U.S. patent application number 10/253117 was filed with the patent office on 2003-06-26 for method for enhancing an immune response.
Invention is credited to Kobayashi, Hiroko, Raz, Eyal R..
Application Number | 20030119773 10/253117 |
Document ID | / |
Family ID | 23363315 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030119773 |
Kind Code |
A1 |
Raz, Eyal R. ; et
al. |
June 26, 2003 |
Method for enhancing an immune response
Abstract
Disclosed is a method for enhancing an immune response to a
substance, such as an antigen or microbial pathogen. The immune
response can be, for example, production of IgG2 antibodies. The
method comprises administering an immunostimulatory nucleotide
sequence (ISS) to a subject at least one hour prior to exposure to
the substance by the subject. The subject may be exposed to the
substance either naturally, as with an environmental pathogen, or
by administration, as with a known antigen. The method can be used
for protecting or immunizing a subject against an antigen or
pathogen, providing more effective immunization than if the ISS
were co-administered with the substance. The method can be used
prophylactically or therapeutically. In preferred embodiments, the
ISS comprises a CG, p(GC) or p(IC) DNA or RNA nucleotide sequence.
Of these, a CG containing nucleotide sequence is preferred. The ISS
can further comprise a pG nucleotide sequence. Examples of an ISS
include sequences comprising 5'-rrcgyy-3', 5'-rycgyy-3',
5'-rrcgyycg-3' or 5'-rycgyycg-3'. The ISS is preferably
administered between about 6 hours and about 6 weeks prior to
exposure to the substance, and more preferably between about 1 day
and about 4 weeks prior. Most preferably, the ISS is administered
between about 3 days and about 8 days prior to exposure to the
substance. The ISS can be administered via a mucosal or systemic
route. The substance can be an antigen or pathogen associated with
an infectious disease, an allergen or a cancer.
Inventors: |
Raz, Eyal R.; (Del Mar,
CA) ; Kobayashi, Hiroko; (Fukushima, JP) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
23363315 |
Appl. No.: |
10/253117 |
Filed: |
September 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10253117 |
Sep 23, 2002 |
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09347343 |
Jul 2, 1999 |
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Current U.S.
Class: |
514/44R ;
424/204.1; 424/234.1; 424/277.1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 39/39 20130101; A61P 37/04 20180101; A61K 2039/55561 20130101;
A61K 2039/57 20130101 |
Class at
Publication: |
514/44 ;
424/204.1; 424/277.1; 424/234.1 |
International
Class: |
A61K 048/00; A61K
039/12; A61K 039/02; A61K 039/00 |
Goverment Interests
[0002] This invention was made with Government support under Grant
No. AI40682, awarded by the National Institutes of Health. The
government has certain rights in this invention.
Claims
What is claimed is:
1. A method for enhancing an immune response to a substance
comprising administering an immunostimulatory nucleotide sequence
(ISS) to a subject at least one hour prior to exposure to the
substance by the subject.
2. The method of claim 1, wherein the immune response is innate
immunity.
3. The method of claim 1, wherein the immune response is a Th1
response.
4. The method of claim 1, wherein the immune response includes
production of an antibody.
5. The method of claim 4, wherein the antibody is of the IgG2
class.
6. The method of claim 1, wherein the immune response includes
release of antigen-specific interferon-.gamma..
7. The method of claim 1, wherein the immune response includes a
cytotoxic T lymphocyte (CTL) response.
8. The method of claim 1, wherein the substance is an antigen or
pathogen associated with an infectious disease, an allergen or a
cancer.
9. The method of claim 8, wherein the infectious disease is
selected from the group comprising viral, bacterial, mycobacterial
and parasitic disease.
10. The method of claim 1, wherein the ISS comprises a CG, p(GC) or
p(IC) DNA or RNA nucleotide sequence.
11. The method of claim 1, wherein the ISS comprises the sequence
5'-rrcgyy-3' (SEQ ID NO: 1), 5'-rycgyy-3' (SEQ ID NO: 2),
5'-rrcgyycg-3' (SEQ ID NO: 3) or 5'-rycgyycg-3' (SEQ ID NO: 4).
12. The method of claim 11, wherein the nucleotide sequence is
selected from a group comprising AACGTT (SEQ ID NO: 5), AGCGTC (SEQ
ID NO: 6), AGCGTT (SEQ ID NO: 7), GACGTT (SEQ ID NO: 8), GGCGTT
(SEQ ID NO: 9), AACGTT (SEQ ID NO: 10), GTCGTT (SEQ ID NO: 24),
AGCGTCCG (SEQ ID NO: 25), AACGTTCG (SEQ ID NO: 26), AGCGTTCG (SEQ
ID NO: 27), GACGTTCG (SEQ ID NO: 28), GGCGTTCG (SEQ ID NO: 29),
AACGTTCG (SEQ ID NO: 30), and AGCGTCCG (SEQ ID NO: 31).
13. The method of claim 1, wherein the ISS is administered between
about 6 hours and about 6 weeks prior to antigen
administration.
14. The method of claim 1, wherein the ISS is administered between
about 1 day and about 4 weeks prior to antigen administration.
15. The method of claim 1, wherein the ISS is administered between
about 3 days and about 8 days prior to antigen administration.
16. The method of claim 1, wherein the ISS is administered via a
mucosal or systemic route.
17. The method of claim 16, wherein the mucosal route is
intranasal, ophthalmic, intratracheal, intravaginal or
intrarectal.
18. The method of claim 16, wherein the systemic route is
intradermal, intramuscular, subcutaneous or intravenous.
19. A method of immunizing a subject against a substance comprising
administering to the subject an ISS at least one hour prior to
exposing the subject to the substance.
20. The method of claim 19, wherein the ISS comprises a CG, p(GC)
or p(IC) DNA or RNA nucleotide sequence.
21. The method of claim 19, wherein the ISS comprises the sequence
5'-rrcgyy-3' (SEQ ID NO: 1), 5'-rycgyy-3' (SEQ ID NO: 2),
5'-rrcgyycg-3' (SEQ ID NO: 3) or 5'-rycgyycg-3' (SEQ ID NO: 4).
22. The method of claim 21, wherein the nucleotide sequence is
selected from a group comprising AACGTT (SEQ ID NO: 5), AGCGTC (SEQ
ID NO: 6), AGCGTT (SEQ ID NO: 7), GACGTT (SEQ ID NO: 8), GGCGTT
(SEQ ID NO: 9), AACGTT (SEQ ID NO: 10), GTCGTT (SEQ ID NO: 24),
AGCGTCCG (SEQ ID NO: 25), AACGTTCG (SEQ ID NO: 26), AGCGTTCG (SEQ
ID NO: 27), GACGTTCG (SEQ ID NO: 28), GGCGTTCG (SEQ ID NO: 29),
AACGTTCG (SEQ ID NO: 30), and AGCGTCCG (SEQ ID NO: 31).
23. The method of claim 19, wherein the ISS is administered between
about 6 hours and about 6 weeks prior to exposure to the
substance.
24. The method of claim 19, wherein the ISS is administered between
about 1 day and about 4 weeks prior to exposure to the
substance.
25. The method of claim 19, wherein the ISS is administered between
about 3 days and about 8 days prior to exposure to the
substance.
26. The method of claim 19, wherein the substance is an antigen or
pathogen associated with an infectious disease, an allergen or a
cancer.
27. The method of claim 26, wherein the infectious disease is
selected from the group comprising viral, bacterial, mycobacterial
and parasitic disease.
28. The method of claim 19, wherein the ISS is administered via a
mucosal or systemic route.
29. The method of claim 28, wherein the mucosal route is
intranasal, ophthalmic, intratracheal, intravaginal or
intrarectal.
30. The method of claim 28, wherein the systemic route is
intradermal, intramuscular, subcutaneous or intravenous.
31. A method of eliciting IgG2 antibody production comprising
administering to a subject an ISS at least one hour prior to
administration of an antigen to the subject.
32. The method of claim 31, wherein the ISS comprises a CG, p(GC)
or p(IC) DNA or RNA nucleotide sequence.
33. The method of claim 31, wherein the ISS comprises the sequence
5'-rrcgyy-3' (SEQ ID NO: 1), 5'-rycgyy-3' (SEQ ID NO: 2),
5'-rrcgyycg-3' (SEQ ID NO: 3) or 5'-rycgyycg-3' (SEQ ID NO: 4).
34. The method of claim 33, wherein the nucleotide sequence is
selected from a group comprising AACGTT (SEQ ID NO: 5), AGCGTC (SEQ
ID NO: 6), AGCGTT (SEQ ID NO: 7), GACGTT (SEQ ID NO: 8), GGCGTT
(SEQ ID NO: 9), AACGTC (SEQ ID NO: 10), GTCGTT (SEQ ID NO: 24),
AGCGTCCG (SEQ ID NO: 25), AACGTTCG (SEQ ID NO: 26), AGCGTTCG (SEQ
ID NO: 27), GACGTTCG (SEQ ID NO: 28), GGCGTTCG (SEQ ID NO: 29),
AACGTTCG (SEQ ID NO: 30), and AGCGTCCG (SEQ ID NO: 31).
35. The method of claim 31, wherein the ISS is administered between
about 6 hours and about 6 weeks prior to antigen
administration.
36. The method of claim 31, wherein the ISS is administered between
about 1 day and about 4 weeks prior to antigen administration.
37. The method of claim 31, wherein the ISS is administered between
about 3 days and about 8 days prior to antigen administration.
38. The method of claim 31, wherein the antigen is associated with
an infectious disease, an allergen or a cancer.
39. The method of claim 38, wherein the infectious disease is
selected from the group comprising viral, bacterial, mycobacterial
and parasitic disease.
40. The method of claim 31, wherein the ISS is administered via a
mucosal or systemic route.
41. The method of claim 40, wherein the mucosal route is
intranasal, ophthalmic, intratracheal, intravaginal or
intrarectal.
42. The method of claim 40, wherein the systemic route is
intradermal, intramuscular, subcutaneous or intravenous.
Description
[0001] This application is related to pending U.S. patent
applications having the Ser. Nos. 09/167,039, filed Oct. 5, 1998,
and 09/235,742, filed Jan. 20, 1999, the latter of which is a
continuing prosecution application based on Ser. No. 08/927,120,
filed Sep. 5, 1997. The entire contents of each of these related
applications are hereby incorporated by reference into this
application. Throughout this application various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to describe more fully the state of the art to
which this invention pertains.
TECHNICAL FIELD OF THE INVENTION
[0003] The invention relates to a method for enhancing an immune
response by administering an immunostimulatory nucleotide sequence
prior to antigen exposure. More particularly, the method is suited
for enhancing antibody production, IFN.gamma. release, CTL activity
and Th1-related effects in response to antigen administration.
BACKGROUND OF THE INVENTION
[0004] Adjuvants are typically administered in conjunction with
antigen in vaccination protocols. Adjuvants serve to amplify or
modulate the immune response to a co-delivered antigen. Currently,
few adjuvants (e.g., alum and MF59) have been approved for use in
human vaccination.
[0005] Immunostimulatory DNA sequences (ISS) delivered in
conjunction with an antigen activate innate immunity and bias the
adaptive immune response toward Th1 differentiation. ISS have been
used as an adjuvant to amplify the immune response to a
co-delivered antigen. See, for example, WO 98/16247, and U.S. Pat.
Nos. 5,736,524 and No. 5,780,448.
[0006] There remains a need for optimization of the nature and
efficacy of vaccination and immunotherapeutic protocols.
SUMMARY OF THE INVENTION
[0007] The invention provides a method for enhancing an immune
response to a substance, such as an antigen administered to a
subject, or a pathogen to which the subject is exposed. The method
can be used to modulate the magnitude, the duration, and the nature
of the immune response to subsequent exposure to a substance. The
method comprises administering an immunostimulatory nucleotide
sequence (ISS) to the subject at least one hour prior to exposure
to the substance by the subject. This "pre-priming" of the subject
with ISS prior to antigen administration or pathogen exposure
results in amplification of the Th1 immune response to the
substance as compared to co-administration of ISS and antigen.
Pre-priming with ISS also shifts the nature of the immune response
from a Th2 type response to a Th1 type response.
[0008] Examples of an immune response that can be enhanced by the
method of the invention include, but are not limited to, activation
of innate immunity (e.g., macrophages, natural killer (NK) cells),
a Th1 response, a cytotoxic T lymphocyte (CTL) response, and
production of an antibody. The antibody response is preferably
increased production of antibodies of the IgG2a subclass. The
method can be used for immunizing a subject against an antigen, and
provides more effective immunization than if the ISS were
co-administered with the antigen. The method can be used
prophylactically or therapeutically.
[0009] In preferred embodiments, the ISS comprises a CG, p(GC) or
p(IC) DNA or RNA nucleotide sequence. Of these, a CG containing
nucleotide sequence is preferred. Preferably, the ISS further
comprises a pG nucleotide sequence. Examples of an ISS include, but
are not limited to, sequences comprising 5'-rrcgyy-3' (SEQ ID NO:
1), such as AACGTT, AGCGTC, AGCGTT, GACGTT, GGCGTT, AACGTC, and
AGCGTC (SEQ ID NOs: 5-11, respectively), 5'-rycgyy-3' (SEQ ID NO:
2) such as GTCGTT (SEQ ID NO: 24), 5'-rrcgyycg-3' (SEQ ID NO: 3),
or 5'-rycgyycg-3' (SEQ ID NO: 4).
[0010] The ISS is preferably administered between about 6 hours and
about 6 weeks prior to antigen administration, and more preferably
between about 1 day and about 4 weeks prior to antigen
administration. Most preferably, the ISS is administered between
about 3 days and about 8 days prior to antigen administration. In a
preferred embodiment of the method, the ISS is administered via a
systemic route such as a dermal or intramuscular route, or via a
mucosal route such as an intranasal, ophthalmic, intrarectal,
intravaginal or intratracheal route.
[0011] Because pre-priming activates innate immunity, the method of
the invention can be used to protect against subsequent infection
by a pathogen, such as a viral, bacterial, parasitic or other
infectious agent. Preferably, the substance is a pathogen or an
antigen associated with an infectious disease, an allergen or a
cancer. Examples of infectious disease include, but are not limited
to, viral, bacterial, mycobacterial and parasitic disease.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 is a schematic representation of the ISS pre-priming
and .beta.-gal immunization protocol described in the examples.
Mice received a single i.d. or i.n. injection with ISS (50 .mu.g)
either the specified days before or with .beta.-gal (50 .mu.g)
immunization via the same route (Day 0). Control mice received
.beta.-gal immunization without ISS. Serial bleeds occurred after
.beta.-gal immunization, and mice were sacrificed during week 7 for
determination of splenocyte cytokine and CTL responses. "i.d."
refers to intradermal delivery; and "i.n." to intranasal
delivery.
[0013] FIG. 2A is graph showing time course of IgG2a production
after i.d. ISS pre-priming and .beta.-gal immunization. Mice
received either no ISS (open squares), ISS on day 0 (open circles),
day-3 (closed diamonds), day-7 (closed circles), day-14 (closed
squares), or day-28 (closed triangles) relative to .beta.-gal. Mice
were bled at serial time points to establish the kinetics of lgG2a
production. Results represent mean values for 4 mice per group and
error bars reflect standard errors of the means. Results are
representative of 3 similar and independent experiments.
[0014] FIG. 2B is graph showing serum IgG2a after i.d. ISS
pre-priming and .beta.-gal immunization at 7 weeks post
immunization. The open diamond represents data for mice receiving
.beta.-gal alone, and the closed circles represent data for mice
receiving ISS at the indicated day relative to .beta.-gal
administration. Results represent mean values for 4 mice per group
and error bars reflect standard errors of the means. Results are
representative of 3 similar and independent experiments. Mice
receiving ISS up to 14 days prior to .beta.-gal demonstrated an
improved IgG2a response when compared to mice immunized with
.beta.-gal alone (; p.ltoreq.0.05). Mice receiving ISS 7 days
before .beta.-gal immunization had a significantly improved IgG2a
response when compared to mice co-administered ISS with .beta.-gal
(*; p.ltoreq.0.05).
[0015] FIG. 2C is graph showing serum IgG1 after i.d. ISS
pre-priming and .beta.-gal immunization at 7 weeks post
immunization. Results represent mean values for 4 mice per group
and error bars reflect standard errors of the means. Results are
representative of 3 similar and independent experiments.
[0016] FIG. 3A is a bar graph showing the splenocyte IFN.gamma.
response after i.d. ISS pre-priming and .beta.-gal immunization.
Results represent the mean for 4 mice in each group and similar
results were obtained in 2 other independent experiments. Error
bars reflect standard errors of the means. Mice receiving ISS up to
14 days prior to .beta.-gal demonstrated an improved IFN.gamma.
response when compared to mice immunized with .beta.-gal alone (;
p.ltoreq.0.05). Delivery of ISS from 3-7 days before .beta.-gal led
to an improved IFN.gamma. response when compared to mice receiving
ISS/.beta.-gal co-immunization (*; p.ltoreq.0.05).
[0017] FIG. 3B is a graph showing the splenocyte CTL response after
i.d. ISS pre-priming and .beta.-gal immunization. Mice received
either no ISS (open squares), ISS on day 0 (open circles), day-3
(closed diamonds), day-7 (closed circles), day-14 (closed squares),
or day-28 (closed triangles) relative to .beta.-gal. Results
represent the mean for 4 mice in each group and similar results
were obtained in 2 other independent experiments. Error bars
reflect standard errors of the means.
[0018] FIG. 3C is a plot showing a comparison of CTL response at an
effector:target ratio of 25:1. The open diamond represents data for
mice receiving .beta.-gal alone, and the closed circles represent
data for mice receiving ISS at the indicated day relative to
.beta.-gal administration. Mice receiving ISS up to 14 days prior
to .beta.-gal demonstrated an improved CTL response when compared
to mice immunized with .beta.-gal alone (; p.ltoreq.0.05).
[0019] FIG. 4 shows splenocyte cytokine mRNA expression. Mice were
i.d. injected with 50 .mu.g of ISS on day 0. Control mice received
either LPS (50 .mu.g) or nothing. At serial time points after
injection, mice were sacrificed, splenocytes were isolated, and
subjected to RT-PCR. PCR products were visualized by
electrophoresis on 2% agarose gels and staining with ethidium
bromide.
[0020] FIG. 5A is a bar graph showing splenocyte IFN.gamma.
response after i.n. ISS pre-priming and .beta.-gal immunization.
Results represent the mean for 4 mice in each group and similar
results were obtained in 2 other independent experiments. Error
bars reflect standard errors of the means. Mice receiving ISS up to
7 days prior to .beta.-gal demonstrated an improved IFN.gamma.
response when compared to mice immunized with .beta.-gal alone (;
p.ltoreq.0.05). Delivery of ISS from 1-3 days before .beta.-gal led
to an improved IFN.gamma. response when compared to mice receiving
ISS/.beta.-gal co-immunization (*; p.ltoreq.0.05).
[0021] FIG. 5B is a graph showing splenocyte CTL response after
i.n. ISS pre-priming and .beta.-gal immunization. Mice received
either no ISS (open squares), ISS on day 0 (open circles), or day-1
(closed triangles), day-3 (closed diamonds), day-7 (closed
circles), or day-14 (closed squares) relative to .beta.-gal.
Results represent the mean for 4 mice in each group and similar
results were obtained in 2 other independent experiments. Error
bars reflect standard errors of the means. Mice receiving ISS up to
7 days prior to .beta.-gal demonstrated statistically improved CTL
responses at effector:target ratios of 5:1 and 25:1 when compared
to mice immunized with .beta.-gal alone (p.ltoreq.0.05).
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention provides an unconventional approach to
amplifying the immune response in vivo. This approach provides a
practical tool to amplify the immune response to subsequent antigen
exposure, to activate innate immunity, to generate CTL activity and
to bias the subsequent immune response toward a Th1 type of
response. The invention is based on the discovery that pre-priming
a subject by dissociating ISS delivery from antigen delivery
significantly amplifies the immune response to antigen. This
pre-priming effect is applicable to both systemic and mucosal
immunization, and can be used for protection against antigens as
well as against pathogens. The invention additionally provides
information about the kinetics of the pre-priming effect and the
optimal timing for ISS delivery for both systemic and mucosal
applications.
[0023] The invention is also based on the discovery that ISS
administration activates innate immunity as evidenced by increased
serum levels of IFN.gamma. and IL-12, which activate macrophages
and natural killer (NK) cells, respectively. The method of the
invention can thus be used for broad protection against
subsequently encountered pathogens as well as against subsequently
administered antigens. The combination of information about
activation of innate immunity and the time course of ISS-induced
enhancement of immune responses enables an effective strategy for
protection against a broad range of substances.
[0024] Definitions
[0025] All scientific and technical terms used in this application
have meanings commonly used in the art unless otherwise specified.
As used in this application, the following words or phrases have
the meanings specified.
[0026] As used herein, "immunostimulatory nucleotide sequence" or
"ISS" means a polynucleotide that includes, or consists of, at
least one immunostimulatory oligonucleotide (ISS-ODN) moiety. The
ISS moiety is a single-or double-stranded DNA or RNA
oligonucleotide having at least six nucleotide bases that may
include, or consist of, a modified oligonucleotide or a sequence of
modified nucleosides. The ISS moieties comprise, or may be flanked
by, a CG containing nucleotide sequence or a p(IC) nucleotide
sequence, which may be palindromic.
[0027] As used herein, "polynucleotide" refers to DNA or RNA and
can include sense and antisense strands as appropriate to the goals
of the therapy practiced according to the invention. Polynucleotide
in this context includes oligonucleotides.
[0028] As used herein, "subject" refers to the recipient of the
therapy to be practiced according to the invention. The subject can
be any vertebrate, but will preferably be a mammal. If a mammal,
the subject will preferably be a human, but may also be a domestic
livestock, laboratory subject or pet animal.
[0029] As used herein, "substance" refers to any substance to which
an immune response may be directed, and includes antigens and
pathogens.
[0030] As used herein, "exposure" to a substance includes both
natural, environmental exposure to the substance as well as
administration of the substance to a subject.
[0031] As used herein, enhancing "innate immunity" includes
enhancing activation of macrophages, NK cells, antigen presenting
cells (APCs), and other elements known to be involved in protection
against subsequent exposure to microbial pathogens. Enhancement of
innate immunity can be determined using conventional assays for
activation of these elements, including but not limited to assays
described in the examples set forth below.
[0032] As used herein, "enhancing a Th1 immune response" in a
subject is evidenced by:
[0033] (1) a reduction in levels of IL-4 or IL-5 measured before
and after antigen challenge; or detection of lower (or even absent)
levels of IL-4 in a treated subject as compared to an
antigen-primed, or primed and challenged, control;
[0034] (2) an increase in levels of IL-12, IL-18 and/or IFN
(.alpha., .beta. or .gamma.) before and after antigen challenge; or
detection of higher levels of IL-12, IL-18 and/or IFN (.alpha.,
.beta. or .gamma.) in an ISS treated subject as compared to an
antigen-primed or, primed and challenged, control;
[0035] (3) production of IgG2a antibody or its human analog in a
treated subject;
[0036] (4) a reduction in levels of antigen-specific IgE as
measured before and after antigen challenge; or detection of lower
(or even absent) levels of antigen-specific IgE in an ISS treated
subject as compared to an antigen-primed, or primed and challenged,
control; and/or
[0037] (5) induction of a cytotoxic T lymphocyte ("CTL") response
in a treated subject.
[0038] Exemplary methods for determining such values are described
further in the Examples. The ISS of the invention provide
relatively safe, effective means of stimulating a robust immune
response in a vertebrate subject against any antigen.
[0039] Methods
[0040] The invention provides a method for enhancing an immune
response. The method can be used to modulate the magnitude, the
duration and/or the quality of the immune response to a
subsequently administered antigen or to subsequent exposure to a
substance such as a pathogen. In one embodiment, the method
enhances the production of antibodies that recognize the substance.
Enhanced antibody production can be determined by detecting
increased antibody levels in a subject or subjects pre-primed with
ISS as compared to antibody levels in a subject or subject not
receiving ISS prior to antigen administration. An example of a
suitable assay for determining enhanced antibody production is
described below in Example 1. Enhanced antibody production can also
include increasing the production of one class of antibody relative
to production of another, less desirable class of antibody. For
example, production of IgG2a antibodies can be enhanced while
levels of IgE antibodies are reduced.
[0041] The immune response can also be enhanced by shifting the
response from a Th2 to a Th1 type response. As used herein,
"Th1/Th2 response(s)" refer to types 1 and 2, respectively, helper
T lymphocyte (Th) mediated immune responses. Th2 responses include
the allergy-associated IgE antibody class as well as elevated
levels of IL-4 and IL-5 cytokines by Th2 lymphocytes. Soluble
protein antigens tend to stimulate relatively strong Th2 responses.
Th1 cells secrete IL-2, interferon-gamma (IFN.gamma.) and tumor
necrosis factor-beta (TNF.beta.) (the latter two of which are
involved in macrophage activation and delayed-type hypersensitivity
in response to antigen stimulation or infection with a
pathogen).
[0042] Accordingly, Th2 associated responses can be suppressed,
thereby reducing the risk of prolonged allergic inflammation and
antigen-induced anaphylaxis. The enhancement of Th1 associated
responses is of particular value in responding to intracellular
infections because cellular immunity is enhanced by activated Th1
(IFN.gamma.) cells. In addition, administration of polynucleotides
helps stimulate production of CTL, further enhancing the immune
response.
[0043] The method of the invention can be used to modulate or
enhance the immune response both prophylactically and
therapeutically. Thus, the invention provides a method of
immunizing a subject as well as a method of immunotherapy.
[0044] The method of the invention comprises administering an ISS
to a subject prior to exposure to the substance. This pre-priming
is typically performed at least one hour prior to antigen
administration or other exposure to a substance. The ISS is
preferably administered between about 6 hours and about 6 weeks
prior to antigen administration or other exposure to a substance,
and more preferably between about 1 day and about 4 weeks prior to
antigen administration. Most preferably, the ISS is administered
between about 3 days and about 8 days prior to antigen
administration. The antigen or other substance can be introduced by
conventional immunization techniques, or by natural exposure.
[0045] Preferably, the substance is an antigen or a pathogen
associated with an infectious disease, an allergen or a cancer.
Examples of infectious disease include, but are not limited to,
viral, bacterial, mycobacterial and parasitic disease. Examples of
allergens include, but are not limited to, plant pollens, dust mite
proteins, animal dander, saliva and fungal spores. Examples of
cancer-associated antigens include, but are not limited to, live or
irradiated tumor cells, tumor cell extracts and protein subunits of
tumor antigens. The antigen can also be a sperm protein for use in
contraception. In some embodiments, the antigen is an environmental
antigen. Examples of environmental antigens include, but are not
limited to, respiratory syncytial virus ("RSV"), flu viruses and
cold viruses.
[0046] Structure and Preparation of ISS
[0047] The ISS of the invention includes an oligonucleotide, which
can be a part of a larger nucleotide construct such as a plasmid or
bacterial DNA. The term "polynucleotide" therefore includes
oligonucleotides, modified oligonucleotides and oligonucleosides,
alone or as part of a larger construct. The polynucleotide can be
single-stranded DNA (ssDNA), double-stranded DNA (dsDNA),
single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA). The ISS
can include bacterial DNA, which provides ISS activity.
[0048] The polynucleotide portion can be linearly or circularly
configured, or the oligonucleotide portion can contain both linear
and circular segments. Modifications of oligonucleotides include,
but are not limited to, modifications of the 3'OH or 5'OH group,
modifications of the nucleotide base, modifications of the sugar
component, and modifications of the phosphate group.
[0049] The ISS can comprise ribonucleotides (containing ribose as
the only or principal sugar component), deoxyribonucleotides
(containing deoxyribose as the principal sugar component), or in
accordance with the established state-of-the-art, modified sugars
or sugar analogs may be incorporated in the oligonucleotide of the
present invention. Examples of a sugar moiety that can be used
include, in addition to ribose and deoxyribose, pentose,
deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose,
lyxose, and a sugar "analog" cyclopentyl group. The sugar may be in
pyranosyl or in a furanosyl form. In the modified oligonucleotides
of the present invention, the sugar moiety is preferably the
furanoside of ribose, deoxyribose, arabinose or 2'-O-methylribose,
and the sugar may be attached to the respective heterocyclic bases
either in I or J anomeric configuration.
[0050] The phosphorous derivative (or modified phosphate group)
that can be attached to the sugar or sugar analog moiety in the
modified oligonucleotides of the present invention can be a
monophosphate, diphosphate, triphosphate, alkylphosphate,
alkanephosphate, phosphoronthioate, phosphorodithioate or the like.
The heterocyclic bases, or nucleic acid bases that are incorporated
in the oligonucleotide base of the ISS can be the naturally
occurring principal purine and pyrimidine bases, (namely uracil or
thymine, cytosine, adenine and guanine, as mentioned above), as
well as naturally occurring and synthetic modifications of said
principal bases. Those skilled in the art will recognize that a
large number of "synthetic" non-natural nucleosides comprising
various heterocyclic bases and various sugar moieties (and sugar
analogs) are available, and that the ISS can include one or several
heterocyclic bases other than the principal five base components of
naturally occurring nucleic acids. Preferably, however, the
heterocyclic base in the ISS is selected from uracil-5-yl,
cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl,
4-aminopyrrolo[2,3-d]pyrimidin-5-y- l,
2-amino-4-oxopyrolo[2,3-d]pyrimidin-5-yl,
2-amino-4-oxopyrrolo[2,3-d]py- rimidin-3-yl groups, where the
purines are attached to the sugar moiety of the oligonucleotides
via the 9-position, the pyrimidines via the 1-position, the
pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines
via the 1-position.
[0051] Structurally, the root oligonucleotide of the ISS is a
non-coding sequence that can include at least one unmethylated CpG
motif. The relative position of any CpG sequence in ISS with
immunostimulatory activity in certain mammalian species (e.g.,
rodents) is 5'-CG-3'(i.e., the C is in the 5' position with respect
to the G in the 3' position).
[0052] Some oligonucleotide ISS (ISS-ODN) are known. In such
ISS-ODN, the CpG motif is flanked by at least two purine
nucleotides (e.g., GA or AA) and at least two pyrimidine
nucleotides (5'-r-r-[C]-[G]-y-y-3'; SEQ ID NO: 1), or flanked by a
purine and a pyrimidine 5' to the CG (5'-r-y-[C]-[G]-y-y-3'; SEQ ID
NO: 2), wherein the pyrimidine 5' to the CG is preferably T. CpG
motif-containing ISS-ODN are believed to stimulate B lymphocyte
proliferation (see, e.g., Krieg, et al., Nature, 374:546-549,
1995).
[0053] The core hexamer structure of the foregoing ISS can be
flanked upstream and/or downstream by any number or composition of
nucleotides or nucleosides. However, ISS are at least 6 bases in
length, and preferably are between 6 and 200 bases in length, to
enhance uptake of the ISS into target tissues. Those of ordinary
skill in the art will be familiar with, or can readily identify,
reported nucleotide sequences of known ISS-ODN for reference in
preparing ISS. For ease of reference in this regard, the following
sources are especially helpful: Yamamoto, et al., Microbiol.
Immunol., 36:983 (1992); Ballas, et al., J. Immunol., 157:1840
(1996); Klinman, et al., J. Immunol., 158:3635 (1997); Sato, et
al., Science, 273:352 (1996).
[0054] In particular, ISS useful in the invention include those
that have hexameric nucleotide sequences having "CpG" motifs.
Although DNA sequences are preferred, RNA ISS can be used, with
inosine and/or uracil substitutions for nucleotides in the hexamer
sequences.
[0055] For example, DNA based ISS useful in the invention include
those that have the following hexameric nucleotide sequences:
1 AACGTT, AGCGTC, AGCGTT, GACGTT, GGCGTT, AACGTC, AGCGTC, (SEQ ID
NOs:5-24, respectively) GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC,
GGCGCC, AGCGCT, GACGCT, GGCGCT, TTCGAA, GGCGTT, AACGCC, and
GTCGTT.
[0056] Also useful are octamers in the form of 5'-rrcgyycg-3' (SEQ
ID NO: 3), such as AGCGTCCG, AACGTTCG, AGCGTTCG, GACGTTCG,
GGCGTTCG, AACGTTCG, and AGCGTCCG (SEQ ID NOs: 25-31, respectively),
and in the form of 5'-rycgyycg-3' (SEQ ID NO: 4), wherein the y is
preferably "t", larger ISS-ODN having a second CG further 3' to the
core hexameric sequence, and bacterial DNA, which are enriched with
ISS.
[0057] The ISS may or may not include palindromic regions. If
present, a palindrome may extend only to a CpG motif, if present,
in the core hexamer sequence, or may encompass more of the hexamer
sequence as well as flanking nucleotide sequences.
[0058] In addition, backbone phosphate group modifications (e.g.,
methylphosphonate, phosphorothioate, phosphoroamidate and
phosphorodithioate internucleotide linkages) can confer
anti-microbial activity on the ISS and enhance their stability in
vivo, making them particularly useful in therapeutic applications.
A particularly useful phosphate group modification is the
conversion to the phosphorothioate or phosphorodithioate forms of
ISS. In addition to their potentially anti-microbial properties,
phosphorothioates and phosphorodithioates are more resistant to
degradation in vivo than their unmodified oligonucleotide
counterparts, making the ISS of the invention more available to the
subject.
[0059] ISS can be synthesized using techniques and nucleic acid
synthesis equipment that are well-known in the art. For reference
in this regard, see, e.g., Ausubel, et al., Current Protocols in
Molecular Biology, Chs. 2 and 4 (Wiley Interscience, 1989);
Maniatis, et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Lab. New York, 1982); U.S. Pat. No. 4,458,066 and
U.S. Pat. No. 4,650,675. Because the ISS is non-coding, there is no
concern about maintaining an open reading frame during
synthesis.
[0060] Alternatively, ISS can be isolated from microbial species
(especially mycobacteria) using techniques well-known in the art,
such as nucleic acid hybridization. Whole or fragmented bacterial
DNA can be used. Preferably, such isolated ISS will be purified to
a substantially pure state; i.e., to be free of endogenous
contaminants, such as lipopolysaccharides.
[0061] Compositions
[0062] The invention provides compositions that are useful for
treating and preventing disease, such as allergy, cancer or
infection. In one embodiment, the composition is a pharmaceutical
composition. The composition is preferably an immunotherapeutic
composition. The composition can comprise a therapeutically or
prophylactically effective amount of an ISS of the invention, as
described above. The composition can optionally include a carrier,
such as a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method
used to administer the composition. Accordingly, there is a wide
variety of suitable formulations of pharmaceutical compositions of
the present invention.
[0063] Administration and Dosage
[0064] In a preferred embodiment of the method, the ISS is
administered via a systemic or mucosal route, or directly into a
specific tissue, such as the liver, bone marrow, or into the tumor
in the case of cancer therapy. Examples of systemic routes include,
but are not limited to, intradermal, intramuscular, subcutaneous
and intravenous administration. Examples of mucosal routes include,
but are not limited to, intranasal, intravaginal, intrarectal,
intratracheal and ophthalmic administration. Mucosal routes,
particularly intranasal, intratracheal and ophthalmic, are
preferred for protection against natural exposure to environmental
pathogens such as RSV, flu viruses and cold viruses or to allergens
such as grass and ragweed pollens and house dust mites. The local
activation of innate immunity by ISS will enhance the protective
effect against a subsequently encountered substance, such as an
antigen, allergen or microbial agent.
[0065] Treatment includes prophylaxis and therapy. Prophylaxis or
therapy can be accomplished by a single direct administration at a
single time point or multiple time points. Administration can also
be delivered to a single or to multiple sites.
[0066] The subject can be any vertebrate, but will preferably be a
mammal. Mammals include human, bovine, equine, canine, feline,
porcine, and ovine animals. If a mammal, the subject will
preferably be a human, but may also be a domestic livestock,
laboratory subject or pet animal.
[0067] The dose of ISS administrated to a subject, in the context
of the present invention, should be sufficient to effect a
beneficial therapeutic response in the subject over time, or to
inhibit growth of cancer cells, to inhibit allergic responses or to
inhibit infection. Thus, ISS is administered to a patient in an
amount sufficient to elicit an effective immune response to the
specific antigens and/or to alleviate, reduce, cure or at least
partially arrest symptoms and/or complications from the disease or
infection. An amount adequate to accomplish this is defined as a
"therapeutically effective dose."
[0068] A particular advantage of the ISS of the invention is their
capacity to exert immunomodulatory activity even at relatively
minute dosages. Although the dosage used will vary depending on the
clinical goals to be achieved, a suitable dosage range is one that
provides up to about 1-1000 .mu.g of ISS/ml of carrier in a single
dosage. Alternatively, a target dosage of ISS can be considered to
be about 1-10 .mu.M in a sample of subject blood drawn within the
first 24-48 hours after administration of ISS. Based on current
studies, ISS are believed to have little or no toxicity at these
dosage levels.
[0069] In this respect, it should be noted that the
anti-inflammatory (anti-allergenic), anti-microbial and
immunotherapeutic activities of ISS in the invention are
essentially dose-dependent. Therefore, to increase ISS potency by a
magnitude of two, each single dose is doubled in concentration.
Clinically, it may be advisable to administer the ISS in a low
dosage (e.g., about 1 .mu.g/ml to about 50 .mu.g/ml), then increase
the dosage as needed to achieve the desired therapeutic goal. Some
routes of administration, such as via ophthalmic drops, will
require higher concentrations. Those skilled in the art can adjust
the dosage and concentration to suit the particular route of
delivery.
[0070] In view of the teaching provided by this disclosure, those
of ordinary skill in the clinical arts will be familiar with, or
can readily ascertain, suitable parameters for administration of
ISS according to the invention.
EXAMPLES
[0071] The following examples are presented to illustrate the
present invention and to assist one of ordinary skill in making and
using the same. The examples are not intended in any way to
otherwise limit the scope of the invention.
Example 1
ISS Pre-Priming Elicits Extended Th1-Biased Immune Responses
[0072] This example demonstrates that ISS provide Th1 adjuvant
activity for an extended period of time after delivery. ISS were
administered intradermally (i.d.) up to 28 days prior to a primary
i.d. immunization with .beta.-galactosidase (.beta.-gal), and
enhanced antibody production, antigen-specific cytokine production
and CTL response were measured.
[0073] Methods
[0074] Immunization Reagents
[0075] .beta.-gal, lipopolysaccharide (LPS), and cholera toxin (CT)
(Sigma, St Louis, Mo.), ISS-ODN and mutated phosphorothioate
oligodeoxynucleotide (M-ODN) (Trilink Biotechnologies, San Diego.
Calif.) were used to immunize mice. The ISS-ODN used in these
studies has the following sequence: 5'-TGACTGTGAACGTTCGAGATGA-3'
(SEQ ID NO: 32). The M-ODN has the sequence
5'-TGACTGTGAACCTTAGAGATGA-3' (SEQ ID NO: 33).
[0076] Immunization Protocols
[0077] Female BALB/c mice, aged 6-8 weeks, were purchased from
Jackson Laboratory (Bar Harbor, Me.) and were used in all
experiments. The i.d. and i.n. immunization protocols used in these
experiments are outlined in FIG. 1. In brief, ISS-ODN was delivered
to mice from 28 days to 1 day prior to or with .beta.-gal. Mice
received a single i.d. injection with ISS (50 .mu.g) either the
specified day before or with i.d. .beta.-gal (50 .mu.g)
immunization. Control mice received i.d. .beta.-gal immunization
alone or with M-ODN. Splenocytes were harvested from sacrificed
mice during week 7. Intradermal injections were performed at the
base of the tail in 50 .mu.l of saline. Mice were anaesthetized for
i.n. delivery of reagents with Metophane (Mallinckrodt Veterinary
Inc., Mundelein, Ill. and 15 .mu.l of saline with reagents was
delivered to each nare. In some experiments 50 .mu.g of ISS-ODN,
M-ODN, or LPS were injected i.d., and serum and, spleens were
collected at times ranging from 1-14 days after injection to assess
in vivo immune activation.
[0078] Collection of Samples
[0079] Blood was obtained by retro-orbital bleed, serum was spun
and then stored at -70.degree. C. until IgG2a, IgG1, or IFN.gamma.
assay.
[0080] Antibody Assays
[0081] Serum was used in ELISA assays for antigen specific
immunoglobulin as described previously. Results are expressed in
units/ml (U/ml) based on pooled high titer anti-.beta.-gal
standards obtained from mice receiving multiple immunizations. The
undiluted serum IgG standards were given an arbitrary concentration
of 400,000 U/ml. Ninety-six-well plates were coated with 5 .mu.g of
.beta.-gal (Sigma) in 50 .mu.l borate buffered saline (BBS; pH 9.2)
overnight at 4.degree. C. Plates were then incubated with 1% BSA in
BBS for 2 hours at 37.degree. C. Plates were washed twice with
BBS/0.5% Tween 20 (Sigma) and incubated with samples overnight at
4.degree. C. Plates were washed 8 times with BBS/Tween 20 and
incubated with alkaline phosphatase-linked anti-IgG1 or IgG2a
(Southern Biotechnologies, Birmingham, Ala.) for 2 hours at room
temperature. The plates were then washed 8 times with BBS/Tween 20
and incubated with a solution of p-nitrophenyl phosphate (1 mg/ml;
Boehringer Mannheim). Absorbance at 405 nm was read at 1 hour and
compared to the standard curve on each plate using the DeltaSOFT II
v. 3.66 program (Biometallics, Princeton, N.J.).
[0082] Cytokine Assays
[0083] Mouse spleens were harvested at week 8 for CTL and cytokine
assays. Three days before setting up cytokine assays mice were i.v.
boosted with 10 .mu.g .beta.-gal. Mice were sacrificed by cervical
dislocation. Spleens were harvested and teased to make single cell
preparations. Splenocyte cytokine profiles were analyzed by
incubation of 5.times.10.sup.5 splenocytes in 96-well plates in a
final volume of 200 .mu.l of supplemented RPMI 1640 with .beta.-gal
added at 10 .mu.g/ml, at 37.degree. C./5% CO.sub.2 as previously
described. Culture supernatants were harvested at 72 hours and
analyzed by ELISA. Pharmagen (San Diego, Calif.) anti-IFN.gamma.
and anti-IL12 capture and detection antibodies, recombinant
IFN.gamma. and IL-12, were all used per the manufacturer's
recommendations. A standard curve was generated on each plate using
known amounts of recombinant IFN.gamma. and each culture
supernatant was compared to the standard curve on the plate using
the DeltaSOFT II v. 3.66 program.
[0084] CTL Assays
[0085] Seven million splenocytes from immunized mice were incubated
with 6.times.10.sup.6 mitomycin-C treated naive splenocytes in the
presence of recombinant human IL-2 and class I H2.sup.d restricted
.beta.-gal nanopeptide (T-P-H-P-A-R-I-G-L; SEQ ID NO: 34). in
supplemented RPMI 1640 with 10% FCS at 37.degree. C./5% CO.sub.2,
as previously described. After 5 days, re-stimulated cells were
harvested and debris was removed by centrifugation on a lympholyte
M (Accurate Chemicals, Westbury, N.Y.) gradient. Specific lysis was
measured by aliquotting effector cells with H2.sup.d restricted
p815 peptide pulsed target cells at 25:1, 5:1, and 1:1 ratios.
Controls for specific lysis included non-pulsed p815 cells, and
p815 cells pulsed with an irrelevant influenza nucleoprotein
peptide. Cells were incubated for 4 hours in clear 2% BSA
supplemented RPMI 1640 in round bottom plates. Total and specific
lysis were measured using the Promega Cytotox 96 kit (Madison,
Wis.). The assay system measures lactate dehydrogenase (LDH)
release using a substrate metabolized by LDH into a colored
by-product. The equation used to calculate specific lysis was
(target lysis-non-specific lysis)/(total lysis).times.100.
[0086] Statistics
[0087] Statistical analysis of results was conducted using Statview
computer software (Abacus Concepts, Grand Rapids, Mich.). A
two-tailed Student's t test was used to establish p values, and
those having p values.ltoreq.0.05 were considered significant.
[0088] Results
[0089] Analysis of the IgG2a response of immunized mice
demonstrated that ISS provides a prolonged window of adjuvant
activity (FIG. 2A). When compared to immunization with .beta.-gal
alone, mice receiving ISS up to 14 days prior to .beta.-gal had a
significant increase in their serum IgG2a response. Furthermore,
the IgG2a response was improved over ISS/.beta.-gal co-immunization
if ISS-ODN was given 3-7 days before antigen, and statistical
significance was reached if ISS-ODN was delivered 7 days before
antigen (p.ltoreq.0.05 for week 6 IgG2a levels) (FIG. 2B). Of note,
the relatively IL-4 dependent IgG1 response was not increased by
ISS pre-priming or by co-delivery with .beta.-gal (FIG. 2C). Mice
immunized with M-ODN either prior to or with .beta.-gal
immunization did not demonstrate an improved IgG2a or IgG1 response
when compared to mice immunized with .beta.-gal alone.
[0090] The effect of ISS pre-priming on cellular immune responses
was next evaluated. Splenocytes were harvested from immunized mice
during week 8, and utilized in both cytokine and CTL assays (FIGS.
3A-3C). Antigen specific IL-4 production was significantly
increased in mice pre-primed up to 14 days before .beta.-gal
immunization compared to mice immunized with antigen alone
(p.ltoreq.0.05). In addition, mice ISS pre-primed 3-7 days before
.beta.-gal immunization demonstrated a 100% increase in their
IFN.gamma. response compared to mice co-immunized with ISS and
.beta.-gal (p.ltoreq.0.05).
[0091] Further studies evaluated the CTL response of mice after ISS
pre-priming. The results again demonstrate a prolonged window of
ISS adjuvant activity. Delivery of ISS up to 14 days before
.beta.-gal vaccination led to a significantly improved CTL response
over .beta.-gal vaccination without adjuvant (p.ltoreq.0.05). ISS
pre-priming at day-7 and day-3 resulted in a trend toward improved
response, but did not lead to a statistically significant increase
in CTL activity when compared to ISS/.beta.-gal co-immunization.
Mice immunized with M-ODN either prior to or with .beta.-gal
immunization did not demonstrate an improved IFN.gamma. or CTL
response when compared to mice immunized with .beta.-gal alone.
[0092] The data presented in this example demonstrate that i.d.
delivery of ISS, up to 2 weeks prior to i.d. .beta.-gal
administration, results in an improved Th1 biased immune response
relative to i.d. vaccination with antigen alone. Anti-.beta.-gal
IgG2a (Th1 isotype), IFN.gamma. release by antigen specific T
cells, and CTL activity against peptide pulsed target cells, are
all higher in mice pre-primed (up to 14 days) with ISS compared to
mice immunized with .beta.-gal alone. The pre-priming effect was
diminished when the interval between ISS and antigen delivery was
extended to 28 days. Interestingly, the optimal immune response is
seen in mice pre-primed with ISS 3-7 days prior to .beta.-gal
injection and not in mice co-delivered ISS and .beta.-gal. A 3-7
day ISS pre-priming interval results in anti-.beta.-gal IgG2a
levels and .beta.-gal specific IFN.gamma. responses which are
approximately twice as high as those seen in ISS/.beta.-gal
co-immunized mice.
Example 2
ISS Activate the Immune System for up to 14 Days
[0093] This example demonstrates that serum levels of the type 1
cytokines IL-12 and IFN.gamma. are elevated for extended periods
following injection of ISS into nave mice. The data presented here
show that the duration and peak expression of these intercellular
signaling molecules correlate well with the duration and peak of
the ISS pre-priming effect.
[0094] Methods
[0095] Serum levels of IL-12 and IFN.gamma. were measured in nave
mice before and 1-14 days after i.d. injection of ISS alone. The
materials and assays used were the same as those described above in
Example 1. In addition, the time course of peak in vivo splenocyte
cytokine production after ISS delivery was evaluated using RT-PCR
to measure IL-12 p40 and IFN.gamma. mRNA expression. Flow cytometry
was used to measure the level of expression of various
co-activation molecules on B cells from mice injected with ISS. The
molecules examined include class I and II, CD40 and B7.2. To
control for auto-fluorescence and non-specific antibody staining,
isotype control antibodies were used.
[0096] Mice were i.d. injected with 50 .mu.g of ISS on day 0.
Control mice received LPS (50 .mu.g) or nothing. At serial time
points after injection, serum was obtained and cytokine levels were
analyzed by ELISA.
[0097] RT-PCR
[0098] For cytokine mRNA analysis, total cellular RNA was extracted
from splenocytes using the Stratagene RNA Isolation kit, (San
Diego, Calif.) and subjected to semi-quantitative RT-PCR.
First-strand cDNA preparation and PCR amplification were performed
using the SuperScript Preamplification system (GibcoBRL,
Gaithersburg, Md.) and Advan Taq Plus DNA Polymerase (Clontech, San
Francisco, Calif.), respectively. The primer sequences used were:
IL-12p4O sense 5'GGGACATCATCAAACCAGACC-3' (SEQ ID NO: 35), and
antisense 5'-GCCAACCAAGCAGAAGACAGC-3' (SEQ ID NO: 36); IFN.gamma.
sense 5'-TGCATCTTGGCTTGCAGCTCTTCCTCATGGC-3' (SEQ ID NO: 37), and
antisense 5'TGGACCTGTGGGTTGTTGACCTCAAACTTGGC-3' (SEQ ID NO: 38);
and GAPDH sense 5'-ACCACAGTCCATGCCATCAC-3' (SEQ ID NO: 39) and
antisense, 5'-TCCACCACCCTGTTGCTGTA-3' (SEQ ID NO: 40).
[0099] PCR products were visualized by electrophoresis on 2%
agarose gels after staining with ethidium bromide.
[0100] Flow Cytometry
[0101] At serial time points after injection, mice were sacrificed
and spleens were harvested and made into single cell suspensions.
Cells were stained with B220 to identify B cells and with FI TC
labeled antibodies to detect co-stimulatory molecules identified in
the Table 2. Live cells (propidium iodide-negative) were analyzed
by flow cytometry (Becton Dickinson, San Jose, Calif.).
[0102] The flow cytometry methods used in this example have been
described by Martin-Orozco E et al., 1999, Enhancement of Antigen
Presenting Cell Surface Molecules Involved in Cognate Interactions
by Immunostimulatory DNA Sequences (ISS), Int. Immunol. 11 (in
press). Briefly, following incubation with Fc block (PharMingen,
San Diego, Calif.), sample cells were stained with PE conjugated
antibodies specific for B cells (anti-B220, PharMingen) and with
FITC conjugated antibodies specific for the following surface
molecules: anti-MHC class 1, class II, CD16/32, CD40, CD80, and
CD86 (PharMingen). Isotype controls for the specific surface
markers are as follows, Hamster IgG, Hamster IgM, Rat IgG2a, Rat
IgG2b, Rat IgM, Mouse IgG2&, Mouse IgG2b (Cal Tag or
PharMingen). Propidium iodide was included in the last wash at a
concentration of 2 .mu.g/ml. Live cells (propidium iodide-negative)
were analyzed on a FACSCalibur flow cytometer (Becton Dickinson,
San lose, Calif.). The data were analyzed with Cell Quest (Becton
Dickinson) and FlowJo (Tree Star, San Carlos, Calif.) software.
[0103] Immunostimulatory oligodeoxynucleotide treatment
significantly increased non-specific antibody binding or
autofluorescence (seen as increases in isotype control
antibody-stained mean fluorescence), so this was controlled for
with the mean fluorescence intensity ratio (MFIR; mean fluorescence
when stained for surface molecule/mean fluorescence when stained
with isotype control antibody). The MFIR represents the fold
increase in surface marker expression relative to background
autofluorescence and nonspecific antibody binding. MFIR provides a
conservative and accurate estimate of expression of surface
molecules when studying cells treated with ISS-containing DNA.
[0104] Results
[0105] Serum levels of cytokines measured in nave mice before and
1-14 days after i.d. injection of ISS alone are shown in Table 1.
The data presented in Table 1 represent means for 2 truce per group
plus or minus the standard error of the mean. The results show that
i.d. injection of ISS into mice leads to elevated serum IL-12 and
IFN.gamma. levels for up to 2 weeks after delivery. These results
are consistent with the 14 day window of ISS adjuvant noted with
the antigen specific immune response. However, peak serum levels of
IL-12 and IFN.gamma. were seen 1 day after ISS delivery and serum
IgG2a levels and splenocyte cytokine and CTL responses were highest
in mice pre-pried with ISS 3-7 days before .beta.-gal
immunization.
2TABLE 1 In vivo cytokine production induced by ISS-ODN ISS-ODN
IL-12 (pg/ml) IFN.gamma. (pg/ml) None <42 <14 Day (1) 4028
.+-. 1878 343 .+-. 83 Day (3) 2356 .+-. 464 312 .+-. 101 Day (7)
2034 .+-. 288 174 .+-. 36 Day (14) 763 .+-. 255 35 .+-. 71 LPS Day
7 <42 <14
[0106] Because many of the antigen specific immune responses which
characterize the ISS pre-priming effect were measured in spleen,
the time course of peak in vivo splenocyte cytokine production
after ISS delivery was examined. Using RT-PCR, the time course for
peak splenocyte IL12 p40 and IFN.gamma. mRNA expression was
assessed. Intradermal ISS delivery led to peak levels of IL12 p40
and IFN.gamma. mRNA at 7 and 3 days, respectively (FIG. 4). These
time points fall within the window identified for the maximal ISS
pre-priming effect on antigen specific immunity.
[0107] Previous reports have shown that a number of co-activation
molecules on B cells and APCs are up-regulated by ISS. The
relatively long ISS pre-priming effect could be mediated by
up-regulation of these surface proteins. Experiments were designed
to establish if i.d. ISS injection would lead to a detectable and
prolonged increase in the expression of these molecules in vivo.
Flow cytometry was used to measure the level of expression of
various co-activation molecules on 13 cells from ISS injected mice.
To control for auto-fluorescence and non-specific antibody
staining, isotype control antibodies were used and the results are
presented as mean fluorescence intensity ratio (MFIR; MFI of
antibody of interest/MFI of isotype control).
[0108] As shown in Table 2, ISS increased the expression of a
number of co-activation molecules such as class I and II, CD40, and
B7.2 on the surface of B cells from ISS injected mice. The time
course for up-regulation of these surface proteins was extended and
peak co-stimulatory molecule expression was seen 3-7 days after ISS
injection. These results were consistent with the splenocyte
cytokine RT-PCR results, and the 3-7 day interval between ISS
pre-priming and .beta.-gal vaccination which led to maximal antigen
specific immunity.
3TABLE 2 Up-regulation of cell surface molecules in vivo by ISS-ODN
H-2K.sup.d I-A.sup.d (MHC (MHC CD16/ CD54 CD80 CD86 ISS class I)
class II) CD32 CD40 (ICAM-1) (B7-1) (B7.2) None 25.3 77.9 7.26 11.4
4.5 2.85 1.75 Day 3 27.1 87.1 9.3 11.7 7.68 2.85 2.18 Day 7 27.7
118 8.23 12.4 7.98 2.64 2.12 Day 14 30.1 94.9 7.26 9.5 6.36 2.37
1.97
Example 3
Mucosal ISS Pre-Priming Enhances Th1 and IgA Adjuvant Activity
[0109] This example shows that other forms of immunity, e.g.
mucosal immunity, can be enhanced by pre-priming with ISS. The
example also shows that intranasal (i.n.) delivery of ISS can
modulate both systemic and mucosal immune responses.
[0110] Methods
[0111] The materials and assays used were as described above in
Example 1. Mice received a single i.n. injection with ISS (50
.mu.g) either the specified day before or with i.n. .beta.-gal (50
.mu.g) immunization. Control mice received i.n. .beta.-gal
immunization alone or in conjunction with M-ODN. Splenocytes were
harvested from sacrificed mice during week 7.
[0112] Bronchoalveolar lavage fluid (BALF) was used in ELISA assays
for antigen specific immunoglobulin. Fecal IgA standards were given
arbitrary concentrations of 20,000 U/ml. BALF was obtained by
cannulation of the trachea of sacrificed mice during week 8. The
lungs were then flushed with 0.8ml of PBS. The return was spun to
remove cellular debris, and frozen at -70.degree. C. until IgA
assay.
[0113] Results
[0114] Table 3 and FIG. 5 show that i.n. pre-priming provides a 7
day window of systemic Th1 and mucosal IgA adjuvant activity. This
window of adjuvant activity is shorter than when reagents are
delivered i.d. Nonetheless, all immune parameters were
significantly higher if mice received ISS within the week preceding
or with .beta.-gal than if mice were immunized with .beta.-gal
alone. Mice immunized with M-ODN either prior to or with .beta.-gal
immunization did not demonstrate an improved IFN.gamma. or CTL
response when compared to mice immunized with .beta.-gal alone. The
pre-priming effect observed on the BALF IgA response was modest but
prolonged (1 week). Recognizing that without adjuvant the immune
response to simple protein antigens such as .beta.-gal is
negligible, the week long ISS pre-priming effect with i.n. delivery
is significant.
4TABLE 3 Anti-.beta.-gal Ig production induced by mucosal
pre-priming and immunization ISS-ODN .beta.-gal Serum IgG2a (U/ml)
BALF IgA (U/ml) -- + <500 <50 Day (0) + 239000 .+-. 71500
2940 .+-. 825 Day(-1) + 322000 .+-. 112000 463 .+-. 47 Day (-3) +
38000 .+-. 3490 398 .+-. 38 Day (-7) + 17800 .+-. 4830 459 .+-. 183
Day (-14) + <500 <50
[0115] A stronger i.n. ISS pre-priming effect was observed on the
anti-.beta.-gal IgG2a levels, CTL activity and antigen specific
IFN.gamma. responses. Immunostimulatory oligodeoxynucleotide
pre-priming at day-1 improved the IgG2a response slightly and the
antigen specific splenocyte IFN.gamma. response was improved
significantly (p.ltoreq.0.05) compared to i.n. co-immunization with
ISS and .beta.-gal. These results demonstrate that ISS pre-priming
is also effective with mucosal delivery, although the duration and
the optimal interval between ISS and .beta.-gal delivery were
different than with i.d. delivery.
[0116] Discussion
[0117] A previous epidemiological study conducted on approximately
1000 Japanese school children documented a correlation between
exposure to Mycobacteria tuberculosis (MTH) and a Th1 biased serum
cytokine profile in study subjects. In addition, purified protein
derivative (PPD) converters demonstrated a significantly lower
incidence of allergic disease, and significantly lower serum IgE
levels versus PPD negative school children (Shirakawa, T. et al.,
1997, Science 275:77-9). Similar observations were made in an
experimental murine system (Erb, K. I. et al., 1998, J. Exp. Med.
187:561-9). In this respect, exposure to MTB may be considered to
pre-prime the host toward Th1 immunity. Moreover, a recent study
demonstrated that the infection of dendritic cells with MTB
resulted in the release of TNF.alpha., and IL-12, as well as the
up-regulation of MHC class I, ICAM-1, CD40, and B7 co-stimulatory
molecules (Henderson, R. A. et al., 1997,J. Immunol. 159:635). This
activation profile is very similar to the pattern induced with ISS.
As ISS DNA was initially identified and isolated from MTB DNA, it
is conceivable that this adjuvant plays a role in biasing the
immune profile of MTB exposed hosts toward a Th1 phenotype, as do
synthetic ISS-ODNs as demonstrated herein.
[0118] In gene vaccinated animals, ISS (CpG motifs) within the
plasmid DNA (pDNA) backbone generate the necessary initial cytokine
milieu (i.e., IL-12 and IFNs) to foster a Th1 response to the
encoded antigen. Thus, gene vaccination plasmids provide both a
source of adjuvant and antigen. These two activities are probably
not simultaneous. The local induction of cytokine by ISS DNA is
rapid (within 24 hours) and probably precedes the expression of
sufficient amounts of antigen to elicit an effective immune
response. A similar argument can be made for the up-regulation of
co-stimulatory ligands. Thus, in gene vaccination, the pre-priming
effects mediated by ISS DNA are likely to contribute to the Th1
biased immune response to the encoded antigen.
[0119] The above examples show that pre-priming enhances a variety
of immune responses and is effective for enhancing both mucosal and
systemic immunity. In summary, the invention provides a novel
paradigm for Th1 biased immunization, called ISS pre-priming.
Immunostimulatory sequence DNA administration biases the host
immune system toward Th1 biased immune responses for up to 2 weeks.
In addition, ISS delivered up to 7 days before antigen produces a
stronger immune response than ISS/antigen co-immunization.
[0120] The data presented herein show that ISS can not only be used
as an adjuvant in the traditional sense, but that it can also be
used as an immuno-modifying therapeutic agent. For example, ISS
might be i.d. injected or applied i.n. for the treatment of
allergic rhinitis or inhaled to treat asthma. This immunologic
strategy would provide relatively prolonged Th1 biased immunologic
protection against continuous exposure to inhaled allergens.
Instead of amplifying a pre-existing allergic Th2 biased immune
response, allergen exposure would down-regulate allergic
inflammation of the nasal mucosa and bronchial surface via the
numerous type 1 cytokines released after ISS exposure. A similar
approach might be effective for preventing or treating infectious
diseases such as influenza or rotavirus gastroenteritis via a Th1
biased and activated immune system post ISS pre-priming. This
invention provides ISS pre-priming as a new vaccination strategy,
and a new paradigm for the prevention and treatment of infectious,
allergic and malignant disease.
[0121] Those skilled in the art will appreciate that the
conceptions and specific embodiments disclosed in the foregoing
description may be readily utilized as a basis for modifying or
designing other embodiments for carrying out the same purposes of
the present invention. Those skilled in the art will also
appreciate that such equivalent embodiments do not depart from the
spirit and scope of the invention as set forth in the appended
claims.
Sequence CWU 1
1
40 1 6 DNA synthetic oligonucleotide 1 rrcgyy 6 2 6 DNA synthetic
oligonucleotide 2 rycgyy 6 3 8 DNA synthetic oligonucleotide 3
rrcgyycg 8 4 8 DNA synthetic oligonucleotide 4 rycgyycg 8 5 6 DNA
synthetic oligonucleotide 5 aacgtt 6 6 6 DNA synthetic
oligonucleotide 6 agcgtc 6 7 6 DNA synthetic oligonucleotide 7
agcgtt 6 8 6 DNA synthetic oligonucleotide 8 gacgtt 6 9 6 DNA
synthetic oligonucleotide 9 ggcgtt 6 10 6 DNA synthetic
oligonucleotide 10 aacgtc 6 11 6 DNA synthetic oligonucleotide 11
agcgtc 6 12 6 DNA synthetic oligonucleotide 12 gacgtc 6 13 6 DNA
synthetic oligonucleotide 13 ggcgtc 6 14 6 DNA synthetic
oligonucleotide 14 aacgcc 6 15 6 DNA synthetic oligonucleotide 15
agcgcc 6 16 6 DNA synthetic oligonucleotide 16 gacgcc 6 17 6 DNA
synthetic oligonucleotide 17 ggcgcc 6 18 6 DNA synthetic
oligonucleotide 18 agcgct 6 19 6 DNA synthetic oligonucleotide 19
gacgct 6 20 6 DNA synthetic oligonucleotide 20 ggcgct 6 21 6 DNA
synthetic oligonucleotide 21 ttcgaa 6 22 6 DNA synthetic
oligonucleotide 22 ggcgtt 6 23 6 DNA synthetic oligonucleotide 23
aacgcc 6 24 6 DNA synthetic oligonucleotide 24 gtcgtt 6 25 8 DNA
synthetic oligonucleotide 25 agcgtccg 8 26 8 DNA synthetic
oligonucleotide 26 aacgttcg 8 27 8 DNA synthetic oligonucleotide 27
agcgttcg 8 28 8 DNA synthetic oligonucleotide 28 gacgttcg 8 29 8
DNA synthetic oligonucleotide 29 ggcgttcg 8 30 8 DNA synthetic
oligonucleotide 30 aacgttcg 8 31 8 DNA synthetic oligonucleotide 31
agcgtccg 8 32 22 DNA synthetic oligonucleotide 32 tgactgtgaa
cgttcgagat ga 22 33 22 DNA synthetic oligonucleotide 33 tgactgtgaa
ccttagagat ga 22 34 9 PRT e. coli 34 Thr Pro His Pro Ala Arg Ile
Gly Leu 1 5 35 21 DNA synthetic oligonucleotide 35 gggacatcat
caaaccagac c 21 36 21 DNA synthetic oligonucleotide 36 gccaaccaag
cagaagacag c 21 37 32 DNA synthetic oligonucleotide 37 tgcatcttgg
ctttgcagct cttcctcatg gc 32 38 32 DNA synthetic oligonucleotide 38
tggacctgtg ggttgttgac ctcaaacttg gc 32 39 20 DNA synthetic
oligonucleotide 39 accacagtcc atgccatcac 20 40 20 DNA synthetic
oligonucleotide 40 tccaccaccc tgttgctgta 20
* * * * *