polysaccharide-F3 (100 mg)
in 0.2 N NaHCO.sub.3/Na.sub.2CO.sub.3 (10 mL) was reacted with
biotinamidohexanoyl-6-amino-hexanoic acid N-hydroxy-succinimide
ester (biotin-XX-NHS) 1.0 mg in DMF (1 mL). The mixture was stirred
at room temperature for 12 h. After completion of the reaction, the
resulting solution was dialyzed using membrane tubing with a MWCO
of 6000-8000 dalton (5.times.500 mL) at 4.degree. C. for 48 h.
After dialysis, the biotinyl-F3 was lyophilized to give a brown
powder 90 mg (90%). The purification of biotinyl-F3 was monitored
by HPLC and streptavidin-FITC was used for the binding assay.
Example 3
Western Blot Analysis of Purified Receptor:Fc Fusion Proteins
[0140] The purified receptor.Fc fusion proteins of Example 1 were
subjected to electrophoresis, transferred onto nitrocellulose
membrane (Hybond-C extra, Amersham Pharmacia Biotech) and reacted
with (1:3000) peroxidase-conjugated goat anti-human IgG Ab
(Jackson, Pa., USA) in TBST (5% non-fat dry milk in Tris-buffered
saline with 0.02% Tween 20) buffer. After washing with TBST, blots
were then incubated with enhanced chemiluminescence reagents
(Amersham Pharmacia Biotech) for visualization.
Example 4
Immunosorbent Dot Binding assay
[0141] Biotinylated F3 was blotted onto methanol-activated PVDF
membranes (2 .mu.L/dot) after 5-fold serial dilution, using a
Bio-Dot Microfiltration Apparatus.TM. (Bio-Rad, CA, USA). After
drying in air, the blot was incubated in TBST, followed by
incubation with 100 .mu.L streptavidin-conjugated horseradish
peroxidase (HRP) (1:2000 dilution) (Chemicon, CA, USA). Binding
reactions were visualized with enhanced chemiluminescence (ECL)
reagents (Amersham Pharmacia Biotech).
[0142] Non-biotinylated polysaccharides were also immobilized onto
methanol-activated PVDF membranes, followed by incubation with 100
.mu.L receptor.Fc fusion protein (1 .mu.g/ml, in 2 mM
CaCl.sub.2/TBST) on a Bio-Dot Microfiltration Apparatus.TM.
(Bio-Rad, CA, USA) for 1 h at room temperature, then followed by
reaction with (1:3000) HRP-conjugated goat anti-human IgG antibody
(Jackson, Pa., USA) in TBST (5% non-fat dry milk in Tris-buffered
saline with 0.02% Tween 20) buffer. After washing with TBST, the
blot was incubated with enhanced chemiluminescence reagents
(Amersham Pharmacia Biotech) for visualization.
Example 5
Expression of Recombinant Receptor.Fc Fusion Protein
[0143] The extracellular domains of several innate immunity
receptors from immune cells were cloned by reverse-transcription
polymerase chain reaction (RT-PCR) according to the method of
Example 1. The amplified DNA fragments were fused with the Fc
portion of human IgG1contained in the pcDNA3/hIgG1-mutant plasmid.
The cloned fusion genes was transfected into 293 FREESTYLE
mammalian cells, and the secreted proteins were purified by protein
beads according to the method of Example 1. As shown in FIG. 1,
sixteen C-type lectin genes were cloned (FIG. 1A). Specifically,
FIG. 1A shows DNA fragments of innate immunity receptors amplified
by RT-PCR, then fractionated on 0.8% agarose and visualized by
ethidium bromide staining. FIG. 1B shows the expressed recombinant
receptor.Fc fusion proteins following electrophoresis on a 12%
SDS-PAGE gel. In both FIG. 1A and FIG. 1B, the following lane
designations are used: Lane 1: CLEC2B/AICL, Lane 2: CLEC4C/BDCA-2,
Lane 3: CLEC13A/BIMLEC, Lane 4: CLEC1A/CLEC-1, Lane 5:
CLEC4D/CLEC-6, Lane 6: CLEC12A/CLL-1, Lane 7: CLEC4A/DCIR, Lane 8:
CLEC4L/DC-SIGN, Lane 9: CLEC4M/DC-SIGNR, Lane 10: CLEC7A/Detin-1,
Lane 11: CLEC6A/Detin-2, Lane 12:CLEC4H2/HBVxAgBP, Lane 13:
CLEC4K/Langerin, Lane 14: KLRG/MAFAL, Lane 15: CLEC5A/MDL-1, Lane
16: CLEC4E/MINCLE. In addition, the human TREM (triggering receptor
expressed on myeloid cells)-1,-2 and TREM-like transcripts
(TLT)-1,-2 (Bouchon et al., 2000, J Immunol 164, 4991-5; Daws et
al., 2003, J Immunol 171, 594-9; Washington et al., 2002, Blood
100, 3822-4) were also cloned and expressed by similar
strategy.
Example 6
Dose-Dependent Interaction Between Immobilized Polysaccharides with
Receptor.Fc Fusion Proteins
[0144] The interaction between polysaccharides and the receptor.Fc
fusion proteins was tested using a dot-binding assay according to
the method of Example 4. The water soluble fraction 3 of Reishi
polysaccharides (F3) (see Example 3) contains the active components
to stimulate cell producing cytokines (Wang et al., 2002, Bioorg
Med Chem 10, 1057-62; Chen et al., 2004, Bioorg Med Chem 12,
5595-601; Chien et al., 2004, Bioorg Med Chem 12, 5603-9; Hsu et
al., 2004, J Immunol 173, 5989-99). Reichi saccharide was known to
contain either a polysaccharide backbone with .beta.-1,3-linkages,
or a polymannose backbone with .alpha.-1,4-linkage (Usui et al.,
1983, Carbohydr. Res., 273; Miyazaki and Nishijime, 1982,
Carbohydr. Res. 109, 290; Bao et al., 2002, Phytochemistry 59,
175-81). The Dectin-1 receptor, a member of the C-type lectin
family, has been shown to interact with .beta.-1,3-D-glycans (Brown
and Gordon, 2001, Nature 413, 36-7). Dectin-1 receptor has been
shown to mediate the biological effects of beta-glucans (Brown et
al., 2003, J Exp Med 197, 1119-24). Thus the F3 portion of Reishi
was tested to determine whether it could interact with the Dectin-1
receptor using the dot-binding assay of Example 4.
[0145] Biotinylated F3 fraction ("Biotin-GLPS F3" in FIG. 2A)
(prepared according to Example 2) was immobilized on a PVDF
membrane after a 5-fold serial dilution and incubated with
streptavidin-conjugated HRP, and the resulting binding reaction was
detected using enhanced chemiluminescence reagents (see Example 4).
As shown in FIG. 2A, the sensitivity of this dot binding assay is
better than about 0.08 .mu.g. FIG. 2A also shows that no background
is seen when unbiotinylated F3 ("GLPS-F3" in FIG. 2A) is
immobilized on the PVDF membrane and then contacted with
streptavidin-conjugated HRP.
[0146] Un-biotinylated F3 fraction was also immobilized on a PVDF
membrane after serial dilution, and incubated with 100 .mu.L of 1
.mu.g/mL Dectin-1 .Fc fusion protein or human IgG1 (as a negative
control), followed by incubation with goat HRP-conjugated
anti-human IgG (see Example 4). As shown in FIG. 2B, Dectin-1.Fc
can detect the presence of less than about 1 ng of F3 in the
dot-binding assay. There is no visible background on the regions of
the blot contacted with human IgG1 instead of Dectin-1. Fe.
[0147] The dot density of the blot of FIG. 2B was determined by a
densitometer (ImageQuant), and the results show that the
Dectin-1.Fc binding signal increased in a dose-dependent manner
(see FIG. 2C).
[0148] In order to determine whether other polysaccharides inhibit
the interaction between F3 and Dectin-1, F3 (10 .mu.g/dot) was
immobilized on PVDF membrane and then contacted with 100 .mu.L
Dectin-1.Fc (1 .mu.g/mL) in the presence of serially diluted
solutions of .beta.-glucan, D-glucose, and D-galactose (0.1
.mu.g-1000 .mu.g), followed by incubation with goat HRP-conjugated
anti-human IgG. FIG. 2D shows dot density analysis of the blot for
competitor .beta.-glucan, and FIG. 2E shows a blot image for all
the competitors. It can be seen that the interaction between
Dectin-1.Fc and the F3 fraction is inhibited by .beta.-1,3-glucan,
but not by D-glucose or D-galactose. This indicates the interaction
between Dectin-1 .Fc with F3 is via recognition of
.beta.-1,3-glucan.
Example 7
Identification of Receptors Capable of Interacting with F3
Fraction
[0149] The interaction of F3 with other members of the C-type
lectin family or with Ig-like receptors was assayed.
Non-biotinylated F3 and non-biotinylated F3C (which is derived from
F3 after passing through 100 kDa MWCO centrifugal tube) (10
.mu.g/dot) was immobilized on PVDF membrane (see Example 4), then
incubated with 100 .mu.L of 1 .mu.g/mL solutions of 25 different
recombinant receptor.Fc fusion proteins (including 19 lectin
receptors, and 8 members of TREM and TLT families) and human IgG1
as control. Binding was detected using goat HRP-conjugated anti-IgG
antibody and ECL reagents. The results are depicted in table form
in FIG. 3 (with relative dot intensities indicated by "+" symbols,
and no detectable binding indicated by "-" symbol) and an image of
the blot is depicted in FIG. 4A. The probe numbering system used in
FIG. 3 is retained in FIG. 4A.
[0150] The results show that in addition to Dectin-1.Fc (probe no.
14 in FIG. 3 and FIG. 4A), F3 also interacted with KCR.Fc (probe
no. 7 in FIG. 3 and FIG. 4A), DC-SIGNR.Fc (probe no. 11 in FIG. 3
and FIG. 4A), and TLT-2.Fc (probe no. 21 in FIG. 3 and FIG. 4A). It
is interesting to note that F3C, which is derived from F3 after
passing through 100 kDa MWCO centrifugal tube, has less binding
affinity to TLT2. This suggests that TLT2 can differentiate the
subtle difference between F3 and F3c.
[0151] Members of the lectin receptor family rely on Ca++ for
interaction; therefore, the ability of EDTA (Ethylene Diamine Tetra
Acetic Acid) to inhibit binding to F3 was studied. It was found
that EDTA (10 mM in TBST) completely abolished the interaction of
F3 with KCR.Fc and with DC-SIGNR.Fc, but not the interaction of F3
with Dectin-1.Fc and TLT2.Fc. FIG. 4B depicts images of the blots
made in the presence and absence of Ca++ (left panel is TBST only;
right panel is 10 mM EDTA+TBST). Binding was detected using goat
HRP-conjugated anti-IgG antibody and ECL reagents. This result
agrees with previous observations that the interaction between
ligands and KCR (Hoyle and Hill, 1988, J Biol Chem 263, 7487-92)
and DC-SIGNR is Ca++-dependent (Soilleux et al., 2000, J Immunol
165, 2937-42), while Ca++ is dispensable for the interaction
between Dectin-1and .beta.-1,3-glucan (Herre et al., 2004, Mol
Immunol 40, 869-76). Thus, F3 apparently contains abundant glycans
which can interact with multiple receptors on immune cells
simultaneously.
[0152] FIG. 4C depicts a dot blot using .beta.-glucan as
polysaccharide (10 .mu.g/dot) and using 100 .mu.L of 1 .mu.g/mL
Dectin-1.Fc, DC-SIGN.Fc, mKCR.Fc, and TLT2.Fc. Binding was detected
using goat HRP-conjugated anti-IgG antibody and ECL reagents. Of
the four receptor.Fc fusion proteins tested, only Dectin-1.Fc can
bind to .beta.-1,3-glucan. This indicates that the other three
receptor.Fc fusion proteins bind to sugar components other than
.beta.-1,3-glucan.
Example 8
Fingerprints of Polysaccharides from Various Sources
[0153] The dot-binding assay of Example 4 was performed using
Dectin1.Fc, mKCR.Fc, DC-SIGNR.Fc, and TLT2.Fc fusion proteins in
order to obtain the fingerprints of polysaccharides isolated from
Cordyceps and other resources on market. Each polysaccharide
composition was immobilized on a PVDF membrane as described above
and then contacted with 100 .mu.L of a 1 .mu.g/mL solution of the
fusion protein. Binding was detected using goat HRP-conjugated
anti-IgG antibody and ECL reagents. FIG. 5A shows the individual
dot blots for each fusion protein and FIG. 5B shows the sample key
numbers and the relative dot intensities in table form. The Reishi
crude extract (spot no. 5 in FIG. 5) only interacts with
Dectin-1.Fc and DC-SIGNR.Fc, while the purified F3 (spot no. 1)
from the crude extract interacts with all the four receptors. This
indicates that the F3 purification process enriches the components
that interact with immune receptors. Polysaccharide from Cordyceps
(spot no. 7) interacts strongly with Dectin-1.Fc, indicating that
the polysaccharide contains .beta.-1,3 glycan, but its interaction
with the other three receptors is much weaker than that of F3.
Polysaccharides isolated from Dendrobiun huoshanense test positive
with the iodine test reaction (see Example 2) suggesting these
fractions comprise mainly .alpha.-D-glucan. In contrast to those
isolated from fungi, the mixture of polysaccharides of D.
huoshanense (spot no. 6) does not react with any of the four
receptor.Fc fusion proteins. Polysaccharides isolated from mushroom
polysaccharides by ddH2O (fraction L, spot no. 8) and 0.25N NaOH
(fraction M, spot no. 9) (see Example 2) bind differentially to
Dectin-1.Fc and DC-SIGNR.Fc. Thus, this approach can produce
distinct fingerprints from polysaccharides isolated from different
sources and preparations,
[0154] Examples 6-8 illustrate that F3 interacts with Dectin-1.Fc,
mKCR.Fc, DC-SIGNR.Fc, and TLT2.Fc. The Kupffer cell receptor (KCR)
has high affinity to D-galactose and N-acetylgalactyosamine (Fadden
et al., 2003, Glycobiology 13, 529-37), and is able to clear serum
D-galactose- or D-fucose-terminated glycoprotein (Lehrman et al.,
1986, J Biol Chem 261, 7426-32). The immunomodulatory function of
F3 is dependent on the presence of fucose, and glycolytic cleavage
by .alpha.1,2-fucosidase abolishes F3 activity. Thus it would be
interesting to ask whether these four receptors can interact with
F3 after glycolytic cleavage. DC-SIGNR/L-SIGN is structurally
similar to DC-SIGN (77% identity), but it is only expressed in the
endothelial cells of liver sinusoid, lymph node and placenta (Van
Liempt et al., 2004, J Biol Chem 279, 33161-7). Both DC-SIGN and
DC-SIGNR can bind to N-linked high-mannose oligosaccharides
(Man.sub.9GlcNAc.sub.2Asn glycopeptide). However, only DC-SIGN, and
not DC-SIGNR, can bind to glycans with a terminal fucose residue
(Guo et al., 2004, Nat Struct Mol Biol 11, 591-8). Even though
DC-SIGNR binds relatively restricted ligands than DC-SIGN, only
DC-SIGNR can interact with F3. This suggests that F3 might contain
a unique structure distinct from Fuc.alpha.1-4GlcNAc, Lewis.sup.X,
Lewis.sup.a and blood group sugar epitopes (the known ligands for
DC-SIGN).
[0155] TLT-2 is a member of TREM-like transcripts family, which
contain a characteristic single V-set immunoglobulin (Ig) domain
and a long cytoplasmic tail with a proline-rich region and an
immune receptor tyrosine-based inhibitory motif (ITIM), the latter
known to be used for interactions with protein tyrosine
phosphatases (Washington et al., 2002, Blood 100, 3822-4;
Washington et al., 2004, Blood 104, 1042-7). Since F3 has potent
immunostimulatory functions, it would be interesting to study
whether the removal of TLT2.Fc.binding components from F3 by
affinity chromatography could further enhance the stimulatory
functions of F3 in the future. Alternatively, F3 can be further
purified by affinity chromatography using Dectin-1.Fc, KCR.Fc, and
DC-SIGNR.Fc to remove other components in F3.
[0156] The differential fingerprints between F3 and F3c; between F3
and Reishi 1-3; and between mushroom polysaccharides fraction L and
M, suggest that these four receptor.Fc fusion proteins exemplified
herein can be used to optimize purification procedures, and to
monitor the variation of polysaccharides from different sources or
from different fermentation conditions.
Example 9
Identification of Human Lectin Receptors that Interact with GLPS-F3
by Enzyme Linked Immunoassay on Microtiter Plates
[0157] The interactions of polysaccharides with receptor.Fc fusion
proteins was further investigated by performing an enzyme-linked
immunoassay (EIA), which was based on immobilizing GLPS-F3 through
both hydrophilic and hydrophobic forces onto microtiter plates
(polysytrene). In this format, the number of different receptor.Fc
fusions for profiling was increased in comparison to Example 7. To
optimize the quantity of GLPS-F3 for immobilization, various
amounts (3-1000 ng/well, diluted in 100 mM Tris buffer, pH9.5) of
biotinylated-GLPS-F3 (Biotin-GLPS-F3) were coated onto MaxiSorp
StarWell microtiter plates (50 .mu.L/well; Nunc). The plates were
incubated overnight at 4.degree. C., and then the wells were washed
twice with TBST, followed by blocking with 200 .mu.L blocking
buffer (2% BSA/TBST) for 1 hour at room temperature.
Peroxidase-conjugated avidin (1:5000 dilution, Vector Laboratories)
and TMB (tetramethylbenzidine) substrate was then used for
detection of immobilized biotinylated GLPS-F3 . As shown in FIG.
6A, the quantity of Biotin-GLPS-F3 for plate coating reached
plateau at 100 ng/well, which was therefore chosen to use for
immobilizing un-biotinylated GLPS-F3 in EIA.
[0158] The interaction between GLPS-F3 and receptor.Fc was then
tested. Unbiotinylated GLPS-F3 was immobilized at 100 ng/well as
described above, and 100 .mu.L receptor.Fc fusion protein (1
.mu.g/ml in 2 mM MgCl.sub.2/2 mM CaCl.sub.2/1% BSA/TBST) was added
into each well and incubated for 1 hour at room temperature. After
washing with TBST, wells were incubated with peroxidase-conjugated
goat anti-human IgG Ab (1:5000 dilution, Jackson ImmunoResearch
Laboratories) in blocking buffer at room temperature for 30 min.
Wells were incubated with 100 .mu.L TMB substrate for 15 min after
TBST washing and read at 450 nm in a Fusion plate reader (Perkin
Elmer). The results were normalized with respect to Fc.Dectin-1
binding (Dectin-1 is a known lectin receptor that binds to
.beta.-1,3-glucan which is the backbone found in GLPS-F3). FIG. 6B
depicts in graphical form the affinity of each receptor for GLPS-F3
relative to Dectin-1. The results show that high binding affinity
to GLPS-F3 was observed for Fc.Langerin, Fc.DC-SIGN, MMR.Fc,
TLR2.Fc, TLR4.Fc, Fc.CLEC-2 (CLEC1B) and Fc.CLEC-6 (CLEC4D) (high
binding was defined in this assay as >50% binding intensity
compared to Fc.Dectin-1). It is noteworthy that TLR2 and TLR4,
which have been demonstrated to play a role in GLPS-induced cell
activation (Hsu et al., J Immunol 173:5989-5999 (2004); Shao et
al., Biochem Biophys Res Commun 323:133-141 (2004)), bound to
GLPS-F3 in the EIA format as well. There was also weaker but
positive GLPS-F3 binding ability (25-50% binding intensity compared
to Fc.dectin-1) found in Fc.NKG2D, Fc.MINCLE, Fc.mKCR, DCAL1.Fc,
DEC205.Fc, Endo180.Fc and NKp30(NCR3).Fc. Other lectin receptors
including Fc.AICL, Fc.BDCA2, Fc.CLEC1, Fc.CLL1, Fc.DCIR,
Fc.DC-SIGNR, Fc.dectin-2, Fc.MDL-1 and Fc.ML2 had minimal binding
ability to GLPS-F3, as did control human IgG1.
Example 10
Competition Assay for GLPS-F3-Interacting Innate Immunity
Receptors
[0159] To understand the interaction of GLPS-F3 with specific
innate immunity receptors, the polysaccharides mannan and
.beta.-glucan and the monosaccharides D-mannose (Man), D-glucose
(Glc), N-acetyl-glucosamine (GlcNAc), D-galactose (Gal),
N-acetyl-galactosamine (GalNAc), L-fucose (Fuc) and sialic acid,
were used in a competition assay. Innate immunity receptors that
showed higher binding ability to GLPS-F3 were examined, including
Fc.Dectin-1, Fc.Langerin, Fc.DC-SIGN, TLR4.Fc, MMR.Fc, Fc.CLEC-2
(CLEC1B) and Fc.CLEC-6 (CLEC4D). The assays were carried out as in
Example 9, with the addition of 1 mg/ml of each polysaccharide or
monosaccharide.
[0160] As shown in FIG. 7 (which shows graphically the % binding
for each receptor/saccharide combination relative to the binding
seen in the absence of saccharide) and Table I (which provides the
data from FIG. 7 in tabular form), the interaction between GLPS-F3
and Fc.Dectin-1could be blocked by .beta.-glucan with 58%
inhibition, which is in accordance with published results (Palma et
al., J Biol Chem 281:5771-5779 (2006); Willment et al., J Biol Chem
276:43818-43823 (2001)). The addition of sialic acid (83%
inhibition) interfered with the binding of Fe.Dectin-1 to GLPS-F3.
The interaction between Fc.Langerin and GLPS-F3 was disrupted by
mannan, Man and GlcNAc (95%, 26% and 84% inhibition), which are
reported as the sugar ligands for Langerin (Stambach & Taylor,
Glycobiology 13:401-410 (2002)); sialic acid (95% inhibition) was
also observed to interfere with the binding of Fc.Langerin to
GLPS-F3. As for the binding of Fc.DC-SIGN to GLPS-F3, mannan, Man,
Fuc and sialic acid showed a potent blocking activity (98%, 72%,
92% and 90% inhibition), while Glc and GlcNAc had a weaker effect
(45% and 27% inhibition, respectively) in blocking the interaction.
Mannan, Man, Glc, GlcNAc, Gal, Fuc and sialic acid blocked the
interaction (98%, 87%, 45%, 78%, 36%, 88% and 93% inhibition)
between GLPS-F3 and MMR.Fc, an important lectin receptor that is
known to bind Man, Fuc, GlcNAc and sialyl Lewis x (sLex) (Letuex et
al., J Exp Med 191:1117-1126 (2000); Stahl, Am J Respir Cell Mol
Biol 2:317-318 (1990)). The interaction of Fc.CLEC-2 to GLPS-F3 was
blocked by the addition of sialic acid (55% inhibition). For
Fc.CLEC-6, no obvious blocking was observed among the sugar tested.
Notably, mannan and Fuc showed a blocking effect (72% and 44%
inhibition, respectively) on TLR4.Fc and GLPS-F3 interaction. The
data obtained here was in line with the results reported by the
study of sugar ligands for Dectin-1, Langerin, DC-SIGN and MMR. It
was also indicated that many lectin receptors could bind to GLPS-F3
with multivalency through different sugar components.
TABLE-US-00002 TABLE I Percentage of binding of innate immunity
receptor. Fc fusions to GLPS-F3 in the presence of sugar
competitors relative to binding seen in absence of sugar
competitor. Innate Immunity Receptor Sugar Dectin-1 Langerin
DC-SIGN TLR4.Fc MMR.Fc CLEC-2 CLEC-6 none 100 .+-. 7.6 100 .+-. 1.0
100 .+-. 0.1 100 .+-. 4.5 100 .+-. 2.2 100 .+-. 8.0 100 .+-. 0.8
mannan 82 .+-. 0.2 5 .+-. 0.9 2 .+-. 0.5 28 .+-. 6.3 2 .+-. 0.5 88
.+-. 6.3 75 .+-. 0.8 Man 89 .+-. 0.5 74 .+-. 2.1 28 .+-. 1.3 89
.+-. 2.6 13 .+-. 6.4 95 .+-. 9.1 98 .+-. 3.7 b-glucan 42 .+-. 0.3
77 .+-. 3.2 81 .+-. 1.4 96 .+-. 4.0 100 .+-. 4.2 95 .+-. 5.1 98
.+-. 7.3 Glc 86 .+-. 1.5 87 .+-. 2.0 55 .+-. 5.4 101 .+-. 4.7 55
.+-. 2.9 108 .+-. 6.8 100 .+-. 5.3 GlcNAc 91 .+-. 2.6 16 .+-. 2.7
73 .+-. 8.6 99 .+-. 4.7 22 .+-. 3.4 103 .+-. 10.2 99 .+-. 8.5 Gal
88 .+-. 0.4 92 .+-. 0.9 82 .+-. 4.0 100 .+-. 2.7 64 .+-. 4.2 104
.+-. 5.1 90 .+-. 3.7 GalNAc 88 .+-. 2.6 97 .+-. 0.7 110 .+-. 3.1 95
.+-. 4.1 80 .+-. 4.2 110 .+-. 11.4 96 .+-. 2.4 Fuc 92 .+-. 5.2 76
.+-. 1.9 8 .+-. 1.8 56 .+-. 1.9 12 .+-. 2.0 91 .+-. 8.5 82 .+-. 6.5
sialic acid 17 .+-. 0.3 5 .+-. 0.3 10 .+-. 1.0 77 .+-. 3.1 7 .+-.
2.4 45 .+-. 6.2 94 .+-. 13.0
[0161] The systems presented in Examples 7-10 are useful tools for
high throughput profiling of not only GLPS, but also other
glycoprotein mixtures including many Chinese herb drugs currently
in use. By using different surfaces for immobilizing
polysaccharides (PVDF and polystyrene), different profiles were
obtained for GLPS-F3. This may be due to preferential binding of
certain polysaccharides within the mixtures to different surfaces.
The results obtained from these two complementary formats provide
"fingerprints" of polysaccharide mixtures. These strategies of
fingerprinting polysaccharide mixtures can be used, for example, to
monitor the contents of herb extracts under different conditions,
from different sources, or from different batches. Moreover, the
information gathered from the profiles of specific polysaccharide
mixtures will be of great importance in understanding the
underlying molecular mechanisms of their biological effects in
vivo.
Example 11
Detection of the Interaction of DVLRL (MDL-1) with Dengue Virus
[0162] The following examples show how the fusion proteins and
methods of the disclosure can be used to identify the innate
immunity receptor(s) that interact with a pathogen, and how that
information can subsequently be used to determine the downstream
effects of pathogen binding to the innate immunity receptor, and
also to design therapeutic agents for the treatment of pathogen
infection.
[0163] Dengue is one of the most important mosquito-borne viral
disease affecting humans. Its global distribution is comparable to
that of malaria, and an estimated 2.5 billion people live in areas
at risk for epidemic transmission. The clinical syndromes after
dengue virus (DV) infection include dengue fever (DF) and dengue
hemorrhagic fever (DHF)/dengue shock syndrome (DSS). However, the
underlying molecular mechanisms leading to DHF and DSS are still
not well elucidated.
[0164] DC-SIGN is known to mediate DV infection of human dendritic
cells (Tassaneetrithepet al., J Exp Med, 2003. 197(7): p. 823-9).
In order to understand the pathogenesis of DV, it is important to
determine whether DV can interact with other membrane-bound C-type
lectin receptors and C-type-like lectin receptors from dendritic
cells, macrophages, natural killer cells, and peripheral blood
mononuclear cells (PBMCs). To this end, the extracellular domains
of DVLR1 (MDL-1/CLEC5A), Dectin-1, KCR, and DC-SIGN (as a positive
control) were fused to the Fc portion of human IgG1. Specifically,
primers for DC-SIGN (SEQ ID NO: 17 and SEQ ID NO: 18), DVLR1 (SEQ
ID NO: 21 and SEQ ID NO:22), Dectin-1 (SEQ ID NO:25 and SEQ ID
NO:26) and KCR (forward: 5'-CAGCCTTGGAGACCTGAGT-3' SEQ ID NO: 37;
reverse 5'-TAGCCTACTCTGGCCGC-3' SEQ ID NO:38) were used to generate
amplified cDNA fragments. Each forward primer had an extra BamHI
site, and each reverse primer had an extra EcoRI site to faciliate
the subcloning of the amplified cDNA into the pCDNA3.1 (Invitrogen)
mammalian expession vector containing the human IgG1 Fc portion.
The resulting vector was then transfected into 293 FreeStyle cells
(Invitrogen) to produce soluble recombinant proteins. All
recombinant receptor.Fc fusion proteins were purified by protein A
Sepharose beads (Pharmacia) and eluted with 0.1M glycine --HCl
(pH0.3).
[0165] One .mu.g of each receptor.Fc fusion protein was coated onto
microtiter plates overnight at 4.degree. C. DV (5.times.10.sup.6
particles) of strain 16681 (a DEN2 strain) in binding buffer
(1%BSA, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2, 50 mM Tris-HCI pH 7.5,
150 mM NaCl) was then added to the plates and the plates were
incubated for 2 hours. After washing non-bound virus, a
biotinylated anti-DEN2 envelope protein antibody (Wu et al., J
Virol, 2002. 76(8): p. 3596-604) was applied to bind to the virus
for 1 hour. Diluted horseradish peroxidase-conjugated streptavidin
was then added to the plates, followed by a 1 hour incubation. TMB
substrate was then added and the plates were read using an ELISA
reader at OD450 nm.
[0166] The results are depicted in FIG. 8A (** indicates
p<0.01,*** indicates p<0.001 (Student's t test)). The results
show that in addition to DC-SIGN (positive control), DV also binds
to DVLR1/MDL-1. To confirm this result, immunoprecipitation studies
were performed with human IgG1 (negative control), DC-SIGN.Fc,
KCR.Fc, and DVLR1.Fc. Specifically, 5.times.10.sup.6 Dengue virus
particles were incubated with 5 .mu.g of each protein, and then
Protein A beads were added. The resulting immunocomplexes were
washed, separated by SDS-PAGE, and transferred onto nitrocellulose
membrane. The membrane was then probed with biotinylated anti-DEN2
envelope protein antibody and developed with horseradish
peroxidase-conjugated streptavidin. The results are shown in FIG.
8B. The results show that only DC-SIGN.Fc and DVLR1.Fc were able to
immunoprecipitate DV.
[0167] The microtiter plate assay was repeated in the presence of
EDTA (10 mM) to chelate Ca.sup.2+ cations. The results (FIG. 8C)
reveal that DVLR1 binding to Dengue virus is Ca.sup.2+ independent,
whereas DC-SIGN binding is Ca.sup.2+ dependent (*** indicates
p<0.001, Student's t test).
[0168] The microtiter plate assay was also repeated for DVLR1.Fc
fusion protein with DV particles (5.times.10.sup.6) that had been
1) preincubated with 500 U of the glycosidase PNGaseF (New England
Biolabs, Inc.) overnight at 37.degree. C.; or 2) treated with
dithiothreitol (DTT) (0.1 M); or 3) incubated at 95.degree. C. for
5 minutes; or 4) UV irradiated for 5 minutes. The results are shown
in FIG. 8D (asterisks indicate where the binding affinity of
DVLR1.Fc fusion protein is altered by modification of the virus
relative to non-treated virus; ** p<0.01, *** p<0.001,
Student's t test). The results indicate that pretreatment of DV
with PNGase F inhibited DVLR1.Fc interaction significantly, and
that pretreatment with either heat or dithiothreitol almost
completely inhibited DVLR1.Fc binding, but not DC-SIGN.Fc binding
to DV. This suggests that both the sugar epitope(s) and the three
dimensional conformation of DV are important for binding to
DVLR1.
[0169] In order to evaluate the expression of DVLR1 on immune
cells, flow cytometric analysis was performed on human
polymorphonuclear (PMN) cells (neutrophils), PBMCs, macrophages,
and dendritic cells. PMNs and PBMCs were isolated from the whole
blood of human healthy donors by dextran sedimentation as described
(Kuan et al., Br. J. Pharmacol., 2005, 145(4):460-468) and standard
density gradient centrifugation with Ficoll-Paque respectively
(Amersham Biosciences, Piscataway, N.J.). Purified neutrophils were
resuspended in phosphate saline buffer (PBS, pH 7.4,) with
hypotonic lysis of erythrocytes. CD14+ cells were subsequently
purified from PBMCs by high-gradient magnetic sorting using the
VARIOMACS technique with anti-CD14 microbeads (Miltenyi Biotec
GmbH, Bergisch Gladbach, Germany), then were cultured in complete
RPMI-1640 medium (Life Technologies, Gaithersburg, Md.)
supplemented with 10 ng/ml human M-CSF (R&D Systems,
Minneapolis, Minn.) for 6 days (Chang et al., J. Leukoc Biol, 2004,
75(3):486-494). Dendritic cells (DC) were generated from adherent
PBMCs by culture in RPMI 1640 medium supplemented with 10% fetal
calf serus, 800 U/ml human GM-CSF (Leucomax; Schering-Plough,
Kenilworth, N.J.), and 500 U/ml human IL-4 (R&D Systems) for 6
days (immature DCs). To prepare mature activated DCs, immature DCs
were further incubated with gamma-irradiated (5500 rad) CD40 ligand
(CD40L)-expressing L cells (DNAX Research Institute, Palo Alto,
Calif.) at a ratio of 3:1 for 36 hr (Hsu et al., J Immunol., 2002,
168(10):4846-4853).
[0170] Flow cytometry was performed on the above-mentioned cell
types using FITC-conjugated anti-DVLR1 monoclonal antibodies
(R&D Systems, Minneapolis, Minn.), or FITC-conjugated
anti-DC-SIGN monoclonal antibodies (ED PharMingen), in conjunction
with Phycoerythrin (PE)-conjugated anti-CD3, CD19, CD56, CD14, and
CD66 antibodies for double staining (BD PharMingen). Matched
isotype controls (IgG2b for DVLR1 mAb, IgG 1 for DC-SIGN; Sigma)
were also performed in this surface staining to provide background
information. Fluorescence was analyzed by FACSCalibur flow
cytometry (Becton Dickinson) with CellQuest software (Becton
Dickinson). CD marker positive cells were gated to determine the
expression of DVLR1 or DC-SIGN. The results are shown in FIG. 9A
(DVLR1) and FIG. 9B (DC-SIGN) (shaded area represents isotype
control). The results indicate that DC-SIGN is mainly expressed on
immature dendritic cells, and is weakly expressed on macrophages.
The results also indicate that DVLR1 was detected on the surface of
CD14+ derived macrophages (M.PHI.), CD66+ PMNs and CD14+ freshly
isolated PBMCs, but not on CD14+ derived immature and mature
dendritic cells. This is in accord with previous observations that
DVLR1/MDL-1 mRNA is expressed in human monocytes and macrophages,
but not in dendritic cells (Bakker et al., Proc. Natl. Acad Sci
USA, 1999, 96(17):9792-9796).
[0171] The results presented in this example show that the
receptor.Fc fusion protein-based methods disclosed herein can be
used to determine the identity of the innate immunity receptors
that bind to a specific pathogen, such as Dengue virus. This in
turn allows one to identify the cell types that interact with the
pathogen, and furthermore provides a new target for treatment or
prevention of infection by the pathogen. For example, the results
disclosed herein suggest that agents that prevent DV from binding
to DVLR1 can be used for prophylactic and/or therapeutic purposes.
For example, monoclonal antibodies against DVLR1 can be generated
by one skilled in the art that prevent the binding of DV to DVLR1.
Moreover, since DV is a member of the family Flaviviridae, this
result suggests that DVLR1 may interact with other viruses within
the same family, for example, viruses within the genus Flavivirus
(such as West Nile Virus, Japanese encephamyelitis virus (JEV),
yellow fever virus, tick-borne encephamyelitis virus) and viruses
within the genus Hepacivirus (such as Hepatitis C virus).
Accordingly, DVLR1 may serve as a therapeutic or prophylactic
target for these viruses also. In addition, since DVLR1 is a
pattern recognition receptor, DVLR1 may serve as a therapeutic or
prophylactic target for other enveloped viruses, including but not
limited to influenza virus.
Example 12
Dengue Virus Induced DAP12 Phosphorylaytion is Mediated Via
DVLR1
[0172] DVLR1/MDL-1 is a type II transmembrane protein comprising
187 aa in length, and it includes a charged residue in the
transmembrane region that enables it to pair with DAP12 (DNAX
activating protein of 12 kDa) (Bakker et al., Proc. Natl. Acad Sci
USA, 1999, 96(17):9792-9796). DAP12 is a disulfide-linked,
homodimeric transmembrane protein with a minimal extracellular
domain, a charged aspartic acid in the transmembrane domain and an
ITAM (immunoreceptor tyrosine-based activation motif) in its
cytoplasmic tail. Because DV binds to DVLR1 on CD14+ macrophages,
and because DAP12 has an ITAM, it was of interest to determine
whether DV can induce DAP12 phosphorylation in CD14+ macrophages.
Accordingly, CD14+ macrophages were infected with DV using the a
slight modification of the method disclosed in Chen et al, J.
Virol. 2002, 76(19):9877-9887. Briefly, terminal differentiated
macrophages were washed once with incomplete RPMI medium to remove
fetal calf serum in culture medium. The cells were then infected
with DV at different multiplicities of infection (MOI). The virus
was incubated with the cells in serum-free RPMI at 37.degree. C.
for 2.5 h to permit viral adsorption. The culture plates were
gently agitated every 30 min for optimal virus-cell contact.
Thereafter, the unabsorbed viruses were removed by washing the cell
monolayers twice with serum-free RPMI and then once with
incubation, the cell-free supernatants were harvested separately
and stored in aliquots at -80.degree. C. until assayed for
infectious-virus production and cytokine secretion (see Example
13). Infectious virus titers were determined by a plaque forming
assay on BHK-21 cells. Plaques were counted by visual inspection at
7 days after crystal violet overlay to determine the number of
plaque-forming units (PFU) per mL of supernant (Lin et al., J.
Virol., 1998, 72(12):9729-9737). To detect intracellular DV
antigens, infected cells were fixed with 1% paraformaldehyde and
permeabilized with 0.1% saponin, followed by staining with NS3 mAb
(Lin et al., J. Virol., 1998, 72(12):9729-9737) or matched isotype
control (IgG1; Sigma). After incubation for 1h, PE-conjugated goat
F(ab)' anti-mouse IgG secondary was added for fluorescence
detection and fluorescence was analyzed by FACSCalibur flow
cytometry with CellQuest software.
[0173] The results are shown in FIG. 10A-D. At 48 h after infection
at MOI=5, DV non-structural protein 3 (NS3) was detected by flow
cytometry in the cytosol of macrophages (FIG. 10A; gray histogram
is antibody isotype control). The extracellular virus titer was
measured at various times following infection, and revealed that
virus particles were released to culture supernatant when
macrophages were infected with live DV, but not with UV-irradiated
DV (UV-DV; 254 nm irradiation for 15 minutes on ice at 5 to 10 cm
distance) (FIG. 10B).
[0174] DAP12 phosphorylation was studied 2 hours after infection at
varying MOIs (MOI=0.05-30, 2 h after infection), and also at a
fixed MOI (MOI=5) over a time course (2-48 h after infection).
Specifically, for detection of phospho-DAP12, macrophages were
stimulated with DV for the appropriate amount of time at the
appropriate MOI and then lysed in lysis buffer (50 mM Tris-HCl
[pH7.5], 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 5mM EDTA, 10 mM
NaF, 1 mM sodium orthovanadate, and proteinase inhibitor cocktail
tablet [Roche]). Equal amount of total cell extracts were
immunoprecipitated with DAP12 rabbit polyclonal antibody (Santa
Cruz Biotechnology Inc, CA) and protein A sepharose (Amersham
Biosciences AB) for 4 h at 4.degree. C. After incubation, the
immunocomplex was washed three times and separated by SDS-PAGE,
followed by transferring onto nitrocellulose membrane and probed
with anti-phosphotyrosine antibody (4G10; Upstate Biotechnology,
Inc). Immunoblots were developed using HRP-conjugated second
antibody and enhanced chemiluminescence (Amersham). For reprobing,
the membrane was stripped with a strong re-probe kit (Chemicon) and
blotted with DAP12 antibody.
[0175] The results obtained at various MOIs are shown in FIG. 10C,
and the time course experiment results are shown in FIG. 10D. The
results show that at 2 h after DV infection, the intensity of DAP12
phosphorylation increased as the MOI was raised from MOI=0.05,
reaching a peak when MOI=5 (FIG. 10C). DAP12 phosphorylation was
detected at 2 h after DV infection, peaked at 12 h, and lasted for
at least 48 h (FIG. 10D). Even though UV-DV could not replicate in
CD14+ macrophages and had no activity in a plaque assay (FIG. 10B),
DAP12 was also phosphorylated at 2 h and phosphorylated DAP12
remained detectable at 12 h, even though the intensity is much
weaker than that induced by live DV (FIG. 10D; UV-DV). This
suggests that DV-induced DAP12 phosphorylation has two phases:
phase I (in the first 6 h) is replication-independent, while phase
II (after 12 h) is replication-dependent.
[0176] To confirm that DAP12 phosphorylation was via DVLR1, RNA
interference (RNAi) with short hairpin RNA (shRNA) was used to
inhibit the expression of DVLR1 in CD14+ macrophages and DAP12
phosphorylation was assayed as above. Specifically, the coding
region of human DVLR1 was targeted with the following DVLR1 siRNA:
TABLE-US-00003 5'-TTGTTGGAATGACCTTAT-3' SEQ ID NO:39
This stretch was adapted with loop sequence (TTCAAGAGA) from
Brummelkamp et al., Science, 2002, 296(5567): 550-553, to create an
shRNA. The polymerase III terminator stretch used here was TTTTTT.
The shRNA was cloned into the pLL3.7 gene silencing vector
(Rubinson et al., Nat. Genet., 2003, 33(3):401-406) which contained
loxP sites, a CMV (cytomegalovirus) promoter driving expression of
enhanced green fluorescent protein (EGFP), and a U6 promoter with
downstream restriction sites (HpaI and XhoI). A DC-SIGN shRNA
construct was also constructed by subcloning the shRNA contained in
the construct pSUPER-siDC-SIGN (Tassancetrithep et al., supra) into
pLL3.7 vector digested with HpaI/XhoI. The constructs were
electroporated into macrophages using the Amaxa kit (Gaithersburg,
Md.) according manufacturer's specifications. Briefly, macrophages
(6.times.10.sup.6) were harvested as described above and
resuspended in 100 .mu.L of nucleofactor solution. After the
addition of siRNA (5 .mu.g) or vector control, cells were
electroporated using Amaxa program Y-001 and allowed to recover for
16 h. The efficiency of DVLR1 and DC-SIGN silencing was analyzed 24
hrs after transfection by immunoblotting using anti-DVLR1 and
DC-SIGN monoclonal antibodies (R&D Systems), respectively.
[0177] The results are shown in FIG. 11. CD14+ macrophages
electroporated with the control vector pLL3.7 or with DC-SIGN-shRNA
did not show a reduction in DAP12 phosphorylation after DV
infection. By contrast, DAP12 phosphorylation decreased
dramatically in CD14+ macrophages electroporated with DVLR1-shRNA
prior to DV infection. Therefore, it was concluded that DV-induced
DAP12 phosphorylation occurs via DVLR1.
Example 13
DVLR1 is Involved in DV-Mediated TNF-.alpha. Release, but not Entry
to CD14+ Macrophages
[0178] Upon DV infection, CD14+ macrophages secrete
pro-inflammatory cytokines and chemokines, including tumor necrosis
factor alpha (TNF-.alpha.), alpha-interferon (IFN-.alpha.),
MIP-1.alpha., and IL-8 (Chen et al, supra). The levels of
TNF-.alpha. in culture supernatant were measured in DV-infected
CD14+ macrophages using a commercial ELISA kit. Measurements were
made at different MOIs and at different times post-infection for
both live DV and UV-DV. The results are shown in FIG. 12A-C (error
bars represent the standard error from the mean of triplicates, and
asterisks indicate statistically different levels of cytokine
production, *=p<0.05;**=p<0.01;***=p<0.001). The results
show that at 6 hours post infection, both live DV and DV-UV had
similar effects on TNF-.alpha. secretion at MOIs ranging from
0.05-30 (FIG. 12A). At 12 hours post infection, TNF-.alpha.
secretion increased in a dose dependent (increasing MOI) manner
only for live DV. For UV-DV infected cells at 12 hours post
infection, TNF-.alpha. levels remained the same at all MOIs (FIG.
12B). FIG. 12C shows a time course measurement of TNF. The results
show that when infected with live DV at MOI=5, TNF-.alpha.
secretion increased rapdily from 6 h (8 pg/ml) to 12 h (85 pg/ml),
and peaked at 48 h (350 pg/ml). When incubated with UV-DV, however,
TNF-.alpha. secretion decreased from 8 pg/ml (at 6 h) to 5 pg/ml
(at 12 h). This suggests that the initial response (at 6 h) is
independent of virus replication, while the later phase of
TNF-.alpha. secretion (after 12 h) correlates with DV
replication.
[0179] DC-SIGN has previously been shown to interact with DV in
order to mediate virus entry into dendritic cells. Using the RNAi
methodology and reagents of the prior examples, the effect of
DC-SIGN-shRNA and DVLR1-shRNA on NS3 expression in DV-infected
CD14+ macrophages was investigated. FIG. 13A shows that
DC-SIGN-shRNA and DVLR1-shRNA can knock down their respective
proteins (PWTSI and pLL3.7 are no insert controls). FIG. 13B
depicts the results of flow cytometry analysis and illustrates that
only DC-SIGN-shRNA could attenuate DV NS3 expression in CD14+
macrophages. This result was confirmed using immunofluorescence
confocal microscopy using anti-DS3 antibodies. FIG. 13C illustrates
real time PCR analysis of virus titer in the supernatant of cells
electroporated with the shRNA constructs. The results indicate that
only DC-SIGN-shRNA is capable of reducing virus titer in the
supernatant of DV-infected cells.
Example 14
DVLR1 is Involved in DV-Induced Proinflammatory Cytokine Release
from CD14+ Macrophages
[0180] The cytokine release profile for CD14+ macrophages infected
with DV (MOI=5) was evaluated using ELISA after knock down of DVLR1
and DC-SIGN according to the methods of the preceeding examples
(2.5 h transfection). In the first 12 h, DC-SIGN-shRNA did not
affect the secretion of TNF-.alpha., MIP-1.alpha., IFN-.alpha.,
IL-6, or IL-8. See FIG. 14A-B (error bars represent the standard
error from the mean of triplicates, and asterisks indicate
statistically significant differences compared to control
experiments; *=p<0.05; **=p<0.01; ***=p<0.001). After 48
h, DC-SIGN-shRNA had a mild inhibitory effect (less than 20%) on
TNF-.alpha., MIP-1.alpha., IFN-.alpha., and IL-6 secretion; IL-8
secretion was not affected. Since DC-SIGN is involved in virus
entry and replication, this observation suggests that initial
cytokine secretion (first 12 h) is independent of DV replication.
In contrast, knock down of DVLR1 dramatically suppressed
(p<0.005) the secretion of TNF-.alpha., MIP-1.alpha.,
IFN-.alpha., IL-8, but not of IL-6. This suggests that DVLR1 is
responsible for DV-induced cytokine release from CD14+ macrophages.
Accordingly, therapeutic agents that prevent the binding of DV to
DVLR1 will be useful for preventing the deleterious effects of
DV-induced cytokine release in humans. For example, monoclonal
antibodies that prevent DVLR1 interaction with DV will be useful
for preventing or treating DV-induced Dengue shock syndrome (DSS)
or Dengue haemorrhagic fever (DHF).
Example 15
Antagonistic Anti-DVLR1 Monoclonal Antibodies (mAbs) Abolish
Inflammatory Cytokine Release by DV Serotypes 1,2,3, and 4
[0181] Monoclonal antibodies against DVLR1 were generated using
standard techniques. Briefly, mice were immunized with DVLR-1.Fc
fusion protein, and hybridomas were formed by fusing splenocytes
from the mice with P3/NSI/1-Ag4-1 [NS-1] myeloma cells (ATCC
TIB-18). Among the mAbs generated, clone 9B12, the subelones of
3E12 (clones 3E12A2, 3E12C1, 3E12G9), and clone 8H8F5 suppressed
TNF-.alpha. release from macrophages after infection with DEN1
(strain 766733A), DEN2 (strain PL046), DEN3 (strain H-87), and DEN4
(strain 866146A) in a dose-dependent manner. See FIG. 15 which
shows ELISA measurements of TNF-.alpha. secreted into culture
supernatants by CD14+ macrophages infected with DV. In accordance
with standard nomenclature, each antibody is referred to via the
clone number of the hybridoma that secretes it. Hence, the
disclosure also provides the hybridomas that secrete the
abovementioned monoclonal antibodies.
[0182] The results demonstrate that anti-DVLR1 antibodies will
serve as useful therapeutic agents for preventing proinflammatory
cytokine release from DV-infected CD14+ macrophages in humans. In
particular, but not exclusively, the monoclonal antibodies of this
Example, or fragments thereof, or antibodies (or fragments thereof)
that bind to the same epitopes as the antibodies of this Example,
may be formulated as pharmaceutical compositions and then
administered for the treatment or prophylaxis of DV infection in
humans, according to the methods provided herein.
Deposit of Materials
[0183] The following hybridoma cultures are being deposited with
______ which is an International Depositary Authority (IDA)
recognized under the Budapest Treaty: clone 9B12, the subclones of
3E12 (clones 3E12A2, 3E12C1, 3E12G9), and clone 8H8F5. The
accession numbers of the clones and the dates of deposit are as
follows: ______. These deposits were made under the provisions of
the Budapest Treaty on the International Recognition of the Deposit
of Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of viable cultures for 30 years from the date of the deposit. The
organisms will be made available by the abovementioned IDA under
the terms of the Budapest Treaty, and subject to an agreement
between the assignee and the abovementioned IDA, which assures
permanent and unrestricted availability of the progeny of the
cultures to the public upon issuance of the pertinent U.S. patent
or upon laying open to the public of any U.S. or foreign patent
application, whichever comes first, and assures availability of the
progeny to one determined by the U.S. Commissioner of Patents and
Trademarks to be entitled thereto according to 35 USC .sctn.122 and
the Commissioner's rules pursuant thereto (including 37 CFR
.sctn.1.12 with particular reference to 8860G 638).
[0184] The assignee of the present application has agreed that if
the cultures on deposit should die or be lost or destroyed when
cultivated under suitable conditions, they will be promptly
replaced on notification with a viable specimen of the same
culture. Availability of the deposited strain is not to be
construed as a license to practice the invention in contravention
of the rights granted under the authority of any government in
accordance with its patent laws.
[0185] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the hybridomas deposited, since the deposited embodiments are
intended to illustrate only certain aspects of the invention and
any antibodies that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that they represent. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
39 1 27 DNA Artificial sequence Synthetic DNA primer sequence 1
gaatcctttc agtactacca gctctcc 27 2 27 DNA Artificial sequence
Synthetic DNA primer sequence 2 gaattctcag tcaccttcgc ctaatgt 27 3
24 DNA Artificial sequence Synthetic DNA primer sequence 3
ggatccctgg ggatttggtc tgtc 24 4 24 DNA Artificial sequence
Synthetic DNA primer sequence 4 gaattcttaa ggtagttggt ccac 24 5 24
DNA Artificial sequence Synthetic DNA primer sequence 5 ggatcctctc
agagtttatg cccc 24 6 24 DNA Artificial sequence Synthetic DNA
primer sequence 6 ggatcccccc attatcttag acat 24 7 30 DNA Artificial
sequence Synthetic DNA primer sequence 7 ggatcctttc aaaaatattc
tcagcttctt 30 8 28 DNA Artificial sequence Synthetic DNA primer
sequence 8 gaattctcat aagtggatct tcatcatc 28 9 30 DNA Artificial
sequence Synthetic DNA primer sequence 9 ggatccttta tgtatagcaa
aactgtcaag 30 10 30 DNA Artificial sequence Synthetic DNA primer
sequence 10 gaattcttat atgtagatct tcttcatctt 30 11 27 DNA
Artificial sequence Synthetic DNA primer sequence 11 gaatcccatc
acaacttttc acgctgt 27 12 27 DNA Artificial sequence Synthetic DNA
primer sequence 12 gaattcctag ttcaatgttg ttccagg 27 13 29 DNA
Artificial sequence Synthetic DNA primer sequence 13 gaagatctac
atttcgcatc tttcaaacc 29 14 27 DNA Artificial sequence Synthetic DNA
primer sequence 14 gcggttaaag agattttcct ttgttca 27 15 24 DNA
Artificial sequence Synthetic DNA primer sequence 15 ggatcccggt
ttatgggcac cata 24 16 24 DNA Artificial sequence Synthetic DNA
primer sequence 16 ggatcctcac ggttctgatg ggac 24 17 26 DNA
Artificial sequence Synthetic DNA primer sequence 17 ggatccaagg
tccccagctc cataag 26 18 23 DNA Artificial sequence Synthetic DNA
primer sequence 18 gaattcctac gcaggagggg ggt 23 19 24 DNA
Artificial sequence Synthetic DNA primer sequence 19 ggatcctcca
aggtccccag ctcc 24 20 26 DNA Artificial sequence Synthetic DNA
primer sequence 20 gaattcctat tcgtctctga agcagg 26 21 26 DNA
Artificial sequence Synthetic DNA primer sequence 21 agatctagta
acgatggttt caccac 26 22 27 DNA Artificial sequence Synthetic DNA
primer sequence 22 gaattcctgt gatcatttgg cattctt 27 23 24 DNA
Artificial sequence Synthetic DNA primer sequence 23 ggatccacat
atggtgaaac tggc 24 24 21 DNA Artificial sequence Synthetic DNA
primer sequence 24 gaattccatc agtcgatggg c 21 25 27 DNA Artificial
sequence Synthetic DNA primer sequence 25 ggatccacca tggctatttg
gagatcc 27 26 30 DNA Artificial sequence Synthetic DNA primer
sequence 26 gaattcttac attgaaaact tcttctcaca 30 27 27 DNA
Artificial sequence Synthetic DNA primer sequence 27 ggatcctcca
aatttcagag ggacctg 27 28 25 DNA Artificial sequence Synthetic DNA
primer sequence 28 gaattctcag tgactctcct ggctg 25 29 30 DNA
Artificial sequence Synthetic DNA primer sequence 29 ggatccgtaa
ctttgaagat agaaatgaaa 30 30 28 DNA Artificial sequence Synthetic
DNA primer sequence 30 gaatcctcat gcctccctaa aatatgta 28 31 23 DNA
Artificial sequence Synthetic DNA primer sequence 31 ggatcctcat
gctccgggcc gcg 23 32 27 DNA Artificial sequence Synthetic DNA
primer sequence 32 gaattcgcta gcaatcacca atgctga 27 33 20 DNA
Artificial sequence Synthetic DNA primer sequence 33 agaggtgaca
gaggatccca 20 34 24 DNA Artificial sequence Synthetic DNA primer
sequence 34 gaattcgtga tcccatcaca gtcc 24 35 22 DNA Artificial
sequence Synthetic DNA primer sequence 35 ggatcctgcc agggctccaa ct
22 36 19 DNA Artificial sequence Synthetic DNA primer sequence 36
atgacagatc tgagggtca 19 37 19 DNA Artificial sequence Synthetic DNA
primer sequence 37 cagccttgga gacctgagt 19 38 17 DNA Artificial
sequence Synthetic DNA primer sequence 38 tagcctactc tggccgc 17 39
18 DNA Artificial sequence Synthetic DNA primer sequence 39
ttgttggaat gaccttat 18
* * * * *