U.S. patent application number 10/888886 was filed with the patent office on 2004-12-30 for methods and products for inducing mucosal immunity.
Invention is credited to Davis, Heather L., McCluskie, Michael J..
Application Number | 20040266719 10/888886 |
Document ID | / |
Family ID | 22198286 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040266719 |
Kind Code |
A1 |
McCluskie, Michael J. ; et
al. |
December 30, 2004 |
Methods and products for inducing mucosal immunity
Abstract
The invention relates methods and products for inducing mucosal
immunity. In particular, the invention relates to the use of
immunostimulatory oligonucleotides containing a CpG motif for
inducing mucosal immunity. The CpG immunostimulatory
oligonucleotides may be administered alone or in combination with
antigen and/or with other adjuvants.
Inventors: |
McCluskie, Michael J.;
(Ottawa, CA) ; Davis, Heather L.; (Dunrobin,
CA) |
Correspondence
Address: |
Maria A. Trevisan
Wolf, Greenfield & Sacks, P.C.
600 Atlantic Avenue
Boston
MA
02210
US
|
Family ID: |
22198286 |
Appl. No.: |
10/888886 |
Filed: |
July 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10888886 |
Jul 9, 2004 |
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09316199 |
May 21, 1999 |
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60086393 |
May 22, 1998 |
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Current U.S.
Class: |
514/44A ;
424/450 |
Current CPC
Class: |
A61K 2039/53 20130101;
A61P 35/00 20180101; A61K 2039/55561 20130101; A61P 31/00 20180101;
A61K 2039/541 20130101; A61K 39/39 20130101; A61P 37/04 20180101;
A61K 2039/55566 20130101; A61K 2039/5555 20130101; A61K 2039/55577
20130101; A61K 2039/55522 20130101; A61K 2039/55544 20130101; A61K
2039/55555 20130101; A61P 37/00 20180101 |
Class at
Publication: |
514/044 ;
424/450 |
International
Class: |
A61K 048/00; A61K
009/127 |
Claims
We claim:
1.-124. (Cancelled)
125. A composition comprising an oral formulation of an effective
amount of an immunostimulatory CpG oligonucleotide 8-100
nucleotides in length comprising
18 5' X1X2CGX3X4 3',
and a pharmaceutically acceptable carrier, wherein at least the C
of the 5'CG3' is unmethylated and X1, X2, X3 and X4 are
nucleotides, wherein the pharmaceutically acceptable carrier
comprises dyestuffs, pigments, sugars, agar, sodium alginate or gum
arabic, and wherein the immunostimulatory CpG oligonucleotide is
isolated or synthetic.
126. The composition of claim 125, wherein the pharmaceutically
acceptable carrier comprises dyestuffs.
127. The composition of claim 125, wherein the pharmaceutically
acceptable carrier comprises pigments.
128. The composition of claim 125, wherein the pharmaceutically
acceptable carrier comprises sugars.
129. The composition of claim 125, wherein the pharmaceutically
acceptable carrier comprises agar.
130. The composition of claim 125, wherein the pharmaceutically
acceptable carrier comprises sodium alginate.
131. The composition of claim 125, wherein the pharmaceutically
acceptable carrier comprises gum arabic.
132. The composition of claim 125, wherein the immunostimulatory
CpG oligonucleotide comprises the sequence
19 5' X1X2CGX3X4 3',
wherein X1 or X2 or both are purines and X3 or X4 or both are
pyrimidines.
133. A method for treating a subject having an infection comprising
administering to a subject having an infection an immunostimulatory
CpG oligonucleotide 8-100 nucleotides in length comprising
20 5' X1X2CGX3X4 3',
and a pharmaceutically acceptable carrier, wherein at least the C
of the 5'CG3' is unmethylated and X1, X2, X3 and X4 are
nucleotides, and wherein the infection is selected from the group
consisting of enterovirus infection, calciviridae infection,
vesicular stomatitis viral infection, rotavirus infection,
Salmonella spp. infection, Helicobacter pyloris infection and M.
intracellulare infection.
134. The method of claim 133, wherein the immunostimulatory CpG
oligonucleotide comprises the sequence
21 5' X1X2CGX3X4 3',
wherein X1 or X2 or both are purines and X3 or X4 or both are
pyrimidines.
135. The method of claim 133, wherein the infection is Helicobacter
pyloris infection.
136. The method of claim 133, wherein the infection is calciviridae
infection.
137. The method of claim 133, wherein the immunostimulatory CpG
oligonucleotide is 8-40 nucleotides in length.
138. The composition of claim 125, wherein the immunostimulatory
CpG oligonucleotide is 8-40 nucleotides in length.
139. The method of claim 133, wherein the `5CG3` is not part of a
palindrome.
140. The composition of claim 125, wherein the `5CG3` is not part
of a palindrome.
141. The method of claim 133, wherein the immunostimulatory CpG
oligonucleotide includes a phosphorothioate backbone
modification.
142. The composition of claim 125, wherein the immunostimulatory
CpG oligonucleotide includes a phosphorothioate backbone
modification.
143. The method of claim 133, wherein the oligonucleotide is orally
administered.
Description
FIELD OF THE INVENTION
[0001] The present invention relates methods and products for
inducing mucosal immunity. In particular, the invention relates to
the use of immunostimulatory oligonucleotides containing a CpG
motif alone or in combination with other mucosal adjuvants for
inducing mucosal immunity.
BACKGROUND OF THE INVENTION
[0002] Two distinct compartments of the immune system have been
identified: (i) the systemic, which comprises the bone marrow,
spleen and lymph nodes, and (ii) the mucosal, which comprises
lymphoid tissue associated with mucosal surfaces and external
secretory glands (McGhee et al, 1992). Mucosal surfaces are
associated with the gastrointestinal (GI), genitourinary (GU) and
respiratory tracts. Each compartment is associated with both
humoral (antibodies) and cell-mediated (cytotoxic T-cells)
responses, however there are differences in the nature of the
immune responses induced in each compartment. Antibodies associated
with the systemic compartment are predominantly of the IgG isotype,
which function to neutralize pathogens in the circulatory system.
In contrast, antibodies in the mucosae are primarily secretory IgA
(S-IgA), which function to prevent entry of the pathogen into the
body via the mucosal surface (Lamm et al., 1992). Systemic immunity
cannot prevent entry of pathogenic organisms at mucosal
surfaces.
[0003] Successful systemic immunization (i.e., delivery of antigen
to the systemic compartment) will induce systemic immunity but does
not usually yield mucosal immune responses. In contrast, antigen
delivered at mucosal surfaces triggers both mucosal (at local and
sometimes at distant sites) and systemic responses (Haneberg et
al., 1994, Gallichan and Rosenthal, 1995).
[0004] Most vaccines developed to date are delivered parenterally,
for example by intramuscular (IM) or intradermal (ID) injection,
and as such induce primarily systemic immunity. However, the
combined mucosal surface area is more than 200 times greater than
that of the skin and is the primary site of transmission of
numerous infectious diseases. Therefore, current vaccination
strategies permit the pathogen to enter the body and only fight it
once it is in circulation. Infection and morbidity rates could be
reduced if effective mucosal immunity could be induced.
Furthermore, there is evidence that mucosal vaccines may have a
broader age range of recipients. Finally, mucosal vaccines are
often administered by non-invasive means (e.g., nose drops, nasal
spray, inhaled nebulizer), thus they are easier and less expensive
to administer, have less need for trained personnel and no risk of
needle stick injury or cross contamination (for reviews see
Mestecky et al., 1992, Staats et al., 1994, O,Hagan 1994).
[0005] As mentioned above, the hallmark of mucosal immunity is
local production of S-IgA antibodies. These constitute >80% of
all antibodies in mucosae-associated tissues and are induced,
transported and regulated by mechanisms quite distinct from those
of the systemic response. IgA is of primary importance to the host
defense because it acts not only to resist strict mucosal pathogens
but also of the many microorganisms which initially colonize
mucosal surfaces but subsequently cause systemic disease. There
appear to be three sites of IgA mediated mucosal defense: (i) in
the lumen, where S-IgA can neutralize viruses, bacterial toxins and
enzymes, and act as a mucosal barrier to prevent viral attachment,
microbial adherence and adsorption of antigen; (ii) within
epithelial cells where dimeric IgA can bind to intracellular
antigen; (iii) within the lamina propria where dimeric IgA can
complex with antigen and the immune complex thus formed transported
to the lumen (Lamm et al., 1992).
[0006] Many vaccines in development are composed of synthetic or
recombinant antigens (peptides or polypeptides). These are
considered safer than traditional attenuated or inactivated whole
pathogens, however they are often poorly immunogenic and require
adjuvants to enhance specific immunity. For systemic
administration, aluminum precipitates (alum) may be added to the
antigens to augment immune responses. Alum is currently the only
adjuvant licensed for human use in most countries including the
USA, however it is not suitable for delivery to mucosal surfaces.
Therefore most mucosal vaccines used today contain live-attenuated
organisms, and little success has been obtained with mucosal
delivery of subunit vaccines.
[0007] Cholera toxin (CT) is the mucosal adjuvant most commonly
used in animal models. CT is the primary enterotoxin produced by
Vibrio cholerae. It is an 84 kilodalton polymeric protein
consisting of two subunits, a monomeric A subunit and a pentameric
ring shaped B subunit. The B subunit binds GM1 gangliosides at the
surface of eukaryotic cells and enables insertion of the A subunit
into the cytosol, where it ADP-ribosylates GTP-binding regulatory
protein associated with adenylate cyclase (Spangler, 1992).
[0008] CT enhances antigen presentation by macrophages, epithelial
cells and B cells, promotes differentiation and isotype switching
in B cells, and has complex inhibitory and stimulatory effects on
T-cell proliferation and lymphokine production (Snider, 1995). Some
groups report that CT can selectively activate Th2-type CD4+ T
cells while inhibiting Th1-type cells (Takahashi et al., 1996,)
while others report activation of both TH1 and Th2-type CD4+ T
cells (Hornquist and Lycke, 1993). Differences may be due to a
number of factors including route of immunization and the nature of
the antigen.
[0009] The Escherichia coli heat-labile enterotoxin (labile toxin,
LT) is structurally and functionally closely related to CT, and has
similar adjuvant properties (Lycke et al., 1992). LT can confer
immunity to co-administered antigens that are on their own
non-immunogenic when administered by mucosal routes; this adjuvant
effect is noted whether LT is simply mixed with or is physically
coupled to the antigen (Holmgren et al., 1993).
[0010] While very effective as mucosal adjuvants in animal models,
CT and LT are highly toxic, and especially so in humans.
Genetically detoxified mutants of both CT and LT have been
developed by using site-directed mutagenesis, which, at least in
animal models appear to be less toxic yet retain some adjuvanticity
(e.g., LTK63 is LT with a single substitution at serine-63)
(Rappuoli et al., 1995, Douce et al., 1994, Pizza et al., 1994, De
Haan et al., 1996). Nevertheless, the level of adjuvanticity
appears to be proportional to the level of retained toxicity, and
thus there is a clear need for an alternative safe and effective
mucosal adjuvant.
SUMMARY OF THE INVENTION
[0011] The present invention relates to methods and products for
inducing immune responses using immunostimulatory CpG dinucleotide
containing oligonucleotides. In one aspect the invention is a
method for inducing a mucosal immune response. The method includes
the step of administering to a mucosal surface of a subject an
effective amount for inducing a mucosal immune response of an
oligonucleotide, having a sequence including at least the following
formula:
1 5' X.sub.1 X.sub.2CGX.sub.3 X.sub.4 3'
[0012] wherein C and G are unmethylated, wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides, and exposing the subject to
an antigen to induce the mucosal immune response, and wherein the
antigen is not encoded in a nucleic acid vector.
[0013] In another aspect the invention is a method for inducing a
mucosal immune response. The method includes the step of
administering to a mucosal surface of a subject an effective amount
for inducing a mucosal immune response of an antigen and a plasmid
vector, having a sequence including at least the following
formula:
2 5' X.sub.1 X.sub.2CGX.sub.3 X.sub.4 3'
[0014] wherein C and G are unmethylated, wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides.
[0015] In one embodiment the antigen is not encoded in a nucleic
acid vector. In another embodiment the antigen is encoded by a
nucleic acid vector, which optionally may be the plasmid vector. In
yet another embodiment the plasmid vector includes a nucleic acid
sequence which operatively encodes for a cytokine. Preferably the
antigen and the plasmid vector are administered orally or
intranasally. In some embodiments at least 50 .mu.g of the plasmid
vector is administered to the subject.
[0016] According to another aspect of the invention a method for
inducing a mucosal immune response is provided. The method includes
the step of administering to a mucosal surface of a subject an
effective amount for inducing a mucosal immune response of an
antigen and of an oligonucleotide, having a sequence including at
least the following formula:
3 5' X.sub.1 X.sub.2CGX.sub.3 X.sub.4 3'
[0017] wherein C and G are unmethylated, wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides, and wherein the antigen is
encoded by a nucleic acid vector. Preferably the antigen and the
oligonucleotide are administered orally or intranasally.
[0018] In some embodiments of the invention the oligonucleotide has
a backbone selected from the group consisting of a phosphodiester
backbone and a chimeric backbone. In other embodiments the
oligonucleotide has a phosphorothioate backbone. In the embodiments
wherein the oligonucleotide has a phosphorothioate backbone and
wherein the antigen is encoded by a nucleic acid vector and the CpG
is an oligonucleotide it is a preferred but not limited embodiment
that the plasmid and oligonucleotides are delivered with a
colloidal dispersion system. In some embodiments the colloidal
dispersion system is selected from the group consisting of
macromolecular complexes, nanocapsules, microspheres, beads, and
lipid-based systems. In other embodiments the plasmid and
oligonucleotide are coated onto gold particles and are delivered
with a gene-gun.
[0019] A method for inducing a mucosal immune response in a subject
is provided in other aspects. The method involves the step of
administering to a subject an antigen and an effective amount for
inducing a mucosal immune response of an oligonucleotide, having a
sequence including at least the following formula:
4 5' X.sub.1 X.sub.2CGX.sub.3 X.sub.4 3'
[0020] wherein C and G are unmethylated, wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides, and administering to the
subject a hormone to induce the mucosal immune response.
[0021] In one embodiment the antigen and the oligonucleotide are
administered to a mucosal surface of the subject. In another
embodiment the hormone is administered systemically. In one
embodiment the hormone is encoded by a nucleic acid vector.
[0022] The invention in other aspects involves methods for inducing
an immune response. The method involves the steps of orally,
intranasally, ocularly, vaginally, or rectally administering to a
subject an effective amount for inducing an immune response of an
oligonucleotide, having a sequence including at least the following
formula:
5 5' X.sub.1 X.sub.2CGX.sub.3 X.sub.4 3'
[0023] wherein C and G are unmethylated, wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides, and exposing the subject to
an antigen to induce the immune response.
[0024] In some embodiments the antigen is administered orally,
intranasally, ocularly, vaginally, or rectally. In other
embodiments the antigen is administered simultaneously with the
oligonucleotide. Preferably the oligonucleotide is administered in
an effective amount for inducing mucosal immunity.
[0025] According to other aspects the invention is a method for
inducing an immune response. The method involves the step of
orally, intranasally, ocularly, vaginally, or rectally
administering to a subject an effective amount for inducing an
immune response of a CpG containing plasmid, having a sequence
including at least the following formula:
6 5' X.sub.1 X.sub.2CGX.sub.3 X.sub.4 3'
[0026] wherein C and G are unmethylated, wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides, and exposing the subject to
an antigen to induce the immune response.
[0027] In some embodiments the antigen is administered orally,
intranasally, ocularly, vaginally, or rectally. In other
embodiments the antigen is administered simultaneously with the CpG
containing plasmid. Preferably the CpG containing plasmid is
administered in an effective amount for inducing mucosal
immunity.
[0028] The methods involve an induction of mucosal immunity.
Mucosal immunity can be induced in a local and/or remote site. In
some embodiments the mucosal immunity is induced in a local site
and in others the mucosal immunity is induced in a remote site, or
both.
[0029] In order to induce a mucosal immune response the CpG
oligonucleotide can be administered with a prime dose, a boost dose
or both. For instance the CpG oligonucleotide may be administered
with a priming dose of antigen. In another embodiment the CpG
oligonucleotide is administered with a boost dose of antigen. In
some embodiments the subject is administered a priming dose of
antigen and CpG oligonucleotide before the boost dose. In yet other
embodiments the subject is administered a boost dose of antigen and
CpG oligonucleotide after the priming dose.
[0030] In another aspect the invention is a method for inducing a
systemic immune response. The method involves administering to a
mucosal surface of a subject an effective amount for inducing a
systemic immune response of an oligonucleotide, having a sequence
including at least the following formula:
7 5' X.sub.1 X.sub.2CGX.sub.3 X.sub.4 3'
[0031] wherein C and G are unmethylated, wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides, and administering to the
mucosal surface of the subject an antigen to induce the systemic
immune response. In one embodiment the antigen is not encoded in a
nucleic acid vector, and wherein the antigen does not produce a
systemic immune response when administered to the mucosal surface
alone.
[0032] According to another aspect of the invention a method for
inducing a systemic immune response is provided. The method
involves the step of administering to a mucosal surface of a
subject an effective amount for inducing a systemic immune response
of a combination of a non-oligonucleotide mucosal adjuvant and an
oligonucleotide, having a sequence including at least the following
formula:
8 5' X.sub.1 X.sub.2CGX.sub.3 X.sub.4 3'
[0033] wherein C and G are unmethylated, wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides, and exposing the subject an
antigen to induce the systemic immune response.
[0034] In one embodiment the antigen is delivered to a mucosal
surface. In another embodiment the antigen is not encoded in a
nucleic acid vector.
[0035] The subject may be actively exposed to the antigen or
passively exposed to the antigen. In one embodiment of the methods
described herein the subject is actively exposed to the antigen and
the antigen is delivered to a mucosal surface. In other embodiments
the antigen is administered concurrently with the oligonucleotide.
The antigen may be delivered alone or in conjunction with a
colloidal dispersion system. In some embodiments the colloidal
dispersion system is selected from the group consisting of
macromolecular complexes, nanocapsules, microspheres, beads, and
lipid-based systems. Lipid-based systems optionally include
oil-in-water emulsions, micelles, mixed micelles, or liposomes.
[0036] In other embodiments the subject is passively exposed to the
antigen through environmental contact. The subject that is
passively exposed to the antigen in some embodiments is a subject
at risk of developing an allergic reaction, an infectious disease,
or a cancer. In other embodiments the subject has an infectious
disease, a cancer, an allergy or is an asthmatic.
[0037] The antigen that is passively or actively administered to
the subject is any type of antigen known in the art and includes
for example cells, cell extracts, proteins, polypeptides, peptides,
polysaccharides, polysaccharide conjugates, peptide mimics of
polysaccharides, lipids, glycolipids, carbohydrates, allergens,
viruses and viral extracts and muticellular organisms such as
parasites. In one embodiment the antigen is derived from an
infectious organism selected from the group consisting of
infectious bacteria, infectious viruses, infectious parasites, and
infectious fungi.
[0038] The method may also include the step of administering a
non-oligonucleotide mucosal adjuvant in conjunction with the
antigen. Non-oligonucleotide mucosal adjuvants may include, for
example, cholera toxin, derivatives of cholera toxin, labile toxin,
derivatives of labile toxin, alum, MLP, MDP, saponins such as QS21,
cytokines, oil-in-water and other emulsion formulations such as
MF59, SAF, Montanide ISA 720 and PROVAX, PCPP polymers, and
ISCOMS.
[0039] In other embodiments the method includes the step of
administering a cytokine or a B-7 costimulatory molecule to the
subject.
[0040] In some embodiments, the oligonucleotide is administered
orally, mucosally, ocularly, vaginally, rectally, or by inhalation
to a subject.
[0041] The oligonucleotide may be modified. For instance, in some
embodiments at least one nucleotide has a phosphate backbone
modification. The phosphate backbone modification may be a
phosphorothioate or phosphorodithioate modification. In some
embodiments the phosphate backbone modification occurs on the 5'
side of the oligonucleotide or the 3' side of the
oligonucleotide.
[0042] The oligonucleotide may be any size. Preferably the
oligonucleotide has 8 to 100 nucleotides. In other embodiments the
oligonucleotide is 8 to 40 nucleotides in length.
[0043] In some embodiments X.sub.1X.sub.2 are nucleotides selected
from the group consisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT,
CpA, CpG, TpA, TpT, and TpG; and X.sub.3X.sub.4 are nucleotides
selected from the group consisting of: TpT, CpT, ApT, TpG, ApG,
CpG, TpC, ApC, CpC, TpA, ApA, and CpA. Preferably X.sub.1X.sub.2
are GpA or GpT and X.sub.3X.sub.4 are TpT. In other preferred
embodiments X.sub.1 or X.sub.2 or both are purines and X.sub.3 or
X.sub.4 or both are pyrimidines or X.sub.1X.sub.2 are GpA and
X.sub.3 or X.sub.4or both are pyrimidines. In one embodiment
X.sub.2 is a T and X.sub.3 is a pyrimidine. The oligonucleotide may
be isolated or synthetic.
[0044] In some embodiments the oligonucleotide has a sequence
including at least the following formula:
9 5' TCNTX.sub.1X.sub.2CGX.sub.3X.sub.4 3'
[0045] wherein X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are
nucleotides, N is a nucleic acid sequence composed of from about
0-25 nucleotides.
[0046] In other aspects, the invention encompasses pharmaceutical
compositions for orally, intranasally, ocularly, vaginally, or
rectally administering CpG oligonucleotides or CpG plasmids. In one
aspect the composition is an oral formulation of a CpG
oligonucleotide in a buffer for neutralizing biological acids. In
another aspect the composition is an intranasal formulation of a
CpG oligonucleotide in an aerosol. In other aspects the composition
is a vaginal or rectal formulation of a CpG oligonucleotide in a
suppository or other vehicle suitable for delivery to vaginal and
rectal tissue. In other aspect the composition is an ocular
formulation of a CpG oligonucleotide in a solution compatible with
the eye. Such formulations are described herein as well as in
Remingtons Pharmaceutical Sciences, which is hereby incorporated by
reference.
[0047] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 is a bar graph depicting the effect of different
adjuvants on total IgG titers of anti-HBS, wherein BALB/c mice were
immunized by IN inhalation with HBsAg (1 or 10 .mu.g) without or in
combination with Cholera toxin (CT) and/or CpG oligonucleotide
(motif #1826, SEQ ID NO. 90) adjuvants.
[0049] FIG. 2 is a graph depicting the effect of different
adjuvants on total IgG titers of anti-HbS, wherein BALB/c mice were
immunized by IN inhalation with HBsAg (1 .mu.g) without or in
combination with Cholera toxin (CT) and/or CpG oligonucleotide
(motif #1826, SEQ ID NO. 90) adjuvants and at 8 weeks mice were
boosted in the same manner as prime.
[0050] FIG. 3 is a bar graph depicting the effect of different
adjuvants on anti-HBs IgG isotype, wherein BALB/c mice were
immunized by IN inhalation with HBsAg (1 .mu.g) without or in
combination with Cholera toxin (CT) and/or CpG oligonucleotide
(motif #1826, SEQ ID NO. 90) adjuvants (1 .mu.g) and at 8 weeks
mice were boosted in the same manner as prime.
[0051] FIG. 4 is a bar graph depicting the effect of different
adjuvants on HBsAg specific CTL response, wherein BALB/c mice were
immunized by IN inhalation with HBsAg (10 .mu.g) without or in
combination with Cholera toxin (CT) and/or CpG oligonucleotide
(motif #1826, SEQ ID NO. 90) adjuvants at different doses (1 or 10
.mu.g) and four weeks after immunization mice were killed by
Halothane overdose, splenocytes isolated and HBsAg specific CTL
activity measured.
[0052] FIG. 5 is a bar graph depicting the effect of different
adjuvants on anti-HBs IgA titers in lung washes, wherein BALB/c
mice were immunized by IN inhalation with HBsAg (1 or 10 .mu.g)
without or in combination with Cholera toxin (CT) and/or CpG
oligonucleotide (motif #1826, SEQ ID NO. 90) adjuvants at different
doses (1 or 10 .mu.g) and four weeks after immunization (or after
boost for group marked by *) mice were killed by Halothane overdose
and lungs were washed with 1 ml TBS.
[0053] FIG. 6 is a bar graph depicting the effect of different
adjuvants on anti-HBs IgA titers in fecal pellet solutions, wherein
BALB/c mice were immunized by IN inhalation with HBsAg (1 or 10
.mu.g) without or in combination with Cholera toxin (CT) and/or CpG
oligonucleotide (motif #1826, SEQ ID NO. 90) at different doses (1
or 10 .mu.g) and four weeks after immunization (or after boost for
group marked by *) mice were isolated for 24 hr and fecal pellets
were collected and resuspended in TBS at 0.1 mg/ml.
[0054] FIG. 7 is a graph depicting the effect of different
adjuvants on total IgG titers of anti-HBs, wherein BALB/c mice were
immunized by IN inhalation with HBsAg (1 .mu.g) without or in
combination with Cholera toxin (CT), Escherichia coli heat-labile
enterotoxin (LT), the B subunit of Cholera toxin (CTB), a
detoxified mutant of Escherichia coli heat-labile enterotoxin
(LTK63), CpG oligonucleotide (motif #1826, SEQ ID NO. 90) or
non-CpG control oligonucleotide (motif #1982, SEQ ID NO. 90) as
adjuvants (1, 10 or 500 .mu.g). In groups which responded, all mice
gave titers >10, except in the case of 10 .mu.g LT where only
1/5 mice responded.
[0055] FIG. 8 is a bar graph depicting the effect of different
prime/boost strategies on total IgG titers of anti-HBs, wherein
BALB/c mice were immunized: (i) by IM injection with HBsAg (1
.mu.g) in combination with alum plus CpG oligonucleotide (motif
#1826, SEQ ID NO. 90) and boosted at 4 weeks as prime, or by IN
inhalation with HBsAg (1 .mu.g) without or in combination with
Cholera toxin (CT) and/or CpG oligonucleotide (motif #1826, SEQ ID
NO. ); or (ii) by IN inhalation with HBsAg (1 .mu.g) without or in
combination with Cholera toxin (CT) and/or CpG oligonucleotide
(motif #1826, SEQ ID NO. 90) and boosted at 4 weeks as prime or by
IM injection with HBsAg (1 .mu.g) in combination with alum plus CpG
oligonucleotide (motif #1826, SEQ ID NO. 90). Numbers at the top of
each bar represent the IgGa/IgG1 ratio.
[0056] FIG. 9 is a bar graph depicting the effect of different
prime/boost strategies with different adjuvants on HBsAg specific
CTL response, wherein BALB/c mice were immunized: (i) by IM
injection with HBsAg (1 .mu.g) in combination with alum plus CpG
oligonucleotide (motif #1826, SEQ ID NO. 90) and boosted at 4 weeks
as prime, or by IN inhalation with HBsAg (1 .mu.g) without or in
combination with Cholera toxin (CT) and/or CpG oligonucleotide
(motif #1826, SEQ ID NO. 90), or (ii) by IN inhalation with HBsAg
(1 .mu.g) without or in combination with Cholera toxin (CT) and/or
CpG oligonucleotide (motif #1826, SEQ ID NO. 90) and boosted at 4
weeks as prime or by IM injection with HBsAg (1 .mu.g) in
combination with alum plus CpG oligonucleotide (motif #1826, SEQ ID
NO. 90), and 4 weeks after boost mice were killed by Halothane
overdose, splenocytes isolated and HBsAg specific CTL activity
measured.
[0057] FIG. 10 is a bar graph depicting the effect of different
prime/boost strategies with different adjuvants on HBsAg specific T
cell proliferation, wherein BALB/c mice were immunized: (i) by IM
injection with HBsAg (1 .mu.g) in combination with alum plus CpG
oligonucleotide (motif #1826, SEQ ID NO. 90) and boosted at 4 weeks
as prime, or by IN inhalation with HBsAg (1 .mu.g) without or in
combination with Cholera toxin (CT) and/or CpG oligonucleotide
(motif #1826, SEQ ID NO. 90), or (ii) by IN inhalation with HBsAg
(1 .mu.g) without or in combination with Cholera toxin (CT) and/or
CpG oligonucleotide (motif #1826, SEQ ID NO. 90) and boosted at 4
weeks as prime or by IM injection with HBsAg (1 .mu.g) in
combination with alum plus CpG oligonucleotide (motif #1826, SEQ ID
NO. 90 ), and 4 weeks after boost mice were killed by Halothane
overdose, splenocytes isolated and HBsAg specific T cell
proliferation measured.
[0058] FIG. 11 is a bar graph depicting the effect of different
prime/boost strategies with different adjuvants on anti-HBs IgA
titers in lung washes, wherein BALB/c mice were immunized: (i) by
IM injection with HBsAg (1 .mu.g) in combination with alum plus CpG
oligonucleotide (motif #1826, SEQ ID NO. 90) and boosted at 4 weeks
as prime, or by IN inhalation with HBsAg (1 .mu.g) without or in
combination with Cholera toxin (CT) and/or CpG oligonucleotide
(motif #1826, SEQ ID NO. 90), or (ii) by IN inhalation with HBsAg
(1 .mu.g) without or in combination with Cholera toxin (CT) and/or
CpG oligonucleotide (motif #1826, SEQ ID NO. 90) and boosted at 4
weeks as prime or by IM injection with HBsAg (1 .mu.g) in
combination with alum plus CpG oligonucleotide (motif #1826, SEQ ID
NO. 90). Four weeks after boost mice were killed by Halothane
overdose and lungs were washed with 1 ml TBS.
[0059] FIG. 12 is a bar graph depicting the effect of different
prime/boost strategies with different adjuvants on anti-HBs IgA
titers in saliva, wherein BALB/c mice were immunized: (i) by IM
injection with HBsAg (1 .mu.g) in combination with alum plus CpG
oligonucleotide (motif #1826, SEQ ID NO. 90) and boosted at 4 weeks
as prime, or by IN inhalation with HBsAg (1 .mu.g) without or in
combination with Cholera toxin (CT) and/or CpG oligonucleotide
(motif #1826, SEQ ID NO. 90), or (ii) by IN inhalation with HBsAg
(1 .mu.g) without or in combination with Cholera toxin (CT) and/or
CpG oligonucleotide (motif #1826, SEQ ID NO. 90) and boosted at 4
weeks as prime or by IM injection with HBsAg (1 .mu.g) in
combination with alum plus CpG oligonucleotide (motif#1826, SEQ ID
NO. 90). Four weeks after boost mice were injected with 100 .mu.l
0.5% Pilocarpine hydrochloride solution and saliva collected.
[0060] FIG. 13 is a bar graph depicting the effect of different
prime/boost strategies with different adjuvants on anti-HBs IgA
titers in fecal pellet solutions, wherein BALB/c mice were
immunized: (i) by IM injection with HBsAg (1 .mu.g) in combination
with alum plus CpG oligonucleotide (motif #1826, SEQ ID NO. 90) and
boosted at 4 weeks as prime, or by IN inhalation with HBsAg (1
.mu.g) without or in combination with Cholera toxin (CT) and/or CpG
oligonucleotide (motif #1826, SEQ ID NO. 90), or (ii) by IN
inhalation with HBsAg (1 .mu.g) without or in combination with
Cholera toxin (CT) and/or CpG oligonucleotide (motif #1826, SEQ ID
NO. 90) and boosted at 4 weeks as prime or by IM injection with
HBsAg (1 .mu.g) in combination with alum plus CpG oligonucleotide
(motif #1826, SEQ ID NO. 90). Four weeks after boost mice were
isolated for 24 hr and fecal pellets were collected and resuspended
in TBS at 0.1 mg/ml.
BRIEF DESCRIPTION OF THE TABLES
[0061] Table 1 lists immunostimulatory oligonucleotide
sequences.
[0062] Table 2 lists the effect of different adjuvants on
HBsAg-specific antibody isotypes.
[0063] .sup.a BALB/c mice were immunized by IN inhalation with
HBsAg (1 .mu.g) without or in combination with Cholera toxin (CT),
Escherichia coli heat-labile enterotoxin (LT), the B subunit of
Cholera toxin (CTB), a detoxified mutant of Escherichia coli
heat-labile enterotoxin (LTK63) and/or CpG oligonucleotide (motif
#1826, SEQ ID NO. 90) (1 or 10 .mu.g) as adjuvants.
[0064] .sup.bValues represent the group geometric mean (GMT) of the
ELISA end-point dilution titer for HBsAg-specific IgG1 or IgG2a
antibodies in plasma taken 4 wk after immunization. Titers were
defined as the highest plasma dilution resulting in an absorbance
value two times that of non-immune plasma, with a cut-off value of
0.05.
[0065] .sup.cThe IgG2a to IgG1 ratios (IgG2a:IgG1) are reported,
with a value >1 indicating a predominantly Th-1 like
response.
[0066] .sup.dN/A: Not applicable since no antibody responses
detected.
[0067] .sup.e=: All mice immunized with these adjuvant combinations
died within 96 hours.
[0068] Table 3 lists the effect of different adjuvants on
HBsAg-specific IgA responses.
[0069] .sup.aBALB/c mice were immunized by IN inhalation with HBsAg
(1 .mu.g) without or in combination with Cholera toxin (CT),
Escherichia coli heat-labile enterotoxin (LT), the B subunit of
Cholera toxin (CTB), a detoxified mutant of Escherichia coli
heat-labile enterotoxin (LTK63) and/or CpG oligonucleotide (motif
#1826, SEQ ID NO. 90) (1 or 10 .mu.g) as adjuvants. All groups
contained 5 mice unless otherwise indicated.
[0070] .sup.bValues represent the geometric mean titers .+-. the
standard error of the mean (GMT.+-.SEM) of the ELISA end-point
dilution titer for HBsAg-specific IgA antibodies in lung wash or
fecal solutions taken 4 wk after immunization.
[0071] .sup.cIgA titers in lung washes were defined as the highest
dilution that resulted in an absorbance value (OD 450) two times
greater than that of non-immune lung wash, with a cut-off value of
0.05.
[0072] .sup.dIgA titers in fecal extracts were expressed as OD
450.times.10.sup.3 above background (non-immune fecal extract).
Seroconversion was defined as an endpoint titer for total
IgG>100.
[0073] .sup.e=: All mice immunized with these adjuvant combinations
died within 96 hours.
[0074] Table 4 shows the different mucosal/parenteral prime
boost/strategies used to immunize BALB/c mice and summarizes the
results as to which approach induced antigen-specific systemic and
mucosal immune responses.
DETAILED DESCRIPTION OF THE INVENTION
[0075] The invention relates to methods and products for inducing
immunity using immunostimulatory CpG oligonucleotides. One aspect
of the invention is based on the finding that CpG oligonucleotides
act as a potent mucosal adjuvants to induce immune responses at
both local and remote sites against an antigen administered to the
mucosal tissue. This finding is striking even in view of previous
findings that CpG oligonucleotide is a potent adjuvant for systemic
delivery, because with systemic delivery the protein alone induces
detectable immune responses but with mucosal delivery the protein
alone does not induce an immune response. As demonstrated in the
Examples below, both systemic and mucosal immunity are induced by
mucosal delivery of CpG oligonucleotides. The systemic immunity
induced in response to CpG oligonucleotides included both humoral
and cell-mediated responses to specific antigens that were not
capable of inducing systemic immunity when administered alone to
the mucosa. Furthermore, both CpG oligonucleotides and cholera
toxin (CT, a mucosal adjuvant that induces a Th2-like response)
induced CTL. This is surprising since with systemic immunization,
the presence of Th2-like antibodies is normally associated with a
lack of CTL (Schirmbeck et al, 1995).
[0076] Additionally, CpG oligonucleotides were found to induce a
mucosal response at both local (e.g., lung) and remote (e.g., lower
digestive tract) mucosal sites. Although CpG oligonucleotide was
similar to CT for induction of systemic antibodies (IgG) and local
mucosal antibodies (IgA), significant levels of IgA antibodies were
induced at a distant mucosal site only by CpG oligonucleotide and
not by CT. This was surprising because CT is generally considered
to be a highly effective mucosal adjuvant. Another manner in which
CpG oligonucleotide was superior to CT was with respect to the
Th-type of response. As has been previously reported (Snider 1995),
CT induces predominantly IgG1 isotype of antibodies, which are
indicative of Th2-type response. In contrast, CpG oligonucleotide
was more Th1 with predominantly IgG2a antibodies, especially after
boost or when the two adjuvants were combined. Th1-type antibodies
in general have better neutralizing capabilities, and furthermore,
a Th2 response in the lung is highly undesirable because it is
associated with asthma (Kay, 1996, Hogg, 1997). Thus the use of CpG
oligonucleotide as a mucosal adjuvant has benefits that other
mucosal adjuvants cannot achieve.
[0077] The discovery of CpG oligonucleotide as a safe and effective
mucosal adjuvant is also advantageous because although CT is a
highly effective mucosal adjuvant, it is too toxic for use in
humans. A mouse (.about.20 g body weight) can tolerate the toxic
effects of up to 10 .mu.g of CT, however a dose as little as 1-5
.mu.g will cause severe diarrhea in a human (.about.70 kg body
weight) (Jertborn et al., 1992). Animals inhaling CpG
oligonucleotide showed no short-term signs of distress over those
receiving HBsAg alone, and all recovered quickly with no apparent
long-lasting effects. CpG oligonucleotide is well tolerated at very
high doses (e.g., greater than 100 .mu.g), when delivered
systemically or mucosally.
[0078] Thus in one aspect the invention is a method for inducing a
mucosal immune response in a subject. The method includes the step
of administering to a mucosal surface of a subject an effective
amount for inducing a mucosal immune response of an
oligonucleotide, having a sequence including at least the following
formula:
10 5' X.sub.1 X.sub.2CGX.sub.3 X.sub.4 3'
[0079] wherein C and G are unmethylated, wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides, and exposing the subject to
an antigen to induce the mucosal immune response. In other aspects
the method involves administering a plasmid vector, having a
sequence including at least the above formula instead of the
oligonucleotide. The oligonucleotide, referred to herein as the
oligonucleotide or the CpG oligonucleotide, is not a plasmid
vector. These distinctions are made clear in the definitions set
forth below. For purposes of brevity, the invention is described
herein with respect to CpG oligonucleotides, but the description
also applies to plasmid vectors.
[0080] The CpG oligonucleotide is particularly useful as a
prophylactic vaccine for the induction of mucosal immunity of a
subject at risk of developing an infection with an infectious
organism or a subject at risk of developing an allergy or cancer. A
"subject at risk" a used herein is a subject who has any risk of
exposure to an infection causing infectious pathogen or an allergen
or of developing cancer. For instance, a subject at risk may be a
subject who is planning to travel to an area where a particular
type of infectious agent or allergen is found or it may be a
subject who through lifestyle or medical procedures is exposed to
bodily fluids which may contain infectious organisms or even any
subject living in an area that an infectious organism or an
allergen has been identified. Subjects at risk of developing
infection also include general populations to which a medical
agency recommends vaccination with a particular infectious organism
antigen. If the antigen is an allergen and the subject develops
allergic responses to that particular antigen and the subject is
exposed to the antigen, i.e., during pollen season, then that
subject is at risk of exposure to the antigen. Subjects at risk of
developing cancer include those with a genetic predisposition or
previously treated for cancer, and those exposed to carcinogens
such as tobacco, asbestos, and other chemical toxins or excessive
sunlight and other types of radiation.
[0081] In addition to the use of the CpG oligonucleotide for
prophylactic treatment, the invention also encompasses the use of
the CpG oligonucleotide for the treatment of a subject having an
infection, an allergy or a cancer.
[0082] A "subject having an infection" is a subject that has been
exposed to an infectious pathogen and has acute or chronic
detectable levels of the pathogen in the body. The CpG
oligonucleotide can be used with an antigen to mount an antigen
specific mucosal immune response that is capable of reducing the
level of or eradicating the infectious pathogen. An infectious
disease, as used herein, is a disease arising from the presence of
a foreign microorganism in the body. It is particularly important
to develop effective vaccine strategies and treatments to protect
the body's mucosal surfaces, which are the primary site of
pathogenic entry.
[0083] A "subject having an allergy" is a subject that has or is at
risk of developing an allergic reaction in response to an allergen.
An "allergy" refers to acquired hypersensitivity to a substance
(allergen). Allergic conditions include but are not limited to
eczema, allergic rhinitis or coryza, hay fever, conjunctivitis,
bronchial asthma, urticaria (hives) and food allergies, and other
atopic conditions.
[0084] Currently, allergic diseases are generally treated by the
injection of small doses of antigen followed by subsequent
increasing dosage of antigen. It is believed that this procedure
induces tolerization to the allergen to prevent further allergic
reactions. These methods, however, can take several years to be
effective and are associated with the risk of side effects such as
anaphylactic shock. The methods of the invention avoid these
problems.
[0085] Allergies are generally caused by IgE antibody generation
against harmless allergens. The cytokines that are induced by
mucosal administration of unmethylated CpG oligonucleotides are
predominantly of a class called "Th1" (examples are IL-12 and
IFN-.gamma.) and these induce both humoral and cellular immune
responses. The types of antibodies associated with a Th1 response
are generally more protective because they have high neutralization
and opsonization capabilities. The other major type of immune
response, which is associated with the production of IL4, IL-5 and
IL-10 cytokines, is termed as Th2 immune response. Th2 responses
involve solely antibodies and these have less protective effect
against infection and some Th2 isotypes (e.g., IgE) are associated
with allergy. In general, it appears that allergic diseases are
mediated by Th2 type immune responses while Th1 responses provide
the best protection against infection, although excessive Th1
responses are associated with autoimmune disease. Based on the
ability of the CpG oligonucleotides to shift the immune response in
a subject from a Th2 (which is associated with production of IgE
antibodies and allergy) to a Th1 response (which is protective
against allergic reactions), an effective dose for inducing a
mucosal immune response of a CpG oligonucleotide can be
administered to a subject to treat or prevent an allergy.
[0086] Thus, the CpG oligonucleotide has significant therapeutic
utility in the treatment of allergic conditions such as asthma. Th2
cytokines, especially IL4 and IL-5 are elevated in the airways of
asthmatic subjects. These cytokines promote important aspects of
the asthmatic inflammatory response, including IgE isotope
switching, eosinophil chemotaxis and activation and mast cell
growth. Th1 cytokines, especially IFN-.gamma. and IL-12, can
suppress the formation of Th2 clones and production of Th2
cytokines. "Asthma" refers to a disorder of the respiratory system
characterized by inflammation, narrowing of the airways and
increased reactivity of the airways to inhaled agents. Asthma is
frequently, although not exclusively associated with a topic or
allergic symptoms.
[0087] A "subject having a cancer" is a subject that has detectable
cancerous cells. The cancer may be a malignant or non-malignant
cancer. Cancers or tumors include but are not limited to biliary
tract cancer; brain cancer; breast cancer; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal
cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver
cancer; lung cancer (e.g. small cell and non-small cell); melanoma;
neuroblastomas; oral cancer; ovarian cancer; pancreas cancer;
prostate cancer; rectal cancer; sarcomas; skin cancer; testicular
cancer; thyroid cancer; and renal cancer, as well as other
carcinomas and sarcomas.
[0088] A "subject" shall mean a human or vertebrate animal
including but not limited to a dog, cat, horse, cow, pig, sheep,
goat, chicken, primate, e.g., monkey, fish (aquaculture species),
e.g. salmon, rat, and mouse.
[0089] A CpG oligonucleotide is an oligonucleotide which includes
at least one unmethylated CpG dinucleotide. An oligonucleotide
containing at least one unmethylated CpG dinucleotide is a nucleic
acid molecule which contains an unmethylated cytosine-guanine
dinucleotide sequence (i.e. "CpG DNA" or DNA containing a 5'
cytosine followed by 3' guanosine and linked by a phosphate bond)
and activates the immune system. The CpG oligonucleotides can be
double-stranded or single-stranded. Generally, double-stranded
molecules are more stable in vivo, while single-stranded molecules
have increased immune activity.
[0090] The terms "nucleic acid" and "oligonucleotide" are used
interchangeably to mean multiple nucleotides (i.e. molecules
comprising a sugar (e.g. ribose or deoxyribose) linked to a
phosphate group and to an exchangeable organic base, which is
either a substituted pyrimidine (e.g. cytosine (C), thymine (T) or
uracil (U)) or a substituted purine (e.g. adenine (A) or guanine
(G)). As used herein, the terms refer to oligoribonucleotides as
well as oligodeoxyribonucleotides. The terms shall also include
polynucleosides (i.e. a polynucleotide minus the phosphate) and any
other organic base containing polymer. Nucleic acid molecules can
be obtained from existing nucleic acid sources (e.g. genomic or
cDNA), but are preferably synthetic (e.g. produced by
oligonucleotide synthesis). The entire CpG oligonucleotide can be
unmethylated or portions may be unmethylated but at least the C of
the 5' CG 3' must be unmethylated.
[0091] The methods of the invention may be accomplished by
administering a CpG containing oligonucleotide or a CpG containing
plasmid vector to the subject to induce a mucosal immune response.
As used herein the terms a "CpG oligonucleotide" and a "plasmid
expression vector" are mutually exclusive. The terms "CpG
oligonucleotide" or "CpG nucleic acid" are used to refer to any CpG
containing nucleic acid except for a CpG containing plasmid vector.
A plasmid expression vector is a nucleic acid molecule which
includes at least a promoter and a gene encoding a peptide or
peptide fragment. The plasmid expression vector includes a nucleic
acid sequence encoding the peptide which is operatively linked to a
gene expression sequence which directs the expression of the
peptide within a eukaryotic cell. The "gene expression sequence" is
any regulatory nucleotide sequence, such as a promoter sequence or
promoter-enhancer combination, which facilitates the efficient
transcription and translation of the peptide to which it is
operatively linked. The gene expression sequence may, for example,
be a mammalian or viral promoter, such as a constitutive or
inducible promoter. Such constructs are well known to those of
skill in the art.
[0092] In one preferred embodiment the invention provides a CpG
oligonucleotide represented by at least the formula:
11 5'N.sub.1X.sub.1CGX.sub.2N.sub.23'
[0093] wherein at least one nucleotide separates consecutive CpGs;
X.sub.1 is adenine, guanine, or thymine; X.sub.2 is cytosine,
adenine, or thymine; N is any nucleotide and N.sub.1 and N.sub.2
are nucleic acid sequences composed of from about 0-25 N's
each.
[0094] In another embodiment the invention provides an isolated CpG
oligonucleotide represented by at least the formula:
12 5'N.sub.1X.sub.1X.sub.2CGX.sub.3X.sub.4N.sub.23'
[0095] wherein at least one nucleotide separates consecutive CpGs;
X.sub.1X.sub.2 are nucleotides selected from the group consisting
of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG;
and X.sub.3X.sub.4 are nucleotides selected from the group
consisting of: TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA,
ApA, and CpA; N is any nucleotide and N.sub.1 and N.sub.2 are
nucleic acid sequences composed of from about 0-25 N's each.
Preferably X.sub.1X.sub.2 are GpA or GpT and X.sub.3X.sub.4 are
TpT. In other preferred embodiments X.sub.1 or X.sub.2 or both are
purines and X.sub.3 or X.sub.4 or both are pyrimidines or
X.sub.1X.sub.2 are GpA and X.sub.3 or X.sub.4 or both are
pyrimidines. In a preferred embodiment N.sub.1 and N.sub.2 of the
nucleic acid do not contain a CCGG or CGCG quadmer or more than one
CCG or CGG trimer. The effect of a a CCGG or CGCG quadmer or more
than one CCG or CGG trimer depends in part on the status of the
oligonucleotide backbone. For instance, if the oligonucleotide has
a phosphodiester backbone or a chimeric backbone the inclusion of
these sequences in the oligonucleotide will only have minimal if
any affect on the biological activity of the oligonucleotide. If
the backbone is completely phosphorothioate or significantly
phosphorothioate then the inclusion of these sequences may have
more influence on the biological activity or the kinetics of the
biological activity. In the case when the CpG oligonucleotide is
administered in conjunction with an antigen which is encoded in a
nucleic acid vector, it is preferred that the backbone of the CpG
oligonucleotide be phosphodiester or chimeric. It can be completely
phosphorothioate if the oligonucleotide is delivered directly to
the cell. The cell may have a problem taking up a completely
phosphorothioate oligonucleotide in the presence of a plasmid
vector. Thus when both a vector and an oligonucleotide are
delivered to a subject, it is preferred that the oligonucleotide
have a phosphodiester or chimeric backbone or have a
phosphorothioate backbone but be associated with a vehicle that
delivers it directly into the cell. Such vehicles are known in the
art and include, for example, liposomes and gene guns.
[0096] In another preferred embodiment the CpG oligonucleotide has
the sequence
13 5'TCN.sub.1TX.sub.1X.sub.2CGX.sub.3X.sub.43'.
[0097] Preferably the CpG oligonucleotides of the invention include
X.sub.1X.sub.2 selected from the group consisting of GpT, GpG, GpA
and ApA and X.sub.3X.sub.4 is selected from the group consisting of
TpT, CpT and TpC. For facilitating uptake into cells, CpG
containing oligonucleotides are preferably in the range of 8 to 30
bases in length. However, nucleic acids of any size greater than 8
nucleotides (even many kb long) are capable of inducing an immune
response according to the invention if sufficient immunostimulatory
motifs are present, since larger nucleic acids are degraded into
oligonucleotides inside of cells. Preferred synthetic
oligonucleotides do not include a CCGG or CGCG quadmer or more than
one CCG or CGG trimer at or near the 5' and/or 3' terminals.
Stabilized oligonucleotides, where the oligonucleotide incorporates
a phosphate backbone modification, as discussed in more detail
below are also preferred. The modification may be, for example, a
phosphorothioate or phosphorodithioate modification. Preferably,
the phosphate backbone modification occurs at the 5' end of the
nucleic acid for example, at the first two nucleotides of the 5'
end of the oligonucleotide. Further, the phosphate backbone
modification may occur at the 3' end of the nucleic acid for
example, at the last five nucleotides of the 3' end of the nucleic
acid. Alternatively the oligonucleotide may be completely or
partially modified.
[0098] Preferably the CpG oligonucleotide is in the range of
between 8 and 100 and more preferably between 8 and 30 nucleotides
in size. Alternatively, CpG oligonucleotides can be produced on a
large scale in plasmids and degraded into oligonucleotides.
[0099] The CpG oligonucleotide may be directly administered to the
subject or may be administered in conjunction with a nucleic acid
delivery complex. A "nucleic acid delivery complex" shall mean a
nucleic acid molecule associated with (e.g. ionically or covalently
bound to; or encapsulated within) a targeting means (e.g. a
molecule that results in higher affinity binding to target cell
(e.g. dendritic cell surfaces and/or increased cellular uptake by
target cells). Examples of nucleic acid delivery complexes include
nucleic acids associated with: a sterol (e.g. cholesterol), a lipid
(e.g. a cationic lipid, virosome or liposome), or a target cell
specific binding agent (e.g. a ligand recognized by target cell
specific receptor). Preferred complexes may be sufficiently stable
in vivo to prevent significant uncoupling prior to internalization
by the target cell. However, the complex can be cleavable under
appropriate conditions within the cell so that the nucleic acid is
released in a functional form.
[0100] Delivery vehicles for delivering antigen to mucosal surfaces
have been described. The CpG oligonucleotide and/or the antigen may
be administered alone (e.g. in saline or buffer) or using any
delivery vehicles known in the art. For instance the following
delivery vehicles have been described: Cochleates (Gould-Fogerite
et al., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et
al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu
et., 1998, Morein et al., 1999); Liposomes (Childers et al., 1999,
Michalek et al., 1989, 1992, de Haan 1995a, 1995b); Live bacterial
vectors (e.g., Salmonella, Escherichia coli, Bacillus
calmatte-guerin, Shigella, Lactobacillus) (Hone et al., 1996,
Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991,
Nugent et al., 1998); Live viral vectors (e.g., Vaccinia,
adenovirus, Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et
al., 1996, Nugent et al., 1998, Flexner et al., 1988, Morrow et
al., 1999); Microspheres (Gupta et al., 1998, Jones et al., 1996,
Maloy et al., 1994, Moore et al., 1995, O'Hagan et al., 1994,
Eldridge et al., 1989); Nucleic acid vaccines (Fynan et al., 1993,
Kuklin et al., 1997, Sasaki et al., 1998, Okada et al., 1997, Ishii
et al., 1997); Polymers (e.g. carboxymethylcellulose, chitosan)
(Hamajima et al., 1998, Jabbal-Gill et al., 1998); Polymer rings
(Wyatt et al., 1998); Proteosomes (Vancott et al., 1998, Lowell et
al., 1988, 1996, 1997); Sodium Fluoride (Hashi et al., 1998);
Transgenic plants (Tacket et al., 1998, Mason et al., 1998, Haq et
al., 1995); Virosomes (Gluck et al., 1992, Mengiardi et al., 1995,
Cryz et al., 1998); Virus-like particles (Jiang et al., 1999, Leibl
et al., 1998). Other delivery vehicles are known in the art and
some additional examples are provided below in the discussion of
vectors.
[0101] "Palindromic sequence" shall mean an inverted repeat (i.e. a
sequence such as ABCDEE'D'C'B'A' in which A and A' are bases
capable of forming the usual Watson-Crick base pairs. In vivo, such
sequences may form double-stranded structures. In one embodiment
the CpG oligonucleotide contains a palindromic sequence. A
palindromic sequence used in this context refers to a palindrome in
which the CpG is part of the palindrome, and preferably is the
center of the palindrome. In another embodiment the CpG
oligonucleotide is free of a palindrome. A CpG oligonucleotide that
is free of a palindrome is one in which the CpG dinucleotide is not
part of a palindrome. Such an oligonucleotide may include a
palindrome in which the CpG is not part of the palindrome.
[0102] A "stabilized nucleic acid molecule" shall mean a nucleic
acid molecule that is relatively resistant to in vivo degradation
(e.g. via an exo- or endo-nuclease). Stabilization can be a
function of length or secondary structure. Unmethylated CpG
oligonucleotides that are tens to hundreds of kbs long are
relatively resistant to in vivo degradation. For shorter CpG
oligonucleotides, secondary structure can stabilize and increase
their effect. For example, if the 3' end of an oligonucleotide has
self-complementarity to an upstream region, so that it can fold
back and form a sort of stem loop structure, then the
oligonucleotide becomes stabilized and therefore exhibits more
activity.
[0103] Preferred stabilized oligonucleotides of the instant
invention have a modified backbone. It has been demonstrated that
modification of the oligonucleotide backbone provides enhanced
activity of the CpG oligonucleotides when administered in vivo. CpG
constructs, including at least two phosphorothioate linkages at the
5' end of the oligonucleotide in multiple phosphorothioate linkages
at the 3' end, preferably 5, provides maximal activity and
protected the oligonucleotide from degradation by intracellular
exo- and endo-nucleases. Other modified oligonucleotides include
phosphodiester modified oligonucleotide, combinations of
phosphodiester and phosphorothioate oligonucleotide,
methylphosphonate, methylphosphorothioate, phosphorodithioate, and
combinations thereof. Each of these combinations and their
particular effects on immune cells is discussed in more detail in
PCT Published Patent Applications claiming priority to U.S. Ser.
Nos. 08/738,652 and 08/960,774, filed on Oct. 30, 1996 and Oct. 30,
1997 respectively, the entire contents of which is hereby
incorporated by reference. It is believed that these modified
oligonucleotides may show more stimulatory activity due to enhanced
nuclease resistance, increased cellular uptake, increased protein
binding, and/or altered intracellular localization.
[0104] Both phosphorothioate and phosphodiester oligonucleotides
containing CpG motifs are active in immune cells. However, based on
the concentration needed to induce CpG specific effects, the
nuclease resistant phosphorothioate backbone CpG oligonucleotides
are more potent (2 .mu.g/ml for the phosphorothioate vs. a total of
90 .mu.g/ml for phosphodiester).
[0105] Other stabilized oligonucleotides include: nonionic DNA
analogs, such as alkyl- and aryl-phosphates (in which the charged
phosphonate oxygen is replaced by an alkyl or aryl group),
phosphodiester and alkylphosphotriesters, in which the charged
oxygen moiety is alkylated. Oligonucleotides which contain diol,
such as tetraethyleneglycol or hexaethyleneglycol, at either or
both termini have also been shown to be substantially resistant to
nuclease degradation.
[0106] The nucleic acid sequences of the invention which are useful
as mucosal adjuvants are those broadly described above and
disclosed in PCT Published Patent Applications claiming priority to
U.S. Ser. Nos. 08/738,652 and 08/960,774, filed on Oct. 30, 1996
and Oct. 30, 1997 respectively. Exemplary sequences include but are
not limited to those immunostimulatory sequences shown in Table
1.
14TABLE 1 sequences GCTAGACGTTAGCGT; (SEQ ID NO: 1)
GCTAGATGTTAGCGT; (SEQ ID NO: 2) GCTAGACGTTAGCGT; (SEQ ID NO: 3)
GCTAGACGTTAGCGT; (SEQ ID NO: 4) GCATGACGTTGAGCT; (SEQ ID NO: 5)
ATGGAAGGTCCAGCGTTCTC; (SEQ ID NO: 6) ATCGACTCTCGAGCGTTCTC; (SEQ ID
NO: 7) ATCGACTCTCGAGCGTTCTC; (SEQ ID NO: 8) ATCGACTCTCGAGCGTTCTC;
(SEQ ID NO: 9) ATGGAAGGTCCAACGTTCTC; (SEQ ID NO: 10)
GAGAACGCTGGACCTTCCAT; (SEQ ID NO: 11) GAGAACGCTCGACCTTCCAT; (SEQ ID
NO: 12) GAGAACGCTCGACCTTCGAT; (SEQ ID NO: 13) GAGAACGCTGGACCTTCCAT;
(SEQ ID NO: 14) GAGAACGATGGACCTTCCAT; (SEQ ID NO: 15)
GAGAACGCTCCAGCACTGAT; (SEQ ID NO: 16) TCCATGTCGGTCCTGATGCT; (SEQ ID
NO: 17) TCCATGTCGGTCCTGATGCT; (SEQ ID NO: 18) TCCATGACGTTCCTGATGCT;
(SEQ ID NO: 19) TCCATGTCGGTCCTGCTGAT; (SEQ ID NO: 20) TCAACGTT;
(SEQ ID NO: 21) TCAGCGCT; (SEQ ID NO: 22) TCATCGAT; (SEQ ID NO: 23)
TCTTCGAA; (SEQ ID NO: 24) CAACGTT; (SEQ ID NO: 25) CCAACGTT; (SEQ
ID NO: 26) AACGTTCT; (SEQ ID NO: 27) TCAACGTC; (SEQ ID NO: 28)
ATGGACTCTCCAGCGTTCTC; (SEQ ID NO: 29) ATGGAAGGTCCAACGTTCTC; (SEQ ID
NO: 30) ATCGACTCTCGAGCGTTCTC; (SEQ ID NO: 31) ATGGAGGCTCCATCGTTCTC;
(SEQ ID NO: 32) ATCGACTCTCGAGCGTTCTC; (SEQ ID NO: 33)
ATCGACTCTCGAGCGTTCTC; (SEQ ID NO: 34) TCCATGTCGGTCCTGATGCT; (SEQ ID
NO: 35) TCCATGCCGGTCCTGATGCT; (SEQ ID NO: 36) TCCATGGCGGTCCTGATGCT;
(SEQ ID NO: 37) TCCATGACGGTCCTGATGCT; (SEQ ID NO: 38)
TCCATGTCGATCCTGATGCT; (SEQ ID NO: 39) TCCATGTCGCTCCTGATGCT; (SEQ ID
NO: 40) TCCATGTCGTCCCTGATGCT; (SEQ ID NO: 41) TCCATGACGTGCCTGATGCT;
(SEQ ID NO: 42) TCCATAACGTTCCTGATGCT; (SEQ ID NO: 43)
TCCATGACGTCCCTGATGCT; (SEQ ID NO: 44) TCCATCACGTGCCTGATGCT; (SEQ ID
NO: 45) GGGGTCAACGTTGACGGGG; (SEQ ID NO: 46) GGGGTCAGTCGTGACGGGG;
(SEQ ID NO: 47) GCTAGACGTTAGTGT; (SEQ ID NO: 48)
TCCATGTCGTTCCTGATGCT; (SEQ ID NO: 49) ACCATGGACGATCTGTTTCCCCTC;
(SEQ ID NO: 50) TCTCCCAGCGTGCGCCAT; (SEQ ID NO: 51)
ACCATGGACGAACTGTTTCCCCTC; (SEQ ID NO: 52) ACCATGGACGAGCTGTTTCCCCTC;
(SEQ ID NO: 53) ACCATGGACGACCTGTTTCCCCTC; (SEQ ID NO: 54)
ACCATGGACGTACTGTTTCCCCTC; (SEQ ID NO: 55) ACCATGGACGGTCTGTTTCCCCTC;
(SEQ ID NO: 56) ACCATGGACGTTCTGTTTCCCCTC; (SEQ ID NO: 57)
CACGTTGAGGGGCAT; (SEQ ID NO: 58) TCAGCGTGCGCC; (SEQ ID NO: 59)
ATGACGTTCCTGACGTT; (SEQ ID NO: 60) TCTCCCAGCGGGCGCAT; (SEQ ID NO:
61) TCCATGTCGTTCCTGTCGTT; (SEQ ID NO: 62) TCCATAGCGTTCCTAGCGTT;
(SEQ ID NO: 63) TCGTCGCTGTCTCCCCTTCTT; (SEQ ID NO: 64)
TCCTGACGTTCCTGACGTT; (SEQ ID NO: 65) TCCTGTCGTTCCTGTCGTT; (SEQ ID
NO: 66) TCCATGTCGTTTTTGTCGTT; (SEQ ID NO: 67) TCCTGTCGTTCCTTGTCGTT;
(SEQ ID NO: 68) TCCTTGTCGTTCCTGTCGTT; (SEQ ID NO: 69)
TCCTGTCGTTTTTTGTCGTT; (SEQ ID NO: 70) TCGTCGCTGTCTGCCCTTCTT; (SEQ
ID NO: 71) TCGTCGCTGTTGTCGTTTCTT; (SEQ ID NO: 72)
TCCATGCGTGCGTGCGTTTT; (SEQ ID NO: 73) TCCATGCGTTGCGTTGCGTT; (SEQ ID
NO: 74) TCCACGACGTTTTCGACGTT; (SEQ ID NO: 75) TCGTCGTTGTCGTTGTCGTT;
(SEQ ID NO: 76) TCGTCGTTTTGTCGTTTTGTCGTT; (SEQ ID NO: 77)
TCGTCGTTGTCGTTTTGTCGTT; (SEQ ID NO: 78) GCGTGCGTTGTCGTTGTCGTT; (SEQ
ID NO: 79) TGTCGTTTGTCGTTTGTCGTT; (SEQ ID NO: 80)
TGTCGTTGTCGTTGTCGTTGTCGTT; (SEQ ID NO: 81) TGTCGTTGTCGTTGTCGTT;
(SEQ ID NO: 82) TCGTCGTCGTCGTT; (SEQ ID NO: 83) TGTCGTTGTCGTT; (SEQ
ID NO: 84) TCCATAGCGTTCCTAGCGTT; (SEQ ID NO: 85)
TCCATGACGTTCCTGACGTT; (SEQ ID NO: 86) GTCGYT; (SEQ ID NO: 87)
TGTCGYT; (SEQ ID NO: 88) AGCTATGACGTTCCAAGG; (SEQ ID NO: 89)
TCCATGACGTTCCTGACGTT; (SEQ ID NO: 90) ATCGACTCTCGAACGTTCTC; (SEQ ID
NO: 92) TCCATGTCGGTCCTGACGCA; (SEQ ID NO: 93) TCTTCGAT; (SEQ ID NO:
94) ATAGGAGGTCCAACGTTCTC; (SEQ ID NO: 95)
[0107] The stimulation index of a particular immunostimulatory CpG
DNA can be tested in various immune cell assays. Preferably, the
stimulation index of the CpG oligonucleotide with regard to B cell
proliferation is at least about 5, preferably at least about 10,
more preferably at least about 15 and most preferably at least
about 20 as determined by incorporation of .sup.3H uridine in a
murine B cell culture, which has been contacted with 20 .mu.M of
oligonucleotide for 20 h at 37.degree. C. and has been pulsed with
1 .mu.Ci of .sup.3H uridine; and harvested and counted 4 h later as
described in detail in PCT Published Patent Applications claiming
priority to U.S. Ser. Nos. 08/738,652 and 08/960,774, filed on Oct.
30, 1996 and Oct. 30, 1997 respectively. For use in vivo, for
example, it is important that the CpG oligonucleotide be capable of
effectively inducing IgA expression.
[0108] The CpG oligonucleotide can be administered in conjunction
with another mucosal adjuvant. It was discovered according to the
invention that the combination of a CpG oligonucleotide and a
mucosal adjuvant produced a synergistic immune response. When the
CpG oligonucleotide is administered in conjunction with another
adjuvant, the CpG oligonucleotide can be administered before,
after, and/or simultaneously with the other mucosal adjuvant. For
instance, the CpG oligonucleotide may be administered with a
priming dose of antigen. Either or both of the adjuvants may then
be administered with the boost dose. Alternatively, the CpG
oligonucleotide may be administered with a boost dose of antigen.
Either or both of the adjuvants may then be administered with the
prime dose.
[0109] Additionally it has been discovered that mucosal immunity
can be induced as long as one of the dosages of CpG oligonucleotide
is administered to a mucosal surface. Other doses can be
administered systemically or mucosally without affecting the
induction of the immune response. For example, the subject may be
primed by mucosal delivery of antigen and CpG oligonucleotide, with
or without other mucosal adjuvants and boosted by a parenteral
(e.g., intramuscular, intradermal or subcutaneous) route of
delivery of antigen alone, with CpG oligonucleotides, with a
non-oligonucleotide adjuvant or a combination of adjuvants that may
or may not include CpG oligonucleotide. Alternatively, the prime
dose may be given parenterally and boosted mucosally using the
invention. All of these approaches can induce strong systemic and
mucosal immune responses. Thus the methods of the invention
encompass the administration of at least one dose, either prime or
boost or both, to the mucosal surface. The other doses of CpG
oligonucleotide may be administered mucosally or systemically.
[0110] A "prime dose" is the first dose of antigen administered to
the subject. In the case of a subject that has an infection the
prime dose may be the initial exposure of the subject to the
infectious microbe (passive exposure) and thus the subsequent
purposeful administration of antigen (active exposure) with CpG
oligonucleotide becomes the boost dose. A "boost dose" is a second
or third, etc, dose of antigen administered to a subject that has
already been exposed to the antigen. In some cases the prime dose
administered with the CpG oligonucleotide is so effective that a
boost dose is not required to protect a subject at risk of
infection from being infected.
[0111] The subject is exposed to the antigen. As used herein, the
term "exposed to" refers to either the active step of contacting
the subject with an antigen or the passive exposure of the subject
to the antigen in vivo. Methods for the active exposure of a
subject to an antigen are well-known in the art. In general, an
antigen is administered directly to the subject by any means such
as intravenous, intramuscular, oral, transdermal, mucosal,
intranasal, intratracheal, or subcutaneous administration. The
antigen can be administered systemically or locally. Methods for
administering the antigen and the CpG oligonucleotide are described
in more detail below. A subject is passively exposed to an antigen
if an antigen becomes available for exposure to the immune cells in
the body. A subject may be passively exposed to an antigen, for
instance, by entry of a foreign pathogen into the body or by the
development of a tumor cell expressing a foreign antigen on its
surface. When a subject is passively exposed to an antigen it is
preferred in some embodiments that the CpG oligonucleotide is an
oligonucleotide of 8-100 nucleotides in length and/or has a
phosphate modified backbone.
[0112] The methods in which a subject is passively exposed to an
antigen can be particularly dependent on timing of CpG
oligonucleotide. For instance, in a subject at risk of developing a
cancer or an infectious disease or an allergic or asthmatic
response, the subject may be administered the CpG oligonucleotide
on a regular basis when that risk is greatest, i.e., during allergy
season or after exposure to a cancer causing agent. Additionally
the CpG oligonucleotide may be administered to travelers before
they travel to foreign lands where they are at risk of exposure to
infectious agents. Likewise the CpG oligonucleotide and may be
administered to soldiers or civilians at risk of exposure to
biowarfare to induce a mucosal immune response to the antigen when
and if the subject is exposed to it.
[0113] An "antigen" as used herein is a molecule capable of
provoking an immune response. Antigens include but are not limited
to cells, cell extracts, proteins, polypeptides, peptides,
polysaccharides, polysaccharide conjugates, peptide mimics of
polysaccharides, lipids, glycolipids, carbohydrates, viruses and
viral extracts and muticellular organisms such as parasites and
allergens. The term antigen broadly includes any type of molecule
which is recognized by a host immune system as being foreign.
Antigens include but are not limited to cancer antigens, microbial
antigens, and allergens.
[0114] A "cancer antigen" as used herein is a compound, such as a
peptide or protein, associated with a tumor or cancer cell surface
and which is capable of provoking an immune response when expressed
on the surface of an antigen presenting cell in the context of an
MHC molecule. Cancer antigens can be prepared from cancer cells
either by preparing crude extracts of cancer cells, for example, as
described in Cohen, et al., 1994, Cancer Research, 54:1055, by
partially purifying the antigens, by recombinant technology, or by
de novo synthesis of known antigens. Cancer antigens include
antigens that are recombinately an immunogenic portion of or a
whole tumor or cancer. Such antigens can be isolated or prepared
recombinately or by any other means known in the art.
[0115] A "microbial antigen" as used herein is an antigen of a
microorganism and includes but is not limited to infectious virus,
infectious bacteria, infectious parasites, and infectious fungi.
Such antigens include the intact microorganism as well as natural
isolates and fragments or derivatives thereof and also synthetic
compounds which are identical to or similar to natural
microorganism antigens and induce an immune response specific for
that microorganism. A compound is similar to a natural
microorganism antigen if it induces an immune response (humoral
and/or cellular) to a natural microorganism antigen. Such antigens
are used routinely in the art and are well known to those of
ordinary skill in the art.
[0116] Examples of infectious virus that have been found in humans
include but are not limited to: Retroviridae (e.g. human
immunodeficiency viruses, such as HIV-1 (also referred to as
HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such
as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus;
enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);
Calciviridae (e.g. strains that cause gastr6enteritis); Togaviridae
(e.g. equine encephalitis viruses, rubella viruses); Flaviridae
(e.g. dengue viruses, encephalitis viruses, yellow fever viruses);
Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g. vesicular
stomatitis viruses, rabies viruses); Coronaviridae (e.g.
coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses,
rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae
(e.g. parainfluenza viruses, mumps virus, measles virus,
respiratory syncytial virus); Orthomyxoviridae (e.g. influenza
viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses,
phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever
viruses); Reoviridae (e.g. reoviruses, orbiviurses and
rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);
Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,
polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae
(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,
cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses,
vaccinia viruses, pox viruses); and Iridoviridae (e.g. African
swine fever virus); and unclassified viruses (e.g. the etiological
agents of Spongiform encephalopathies, the agent of delta hepatitis
(thought to be a defective satellite of hepatitis B virus), the
agents of non-A, non-B hepatitis (class 1=internally transmitted;
class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and
related viruses, and astroviruses).
[0117] Both gram negative and gram positive bacteria serve as
antigens in vertebrate animals. Such gram positive bacteria
include, but are not limited to Pasteurella species, Staphylococci
species, and Streptococcus species. Gram negative bacteria include,
but are not limited to, Escherichia coli, Pseudomonas species, and
Salmonella species. Specific examples of infectious bacteria
include but are not limited to: Helicobacterpyloris, Borelia
burgdorferi, Legionellapneumophilia, Mycobacteria sps (e.g. M
tuberculosis, M avium, M intracellulare, M kansaii, M gordonae),
Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria
meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group), Streptococcus faecalis,
Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus
pneumoniae, pathogenic Campylobacter sp., Enterococcus sp.,
Haemophilus influenzae, Bacillus antracis, corynebacterium
diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,
Clostridium perfringers, Clostridium tetani, Enterobacter
aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides
sp., Fusobacterium nucleatum, Streptobacillus moniliformis,
Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia,
and Actinomyces israelli.
[0118] Examples of infectious fungi include: Cryptococcus
neoformans, Histoplasma capsulatum, Coccidioides immitis,
Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.
Other infectious organisms (i.e., protists) include: Plasmodium
such as Plasmodiumfalciparum, Plasmodium malariae, Plasmodium
ovale, and Plasmodium vivax and Toxoplasma gondii.
[0119] Other medically relevant microorganisms have been descried
extensively in the literature, e.g., see C. G. A Thomas, Medical
Microbiology, Bailliere Tindall, Great Britain 1983, the entire
contents of which is hereby incorporated by reference.
[0120] Although many of the microbial antigens described above
relate to human disorders, the invention is also useful for
treating other nonhuman vertebrates. Nonhuman vertebrates are also
capable of developing infections which can be prevented or treated
with the CpG oligonucleotides disclosed herein. For instance, in
addition to the treatment of infectious human diseases, the methods
of the invention are usefull for treating infections of
animals.
[0121] As used herein, the term "treat", "treated", or "treating"
when used with respect to an infectious disease refers to a
prophylactic treatment which increases the resistance of a subject
(a subject at risk of infection) to infection with a pathogen or,
in other words, decreases the likelihood that the subject will
become infected with the pathogen as well as a treatment after the
subject (a subject who has been infected) has become infected in
order to fight the infection, e.g., reduce or eliminate the
infection or prevent it from becoming worse.
[0122] Many vaccines for the treatment of non-human vertebrates are
disclosed in Bennett, K. Compendium of Veterinary Products, 3rd ed.
North American Compendiums, Inc., 1995. As discussed above,
antigens include infectious microbes such as virus, bacteria and
fungi and fragments thereof, derived from natural sources or
synthetically. Infectious virus of both human and non-human
vertebrates, include retroviruses, RNA viruses and DNA viruses.
This group of retroviruses includes both simple retroviruses and
complex retroviruses. The simple retroviruses include the subgroups
of B-type retroviruses, C-type retroviruses and D-type
retroviruses. An example of a B-type retrovirus is mouse mammary
tumor virus (MMTV). The C-type retroviruses include subgroups
C-type group A (including Rous sarcoma virus (RSV), avian leukemia
virus (ALV), and avian myeloblastosis virus (AMV)) and C-type group
B (including murine leukemia virus (MLV), feline leukemia virus
(FeLV), murine sarcoma virus (MSV), gibbon ape leukemia virus
(GALV), spleen necrosis virus (SNV), reticuloendotheliosis virus
(RV) and simian sarcoma virus (SSV)). The D-type retroviruses
include Mason-Pfizer monkey virus (PMV) and simian retrovirus type
1 (SRV-1). The complex retroviruses include the subgroups of
lentiviruses, T-cell leukemia viruses and the foamy viruses.
Lentiviruses include HIV-1, but also include HIV-2, SIV, Visna
virus, feline immunodeficiency virus (FIV), and equine infectious
anemia virus (EIAV). The T-cell leukemia viruses include HTLV-1,
HTLV-II, simian T-cell leukemia virus (STLV), and bovine leukemia
virus (BLV). The foamy viruses include human foamy virus (HFV),
simian foamy virus (SFV) and bovine foamy virus (BFV).
[0123] Examples of other RNA viruses that are antigens in
vertebrate animals include, but are not limited to, the following:
members of the family Reoviridae, including the genus Orthoreovirus
(multiple serotypes of both mammalian and avian retroviruses), the
genus Orbivirus (Bluetongue virus, Eugenangee virus, Kemerovo
virus, African horse sickness virus, and Colorado Tick Fever
virus), the genus Rotavirus (human rotavirus, Nebraska calf
diarrhea virus, murine rotavirus, simian rotavirus, bovine or ovine
rotavirus, avian rotavirus); the family Picomaviridae, including
the genus Enterovirus (poliovirus, Coxsackie virus A and B, enteric
cytopathic human orphan (ECHO) viruses, hepatitis A virus, Simian
enteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirus
muris, Bovine enteroviruses, Porcine enteroviruses , the genus
Cardiovirus (Encephalomyocarditis virus (EMC), Mengovirus), the
genus Rhinovirus (Human rhinoviruses including at least 113
subtypes; other rhinoviruses), the genus Apthovirus (Foot and Mouth
disease (FMDV); the family Calciviridae, including Vesicular
exanthema of swine virus, San Miguel sea lion virus, Feline
picornavirus and Norwalk virus; the family Togaviridae, including
the genus Alphavirus (Eastern equine encephalitis virus, Semliki
forest virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong
virus, Ross river virus, Venezuelan equine encephalitis virus,
Western equine encephalitis virus), the genus Flavirius (Mosquito
borne yellow fever virus, Dengue virus, Japanese encephalitis
virus, St. Louis encephalitis virus, Murray Valley encephalitis
virus, West Nile virus, Kunjin virus, Central European tick borne
virus, Far Eastern tick borne virus, Kyasanur forest virus, Louping
III virus, Powassan virus, Omsk hemorrhagic fever virus), the genus
Rubivirus (Rubella virus), the genus Pestivirus (Mucosal disease
virus, Hog cholera virus, Border disease virus); the family
Bunyaviridae, including the genus Bunyvirus (Bunyamwera and related
viruses, California encephalitis group viruses), the genus
Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever
virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever
virus, Nairobi sheep disease virus), and the genus Uukuvirus
(Uukuniemi and related viruses); the family Orthomyxoviridae,
including the genus Influenza virus (Influenza virus type A, many
human subtypes); Swine influenza virus, and Avian and Equine
Influenza viruses; influenza type B (many human subtypes), and
influenza type C (possible separate genus); the family
paramyxoviridae, including the genus Paramyxovirus (Parainfluenza
virus type 1, Sendai virus, Hemadsorption virus, Parainfluenza
viruses types 2 to 5, Newcastle Disease Virus, Mumps virus), the
genus Morbillivirus (Measles virus, subacute sclerosing
panencephalitis virus, distemper virus, Rinderpest virus), the
genus Pneumovirus (respiratory syncytial virus (RSV), Bovine
respiratory syncytial virus and Pneumonia virus of mice); forest
virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus, Ross
river virus, Venezuelan equine encephalitis virus, Western equine
encephalitis virus), the genus Flavirius (Mosquito borne yellow
fever virus, Dengue virus, Japanese encephalitis virus, St. Louis
encephalitis virus, Murray Valley encephalitis virus, West Nile
virus, Kunjin virus, Central European tick borne virus, Far Eastern
tick borne virus, Kyasanur forest virus, Louping III virus,
Powassan virus, Omsk hemorrhagic fever virus), the genus Rubivirus
(Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog
cholera virus, Border disease virus); the family Bunyaviridae,
including the genus Bunyvirus (Bunyamwera and related viruses,
California encephalitis group viruses), the genus Phlebovirus
(Sandfly fever Sicilian virus, Rift Valley fever virus), the genus
Nairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep
disease virus), and the genus Uukuvirus (Uukuniemi and related
viruses); the family Orthomyxoviridae, including the genus
Influenza virus (Influenza virus type A, many human subtypes);
Swine influenza virus, and Avian and Equine Influenza viruses;
influenza type B (many human subtypes), and influenza type C
(possible separate genus); the family paramyxoviridae, including
the genus Paramyxovirus (Parainfluenza virus type 1, Sendai virus,
Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle
Disease Virus, Mumps virus), the genus Morbillivirus (Measles
virus, subacute sclerosing panencephalitis virus, distemper virus,
Rinderpest virus), the genus Pneumovirus (respiratory syncytial
virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus
of mice); the family Rhabdoviridae, including the genus
Vesiculovirus (VSV), Chandipura virus, Flanders-Hart Park virus),
the genus Lyssavirus (Rabies virus), fish Rhabdoviruses, and two
probable Rhabdoviruses (Marburg virus and Ebola virus); the family
Arenaviridae, including Lymphocytic choriomeningitis virus (LCM),
Tacaribe virus complex, and Lassa virus; the family Coronoaviridae,
including Infectious Bronchitis Virus (IBV), Mouse Hepatitis virus,
Human enteric corona virus, and Feline infectious peritonitis
(Feline coronavirus).
[0124] Illustrative DNA viruses that are antigens in vertebrate
animals include, but are not limited to: the family Poxviridae,
including the genus Orthopoxvirus (Variola major, Variola minor,
Monkey pox Vaccinia, Cowpox, Buffalopox, Rabbitpox, Ectromelia),
the genus Leporipoxvirus (Myxoma, Fibroma), the genus Avipoxvirus
(Fowlpox, other avian poxvirus), the genus Capripoxvirus (sheeppox,
goatpox), the genus Suipoxvirus (Swinepox), the genus Parapoxvirus
(contagious postular dermatitis virus, pseudocowpox, bovine papular
stomatitis virus); the family Iridoviridae (African swine fever
virus, Frog viruses 2 and 3, Lymphocystis virus of fish); the
family Herpesviridae, including the alpha-Herpesviruses (Herpes
Simplex Types 1 and 2, Varicella-Zoster, Equine abortion virus,
Equine herpes virus 2 and 3, pseudorabies virus, infectious bovine
keratoconjunctivitis virus, infectious bovine rhinotracheitis
virus, feline rhinotracheitis virus, infectious laryngotracheitis
virus) the Beta-herpesviruses (Human cytomegalovirus and
cytomegaloviruses of swine, monkeys and rodents); the
gamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease
virus, Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus,
guinea pig herpes virus, Lucke tumor virus); the family
Adenoviridae, including the genus Mastadenovirus (Human subgroups
A,B,C,D,E and ungrouped; simian adenoviruses (at least 23
serotypes), infectious canine hepatitis, and adenoviruses of
cattle, pigs, sheep, frogs and many other species, the genus
Aviadenovirus (Avian adenoviruses); and non-cultivatable
adenoviruses; the family Papoviridae, including the genus
Papillomavirus (Human papilloma viruses, bovine papilloma viruses,
Shope rabbit papilloma virus, and various pathogenic papilloma
viruses of other species), the genus Polyomavirus (polyomavirus,
Simian vacuolating agent (SV-40), Rabbit vacuolating agent (RKV), K
virus, BK virus, JC virus, and other primate polyoma viruses such
as Lymphotrophic papilloma virus); the family Parvoviridae
including the genus Adeno-associated viruses, the genus Parvovirus
(Feline panleukopenia virus, bovine parvovirus, canine parvovirus,
Aleutian mink disease virus, etc). Finally, DNA viruses may include
viruses which do not fit into the above families such as Kuru and
Creutzfeldt-Jacob disease viruses and chronic infectious
neuropathic agents (CHINA virus).
[0125] Each of the foregoing lists is illustrative, and is not
intended to be limiting.
[0126] In addition to the use of the CpG oligonucleotides to induce
an antigen specific immune response in humans, the methods of the
preferred embodiments are particularly well suited for treatment of
birds such as hens, chickens, turkeys, ducks, geese, quail, and
pheasant. Birds are prime targets for many types of infections.
[0127] Hatching birds are exposed to pathogenic microorganisms
shortly after birth. Although these birds are initially protected
against pathogens by maternal derived antibodies, this protection
is only temporary, and the bird's own immature immune system must
begin to protect the bird against the pathogens. It is often
desirable to prevent infection in young birds when they are most
susceptible. It is also desirable to prevent against infection in
older birds, especially when the birds are housed in closed
quarters, leading to the rapid spread of disease. Thus, it is
desirable to administer the CpG oligonucleotide and the non-nucleic
acid adjuvant of the invention to birds to enhance an
antigen-specific immune response when antigen is present.
[0128] An example of a common infection in chickens is chicken
infectious anemia virus (CIAV). CIAV was first isolated in Japan
in. 1979 during an investigation of a Marek's disease vaccination
break (Yuasa et al., 1979, Avian Dis. 23:366-385). Since that time,
CIAV has been detected in commercial poultry in all major poultry
producing countries (van Bulow et al., 1991, pp.690-699) in
Diseases of Poultry, 9th edition, Iowa State University Press).
[0129] CIAV infection results in a clinical disease, characterized
by anemia, hemorrhage and immunosuppression, in young susceptible
chickens. Atrophy of the thymus and of the bone marrow and
consistent lesions of CIAV-infected chickens are also
characteristic of CIAV infection. Lymphocyte depletion in the
thymus, and occasionally in the bursa of Fabricius, results in
immunosuppression and increased susceptibility to secondary viral,
bacterial, or fungal infections which then complicate the course of
the disease. The immunosuppression may cause aggravated disease
after infection with one or more of Marek's disease virus (MDV),
infectious bursal disease virus, reticuloendotheliosis virus,
adenovirus, or reovirus. It has been reported that pathogenesis of
MDV is enhanced by CIAV (DeBoer et al., 1989, p. 28 In Proceedings
of the 38th Western Poultry Diseases Conference, Tempe, Ariz.).
Further, it has been reported that CIAV aggravates the signs of
infectious bursal disease (Rosenberger et al., 1989, Avian Dis.
33:707-713). Chickens develop an age resistance to experimentally
induced disease due to CAA. This is essentially complete by the age
of 2 weeks, but older birds are still susceptible to infection
(Yuasa, N. et al., 1979 supra; Yuasa, N. et al., Arian Diseases 24,
202-209, 1980). However, if chickens are dually infected with CAA
and an immunosuppressive agent (IBDV, MDV etc.) age resistance
against the disease is delayed (Yuasa, N. et al., 1979 and 1980
supra; Bulow von V. et al., J. Veterinary Medicine 33, 93-116,
1986). Characteristics of CIAV that may potentiate disease
transmission include high resistance to environmental inactivation
and some common disinfectants. The economic impact of CIAV
infection on the poultry industry is clear from the fact that 10%
to 30% of infected birds in disease outbreaks die.
[0130] Vaccination of birds, like other vertebrate animals can be
performed at any age. Normally, vaccinations are performed at up to
12 weeks of age for a live microorganism and between 14-18 weeks
for an inactivated microorganism or other type of vaccine. For in
ovo vaccination, vaccination can be performed in the last quarter
of embryo development. The vaccine may be administered
subcutaneously, by spray, orally, intraocularly, intratracheally,
nasally, or by other mucosal delivery methods described herein.
Thus, the CpG oligonucleotide of the invention can be administered
to birds and other non-human vertebrates using routine vaccination
schedules and the antigen is administered after an appropriate time
period as described herein.
[0131] Cattle and livestock are also susceptible to infection.
Disease which affect these animals can produce severe economic
losses, especially amongst cattle. The methods of the invention can
be used to protect against infection in livestock, such as cows,
horses, pigs, sheep, and goats.
[0132] Cows can be infected by bovine viruses. Bovine viral
diarrhea virus (BVDV) is a small enveloped positive-stranded RNA
virus and is classified, along with hog cholera virus (HOCV) and
sheep border disease virus (BDV), in the pestivirus genus.
Although, Pestiviruses were previously classified in the
Togaviridae family, some studies have suggested their
reclassification within the Flaviviridae family along with the
flavivirus and hepatitis C virus (HCV) groups (Francki, et al.,
1991).
[0133] BVDV, which is an important pathogen of cattle can be
distinguished, based on cell culture analysis, into cytopathogenic
(CP) and noncytopathogenic (NCP) biotypes. The NCP biotype is more
widespread although both biotypes can be found in cattle. If a
pregnant cow becomes infected with an NCP strain, the cow can give
birth to a persistently infected and specifically immunotolerant
calf that will spread virus during its lifetime. The persistently
infected cattle can succumb to mucosal disease and both biotypes
can then be isolated from the animal. Clinical manifestations can
include abortion, teratogenesis, and respiratory problems, mucosal
disease and mild diarrhea. In addition, severe thrombocytopenia,
associated with herd epidemics, that may result in the death of the
animal has been described and strains associated with this disease
seem more virulent than the classical BVDVs.
[0134] Equine herpesviruses (EHV) comprise a group of antigenically
distinct biological agents which cause a variety of infections in
horses ranging from subclinical to fatal disease. These include
Equine herpesvirus-1 (EHV-1), a ubiquitous pathogen in horses.
EHV-1 is associated with epidemics of abortion, respiratory tract
disease, and central nervous system disorders. Primary infection of
upper respiratory tract of young horses results in a febrile
illness which lasts for 8 to 10 days. Immunologically experienced
mares may be reinfected via the respiratory tract without disease
becoming apparent, so that abortion usually occurs without warning.
The neurological syndrome is associated with respiratory disease or
abortion and can affect animals of either sex at any age, leading
to incoordination, weakness and posterior paralysis (Telford, E. A.
R. et al., Virology 189, 304-316, 1992). Other EHV's include EHV-2,
or equine cytomegalovirus, EHV-3, equine coital exanthema virus,
and EHV-4, previously classified as EHV-1 subtype 2.
[0135] Sheep and goats can be infected by a variety of dangerous
microorganisms including visna-maedi.
[0136] Primates such as monkeys, apes and macaques can be infected
by simian immunodeficiency virus. Inactivated cell-virus and
cell-free whole simian immunodeficiency vaccines have been reported
to afford protection in macaques (Stott et al. (1990) Lancet
36:1538-1541; Desrosiers et al. PNAS USA (1989) 86:6353-6357;
Murphey-Corb et al. (1989) Science 246:1293-1297; and Carlson et
al. (1990) AIDS Res. Human Retroviruses io 6:1239-1246). A
recombinant HIV gp120 vaccine has been reported to afford
protection in chimpanzees (Berman et al. (1990) Nature
345:622-625).
[0137] Cats, both domestic and wild, are susceptible to infection
with a variety of microorganisms. For instance, feline infectious
peritonitis is a disease which occurs in both domestic and wild
cats, such as lions, leopards, cheetahs, and jaguars. When it is
desirable to prevent infection with this and other types of
pathogenic organisms in cats, the methods of the invention can be
used to vaccinate cats to protect them against infection.
[0138] Domestic cats may become infected with several retroviruses,
including but not limited to feline leukemia virus (FeLV), feline
sarcoma virus (FeSV), endogenous type C oncomavirus (RD-114), and
feline syncytia-forming virus (FeSFV). Of these, FeLV is the most
significant pathogen, causing diverse symptoms, including
lymphoreticular and myeloid neoplasms, anemias, immune mediated
disorders, and an immunodeficiency syndrome which is similar to
human acquired immune deficiency syndrome (AIDS). Recently, a
particular replication-defective FeLV mutant, designated FeLV-AIDS,
has been more particularly associated with immunosuppressive
properties.
[0139] The discovery of feline T-lymphotropic lentivirus (also
referred to as feline immunodeficiency) was first reported in
Pedersen et al. (1987) Science 235:790-793. Characteristics of FIV
have been reported in Yamamoto et al. (1988) Leukemia, December
Supplement 2:204S-215S; Yamamoto et al. (1988) Am. J. Vet. Res.
49:1246-1258; and Ackley et al. (1990) J. Virol. 64:5652-5655.
Cloning and sequence analysis of FIV have been reported in Olmsted
et al. (1989) Proc. Natl. Acad. Sci. USA 86:2448-2452 and
86:43554360.
[0140] Feline infectious peritonitis (FIP) is a sporadic disease
occurring unpredictably in domestic and wild Felidae. While FIP is
primarily a disease of domestic cats, it has been diagnosed in
lions, mountain lions, leopards, cheetahs, and the jaguar. Smaller
wild cats that have been afflicted with FIP include the lynx and
caracal, sand cat, and pallas cat. In domestic cats, the disease
occurs predominantly in young animals, although cats of all ages
are susceptible. A peak incidence occurs between 6 and 12 months of
age. A decline in incidence is noted from 5 to 13 years of age,
followed by an increased incidence in cats 14 to 15 years old.
[0141] Viral, bacterial, and parasitic diseases in fin-fish,
shellfish or other aquatic life forms pose a serious problem for
the aquaculture industry. Owing to the high density of animals in
the hatchery tanks or enclosed marine farming areas, infectious
diseases may eradicate a large proportion of the stock in, for
example, a fin-fish, shellfish, or other aquatic life forms
facility. Prevention of disease is a more desired remedy to these
threats to fish than intervention once the disease is in progress.
Vaccination of fish is the only preventative method which may offer
long-term protection through immunity. Nucleic acid based
vaccinations are described in U.S. Pat. No. 5,780,448 issued to
Davis.
[0142] The fish immune system has many features similar to the
mammalian immune system, such as the presence of B cells, T cells,
lymphokines, complement, and immunoglobulins. Fish have lymphocyte
subclasses with roles that appear similar in many respects to those
of the B and T cells of mammals. Vaccines can be administered by
immersion or orally.
[0143] Aquaculture species include but are not limited to fin-fish,
shellfish, and other aquatic animals. Fin-fish include all
vertebrate fish, which may be bony or cartilaginous fish, such as,
for example, salmonids, carp, catfish, yellowtail, seabream, and
seabass. Salmonids are a family of fin-fish which include trout
(including rainbow trout), salmon, and Arctic char. Examples of
shellfish include, but are not limited to, clams, lobster, shrimp,
crab, and oysters. Other cultured aquatic animals include, but are
not limited to eels, squid, and octopi.
[0144] Polypeptides of viral aquaculture pathogens include but are
not limited to glycoprotein (G) or nucleoprotein (N) of viral
hemorrhagic septicemia virus (VHSV); G or N proteins of infectious
hematopoietic necrosis virus (IHNV); VP1, VP2, VP3 or N structural
proteins of infectious pancreatic necrosis virus (IPNV); G protein
of spring viremia of carp (SVC); and a membrane-associated protein,
tegumin or capsid protein or glycoprotein of channel catfish virus
(CCV).
[0145] Polypeptides of bacterial pathogens include but are not
limited to an iron-regulated outer membrane protein, (IROMP), an
outer membrane protein (OMP), and an A-protein of Aeromonis
salmonicida which causes furunculosis, p57 protein of Renibacterium
salmoninarum which causes bacterial kidney disease (BKD), major
surface associated antigen (msa), a surface expressed cytotoxin
(mpr), a surface expressed hemolysin (ish), and a flagellar antigen
of Yersiniosis; an extracellular protein (ECP), an iron-regulated
outer membrane protein (IROMP), and a structural protein of
Pasteurellosis; an OMP and a flagellar protein of Vibrosis
anguillarum and V. ordalii; a flagellar protein, an OMP
protein,aroA, and purA of Edwardsiellosis ictaluri and E. tarda;
and surface antigen of Ichthyophthirius; and a structural and
regulatory protein of Cytophaga columnari; and a structural and
regulatory protein of Rickettsia.
[0146] Polypeptides of a parasitic pathogen include but are not
limited to the surface antigens of Ichthyophthirius.
[0147] An "allergen" refers to a substance (antigen) that can
induce an allergic or asthmatic response in a susceptible subject.
The list of allergens is enormous and can include pollens, insect
venoms, animal dander dust, fungal spores and drugs (e.g.
penicillin). Examples of natural, animal and plant allergens
include but are not limited to proteins specific to the following
genuses: Canine (Canis familiaris); Dermatophagoides (e.g.
Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia
(Ambrosia artemiisfolia; Lolium (e.g. Lolium perenne or Lolium
multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria
(Alternaria alternata); Alder; Alnus (Alnus gultinoasa); Betula
(Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa);
Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago
lanceolata); Parietaria (e.g. Parietaria officinalis or Parietaria
judaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apis
multiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressus
arizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperus
sabinoides, Juniperus virginiana, Juniperus communis and Juniperus
ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g.
Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana);
Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale);
Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis
glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poapratensis
or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus
lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum
(e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum
(e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea);
Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghum
halepensis); and Bromus (e.g. Bromus inermis).
[0148] The antigen may be an antigen that is encoded by a nucleic
acid vector or it may be not encoded in a nucleic acid vector. In
the former case the nucleic acid vector is administered to the
subject and the antigen is expressed in vivo. In the latter case
the antigen is administered directly to the subject. An "antigen
not encoded in a nucleic acid vector" as used herein refers to any
type of antigen that is not a nucleic acid. For instance, in some
aspects of the invention the antigen not encoded in a nucleic acid
vector is a polypeptide. Minor modifications of the primary amino
acid sequences of polypeptide antigens may also result in a
polypeptide which has substantially equivalent antigenic activity
as compared to the unmodified counterpart polypeptide. Such
modifications may be deliberate, as by site-directed mutagenesis,
or may be spontaneous. All of the polypeptides produced by these
modifications are included herein as long as antigenicity still
exists. The polypeptide may be, for example, a viral polypeptide.
One non-limiting example of an antigen useful according to the
invention is the hepatitis B surface antigen. Experiments using
this antigen are described in the Examples below.
[0149] The term "substantially purified" as used herein refers to a
polypeptide which is substantially free of other proteins, lipids,
carbohydrates or other materials with which it is naturally
associated. One skilled in the art can purify viral or bacterial
polypeptides using standard techniques for protein purification.
The substantially pure polypeptide will often yield a single major
band on a non-reducing polyacrylamide gel. In the case of partially
glycosylated polypeptides or those that have several start codons,
there may be several bands on a non-reducing polyacrylamide gel,
but these will form a distinctive pattern for that polypeptide. The
purity of the viral or bacterial polypeptide can also be determined
by amino-terminal amino acid sequence analysis.
[0150] The invention also utilizes polynucleotides encoding the
antigenic polypeptides. It is envisioned that the antigen may be
delivered to the subject in a nucleic acid molecule which encodes
for the antigen such that the antigen must be expressed in vivo.
Such antigens delivered to he subject in a nucleic acid vector are
referred to as "antigens encoded by a nucleic acid vector." The
nucleic acid encoding the antigen is operatively linked to a gene
expression sequence which directs the expression of the antigen
nucleic acid within a eukaryotic cell. The "gene expression
sequence" is any regulatory nucleotide sequence, such as a promoter
sequence or promoter-enhancer combination, which facilitates the
efficient transcription and translation of the antigen nucleic acid
to which it is operatively linked. The gene expression sequence
may, for example, be a mammalian or viral promoter, such as a
constitutive or inducible promoter. Constitutive mammalian
promoters include, but are not limited to, the promoters for the
following genes: hypoxanthine phosphoribosyl transferase (HPTR),
adenosine deaminase, pyruvate kinase, .beta.-actin promoter and
other constitutive promoters. Exemplary viral promoters which
function constitutively in eukaryotic cells include, for example,
promoters from the cytomegalovirus (CMV), simian virus (e.g.,
SV40), papilloma virus, adenovirus, human immunodeficiency virus
(HIV), Rous sarcoma virus, cytomegalovirus, the long terminal
repeats (LTR) of Moloney leukemia virus and other retroviruses, and
the thymidine kinase promoter of herpes simplex virus. Other
constitutive promoters are known to those of ordinary skill in the
art. The promoters useful as gene expression sequences of the
invention also include inducible promoters. Inducible promoters are
expressed in the presence of an inducing agent. For example, the
metallothionein promoter is induced to promote transcription and
translation in the presence of certain metal ions. Other inducible
promoters are known to those of ordinary skill in the art.
[0151] In general, the gene expression sequence shall include, as
necessary, 5' non-transcribing and 5' non-translating sequences
involved with the initiation of transcription and translation,
respectively, such as a TATA box, capping sequence, CAAT sequence,
and the like. Especially, such 5' non-transcribing sequences will
include a promoter region which includes a promoter sequence for
transcriptional control of the operably joined antigen nucleic
acid. The gene expression sequences optionally include enhancer
sequences or upstream activator sequences as desired.
[0152] The antigen nucleic acid is operatively linked to the gene
expression sequence. As used herein, the antigen nucleic acid
sequence and the gene expression sequence are said to be "operably
linked" when they are covalently linked in such a way as to place
the expression or transcription and/or translation of the antigen
coding sequence under the influence or control of the gene
expression sequence. Two DNA sequences are said to be operably
linked if induction of a promoter in the 5' gene expression
sequence results in the transcription of the antigen sequence and
if the nature of the linkage between the two DNA sequences does not
(1) result in the introduction of a frame-shift mutation, (2)
interfere with the ability of the promoter region to direct the
transcription of the antigen sequence, or (3) interfere with the
ability of the corresponding RNA transcript to be translated into a
protein. Thus, a gene expression sequence would be operably linked
to an antigen nucleic acid sequence if the gene expression sequence
were capable of effecting transcription of that antigen nucleic
acid sequence such that the resulting transcript is translated into
the desired protein or polypeptide.
[0153] The antigen nucleic acid of the invention may be delivered
to the immune system alone or in association with a vector. In its
broadest sense, a "vector" is any vehicle capable of facilitating
the transfer of the antigen nucleic acid to the cells of the immune
system so that the antigen can be expressed and presented on the
surface of the immune cell. The vector generally transports the
nucleic acid to the immune cells with reduced degradation relative
to the extent of degradation that would result in the absence of
the vector. The vector optionally includes the above-described gene
expression sequence to enhance expression of the antigen nucleic
acid in immune cells. In general, the vectors useful in the
invention include, but are not limited to, plasmids, phagemids,
viruses, other vehicles derived from viral or bacterial sources
that have been manipulated by the insertion or incorporation of the
antigen nucleic acid sequences. Viral vectors are a preferred type
of vector and include, but are not limited to nucleic acid
sequences from the following viruses: retrovirus, such as Moloney
murine leukemia virus, Harvey murine sarcoma virus, murine mammary
tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated
virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses;
papilloma viruses; herpes virus; vaccinia virus; polio virus; and
RNA virus such as a retrovirus. One can readily employ other
vectors not named but known to the art.
[0154] Preferred viral vectors are based on non-cytopathic
eukaryotic viruses in which non-essential genes have been replaced
with the gene of interest. Non-cytopathic viruses include
retroviruses, the life cycle of which involves reverse
transcription of genomic viral RNA into DNA with subsequent
proviral integration into host cellular DNA. Retroviruses have been
approved for human gene therapy trials. Most useful are those
retroviruses that are replication-deficient (i.e., capable of
directing synthesis of the desired proteins, but incapable of
manufacturing an infectious particle). Such genetically altered
retroviral expression vectors have general utility for the
high-efficiency transduction of genes in vivo. Standard protocols
for producing replication-deficient retroviruses (including the
steps of incorporation of exogenous genetic material into a
plasmid, transfection of a packaging cell lined with plasmid,
production of recombinant retroviruses by the packaging cell line,
collection of viral particles from tissue culture media, and
infection of the target cells with viral particles) are provided in
Kriegler, M., "Gene Transfer and Expression, A Laboratory Manual,"
W. H. Freeman C.O., New York (1990) and Murry, E. J. Ed. "Methods
in Molecular Biology," vol. 7, Humana Press, Inc., Cliffton, N.J.
(1991).
[0155] A preferred virus for certain applications is the
adeno-associated virus, a double-stranded DNA virus. The
adeno-associated virus can be engineered to be replication
-deficient and is capable of infecting a wide range of cell types
and species. It further has advantages such as, heat and lipid
solvent stability; high transduction frequencies in cells of
diverse lineages, including hemopoietic cells; and lack of
superinfection inhibition thus allowing multiple series of
transductions. Reportedly, the adeno-associated virus can integrate
into human cellular DNA in a site-specific manner, thereby
minimizing the possibility of insertional mutagenesis and
variability of inserted gene expression characteristic of
retroviral infection. In addition, wild-type adeno-associated virus
infections have been followed in tissue culture for greater than
100 passages in the absence of selective pressure, implying that
the adeno-associated virus genomic integration is a relatively
stable event. The adeno-associated virus can also function in an
extrachromosomal fashion.
[0156] Other vectors include plasmid vectors. Plasmid vectors have
been extensively described in the art and are well-known to those
of skill in the art. See e.g., Sambrook et al., "Molecular Cloning:
A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory
Press, 1989. In the last few years, plasmid vectors have been found
to be particularly advantageous for delivering genes to cells in
vivo because of their inability to replicate within and integrate
into a host genome. These plasmids, however, having a promoter
compatible with the host cell, can express a peptide from a gene
operatively encoded within the plasmid. Some commonly used plasmids
include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other
plasmids are well-known to those of ordinary skill in the art.
Additionally, plasmids may be custom designed using restriction
enzymes and ligation reactions to remove and add specific fragments
of DNA.
[0157] It has recently been discovered that gene carrying plasmids
can be delivered to the immune system using bacteria. Modified
forms of bacteria such as Salmonella can be transfected with the
plasmid and used as delivery vehicles. The bacterial delivery
vehicles can be administered to a host subject orally or by other
administration means. The bacteria deliver the plasmid to immune
cells, e.g. dendritic cells, probably by passing through the gut
barrier. High levels of immune protection have been established
using this methodology. Such methods of delivery are useful for the
aspects of the invention utilizing systemic delivery of antigen,
CpG oligonucleotide and/or hormone.
[0158] CpG oligonucleotide can act in a synergistic manner with
other mucosal adjuvants to enhance immune responses. The CpG
oligonucleotide and mucosal adjuvant may be administered
simultaneously or sequentially. When the adjuvants are administered
simultaneously they can be administered in the same or separate
formulations, but are administered at the same time. The adjuvants
are administered sequentially, when the administration of the at
least two adjuvants is temporally separated. The separation in time
between the administration of the two adjuvants may be a matter of
minutes or it may be longer.
[0159] As shown in the Examples section, titers of serum anti-HBs
IgG, which is associated with systemic immunity, in mice immunized
with CpG oligonucleotide plus CT were at least 50-fold higher than
with CT or CpG oligonucleotide alone (FIG. 1). Furthermore, titers
with 1 .mu.g of the two adjuvants together gave better results than
10 .mu.g of either adjuvant alone. These results indicate a
synergistic action of the two adjuvants. Similar results were also
obtained with CpG and LT. Such synergy was seen for both humoral
(FIGS. 1-3) and cell-mediated (CTL and T-cell proliferation) (FIG.
4) responses. As well, the proportion of IgG2a isotype of
antibodies, was about 10-times greater with CpG ODN than CT,
indicating a greater Th1 influence of CpG ODN compared to CT.
Furthermore, the combination of CpG ODN and CT gave a 50-times
higher IgG2a:IgG1 ratio than CT alone. Taken together, these
results indicate a strong synergy of the adjuvant combination
humoral immune responses, with respect to both strength and
Th1-bias, and cellular immune responses (FIG. 3).
[0160] The hallmark of mucosal immunity is the presence of
secretory IgA antibodies in association with mucosal surfaces. IgA
antibodies are essential to prevent entry of the pathogen into the
body. IN immunization of mice with HBsAg alone, 1 or 10 .mu.g,
failed to induce any detectable IgA in lung washes. Nor was there
any IgA with the low dose of antigen and a low dose (1 .mu.g) of CT
or CpG ODN. However there was significant IgA with a high dose of
antigen and low dose of either CT or CpG ODN or a low dose of
antigen and a low dose of combined adjuvants. In fact, IgA levels
with 1 .mu.g of each of CpG ODN and CT combined were higher than
with 10 .mu.g of either alone, when administered with 10 .mu.g
HBsAg (FIG. 5). Furthermore, IgA in fecal extracts, which indicates
induction of mucosal immunity at distant sites, was detected only
with the combined adjvuants (FIG. 6). These results indicate that
CpG ODN is a potent adjuvant for induction of mucosal immunity and
that there is a strong synergistic response when used with another
mucosal adjuvant such as CT.
[0161] Similar results were found when LT was used in place of CT
(FIG. 7, Tables 2 and 3). CT and LT, which are closely related with
considerable structural and functional homology, are both too toxic
for use in humans. However there are a number of derivations of CT
and LT that retain some adjuvant activity yet are much less toxic.
One example is the B-subunit of CT (CTB) which is non-toxic since
the toxicity is associated with the A subunit. Another example is
LTK63, a genetically detoxified mutant of LT with no toxic
enzymatic activity. Although these adjuvants are being used in
human clinical trials, neither was a strong as CpG ODN for
induction of systemic immunity (serum IgG) when each was used at 1
.mu.g (FIG. 7). There was also a synergistic effect when CpG ODN
and CTB or LTK63 were used together, however this was more
noticeable for Th1-bias than for strength of the antibody response
(FIG. 7 and Table 2). The combination of CpG ODN and LTK63 also
induced IgA in lung washes, even though neither adjuvant on its own
induced IgA at low concentrations (Table 3).
[0162] The strong adjuvanticity and low toxicity of CpG
oligonucleotide when delivered to a mucosal surface has important
implications. It will allow many antigens to be delivered to
mucosal surfaces for the induction of strong systemic immune
responses. Non-invasive vaccine delivery is desirable for
immunization of children, animals, mass vaccination programs and
also to avoid the risk of needle-stick injury. Such vaccines could
be delivered intranasally by nose-drops or nasal spray or with a
delivery system, or they could be delivered by other routes (oral,
rectal, ocular) to other mucosal surfaces, including with different
delivery systems.
[0163] The synergistic interaction of CpG oligonucleotide with
mucosal adjuvants has important implications in vaccine
development. Because of the synergistic response it is now possible
to use lower and less toxic doses of mucosal adjuvants such as CT,
or other related toxins or subunits thereof, in conjunction with
CpG oligonucleotide to obtain even better immune responses with
less toxicity. For example, it would be possible to use CpG
oligonucleotide in combination with a less toxic genetically
modified mutants of CT or LT, for a highly effective vaccine of
acceptable toxicity. Not only could the combined adjuvant approach
be used to advantage with different toxins, but also with different
forms of antigen, and different delivery systems to various mucosal
routes. An effective amount as used with respect to this aspect of
the invention is an amount that produces a synergistic immune
response. A synergistic amount is that amount which produces an
immune response against a specific antigen that is greater than the
sum of the individual effects of either the CpG or the mucosal
adjuvant alone.
[0164] The invention can also be used in combination with
parenteral immunization strategies (e.g., intramuscular,
intradermal or subcutaneous injection), which are normally used for
the induction of systemic immune responses. Remarkably, mice
immunized with HBsAg and having CpG oligonucleotide as at least one
adjuvant, when primed by a parenteral route (IM) and boosted by a
mucosal route (IN) or primed IN and boosted IM had up to 10-fold
higher IgG (i.e., systemic humoral response) than when both prime
and boost were by the IM route (FIG. 8). Cellular immune responses
were also stronger with the parenteral/mucosal combined approaches
than with only IN or only IM, as indicated by stronger CTL (FIG. 9)
and higher T-cell proliferation (FIG. 10). While the IN prime and
boost gives good mucosal responses the IM prime and boost gives no
detectable mucosal responses (FIGS. 11-13). The IM prime and IN
boost approach also gave significant IgA in lung washes (FIG. 11)
and saliva (FIG. 12) but not feces (FIG. 13).
[0165] The mucosal adjuvants useful according to the invention are
non-oligonucleotide mucosal adjuvants. A "non-oligonucleotide
mucosal adjuvant" as used herein is an adjuvant other than a CpG
oligonucleotide that is capable of inducing a mucosal immune
response in a subject when administered to a mucosal surface in
conjunction with an antigen. Mucosal adjuvants include but are not
limited to Bacterial toxins: e.g., Cholera toxin (CT), CT
derivatives including but not limited to CT B subunit (CTB) (Wu et
al., 1998, Tochikubo et al., 1998); CTD53 (Val to Asp) (Fontana et
al., 1995); CTK97 (Val to Lys) (Fontana et al., 1995); CTK104 (Tyr
to Lys) (Fontana et al., 1995); CTD53/K63 (Val to Asp, Ser to Lys)
(Fontana et al., 1995); CTH54 (Arg to His) (Fontana et al., 1995);
CTN107 (His to Asn) (Fontana et al., 1995); CTE114 (Ser to Glu)
(Fontana et al., 1995); CTE112K (Glu to Lys) (Yamamoto et al.,
1997a); CTS61F (Ser to Phe) (Yamamoto et al., 1997a, 1997b); CTS106
(Pro to Lys) (Douce et al., 1997, Fontana et al., 1995); and CTK63
(Ser to Lys) (Douce et al., 1997, Fontana et al., 1995), Zonula
occludens toxin, zot, Escherichia coli heat-labile enterotoxin,
Labile Toxin (LT), LT derivatives including but not limited to LT B
subunit (LTB) (Verweij et al., 1998); LT7K (Arg to Lys) (Komase et
al., 1998, Douce et al., 1995); LT61F (Ser to Phe) (Komase et al.,
1998); LT112K (Glu to Lys) (Komase et al., 1998); LT118E (Gly to
Glu) (Komase et al., 1998); LT146E (Arg to Glu) (Komase et al.,
1998); LT192G (Arg to Gly) (Komase et al., 1998); LTK63 (Ser to
Lys) (Marchetti et al., 1998, Douce et al., 1997, 1998, Di Tommaso
et al., 1996); and LTR72 (Ala to Arg) (Giuliani et al., 1998),
Pertussis toxin, PT. (Lycke et al., 1992, Spangler BD, 1992,
Freytag and Clemments, 1999, Roberts et al., 1995, Wilson et al.,
1995) including PT-9K/129G (Roberts et al., 1995, Cropley et al.,
1995); Toxin derivatives (see below) (Holmgren et al., 1993,
Verweij et al., 1998, Rappuoli et al., 1995, Freytag and Clements,
1999); Lipid A derivatives (e.g., monophosphoryl lipid A, MPL)
(Sasaki et al., 1998, Vancott et al., 1998; Muramyl Dipeptide (MDP)
derivatives (Fukushima et al., 1996, Ogawa et al., 1989, Michalek
et al., 1983, Morisaki et al., 1983); Bacterial outer membrane
proteins (e.g., outer surface protein A (OspA) lipoprotein of
Borrelia burgdorferi, outer membrane protine of Neisseria
meningitidis)(Marinaro et al., 1999, Van de Verg et al., 1996);
Oil-in-water emulsions (e.g., MF59) (Barchfield et al., 1999,
Verschoor et al., 1999, O'Hagan, 1998); Aluminum salts (Isaka et
al., 1998, 1999); and Saponins (e.g., QS21) Aquila
Biopharmaceuticals, Inc., Worster, Mass.) (Sasaki et al., 1998,
MacNeal et al., 1998), ISCOMS, MF-59 (a squalene-in-water emulsion
stabilized with Span 85 and Tween 80; Chiron Corporation,
Emeryville, Calif.); the Seppic ISA series of Montanide adjuvants
(e.g., Montanide ISA 720; AirLiquide, Paris, France); PROVAX (an
oil-in-water emulsion containing a stabilizing detergent and a
micell-forming agent; IDEC Pharmaceuticals Corporation, San Diego,
Calif.); Syntext Adjuvant Formulation (SAF; Syntex Chemicals, Inc.,
Boulder, Colo.); poly[di(carboxylatophenoky)phosphazene (PCPP
polymer; Virus Research Institute, USA) and Leishmania elongation
factor (Corixa Corporation, Seattle, Wash.).
[0166] Although mucosal delivery of the antigen is considered a
prerequisite for induction of strong mucosal immune responses, it
is possible to induce strong mucosal immunity to systemically
delivered antigens by modulating the immune response with steroid
hormones, such as described for 1,25-Dihydroxy vitamin D.sub.3
[1,25(OH).sub.2D.sub.3] (Daynes et al., 1996). The invention also
includes methods for the administration of CpG oligonucleotide
alone or in combination with other mucosal adjuvants and antigen to
hormonally treated individuals. Each of the compounds may be
administered together or separately, systemically or mucosally. In
some embodiments the CpG oligonucleotide and antigen and optionally
other mucosal adjuvants are administered mucosally and the hormone
is administered systemically. The hormone may be given parenterally
(e.g., subcutaneous injection) or mucosally (e.g., orally).
[0167] Mucosal immune responses can also be induced with the
co-administration of cytokines with the CpG oligonucleotides.
Immune responses can also be augmented by co-linear expression of
cytokines (Bueler & Mulligan, 1996; Chow et at, 1997; Geissler
et al., 1997; Iwasaki et al., 1997; Kim et al., 1997) or B-7
co-stimulatory molecules (Iwasaki et al., 1997; Tsuji et at, 1997).
The cytokines can be administered directly with CpG
oligonucleotides or may be administered in the form of a nucleic
acid vector that encodes the cytokine, such that the cytokine can
be expressed in vivo. In one embodiment, when the CpG is
administered in the form of a plasmid expression vector, the vector
may encode the cytokine, and a separate administration of cytokine
is not required. The term "cytokine" is used as a generic name for
a diverse group of soluble proteins and peptides which act as
humoral regulators at nano- to picomolar concentrations and which,
either under normal or pathological conditions, modulate the
functional activities of individual cells and tissues. These
proteins also mediate interactions between cells directly and
regulate processes taking place in the extracellular environment.
Examples of cytokines include, but are not limited to IL-1, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, granulocyte-macrophage
colony stimulating factor (GM-CSF), granulocyte colony stimulating
factor (GCSF), interferon-.gamma. (.gamma.-INF), tumor necrosis
factor (TNF), TGF-.beta., FLT-3 ligand, and CD40 ligand.
[0168] Cytokines play a role in directing the T cell response.
Helper (CD4+) T cells orchestrate the immune response of mammals
through production of soluble factors that act on other immune
system cells, including other T cells. Most mature CD4+ T helper
cells express one of two cytokine profiles: Th1 or Th2. Th1 cells
express IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low
levels of TNF-.alpha.. The TH1 subset promotes delayed-type
hypersensitivity, cell-mediated immunity, and immunoglobulin class
switching to IgG.sub.2a. The Th2 subset induces humoral immunity by
activating B cells, promoting antibody production, and inducing
class switching to IgG.sub.1 and IgE. In some embodiments it is
preferred that the cytokine be a Th1 cytokine.
[0169] CpG oligonucleotides were found, surprisingly, to induce
mucosal immunity in remote sites as well as local sites. A "remote
site" as used herein is a mucosal tissue that is located in a
different region of the body than the mucosal tissue to which the
CpG oligonucleotide has been administered. For instance if the CpG
oligonucleotide is administered intranasally, a remote site would
be the mucosal lining of the gut.
[0170] For use in the instant invention, the nucleic acids can be
synthesized de novo using any of a number of procedures well known
in the art. For example, the b-cyanoethyl phosphoramidite method
(Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981);
nucleoside H-phosphonate method (Garegg et al., Tet. Let.
27:4051-4054, 1986; Froehler et al., Nucl. Acid. Res. 14:5399-5407,
1986, ; Garegg et al., Tet. Let. 27:4055-4058, 1986, Gaffney et
al., Tet. Let. 29:2619-2622, 1988). These chemistries can be
performed by a variety of automated oligonucleotide synthesizers
available in the market. Alternatively, CpG dinucleotides can be
produced on a large scale in plasmids, (see Sambrook, T., et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
laboratory Press, N.Y., 1989) and separated into smaller pieces or
administered whole. After being administered to a subject the
plasmid can be degraded into oligonucleotides. Oligonucleotides can
be prepared from existing nucleic acid sequences (e.g., genomic or
cDNA) using known techniques, such as those employing restriction
enzymes, exonucleases or endonucleases.
[0171] For use in vivo, nucleic acids are preferably relatively
resistant to degradation (e.g., via endo-and exo-nucleases).
Secondary structures, such as stem loops, can stabilize nucleic
acids against degradation. Alternatively, nucleic acid
stabilization can be accomplished via phosphate backbone
modifications. One type of stabilized nucleic acid has at least a
partial phosphorothioate modified backbone. Phosphorothioates may
be synthesized using automated techniques employing either
phosphoramidate or H-phosphonate chemistries. Aryl-and
alkyl-phosphonates can be made, e.g., as described in U.S. Pat. No.
4,469,863; and alkylphosphotriesters (in which the charged oxygen
moiety is alkylated as described in U.S. Pat. No. 5,023,243 and
European Patent No. 092,574) can be prepared by automated solid
phase synthesis using commercially available reagents. Methods for
making other DNA backbone modifications and substitutions have been
described (Uhlmann, E. and Peyman, A., Chem. Rev. 90:544, 1990;
Goodchild, J., Bioconjugate Chem. 1:165, 1990).
[0172] Nucleic acids containing an appropriate unmethylated CpG can
be effective in any vertebrate. Different nucleic acids containing
an unmethylated CpG can cause optimal immune stimulation depending
on the mammalian species. Thus an oligonucleotide causing optimal
stimulation in humans may not cause optimal stimulation in a mouse
and vice versa. One of skill in the art can identify the optimal
oligonucleotides useful for a particular mammalian species of
interest using routine assays described herein and/or known in the
art, using the guidance supplied herein.
[0173] The term "effective amount" of a CpG oligonucleotide refers
to the amount necessary or sufficient to realize a desired biologic
effect. For example, an effective amount of an oligonucleotide
containing at least one unmethylated CpG for inducing mucosal
immunity is that amount necessary to cause the development of IgA
in response to an antigen upon exposure to the antigen. Combined
with the teachings provided herein, by choosing among the various
active compounds and weighing factors such as potency, relative
bioavailability, patient body weight, severity of adverse
side-effects and preferred mode of administration, an effective
prophylactic or therapeutic treatment regimen can be planned which
does not cause substantial toxicity and yet is entirely effective
to treat the particular subject. The effective amount for any
particular application can vary depending on such factors as the
disease or condition being treated, the particular CpG
oligonucleotide being administered (e.g. the number of unmethylated
CpG motifs or their location in the nucleic acid), the antigen, the
size of the subject, or the severity of the disease or condition.
One of ordinary skill in the art can empirically determine the
effective amount of a particular CpG oligonucleotide and antigen
without necessitating undue experimentation.
[0174] Subject doses of the compounds described herein typically
range from about 80 mg/day to 16,000 mg/day, more typically from
about 800 mg/day to 8000 mg/day, and most typically from about 800
mg/day to 4000 mg/day. Stated in terms of subject body weight,
typical dosages range from about 1 to 200 mg/kg/day, more typically
from about 10 to 100 mg/kg/day, and most typically from about 10 to
50 mg/kg/day. Stated in terms of subject body surface areas,
typical dosages range from about 40 to 8000 mg/m.sup.2/day, more
typically from about 400 to 4000 mg/m.sup.2/day, and most typically
from about 400 to 2000 mg/m.sup.2/day.
[0175] In some embodiments, particularly when the CpG is in a
plasmid vector, at least 50 .mu.g of the CpG is administered to a
subject. In other embodiments at least 75 .mu.g, 100 .mu.g, 200
.mu.g, 300 .mu.g, 400 .mu.g, 500 .mu.g and every integer in between
of the CpG is administered to the subject.
[0176] For any compound described herein the therapeutically
effective amount can be initially determined from cell culture
assays. For instance the effective amount of CpG oligonucleotide
useful for inducing mucosal immunity can be assessed using the in
vitro assays described above with respect to stimulation index. The
stimulation index can be used to determine as effective amount of
the particular oligonucleotide for the particular subject, and the
dosage can be adjusted upwards or downwards to achieve the desired
levels in the subject. Therapeutically effective amounts can also
be determined from animal models. A therapeutically effective dose
can also be determined from human data for CpG oligonucleotides
which have been tested in humans (human clinical trials have been
initiated) and for compounds which are known to exhibit similar
pharmacological activities, such as other mucosal adjuvants, e.g.,
LT and other antigens for vaccination purposes. The applied dose
can be adjusted based on the relative bioavailability and potency
of the administered compound. Adjusting the dose to achieve maximal
efficacy based on the methods described above and other methods as
are well-known in the art is well within the capabilities of the
ordinarily skilled artisan.
[0177] The formulations of the invention are administered in
pharmaceutically acceptable solutions, which may routinely contain
pharmaceutically acceptable concentrations of salt, buffering
agents, preservatives, compatible carriers, adjuvants, and
optionally other therapeutic ingredients.
[0178] For use in therapy, an effective amount of the CpG
oligonucleotide can be administered to a subject by any mode that
delivers the oligonucleotide to a mucosal surface. "Administering"
the pharmaceutical composition of the present invention may be
accomplished by any means known to the skilled artisan. Preferred
routes of administration include but are not limited to oral,
intranasal, intratracheal, inhalation, ocular, vaginal, and
rectal.
[0179] For oral administration, the compounds (i.e., CpG
oligonucleotides, antigen, mucosal adjuvant) can be formulated
readily by combining the active compound(s) with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a subject to be treated.
Pharmaceutical preparations for oral use can be obtained as solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients
are, in particular, fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate. Optionally the oral formulations may also be formulated
in saline or buffers for neutralizing internal acid conditions.
[0180] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0181] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. Microspheres formulated for oral
administration may also be used. Such microspheres have been well
defined in the art. All formulations for oral administration should
be in dosages suitable for such administration.
[0182] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0183] For administration by inhalation, the compounds for use
according to the present invention may be conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0184] The compounds, when it is desirable to deliver them
systemically, may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion.
Formulations for injection may be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents.
[0185] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0186] Alternatively, the active compounds may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0187] The compounds may also be formulated in rectal or vaginal
compositions such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0188] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0189] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0190] Suitable liquid or solid pharmaceutical preparation forms
are, for example, aqueous or saline solutions for inhalation,
microencapsulated, encochleated, coated onto microscopic gold
particles, contained in liposomes, nebulized, aerosols, pellets for
implantation into the skin, or dried onto a sharp object to be
scratched into the skin. The pharmaceutical compositions also
include granules, powders, tablets, coated tablets,
(micro)capsules, suppositories, syrups, emulsions, suspensions,
creams, drops or preparations with protracted release of active
compounds, in whose preparation excipients and additives and/or
auxiliaries such as disintegrants, binders, coating agents,
swelling agents, lubricants, flavorings, sweeteners or solubilizers
are customarily used as described above. The pharmaceutical
compositions are suitable for use in a variety of drug delivery
systems. For a brief review of present methods for drug delivery,
see Langer, Science 249:1527-1533, 1990, which is incorporated
herein by reference.
[0191] The CpG oligonucleotides and antigens may be administered
per se (neat) or in the form of a pharmaceutically acceptable salt.
When used in medicine the salts should be pharmaceutically
acceptable, but non-pharmaceutically acceptable salts may
conveniently be used to prepare pharmaceutically acceptable salts
thereof. Such salts include, but are not limited to, those prepared
from the following acids: hydrochloric, hydrobromic, sulphuric,
nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,
tartaric, citric, methane sulphonic, formic, malonic, succinic,
naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts
can be prepared as alkline metal or alkaline earth salts, such as
sodium, potassium or calcium salts of the carboxylic acid
group.
[0192] Suitable buffering agents include: acetic acid and a salt
(1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a
salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
Suitable preservatives include benzalkonium chloride (0.003-0.03%
w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and
thimerosal (0.004-0.02% w/v).
[0193] The pharmaceutical compositions of the invention contain an
effective amount of a CpG oligonucleotide and antigens optionally
included in a pharmaceutically-acceptable carrier. The term
"pharmaceutically-acceptable carrier" means one or more compatible
solid or liquid filler, dilutants or encapsulating substances which
are suitable for administration to a human or other vertebrate
animal. The term "carrier" denotes an organic or inorganic
ingredient, natural or synthetic, with which the active ingredient
is combined to facilitate the application. The components of the
pharmaceutical compositions also are capable of being commingled
with the compounds of the present invention, and with each other,
in a manner such that there is no interaction which would
substantially impair the desired pharmaceutical efficiency.
[0194] The CpG oligonucleotides or antigens useful in the invention
may be delivered in mixtures with additional mucosal adjuvant(s) or
antigen(s). A mixture may consist of several mucosal adjuvants in
addition to the CpG oligonucleotide or several antigens.
[0195] A variety of administration routes are available. The
particular mode selected will depend, of course, upon the
particular adjuvants or antigen selected, the particular condition
being treated and the dosage required for therapeutic efficacy. The
methods of this invention, generally speaking, may be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of an immune
response without causing clinically unacceptable adverse effects.
Preferred modes of administration are discussed above.
[0196] The compositions may conveniently be presented in unit
dosage form and may be prepared by any of the methods well known in
the art of pharmacy. All methods include the step of bringing the
compounds into association with a carrier which constitutes one or
more accessory ingredients. In general, the compositions are
prepared by uniformly and intimately bringing the compounds into
association with a liquid carrier, a finely divided solid carrier,
or both, and then, if necessary, shaping the product. Liquid dose
units are vials or ampoules. Solid dose units are tablets, capsules
and suppositories. For treatment of a patient, depending on
activity of the compound, manner of administration, purpose of the
immunization (i.e., prophylactic or therapeutic), nature and
severity of the disorder, age and body weight of the patient,
different doses may be necessary. The administration of a given
dose can be carried out both by single administration in the form
of an individual dose unit or else several smaller dose units.
Multiple administration of doses at specific intervals of weeks or
months apart is usual for boosting the antigen-specific
responses.
[0197] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the compounds, increasing
convenience to the subject and the physician. Many types of release
delivery systems are available and known to those of ordinary skill
in the art. They include polymer base systems such as
poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are: lipids
including sterols such as cholesterol, cholesterol esters and fatty
acids or neutral fats such as mono-di-and tri-glycerides; hydrogel
release systems; sylastic systems; peptide based systems; wax
coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which an agent of the invention is contained in a form within a
matrix such as those described in U.S. Pat. Nos. 4,452,775,
4,675,189, and 5,736,152, and (b) diffusional systems in which an
active component permeates at a controlled rate from a polymer such
as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.
In addition, pump-based hardware delivery systems can be used, some
of which are adapted for implantation.
[0198] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLE
Example 1
Materials and Methods
[0199] 1. Materials and Animals
[0200] Mice. All experiments were carried out using female BALB/c
mice aged 6-8 weeks with 5-10 mice per experimental or control
group. For intranasal immunizations, mice were lightly
anaesthetized with Halothane.RTM. (Halocarbon Laboratories, River
Edge, N.J.).
[0201] Adjuvants: Mice were immunized by IN administration of 1
.mu.g HBsAg (plasma-derived HBV S protein, ad subtype, Genzyme
Diagnostics, San Carlos, Calif.), alone or combined with 1 or 10
.mu.g of CT (purified from Vibrio cholerae, Sigma, St. Louis, Mo.),
LT (purified from Escherichia coli, Sigma), CTB (purified from
Vibrio cholerae, Sigma), LTK63 (mutant of LT bearing an
Ser.fwdarw.Lys at position 63, generously provided by Dr. Rino
Rappuoli, IRIS, Chiron S.p.A., Italy) and/or CpG ODN
(5'-TCCATGACGTTCCTGACGTT-3', CpG ODN #1826 SEQ ID NO. 90) or
non-CpG control ODN (5'TCCAGGACTTCTCTCAGGTT-3', CpG ODN #1982 SEQ
ID NO. 91) (Hybridon Specialty Products, Milford, Mass.). The
antigen and adjuvant(s) were made up to a total volume of 150 .mu.l
with 0.15 M NaCl, and were administered by IN inhalation. ODN were
resuspended in 10 mM Tris (pH 7.0), 1 mM EDTA for storage at +4EC
before dilution into saline for immunization. LPS level in ODN was
undetectable (<1 ng/mg) by Limulus assay (Whittaker Bioproducts,
Walkersville, Md.).
[0202] 2. Mucosal Immunization
[0203] Each animal was immunized with 1 or 10 Fg plasma-derived HBV
S protein (HBsAg, ad subtype, Genzyme Diagnostics, San Carlos,
Calif.), which was administered alone or in combination with 1 or
10 .mu.g of CT or LT or derivative of them and/or CpG
oligonucleotide #1826. The derivatives of CT were the B subunit of
CT (CTB). The detoxified derivatives of LT were all produced by
genetic mutations that affected the A subunit or enzymatic activity
and included LTK63. All vaccines were delivered in a total volume
of 150 .mu.l, which was applied as droplets directly over both
external nares of lightly anaesthetized mice. Some mice were
boosted in the identical manner at 8 weeks after prime. All
experimental groups contained 5 or 10 mice.
[0204] 3. Collection of Samples
[0205] Plasma: Plasma was recovered from mice at various times
after immunization (1, 2, 4 and 8 wk post-prime and 1, 2 and 4 wk
post-boost) by retro-orbital bleeding and stored at -20.degree. C.
until assayed.
[0206] Fecal pellets: Fecal pellets were collected from mice at
various times after immunization (1, 2, 4 and 8 wk post-prime and
1, 2 and 4 wk post-boost). Mice were isolated in individual cages
without bedding for a 24 hr period, following which fecal pellets
were collected and weighed into 0.1 mg aliquots. One ml TBS (0.05 M
Tris-HCI, 0.15 M NaCl, pH 7.5) and 0.1 Fg sodium azide (Sigma) were
added per 0.1 mg of fecal material. Samples were allowed to
rehydrate for 30 min at RT, then were centrifuged at 6000 rpm for
15 min. to remove fecal debris and supernatants were collected and
stored at -20E C until assayed for S-IgA by ELISA.
[0207] Lung washes: Lung washes were carried out on mice 4 wk after
primary immunization or boost. A 0.33 cc Insulin syringe with a
29G1/2 needle attached (Becton Dickenson, Franklin Lakes, N.J.) was
used for carrying out lung washes. One ml TBS was drawn into the
syringe and a length of polyethylene (PE) tubing that was 1 cm
longer than the needle was attached (PE20, ID=0.38 mm, Becton
Dickinson). The mouse was killed by anesthetic overdose and the
trachea was immediately exposed through an anterior midline
incision made using fine-tipped surgical scissors (Fine Science
Tools Inc., North Vancouver, BC). A small incision was then made in
the trachea and a clamp (Fine Science Tools Inc., North Vancouver,
BC) was placed above it. The PE tubing was passed a few mm down the
trachea through the incision and a second clamp was placed just
below the incision to hold the PE tubing in place in the trachea.
The TBS solution was slowly instilled in the lungs then withdrawn
three times (80% recovery expected). Recovered samples were
centrifuge at 13,000 rpm for 7 min., and the supernatants were
collected and stored at -20E C until assayed by ELISA.
[0208] 4. Evaluation of Immune Responses
[0209] Systemic humoral response: HBsAg-specific antibodies
(anti-HBs) in the mouse plasma were detected and quantified by
end-point dilution ELISA assay (in triplicate) for individual
animals as described previously (Davis et al., 1998). Briefly,
96-well polystyrene plates (Corning) coated overnight (RT) with
plasma derived HBsAg particles (as used for immunization) (100 Fl
of 1 Fg/ml in 0.05 M sodium carbonate-bicarbonate buffer, pH 9.6)
were incubated with the plasma for 1 hr at 37 E C. Captured
antibodies were then detected with horseradish peroxidase
(HRP)-conjugated goat anti-mouse IgG, IgG1 or IgG2a (1:4000 in
PBS-Tween, 10% PBS: 100 Fl/well; Southern Biotechnology Inc.,
Birmingham, Ala.), followed by addition of o-phenylenediamine
dihydrochloride solution (OPD, Sigma), 100 Fl/well, for 30 min at
RT in the dark. The reaction was stopped by the addition of 4 N
H.sub.2SO.sub.4, 50 Fl/well.
[0210] End-point dilution titers were defined as the highest plasma
dilution that resulted in an absorbance value (OD 450) two times
greater than that of non-immune plasma, with a cut-off value of
0.05. Anti-HBs titers of responding mice (endpoint titers >10)
were expressed as means SEM of individual animal values, which were
themselves the average of triplicate assays.
[0211] Mucosal humoral response: This was carried out on fecal
supematants or recovered lung washes as for plasma (above) except
samples were incubated on coated plates for 2 hr at 37.degree. C.
and captured antibodies were detected with HRP-conjugated goat
anti-mouse IgA (1:1000 in PBS-Tween. 10% PBS: 100 Fl/well; Southern
Biotechnology Inc). Non-immune fecal pellet or lung wash solutions
were used to determine negative control values. For lung wash
solutions, anti-HBs endpoint dilution titers were reported (as
described above), whereas for fecal pellet solutions, absorbance
values (OD 450) greater than that of non-immune fecal pellet
solution were calculated and expressed as mean SEM of individual OD
450 values, which were themselves the average of triplicate
assays.
[0212] Evaluation of CTL responses: Spleens were removed from mice
4 wk after primary immunization or boost. In vitro assay of
HBsAg-specific cytolytic activity was carried out as previously
described (Davis et al., 1998). In brief, single cell suspensions
were prepared and suspended in tissue culture medium (RPMI 1640,
10% FBS, Life Technologies, Grand Island, N.Y., supplemented with
penicillin-streptomycin solution, 1000 U/ml, 1 mg/ml final
concentrations respectively, Sigma). Splenocytes (3.times.10.sup.7)
were co-cultured for 5 days (37EC, 5% CO2) with 1.5.times.10.sup.6
syngeneic HBsAg-expressing stimulator cells (P815-preS, generously
provided by F. V. Chisari, Scripps Institute, La Jolla, Calif.)
that had been previously inactivated by irradiation (20 000 rad).
Effector cells were harvested, washed, serially diluted and
cultured with 5.times.10.sup.4 .sup.51Cr-labeled HBsAg-expressing
target cells (P815S) in round bottom 96-well culture plates (37EC,
5% CO2, 4 hr). Supernatant (100 Fl) was removed for radiation
(gamma) counting. Spontaneous release was determined by incubating
target cells without effector cells and total release by addition
of 100 Fl 2 N HCl to the target cells. The percent lysis was
calculated as [(experimental release-spontaneous release)/(total
release-spontaneous release)].times.100. The percent specific lysis
was calculated as % lysis with P815S- % lysis with P815 cells. CTL
activity for responding mice [% specific lysis>10 at
effector:target (E:T) of 25:1] were expressed as mean SEM of
individual animal values, which were themselves the average of
triplicate assays.
[0213] 5. Statistical Analysis
[0214] Data were analyzed using the GraphPAD InStat program (Graph
PAD Software, San Diego). The statistical significance of the
difference between two groups was determined from the means and
standard deviations by Student's 2-tailed t-test and between three
or more groups by 1-factor analysis of variance (ANOVA) followed by
Tukey's test for multiple range testing. Differences were
considered to be not significant with p>0.05.
Example 2
Systemic Humoral Responses After Mucosal Immunization
[0215] BALB/c mice immunized on a single occasion by IN inhalation
of HBsAg without adjuvant did not have any detectable anti-HBs IgG
antibodies in their plasma with 1 .mu.g HBsAg and only extremely
low titers (<20) in a few mice with 10 .mu.g of antigen (FIG.
1).
[0216] In contrast, titers of anti-HBs IgG were considerably
greater when HBsAg was administered in combination with either CpG
oligonucleotide or CT as adjuvant (FIG. 1). With a low dose of
adjuvant (1 .mu.g) and either a low or high dose of antigen (1 or
10 .mu.g HBsAg), CpG oligonucleotide was found to be equivalent to
CT for induction of plasma anti-HBs IgG (p=0.73 with 1 .mu.g HBsAg,
and 0.13 with 10 .mu.g HBsAg). CpG oligonucleotide and CT were also
equivalent with a high dose of adjuvant (10 .mu.g) and high dose of
antigen (10 .mu.g HBsAg) (p=0.08), however with a lower dose of
antigen, the higher dose of CT was superior to the CpG
oligonucleotide (p=0.01) (FIG. 1). These results indicate that CpG
oligonucleotide is essentially equal to CT for enhancement of
systemic immune responses with mucosal delivery (IN) of a protein
antigen.
[0217] A combined low dose of CpG oligonucleotide and CT (1 .mu.g
of each) gave a better systemic humoral response than 10 .mu.g CpG
oligonucleotide alone (p=0.01) and was equal to that with 10 .mu.g
CT alone (p=0.22), when added to a 1 .mu.g dose of HBsAg.
Furthermore, with a 10 .mu.g dose of HBsAg, the combined adjuvants
(1 .mu.g each) induced anti-HBs IgG titers as high as those with 10
.mu.g of either adjuvant alone (CT, p=0.27; CpG oligonucleotide,
p=0.09) (FIG. 1). These finding indicate that CpG oligonucleotide
can act synergistically with CT when administered to mucosal tissue
to induce strong systemic humoral responses, and thereby permit a
lower dose of adjuvant to be administered.
[0218] Antibody titers were further increased about 10-fold by
boosting at 8 wks. Post-boost titers of plasma IgG were equivalent
for CT and CpG oligonucleotide used alone, and were 5-10 times
higher than that with both adjuvants together (FIG. 2). These
results indicate that the adjuvant effect of CpG oligonucleotide
alone or in combination with CT can be enhanced by boosting.
[0219] Evaluation of plasma for IgG antibody isotypes after a
single mucosal immunization showed predominantly IgG1 antibodies
(Th2-like) with CT and mixed IgG1/IgG2a antibodies (Th0) with CpG
oligonucleotide alone or in combination with CT. The proportion of
IgG2a isotype of antibodies, was about 10-times greater with CpG
ODN than CT, indicating a greater Th1 influence of CpG ODN compared
to CT. Furthermore, the combination of CpG ODN and CT gave a
50-times higher IgG2a:IgG1 ratio than CT alone (FIG. 3). Following
boost, anti-HBs were still predominantly IgG1 with CT and mixed
with CpG oligonucleotide, although in the latter case, the
proportion of IgG2a was now higher. Surprisingly, plasma anti-HBs
after boost with CpG oligonucleotide and CT were now predominantly
IgG2a (Th-1 like) (FIG. 3). These findings indicate that CpG
oligonucleotide as a mucosal adjuvant stimulates a Th1-like
response, even in the presence of a strong Th2-like adjuvant like
CT.
[0220] Similar results were found when LT was used in place of CT
(FIG. 7, Tables 2 and 3). CT and LT, which are closely related with
considerable structural and functional homology, are both too toxic
for use in humans. However there are a number of derivations of CT
and LT that retain some adjuvant activity yet are much less toxic.
One example is the B-subunit of CT (CTB) which is non-toxic since
the toxicity is associated with the A subunit. Another example is
LTK63, a genetically detoxified mutant of LT with no toxic
enzymatic activity. Although these adjuvants are being used in
human clinical trials, neither was a strong as CpG ODN for
induction of systemic immunity (serum IgG) when each was used at 1
.mu.g (FIG. 7). There was also a synergistic effect when CpG ODN
and CTB or LTK63 were used together, however this was more
noticeable for Th1-bias than for strength of the antibody response
(FIG. 7 and Table 2).
Example 3
Systemic CTL Response After Mucoal Immunization
[0221] Only low levels of CTL were induced with HBsAg alone,
however the addition of either CpG oligonucleotide or CT
significantly increased HBsAg-specific CTL activity. CTL responses
were equivalent for CT and CpG oligonucleotide, regardless of dose.
However, a combination of CT and CpG oligonucleotide (1 .mu.g of
each) increased CTL responses approximately two-fold. (FIG. 4).
Example 4
Mucosal Humoral Responses After Mucosal Immunization
[0222] No anti-HBs S-IgA were detected in lung washes of mice
immunized with 1 or 10 .mu.g HBsAg alone. Nor were anti-HBs IgA
detected with the low dose of antigen combined with a low dose (1
.mu.g) of either CpG oligonucleotide or CT or with a high dose of
CpG oligonucleotide; only low titers were detected with low dose
antigen and high dose CT (FIG. 5). However when low doses of both
CpG oligonucleotide and CT (1 .mu.g each) were used together with
the low dose of antigen, significant levels of HBsAg-specific S-IgA
could be detected in lung washes (FIG. 5).
[0223] With a higher antigen dose (10 .mu.g), S-IgA was detected in
lung washes of mice administered the either low or high doses of CT
and/or CpG oligonucleotide. Titers of IgA were significantly higher
with 1 .mu.g of the two adjuvants together than with 10 .mu.g of CT
or CpG oligonucleotide alone (p=0.0003 and <0.0001 respectively)
(FIG. 5). IgA titers increased approximately ten-fold after
boosting with both adjuvants. Thus CpG oligonucleotide can induce
specific local mucosal immunity against antigen administered
intranasally. Furthermore, similar to as was found for systemic
response (above) CpG oligonucleotide acts in a synergistic fashion
with CT for the induction of mucosal immunity.
[0224] IgA was also detected in fecal pellets of mice immunized
with HBsAg and 10 .mu.g CpG oligonucleotide. In contrast, only very
low levels were detected in mice immunized with HBsAg in
combination with CT (1 or 10 .mu.g) (FIG. 6). Thus, CpG
oligonucleotide can induce mucosal immunity at distant mucosal
sites.
Example 5
Mucosal and Systemic Immune Response to Other Mucosal Adjuvants
[0225] Systemic Immune Responses
[0226] IN delivery of HBsAg (1 .mu.g) without adjuvant did not
induce detectable anti-HBs IgG antibodies in the plasma of any mice
(0/15). In contrast, high titers of anti-HBs IgG were induced in
all mice when HBsAg was administered in combination with CpG, CT or
LT as adjuvant (FIG. 7, Table 2). At a low dose (1 .mu.g) LT, CT
and CpG gave equivalent anti-HBs IgG titers (p=0.22). At a high
dose (10 .mu.g) CT and LT gave higher titers than CpG, however 5/10
mice receiving this dose of LT died within 10 days. No detectable
anti-HBs IgG was detected with a low dose (1 .mu.g) of CTB or
LTK63, however a high dose (10 .mu.g) of CTB gave low anti-HBs IgG
endpoint ELISA titers and a high dose (10 .mu.g) of LTK63 gave as
good levels of anti-HBs IgG as a high dose (10 .mu.g) of CpG
(p=0.97) (FIG. 7, Tables 2 and 3).
[0227] When used together, CpG and either LT or CT (1 .mu.g each)
appeared to have a synergistic effect since anti-HBs titers were 5
to 10 times higher than with any one of the three adjuvants alone
(FIG. 7). Indeed, CpG plus LT (1 .mu.g each) gave a better response
than 10 .mu.g of CpG or LT alone (p=0.007, 0.015 respectively) and
the response with CpG plus CT (1 .mu.g each) was equal to that with
10 .mu.g CT alone (p=0.65). In contrast, there was no synergistic
effect with LTK63 plus CpG (1 .mu.g each) for anti-HBs IgG titers,
which were equivalent to those with 1 .mu.g CpG alone (p=0.40).
Surprisingly, CTB plus CpG (1 .mu.g each) gave lower anti-HBs
titers than 1 .mu.g CpG alone (p=0.007) (FIG. 7). Adjuvant effects
with CpG ODN were due to the CpG motif rather than a non-specific
effect of the ODN backbone since mice immunized with 1 .mu.g of
HBsAg plus 10 .mu.g of non-CpG ODN had no (7/10)or very low (3/10)
titers of anti-HBs IgG antibodies (data not shown).
[0228] Antibodies were predominantly IgG1 (Th2-like) with CT, CTB
and LT and mixed IgG1/IgG2a (Th1/Th2) with LTK63. At a low dose (1
.mu.g) responses with CpG were mixed IgG1/IgG2a (Th1/Th2), but at a
higher dose (10 .mu.g) were more Th1 (IgG2a>>IgG1). Responses
were mixed Th1/T2 with CT/CpG or CTB/CpG and more Th1 with LT/CpG.
At a low dose (1 .mu.g each) LTK63/CpG responses were Th1/Th2, but
at a higher dose (10 .mu.g each) were more Th1 (Table 3). Thus
coadministration of CpG with other adjuvants shifted responses
towards a more Th1-like response as indicated by a greater
proportion of IgG2a antibodies.
[0229] Mucosal Immune Responses
[0230] When adjuvants were used alone, only mice receiving LT or
LTK63 had detectable IgA in lung washes, however when CpG ODN was
also included with CT or LT a greater number of animals responded
or titers were higher than with comparable doses alone, suggesting
a synergistic effect. CpG alone did not induce IgA. Neither did
CTB, alone or combined with CpG (Table 3).
[0231] Only a few adjuvants on their own (LT and CpG) induced IgA
in the feces, and then only in some animals. No significant IgA was
detected with CT, CTB, LTK63 or non-CpG ODN. CpG and LT together
resulted in IgA in the feces of a greater proportion of animals
than either adjuvant alone suggesting an additive or synergistic
effect. No such effects were evident with other combinations (Table
3).
15TABLE 2 Effect of adjuvant on HBsAg-specific antibody isotypes
Anti-HBs response Adjuvant.sup.a dose (.mu.g) IgG2a.sup.b
IgG1.sup.b IgG2a:IgG1.sup.c none -- 0 0 N/A.sup.d CT 1 36 1632 0.02
CT 10 406 3849 0.1 CTB 1 0 0 N/A.sup. CTB 10 6 59 0.1 LT 1 226 6457
0.04 LT 10 895 2024 0.44 LTK63 1 0 0 N/A.sup. LTK63 10 231 455 0.5
CpG ODN 1 146 403 0.4 CpG ODN 10 549 41 13.4 control ODN 1 0 0
N/A.sup. control ODN 10 0 0 N/A.sup. CT + CpG ODN 1 each 3376 2374
1.4 CTB + CpG ODN 1 each 0 0 N/A.sup. LT + CpG ODN 1 each 6268 1438
4.4 LTK63 + CpG ODN 1 each 185 272 0.7 CT + control ODN 1 each 402
5087 0.08 CT + CpG ODN 10 each =.sup.e = = CTB + CpG ODN 10 each
227 208 1.1 LT + CpG ODN 10 each = = = LTK63 + CpG ODN 10 each 3170
413 7.7
[0232]
16TABLE 3 Effect of adjuvant on HBsAg-specific IgA responses
Anti-HBs response.sup.b lung fecal Adjuvaut.sup.a dose (.mu.g)
IgA.sup.c no. of responders IgA.sup.d no. of responders none -- 0 0
0 0 CT 1 0 0 0 0 CT 10 0 0 0 0 CTB 1 0 0 0 0 CTB 10 0 0 0 0 LT 1
160 .+-. 68 5 100, 200 2 LT 10 17 .+-. 5 3/3 (2 dead) 200 .+-. 50
3/3 (2 dead) LTK63 1 0 0 0 0 LTK63 10 26 .+-. 6 4 0 0 CpG ODN 1 0 0
100 1 CpG ODN 10 0 0 0 0 control ODN 1 0 0 0 0 control ODN 10 0 0 0
0 CT + CpG ODN 1 each 17, 49 2 120 1 CTB + CpG ODN 1 each 0 0 0 0
LT + CpG ODN 1 each 232 .+-. 34 5 150 .+-. 20 4 LTK63 + CpG ODN 1
each 14 1 0 0 CT+ control ODN 1 each 0 0 0 0 CT + CpG ODN 10 each
=.sup.e = = = CTB + CpG ODN 10 each 17 1 0 0 LT + CpG ODN 10 each =
= = = LTK63 + CpG ODN 10 each 28 .+-. 46 3/4 130 1/4
[0233]
17TABLE 4 summary of effects of different prime/boost strategies on
HBsAg-specific immune responses IgA PRIME BOOST L S F IgG CTL TCP
IM Ag + alum + none X X CpG IM Ag + X X X X alum + CpG IN Ag X X X
X X IN Ag + CT X X X X X IN Ag + CpG X X X X X IN Ag + CT + X X X X
X X CpG IN Ag X IN Ag + CT IM Ag + X X X X alum + CpG IN Ag + CpG X
X X IN Ag + CT + CpG X X X X X X IN Ag + CT + CpG IN Ag + CT + X X
X X X X CpG IN Ag + CT + CpG none X X Ag: 1 .mu.g HBsAg CpG: 1
.mu.g #1826, CT: 1 .mu.g, alum: 25 .mu.g L: lung, cut-off GMT = 10
S: saliva, cut-off OD.sub.450 .times. 10.sup.3 = 100 F: fecal,
cut-off OD.sub.450 .times. 10.sup.3 = 100 CTL, cut-off 20% at E:T
100:1 TCP, cut-off 2500 cpm
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[0288] 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
examples provided, since the examples are intended as a single
illustration of one aspect of the invention and other fimctionally
equivalent embodiments are within the scope of the invention.
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. The advantages and objects of the invention are
not necessarily encompassed by each embodiment of the invention.
Sequence CWU 1
1
95 1 15 DNA Artificial Sequence Synthetic Sequence 1 gctagacgtt
agcgt 15 2 15 DNA Artificial Sequence Synthetic Sequence 2
gctagatgtt agcgt 15 3 15 DNA Artificial Sequence modified_base
(7)...(7) m5c 3 gctagacgtt agcgt 15 4 15 DNA Artificial Sequence
modified_base (13)...(13) m5c 4 gctagacgtt agcgt 15 5 15 DNA
Artificial Sequence Synthetic Sequence 5 gcatgacgtt gagct 15 6 20
DNA Artificial Sequence Synthetic Sequence 6 atggaaggtc cagcgttctc
20 7 20 DNA Artificial Sequence Synthetic Sequence 7 atcgactctc
gagcgttctc 20 8 20 DNA Artificial Sequence modified_base (3)...(3)
m5c 8 atcgactctc gagcgttctc 20 9 20 DNA Artificial Sequence
modified_base (18)...(18) m5c 9 atcgactctc gagcgttctc 20 10 20 DNA
Artificial Sequence Synthetic Sequence 10 atggaaggtc caacgttctc 20
11 20 DNA Artificial Sequence Synthetic Sequence 11 gagaacgctg
gaccttccat 20 12 20 DNA Artificial Sequence Synthetic Sequence 12
gagaacgctc gaccttccat 20 13 20 DNA Artificial Sequence Synthetic
Sequence 13 gagaacgctc gaccttcgat 20 14 20 DNA Artificial Sequence
modified_base (14)...(14) m5c 14 gagaacgctg gaccttccat 20 15 20 DNA
Artificial Sequence Synthetic Sequence 15 gagaacgatg gaccttccat 20
16 20 DNA Artificial Sequence Synthetic Sequence 16 gagaacgctc
cagcactgat 20 17 20 DNA Artificial Sequence Synthetic Sequence 17
tccatgtcgg tcctgatgct 20 18 20 DNA Artificial Sequence
modified_base (12)...(12) m5c 18 tccatgtcgg tcctgatgct 20 19 20 DNA
Artificial Sequence Synthetic Sequence 19 tccatgacgt tcctgatgct 20
20 20 DNA Artificial Sequence Synthetic Sequence 20 tccatgtcgg
tcctgctgat 20 21 8 DNA Artificial Sequence Synthetic Sequence 21
tcaacgtt 8 22 8 DNA Artificial Sequence Synthetic Sequence 22
tcagcgct 8 23 8 DNA Artificial Sequence Synthetic Sequence 23
tcatcgat 8 24 8 DNA Artificial Sequence Synthetic Sequence 24
tcttcgaa 8 25 7 DNA Artificial Sequence Synthetic Sequence 25
caacgtt 7 26 8 DNA Artificial Sequence Synthetic Sequence 26
ccaacgtt 8 27 8 DNA Artificial Sequence Synthetic Sequence 27
aacgttct 8 28 8 DNA Artificial Sequence Synthetic Sequence 28
tcaacgtc 8 29 20 DNA Artificial Sequence Synthetic Sequence 29
atggactctc cagcgttctc 20 30 20 DNA Artificial Sequence Synthetic
Sequence 30 atggaaggtc caacgttctc 20 31 20 DNA Artificial Sequence
Synthetic Sequence 31 atcgactctc gagcgttctc 20 32 20 DNA Artificial
Sequence Synthetic Sequence 32 atggaggctc catcgttctc 20 33 20 DNA
Artificial Sequence modified_base (14)...(14) m5c 33 atcgactctc
gagcgttctc 20 34 20 DNA Artificial Sequence modified_base
(18)...(18) m5c 34 atcgactctc gagcgttctc 20 35 20 DNA Artificial
Sequence Synthetic Sequence 35 tccatgtcgg tcctgatgct 20 36 20 DNA
Artificial Sequence Synthetic Sequence 36 tccatgccgg tcctgatgct 20
37 20 DNA Artificial Sequence Synthetic Sequence 37 tccatggcgg
tcctgatgct 20 38 20 DNA Artificial Sequence Synthetic Sequence 38
tccatgacgg tcctgatgct 20 39 20 DNA Artificial Sequence Synthetic
Sequence 39 tccatgtcga tcctgatgct 20 40 20 DNA Artificial Sequence
Synthetic Sequence 40 tccatgtcgc tcctgatgct 20 41 20 DNA Artificial
Sequence Synthetic Sequence 41 tccatgtcgt ccctgatgct 20 42 20 DNA
Artificial Sequence Synthetic Sequence 42 tccatgacgt gcctgatgct 20
43 20 DNA Artificial Sequence Synthetic Sequence 43 tccataacgt
tcctgatgct 20 44 20 DNA Artificial Sequence Synthetic Sequence 44
tccatgacgt ccctgatgct 20 45 20 DNA Artificial Sequence Synthetic
Sequence 45 tccatcacgt gcctgatgct 20 46 19 DNA Artificial Sequence
Synthetic Sequence 46 ggggtcaacg ttgacgggg 19 47 19 DNA Artificial
Sequence Synthetic Sequence 47 ggggtcagtc gtgacgggg 19 48 15 DNA
Artificial Sequence Synthetic Sequence 48 gctagacgtt agtgt 15 49 20
DNA Artificial Sequence Synthetic Sequence 49 tccatgtcgt tcctgatgct
20 50 24 DNA Artificial Sequence Synthetic Sequence 50 accatggacg
atctgtttcc cctc 24 51 18 DNA Artificial Sequence Synthetic Sequence
51 tctcccagcg tgcgccat 18 52 24 DNA Artificial Sequence Synthetic
Sequence 52 accatggacg aactgtttcc cctc 24 53 24 DNA Artificial
Sequence Synthetic Sequence 53 accatggacg agctgtttcc cctc 24 54 24
DNA Artificial Sequence Synthetic Sequence 54 accatggacg acctgtttcc
cctc 24 55 24 DNA Artificial Sequence Synthetic Sequence 55
accatggacg tactgtttcc cctc 24 56 24 DNA Artificial Sequence
Synthetic Sequence 56 accatggacg gtctgtttcc cctc 24 57 24 DNA
Artificial Sequence Synthetic Sequence 57 accatggacg ttctgtttcc
cctc 24 58 15 DNA Artificial Sequence Synthetic Sequence 58
cacgttgagg ggcat 15 59 12 DNA Artificial Sequence Synthetic
Sequence 59 tcagcgtgcg cc 12 60 17 DNA Artificial Sequence
Synthetic Sequence 60 atgacgttcc tgacgtt 17 61 17 DNA Artificial
Sequence Synthetic Sequence 61 tctcccagcg ggcgcat 17 62 20 DNA
Artificial Sequence Synthetic Sequence 62 tccatgtcgt tcctgtcgtt 20
63 20 DNA Artificial Sequence Synthetic Sequence 63 tccatagcgt
tcctagcgtt 20 64 21 DNA Artificial Sequence Synthetic Sequence 64
tcgtcgctgt ctccccttct t 21 65 19 DNA Artificial Sequence Synthetic
Sequence 65 tcctgacgtt cctgacgtt 19 66 19 DNA Artificial Sequence
Synthetic Sequence 66 tcctgtcgtt cctgtcgtt 19 67 20 DNA Artificial
Sequence Synthetic Sequence 67 tccatgtcgt ttttgtcgtt 20 68 20 DNA
Artificial Sequence Synthetic Sequence 68 tcctgtcgtt ccttgtcgtt 20
69 20 DNA Artificial Sequence Synthetic Sequence 69 tccttgtcgt
tcctgtcgtt 20 70 20 DNA Artificial Sequence Synthetic Sequence 70
tcctgtcgtt ttttgtcgtt 20 71 21 DNA Artificial Sequence Synthetic
Sequence 71 tcgtcgctgt ctgcccttct t 21 72 21 DNA Artificial
Sequence Synthetic Sequence 72 tcgtcgctgt tgtcgtttct t 21 73 20 DNA
Artificial Sequence Synthetic Sequence 73 tccatgcgtg cgtgcgtttt 20
74 20 DNA Artificial Sequence Synthetic Sequence 74 tccatgcgtt
gcgttgcgtt 20 75 20 DNA Artificial Sequence Synthetic Sequence 75
tccacgacgt tttcgacgtt 20 76 20 DNA Artificial Sequence Synthetic
Sequence 76 tcgtcgttgt cgttgtcgtt 20 77 24 DNA Artificial Sequence
Synthetic Sequence 77 tcgtcgtttt gtcgttttgt cgtt 24 78 22 DNA
Artificial Sequence Synthetic Sequence 78 tcgtcgttgt cgttttgtcg tt
22 79 21 DNA Artificial Sequence Synthetic Sequence 79 gcgtgcgttg
tcgttgtcgt t 21 80 21 DNA Artificial Sequence Synthetic Sequence 80
tgtcgtttgt cgtttgtcgt t 21 81 25 DNA Artificial Sequence Synthetic
Sequence 81 tgtcgttgtc gttgtcgttg tcgtt 25 82 19 DNA Artificial
Sequence Synthetic Sequence 82 tgtcgttgtc gttgtcgtt 19 83 14 DNA
Artificial Sequence Synthetic Sequence 83 tcgtcgtcgt cgtt 14 84 13
DNA Artificial Sequence Synthetic Sequence 84 tgtcgttgtc gtt 13 85
20 DNA Artificial Sequence Synthetic Sequence 85 tccatagcgt
tcctagcgtt 20 86 20 DNA Artificial Sequence Synthetic Sequence 86
tccatgacgt tcctgacgtt 20 87 6 DNA Artificial Sequence Synthetic
Sequence 87 gtcgyt 6 88 7 DNA Artificial Sequence Synthetic
Sequence 88 tgtcgyt 7 89 18 DNA Artificial Sequence Synthetic
Sequence 89 agctatgacg ttccaagg 18 90 20 DNA Artificial Sequence
Synthetic Sequence 90 tccatgacgt tcctgacgtt 20 91 20 DNA Artificial
Sequence Synthetic Sequence 91 tccaggactt ctctcaggtt 20 92 20 DNA
Artificial Sequence Synthetic Sequence 92 atcgactctc gaacgttctc 20
93 20 DNA Artificial Sequence Synthetic Sequence 93 tccatgtcgg
tcctgacgca 20 94 8 DNA Artificial Sequence Synthetic Sequence 94
tcttcgat 8 95 20 DNA Artificial Sequence Synthetic Sequence 95
ataggaggtc caacgttctc 20
* * * * *