U.S. patent application number 10/426237 was filed with the patent office on 2004-01-15 for methods of preventing and treating respiratory viral infection using immunomodulatory polynucleotide sequences.
Invention is credited to Van Nest, Gary.
Application Number | 20040009942 10/426237 |
Document ID | / |
Family ID | 26884244 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009942 |
Kind Code |
A1 |
Van Nest, Gary |
January 15, 2004 |
Methods of preventing and treating respiratory viral infection
using immunomodulatory polynucleotide sequences
Abstract
The invention provides methods of preventing and/or treating
infection by a respiratory virus such as respiratory syncytial
virus (RSV) and SARS-associated coronavirus, particularly reducing
infection and/or one or more symptoms of respiratory virus
infection. A polynucleotide comprising an immunostimulatory
sequence (an "ISS") is administered to an individual which is at
risk of being exposed to a respiratory virus, has been exposed to a
respiratory virus or is infected with a respiratory virus. The ISS
is administered without any antigens of the respiratory virus.
Administration of the ISS results in reduced incidence and/or
severity of one or more symptoms of respiratory virus
infection.
Inventors: |
Van Nest, Gary; (Martinez,
CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
Attn: Catherine M. Polizzi
755 Page Mill Road
Palo Alto
CA
94304
US
|
Family ID: |
26884244 |
Appl. No.: |
10/426237 |
Filed: |
April 29, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10426237 |
Apr 29, 2003 |
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09802686 |
Mar 9, 2001 |
|
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60188583 |
Mar 10, 2000 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K 2039/55561
20130101; A61P 31/12 20180101; A61K 31/711 20130101; A61P 31/14
20180101; A61K 39/39 20130101; A61K 31/7115 20130101; A61P 31/16
20180101; A61K 31/7105 20130101 |
Class at
Publication: |
514/44 |
International
Class: |
A61K 048/00 |
Claims
What is claimed is:
1. A method of suppressing a respiratory virus infection in an
individual who has been exposed to a respiratory virus, comprising
administering a composition comprising a polynucleotide comprising
an immunostimulatory sequence (ISS) to said individual, wherein the
ISS comprises the sequence 5'-C, G-3', wherein a respiratory virus
antigen is not administered in conjunction with administration of
said composition, and wherein said composition is administered in
an amount sufficient to suppress a respiratory virus infection.
2. The method of claim 1, wherein the ISS comprises the sequence
5'-T, C, G-3'.
3. The method of claim 1, wherein the ISS comprises the sequence
5'-purine, purine, C, G, pyrimidine, pyrimidine, C, G-3' or
5'-purine, purine, C, G, pyrimidine, pyrimidine, C, C-3'.
4. The method of claim 3, wherein the ISS comprises a sequence
selected from the group consisting of
5'-AACGTTCC-3',5'-AACGTTCG-3',5'-GACGTTCC-3'- , and
5'-GACGTTCG-3'.
5. The method of claim 1, wherein the ISS comprises the sequence
5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1).
6. The method of claim 1, wherein the individual is a mammal.
7. The method of claim 1, wherein administration is at a site of
exposure.
8. The method of claim 7, wherein the site of exposure is
respiratory mucosa.
9. The method of claim 7, wherein the site of exposure is the nasal
passages.
10. The method of claim 1, wherein the respiratory virus is
SARS-associated coronavirus.
11. A kit for use in suppressing a respiratory virus infection in
an individual infected with or exposed to respiratory virus,
comprising: a composition comprising a polynucleotide comprising an
immunostimulatory sequence (ISS), wherein the ISS comprises the
sequence 5'-C, G-3' and wherein said kit does not comprises a
respiratory virus antigen; and instructions for administration of
said composition to an individual infected with or exposed to
respiratory virus to suppress respiratory virus infection.
12. The kit of claim 11, wherein the ISS comprises the sequence
5'-T, C, G-3'.
13. The kit of claim 11, wherein the ISS comprises the sequence
5'-purine, purine, C, G, pyrimidine, pyrimidine, C, G-3' or
5'-purine, purine, C, G, pyrimidine, pyrimidine, C, C-3'.
14. The kit of claim 13, wherein the ISS comprises a sequence
selected from the group consisting of
5'-AACGTTCC-3',5'-AACGTTCG-3',5'-GACGTTCC-3' and
5'-GACGTTCG-3'.
15. The kit of claim 11, wherein the ISS comprises the sequence
5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part
application of copending U.S. application Ser. No. 09/802,686,
filed Mar. 9, 2001, which in turn claims the priority benefit of
U.S. Provisional application No. 60/188,583, filed Mar. 10, 2000,
each of which is hereby incorporated in their entirety by
reference.
TECHNICAL FIELD
[0002] The invention is in the field of immunostimulatory
poynucleotides, more particularly their use in ameliorating or
preventing respiratory viral infection and symptoms of respiratory
viral infection.
BACKGROUND ART
[0003] Despite massive research efforts to find cures, respiratory
virus infection remains a major health problem worldwide. Influenza
and rhinovirus are causative agents for flu and common cold,
respectively, lead to significant lost productivity annually as
well as discomfort and even death due to illness. While many
over-the-counter remedies are available, these drugs merely treat
symptoms, often with only limited success, leaving the patient
still debilitated from the infection. Respiratory syncytial virus
(RSV) is a leading cause of serious lower respiratory treat
infections and children under two years and has more recently been
identified as an important cause of lower respiratory tract
infection in adults. No vaccines are currently available for
preventing RSV infection. The only antiviral currently approved for
treatment of the disease, ribavirin, suffers from the drawbacks of
being licensed only for administration as continuous small particle
aerosol and being a potential teratogen. A challenge facing
treatment of these infections is to discover an anti-viral agent
which effectively suppresses viral infection, while not producing
unpleasant and unacceptable side-effects.
[0004] Administration of certain DNA sequences, generally known as
immunostimulatory sequences or "ISS," induces an immune response
with a Th1-type bias as indicated by secretion of Th1-associated
cytokines. The Th1 subset of helper cells is responsible for
classical cell-medicated functions such as delayed-type
hypersensitivity and activation of cytotoxic T lymphocytes (CTLs),
whereas the Th2 subset functions more effectively as a helper for
B-cell activation. The type of immune response to an antigen is
generally influenced by the cytokines produced by the cells
responding to the antigen. Differences in the cytokines secreted by
Th1 and Th2 cells are believed to reflect different biological
functions of these two subsets. See, for example, Romagnani (2000)
Ann. Allergy Asthma Immunol. 85:9-18.
[0005] Administration of an immunostimulatory polynucleotide with
an antigen results in a Th1-type immune response to the
administered antigen. Roman et al. (1997) Nature Med. 3:849-854.
For example, mice injected intradernally with Escherichia coli (E.
coli) .beta.-galactosidase (.beta.-Gal) in saline or in the
adjuvant alum responded by producing specific IgG1 and IgE
antibodies, and CD4.sup.+ cells that secreted IL-4 and IL-5, but
not IFN-.gamma., demonstrating that the T cells were predominantly
of the Th2 subset. However, mice injected intradermally (or with a
tyne skin scratch applicator) with plasmid DNA (in saline) encoding
.beta.-Gal and containing an ISS responded by producing IgG2a
antibodies and CD4.sup.+ cells that secreted IFN-.gamma., but not
IL-4 and IL-5, demonstrating that the T cells were predominantly of
the Th1 subset. Moreover, specific IgE production by the plasmid
DNA-injected mice was reduced 66-75%. Raz et al. (1996) Proc. Natl.
Acad. Sci. USA 93:5141-5145. In general, the response to naked DNA
immunization is characterized by production of IL-2, TNF.alpha. and
IFN-.gamma. by antigen-stimulated CD4.sup.+ T cells, which is
indicative of a Th1-type response. This is particularly important
in treatment of allergy and asthma as shown by the decreased IgE
production. The ability of immunostimulatory polynucleotides to
stimulate a Th1-type immune response has been demonstrated with
bacterial antigens, viral antigens and with allergens (see, for
example, WO 98/55495).
[0006] Other references describing ISS include: Krieg et al. (1989)
J. Immunol. 143:2448-2451; Tokunaga et al. (1992) Microbiol.
Immunol. 36:55-66; Kataoka et al. (1992) Jpn. J. Cancer Res.
83:244-247; Yamamoto et al. (1992) J. Immunol. 148:4072-4076;
Mojcik et al. (1993) Clin. Immuno. and Immunopathol. 67:130-136;
Branda et al. (1993) Biochem. Pharmacol. 45:2037-2043; Pisetsky et
al. (1994) Life Sci. 54(2):101-107; Yamamoto et al. (1994a)
Antisense Research and Development. 4:119-122; Yamamoto et al.
(1994b) Jpn. J. Cancer Res. 85:775-779; Raz et al. (1994) Proc.
Natl. Acad. Sci. USA 91:9519-9523; Kimura et al. (1994) J. Biochem.
(Tokyo) 116:991-994; Krieg et al. (1995) Nature 374:546-549;
Pisetsky et al. (1995) Ann. N.Y. Acad. Sci. 772:152-163; Pisetsky
(1996a) J. Immunol. 156:421-423; Pisetsky (1996b) Immunity
5:303-310; Zhao et al. (1996) Biochem. Pharmacol. 51:173-182; Yi et
al. (1996) J. Immunol. 156:558-564; Krieg (1996) Trends Microbiol.
4(2):73-76; Krieg et al. (1996) Antisense Nucleic Acid Drug Dev.
6:133-139; Klinman et al. (1996) Proc. Natl. Acad. Sci. USA.
93:2879-2883; Raz et al. (1996); Sato et al. (1996) Science
273:352-354; Stacey et al. (1996) J. Immunol. 157:2116-2122; Ballas
et al. (1996) J. Immunol. 157:1840-1845; Branda et al. (1996) J.
Lab. Clin. Med. 128:329-338; Sonehara et al. (1996) J. Interferon
and Cytokine Res. 16:799-803; Klinman et al. (1997) J. Immunol.
158:3635-3639; Sparwasser et al. (1997) Eur. J. Immunol.
27:1671-1679; Roman et al. (1997); Carson et al. (1997) J. Exp.
Med. 186:1621-1622; Chace et al. (1997) Clin. Immunol. and
Immunopathol 84:185-193; Chu et al. (1997) J. Exp. Med.
186:1623-1631; Lipford et al. (1997a) Eur. J. Immunol.
27:2340-2344; Lipford et al. (1997b) Eur. J. Immunol 27:3420-3426;
Weiner et al. (1997) Proc. Natl. Acad. Sci. USA 94:10833-10837;
Macfarlane et al. (1997) Immunology 91:586-593; Schwartz et al.
(1997) J. Clin. Invest. 100:68-73; Stein et al. (1997) Antisense
Technology, Ch. 11 pp. 241-264, C. Lichtenstein and W. Nellen,
Eds., IRL Press; Wooldridge et al. (1997) Blood 89:2994-2998;
Leclerc et al. (1997) Cell. Immunol. 179:97-106; Kline et al.
(1997) J. Invest. Med. 45(3):282A; Yi et al. (1998a) J. Immunol.
160:1240-1245; Yi et al. (1998b) J. Immunol. 160:4755-4761; Yi et
al. (1998c) J. Immunol. 160:5898-5906; Yi et al. (1998d) J.
Immunol. 161:4493-4497; Krieg (1998) Applied Antisense
Oligonucleotide Technology Ch. 24, pp. 431-448, C. A. Stein and A.
M. Krieg, Eds., Wiley-Liss, Inc.; Krieg et al. (1998a) Trends
Microbiol. 6:23-27; Krieg et al. (1998b) J. Immunol. 161:2428-2434;
Krieg et al. (1998c) Proc. Natl. Acad. Sci. USA 95:12631-12636;
Spiegelberg et al. (1998) Allergy 53(45S):93-97; Homer et al.
(1998) Cell Immunol. 190:77-82; Jakob et al. (1998) J. Immunol.
161:3042-3049; Redford et al. (1998) J. Immunol. 161:3930-3935;
Weeratna et al. (1998) Antisense & Nucleic Acid Drug
Development 8:351-356; McCluskie et al. (1998) J. Immunol.
161(9):4463-4466; Gramzinski et al. (1998) Mol. Med. 4:109-118; Liu
et al. (1998) Blood 92:3730-3736; Moldoveanu et al. (1998) Vaccine
16: 1216-1224; Brazolot Milan et al. (1998) Proc. Natl. Acad. Sci.
USA 95:15553-15558; Briode et al. (1998) J. Immunol. 161:7054-7062;
Briode et al. (1999) Int. Arch. Allergy Immunol. 118:453-456;
Kovarik et al. (1999) J. Immunol. 162:1611-1617; Spiegelberg et al.
(1999) Pediatr. Pulmonol. Suppl. 18:118-121; Martin-Orozco et al.
(1999) Int. Immunol. 11:1111-1118; EP 468,520; WO 96/02555; WO
97/28259; WO 98/16247; WO 98/18810; WO 98/37919; WO 98/40100; WO
98/52581; WO 98/55495; WO 98/55609 and WO 99/11275. See also Elkins
et al. (1999) J. Immunol. 162:2291-2298, WO 98/52962, WO 99/33488,
WO 99/33868, WO 99/51259 and WO 99/62923. See also Zimmermann et
al. (1998) J. Immunol. 160:3627-3630; Krieg (1999) Trends
Microbiol. 7:64-65; U.S. Pat. Nos. 5,663,153, 5,723,335, 5,849,719
and 6,174,872. See also WO 99/56755, WO 00/06588, WO 00/16804; WO
00/21556; WO 00/67023 and WO 01/12223.
[0007] There remains a serious need to develop effective therapies
and preventive strategies for respiratory viruses.
[0008] All publications and patent applications cited herein are
hereby incorporated by reference in their entirety.
DISCLOSURE OF THE INVENTION
[0009] The invention provides methods of suppressing, ameliorating,
and/or preventing viral infection by a respiratory virus in an
individual (either before or after exposure or infection) using
immunostimulatory polynucleotide sequences. Accordingly, in one
aspect, the invention provides methods of preventing, palliating,
ameliorating, reducing and/or eliminating one or more symptoms of
respiratory virus infection without administering a respiratory
virus antigen. A polynucleotide comprising an immunostimulatory
sequence (an "ISS") is administered to an individual who is at risk
of being exposed to respiratory virus, has been exposed to
respiratory virus or is infected with respiratory virus. The
ISS-containing polynucleotide is administered without any
respiratory virus antigens (i.e., respiratory virus antigen is not
co-administered). Administration of the ISS results in reduced
incidence and/or severity of one or more symptoms of respiratory
virus infection.
[0010] In one embodiment, the invention provides methods of
preventing a symptom of respiratory virus infection in an
individual at risk of being exposed to a respiratory virus which
entail administering an effective amount of a composition
comprising a polynucleotide comprising an immunostimulatory
sequence (ISS) (i.e., an amount of the composition sufficient to
prevent a symptom of respiratory virus infection) to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein respiratory virus antigen is not administered in
conjunction with administration of the composition (i.e., antigen
is not administered with the ISS-containing polynucleotide),
thereby preventing a symptom of respiratory virus infection.
[0011] Another embodiment of the invention provides methods of
preventing a symptom of respiratory virus infection in an
individual which entail administering an effective amount of a
composition comprising a polynucleotide comprising an ISS to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein respiratory virus antigen is not administered in
conjunction with administration of the composition, thereby
preventing a symptom of respiratory virus infection. The individual
may have been exposed to or infected by a respiratory virus.
[0012] Another embodiment of the invention provides methods of
suppressing a respiratory virus infection in an individual which
entail administering a composition comprising a polynucleotide
comprising an ISS to the individual in an amount sufficient to
reduce viral titer of the respiratory virus in a biological sample
from the individual, wherein the ISS comprises the sequence 5'-C,
G-3' and wherein respiratory virus antigen is not administered in
conjunction with administration of the composition, thereby
suppressing a respiratory virus infection. The individual may have
been exposed to or may be infected by a respiratory virus.
[0013] In another embodiment, the invention provides methods of
reducing severity of a symptom of respiratory virus infection in an
individual which entail administering an effective amount of a
composition comprising a polynucleotide comprising an ISS to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein respiratory virus antigen is not administered in
conjunction with administration of the composition, thereby
reducing severity of a symptom of respiratory virus infection. The
individual may be exposed to, at risk of exposure to, or infected
by a respiratory virus.
[0014] In another embodiment, the invention provides methods of
delaying development of a symptom of respiratory virus infection in
an individual which entail administering effective amount of a
composition comprising a polynucleotide comprising an ISS to the
individual, wherein the ISS comprises the sequence 5'-C, G-3' and
wherein respiratory virus antigen is not administered in
conjunction with administration of the composition, thereby
delaying development of a symptom of respiratory virus
infection.
[0015] In another embodiment, the invention provides methods of
reducing duration of a respiratory virus infection in an individual
which entail administering effective amount of a composition
comprising a polynucleotide comprising an ISS to the individual,
wherein the ISS comprises the sequence 5'-C, G-3' and wherein
respiratory virus antigen is not administered in conjunction with
administration of the composition, thereby reducing duration of a
respiratory virus infection. The individual may be exposed to, at
risk of exposure to, or infected by a respiratory virus.
[0016] In further aspect, the invention provides kits for use in
accordance with the methods of the invention. Accordingly, another
embodiment of the invention provides kits for use in ameliorating
and/or preventing a symptom of respiratory virus infection in an
individual infected with, exposed to or at risk of being exposed to
a respiratory virus. The kits comprise a composition comprising a
polynucleotide comprising an ISS, wherein the ISS comprises the
sequence 5'-C, G-3', wherein the kit does not comprise a
respiratory virus antigen, and wherein the kits comprise
instructions for administration of the composition to an individual
infected with, exposed to or at risk of being exposed to a
respiratory virus.
[0017] In some embodiments of the methods and kits of the
invention, the ISS comprises the sequence 5'-purine, purine, C, G,
pyrimidine, pyrimidine, C, G-3' or 5'-purine, purine, C, G,
pyrimidine, pyrimidine, C, C-3'. In further embodiments of the
methods and kits, the ISS comprises a sequence selected from the
group consisting of AACGTTCC, AACGTTCG, GACGTTCC and GACGTTCG.
[0018] In some embodiments of the methods and kits of the
invention, the ISS comprises the sequence 5'-T, C, G-3'. In some
embodiments of the methods and kits of the invention, the ISS
comprises the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID
NO:1).
[0019] In some embodiments of the methods and kits of the
invention, the individual is a mammal. In further embodiments, the
mammal is human.
[0020] In some embodiments of the methods and kits of the
invention, the respiratory virus is respiratory syncytial virus
(RSV). In other embodiments of the methods and kits of the
invention, the respiratory virus is adenovirus. In other
embodiments of the methods and kits of the invention, the
respiratory virus is rhinovirus. In other embodiments of the
methods and kits of the invention, the respiratory virus is
coronavirus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a bar graph depicting lung RSV titer in rats which
received intranasally: PBS (first bar); ISS three days before viral
infection (second bar) non-ISS control sequence three days before
viral infection (third bar); ISS 30 minutes before viral infection
(fourth bar); non-ISS control sequence 30 minutes before viral
infection.
MODES FOR CARRYING OUT The INVENTION
[0022] We have discovered that immunostimulatory polynucleotide
sequences (ISS) are effective anti-viral agents against respiratory
viruses. A polynucleotide comprising an immunostimulatory sequence
(an "ISS") is administered to an individual who is at risk of being
exposed to respiratory virus, has been exposed to respiratory virus
or is infected with respiratory virus. The ISS is administered
without any respiratory virus antigens. Administration of the ISS
without co-administration of a respiratory virus antigen results in
reduced incidence and/or severity of one or more symptoms of
respiratory virus infection.
[0023] The invention also relates to kits for ameliorating and/or
preventing a symptom of respiratory virus infection in exposed
individuals. The kits, which do not contain a respiratory virus
antigen, comprise a polynucleotide comprising an ISS and
instructions describing the administration of an ISS-containing
polynucleotide to an individual for the intended treatment.
[0024] In an art-accepted model of a respiratory virus, namely
cotton rat infected with respiratory syncytial virus (RSV), we have
shown that ISS is effective at reducing viral titers especially if
administered locally (i.e., at a site of infection) and at a
sufficient time before viral infection. For example, rats
pre-treated with an ISS non-locally (i.e., by IP injection) did not
display significant reduction in viral titer although higher doses
may have been efficacious. Further, there is no apparent toxicity
at the therapeutic dosages (i.e., dosages sufficient to reduce
viral titer). Significantly, in contrast to previous reports of
immune modulation by ISS, we report clinical efficacy of ISS in
this viral context.
[0025] General Techniques
[0026] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook et
al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984);
Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in
Enzymology (Academic Press, Inc.); Handbook of Experimental
Immunology (D. M. Weir & C. C. Blackwell, eds.); Gene Transfer
Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds.,
1987); Current Protocols in Molecular Biology (F. M. Ausubel et
al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et
al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et
al., eds., 1991); The Immunoassay Handbook (David Wild, ed.,
Stockton Press NY, 1994); and Methods of Immunological Analysis (R.
Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH
Verlags gesellschaft mbH, 1993).
[0027] Definitions
[0028] The term "respiratory virus" refers to a virus which infects
cells of the respiratory tract, such as cells lining the oral
cavity, nasopharynx, throat, larynx, bronchi and bronchioles, etc.
Respiratory viruses include influenza virus, rhinovirus,
adenovirus, respiratory syncytial virus (RSV), coronavirus, severe
acute respiratory syndrome (SARS)-associated coronavirus, measles
virus, mumps virus, parainfluenza virus, rubella virus, poxvirus,
parvovirus, hantavirus and varicella virus. Statements and
description which use the term "respiratory virus" indicate, and
refer to, any one or more of the respiratory viruses listed
herein.
[0029] "Exposure" to a virus denotes encounter with virus which
allows infection, such as, for example, upon contact with an
infected individual.
[0030] An individual is "seronegative" for a virus if antibodies
specific to the virus cannot be detected in blood or serum samples
from the individual using methods standard in the art, such as
ELISA. Conversely, an individual is "seropositive" for a virus if
antibodies specific for the virus can be detected in blood or serum
samples from the individual using methods standard in the art, such
as ELISA. An individual is said to "seroconvert" for a virus when
antibodies to the virus can be detected in blood or serum from an
individual who was previously seronegative.
[0031] An individual who is "at risk of being exposed" to a virus
is an individual who may encounter the virus such that the virus
infects the individual (i.e., virus enters cells and replicates).
In the context of respiratory virus, which cause acute infection
and resolution of infection and symptoms, the individual may or may
not have previously been exposed to virus, but it is understood
that, at the time of at least one administration of ISS-containing
polynucleotide, the individual is symptom-free and has not been
exposed to virus within about 5 days of administration of ISS.
Because respiratory viruses are ubiquitous, generally any
individual is at risk for exposure to the virus. In some contexts,
an individual is determined to be "at risk" because exposure to the
virus has higher probability of leading to infection (such as with
immunocompromised, elderly and/or very young children and infants)
which can further result in serious symptoms, conditions, and/or
complications. In some settings, including, but not limited to,
institutions such as hospitals, schools, day care facilities,
military facilities, nursing homes and convalescent homes, an
individual is determined to be "at risk" because of time spent in
close proximity to others who may be infected.
[0032] "Suppressing" viral infection indicates any aspect of viral
infection, such as viral replication, time course of infection,
amount (titer) of virus, lesions, and/or one or more symptoms is
curtailed, inhibited, or reduced (in terms of severity and/or
duration) in an individual or a population of individuals treated
with an ISS-containing polynucleotide in accordance with the
invention as compared to an aspect of viral infection in an
individual or a population of individuals not treated in accordance
with the invention. Reduction in viral titer includes, but is not
limited to, elimination of the virus from an infected site or
individual. Viral infection can be assessed by any means known in
the art, including, but not limited to, measurement of virus
particles, viral nucleic acid or viral antigens, detection of
symptoms and detection and/or measurement of anti-virus antibodies.
Anti-virus antibodies are widely used to detect and monitor viral
infection and generally are commercially available.
[0033] "Palliating" a disease or one or more symptoms of a disease
or infection means lessening the extent and/or time course of
undesirable clinical manifestations of a disease state or infection
in an individual or population of individuals treated with an ISS
in accordance with the invention.
[0034] As used herein, "delaying" development of a viral infection
or a symptom of viral infection means to defer, hinder, slow,
retard, stabilize, and/or postpone development of the disease or
symptom when compared to not using the method(s) of the invention.
This delay can be of varying lengths of time, depending on the
history of the disease and/or individual being treated. As is
evident to one skilled in the art, a sufficient or significant
delay can, in effect, encompass prevention, in that the individual
does not develop the disease.
[0035] "Reducing severity of a symptom" or "ameliorating a symptom"
of viral infection means a lessening or improvement of one or more
symptoms of viral infection as compared to not administering an
ISS-containing polynucleotide. "Reducing severity" also includes
shortening or reduction in duration of a symptom. For respiratory
viruses, these symptoms are well known in the art and include, but
are not limited to, inflammation of respiratory mucosa, fever, body
aches, coughing, wheezing, sneezing, nasal discharge and chest
pain.
[0036] "Preventing a symptom of infection" by a respiratory virus
means that the symptom does not appear after exposure to the virus.
Examples of symptoms have been described above.
[0037] "Reducing duration of viral infection" means the length of
time of viral infection (usually indicated by symptoms) is reduced,
or shortened, as compared to not administering an ISS-containing
polynucleotide.
[0038] The term "infected individual", as used herein, refers to an
individual who has been infected by a respiratory virus. Symptoms
of respiratory virus infection are well known in the art and have
been described herein.
[0039] A "biological sample" encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived therefrom, and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as proteins or polynucleotides. The term
"biological sample" encompasses a clinical sample, and also
includes cells in culture, cell supernatants, cell lysates, serum,
plasma, biological fluid, and tissue samples.
[0040] "Viral titer" is a term well known in the art and indicates
the amount of virus in a given biological sample. "Viremia" is a
term well-known in the art as the presence of virus in the blood
stream and/or viral titer in a blood or serum sample. Amount of
virus are indicated by various measurements, including, but not
limited to, amount of viral nucleic acid; presence of viral
particles; replicating units (RU); plaque forming units (PFU).
Generally, for fluid samples such as blood and urine, amount of
virus is determined per unit fluid, such as milliliters. For solid
samples such as tissue samples, amount of virus is determined per
weight unit, such as grams. Methods for determining amount of virus
are known in the art and described herein.
[0041] An "individual" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to,
humans, farm animals, sport animals, rodents, primates and certain
pets. Vertebrates also include, but are not limited to, birds
(i.e., avian individuals) and reptiles (i.e., reptilian
individuals).
[0042] The term "ISS" as used herein refers to polynucleotide
sequences that effect a measurable immune response as measured in
vitro, in vivo and/or ex vivo. Examples of measurable immune
responses include, but are not limited to, antigen-specific
antibody production, secretion of cytokines, activation or
expansion of lymphocyte populations such as NK cells, CD4.sup.+ T
lymphocytes, CD8.sup.+ T lymphocytes, B lymphocytes, and the like.
Preferably, the ISS sequences preferentially activate a Th1-type
response. A polynucleotide for use in methods of the invention
contains at least one ISS.
[0043] As used interchangeably herein, the terms "polynucleotide"
and "oligonucleotide" include single-stranded DNA (ssDNA),
double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and
double-stranded RNA (dsRNA), modified oligonucleotides and
oligonucleosides or combinations thereof. The oligonucleotide can
be linearly or circularly configured, or the oligonucleotide can
contain both linear and circular segments.
[0044] "Adjuvant" refers to a substance which, when added to an
immunogenic agent such as antigen, nonspecifically enhances or
potentiates an immune response to the agent in the recipient host
upon exposure to the mixture.
[0045] An "effective amount" or a "sufficient amount" of a
substance is an amount sufficient to effect beneficial or desired
results, including clinical results. An effective amount can be
administered in one or more administrations. A "therapeutically
effective amount" is an amount to effect beneficial clinical
results, including, but not limited to, alleviation of one or more
symptoms associated with viral infection as well as prevention of
disease (e.g., prevention of one or more symptoms of
infection).
[0046] A microcarrier is considered "biodegradable" if it is
degradable or erodable under normal mammalian physiological
conditions. Generally, a microcarrier is considered biodegradable
if it is degraded (i.e., loses at least 5% of its mass or average
and/or length) after a 72 hour incubation at 37.degree. C. in
normal human serum. Conversely, a microcarrier is considered
"nonbiodegradable" if it is not degraded or eroded under normal
mammalian physiological conditions. Generally, a microcarrier is
considered nonbiodegradable if it not degraded (i.e., loses less
than 5% of its mass and/or average polymer length) after at 72 hour
incubation at 37.degree. C. in normal human serum.
[0047] The term "immunostimulatory sequence-microcarrier complex"
or "ISS-MC complex" refers to a complex of an ISS-containing
polynucleotide and a microcarrier. The components of the complex
may be covalently or non-covalently linked. Non-covalent linkages
may be mediated by any non-covalent bonding force, including by
hydrophobic interaction, ionic (electrostatic) bonding, hydrogen
bonds and/or van der Waals attractions. In the case of hydrophobic
linkages, the linkage is generally via a hydrophobic moiety (e.g.,
cholesterol) covalently linked to the ISS.
[0048] As used herein, the term "comprising" and its cognates are
used in their inclusive sense; that is, equivalent to the term
"including" and its corresponding cognates.
[0049] As used herein, the singular form "a", "an", and "the"
includes plural references unless indicated otherwise. For example,
"a" symptom of viral infection includes one or more additional
symptoms.
Methods of the Invention
[0050] The invention provides methods of ameliorating (i.e.,
reducing severity) and/or preventing symptoms of respiratory virus
infection as well as methods of suppression of infection by a
respiratory virus which entail administering an ISS-containing
polynucleotide (used interchangeably herein with "ISS") to an
individual without administering a respiratory virus antigen. An
ISS-containing composition which does not include a respiratory
virus antigen is administered to an individual at risk of exposure
to, exposed to, infected with, and/or exhibiting symptoms of
infection by a respiratory virus. Individuals receiving ISS are
preferably mammal, more preferably human. In accordance with the
invention, respiratory virus antigen is not administered to the
individual in conjunction with administration of an ISS (i.e., is
not administered in a separate administration at or about the time
of administration of the ISS).
[0051] The respiratory virus may be any virus that infects the
respiratory tract, including, but not limited to, influenza,
respiratory syncytial virus (RSV), parainfluenza type 3 (PIV-3),
adenovirus, rhinovirus, coronavirus, SARS-associated coronavirus,
measles virus, mumps virus, other parainfluenza virus, rubella
virus, poxvirus, parvovirus, hantavirus, varicella virus,
paramyxovirus and myxovirus. In some embodiments, the respiratory
virus is RSV. In some embodiments, the respiratory virus is other
than influenza. In some embodiments, the respiratory virus is
SARS-associated coronavirus.
[0052] In some embodiments, the individual is at risk of being
exposed to virus. Determination of an at risk individual is based
on one or more factors that are associated with disease development
and are generally known by, or can be assessed by, a skilled
clinician. At risk individuals may be especially suitable
candidates to receive ISS, as these individuals are generally
considered to be particularly susceptible to developing symptoms of
infection, which could also further lead to other complications.
For example, in the context of RSV infection, age groups of about 2
years or less and the elderly would be considered at risk. Other
individuals at risk include those that are in close proximity to
individuals who may be infected including, but not limited to,
health care workers, and individuals in institutions such as
hospitals, schools, day care facilities, military facilities,
nursing homes and convalescent homes. Other examples of at risk
individuals are those who are immunocompromised. In the context of
SARS-associated coronavirus infection, individuals at risk include
those that are in close proximity to individuals who may be
infected including, but not limited to, health care workers and
household members (Ksiazek et al., "A novel coronavirus associated
with severe acute respiratory syndrome," N. Engl. J. Med.,
published at www.nejm.org on Apr. 10, 2003).
[0053] In the context of influenza infection, generally the elderly
(for example, aged 60 years and older) are considered at risk,
although, as noted above, many other conditions put an individual
at risk for this infection, such as during what is generally
denoted "flu season" (November through March); conditions which
involve significant contact with other people, such as family
members of infected people and in office buildings, schools,
airplanes, hospitals, schools, day care facilities, military
facilities, nursing homes and convalescent homes. RSV season
generally occurs during winter and early spring. The same general
principles apply to the rhinovirus context.
[0054] In other embodiments, the individual is, or has been,
exposed to and/or infected by virus. Exposure to virus is generally
indicated by sufficient contact with an infected individual or
infected location. Exposure can also be indicated by development of
one or more symptoms associated with viral infection. Infection by
virus may be indicated by any of the above, as well as detection of
virus or anti-virus antibodies (i.e., the individual becomes
seropositive) in a biological sample from the individual.
[0055] ISS
[0056] The methods of this invention entail administering a
polynucleotide comprising an ISS (or a composition comprising such
a polynucleotide). In accordance with the present invention, the
immunomodulatory polynucleotide contains at least one ISS, and can
contain multiple ISSs. The ISSs can be adjacent within the
polynucleotide, or they can be separated by additional nucleotide
bases within the polynucleotide. Alternately, multiple ISSs may be
delivered as individual polynucleotides.
[0057] ISS have been described in the art and may be readily
identified using standard assays which indicate various aspects of
the immune response, such as cytokine secretion, antibody
production, NK cell activation and T cell proliferation. See, e.g.,
WO 97/28259; WO 98/16247; WO 99/11275; Krieg et al. (1995);
Yamamoto et al. (1992); Ballas et al. (1996); Klinman et al.
(1997); Sato et al. (1996); Pisetsky (1996a); Shimada et al. (1986)
Jpn. J. Cancer Res. 77:808-816; Cowdery et al. (1996) J. Immunol.
156:4570-4575; Roman et al. (1997); and Lipford et al. (1997a).
[0058] The ISS can be of any length greater than 6 bases or base
pairs and generally comprises the sequence 5'-cytosine, guanine-3',
preferably greater than 15 bases or base pairs, more preferably
greater than 20 bases or base pairs in length. As is well-known in
the art, the cytosine of the 5'-cytosine, guanine-3' sequence is
unmethylated. An ISS may also comprise the sequence 5'-purine,
purine, C, G, pyrimidine, pyrimidine, C, G-3'. An ISS may also
comprise the sequence 5'-purine, purine, C, G, pyrimidine,
pyrimidine, C, C-3'. As indicated in polynucleotide sequences
below, an ISS may comprise (i.e., contain one or more of) the
sequence 5'-T, C, G-3'. In some embodiments, an ISS may comprise
the sequence 5'-C, G, pyrimidine, pyrimidine, C, G-3' (such as
5'-CGTTCG-3'). In some embodiments, an ISS may comprise the
sequence 5'-C, G, pyrimidine, pyrimidine, C, G, purine, purine-3'.
In some embodiments, an ISS comprises the sequence 5'-purine,
purine, C, G, pyrimidine, pyrimidine-3' (such as 5'-AACGTT-3').
[0059] In some embodiments, an ISS may comprise the sequence
5'-purine, T, C, G, pyrimidine, pyrimidine-3'.
[0060] In some embodiments, an ISS-containing polynucleotide is
less than about any of the following lengths (in bases or base
pairs): 10,000; 5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300;
250; 200; 175; 150; 125; 100; 75; 50; 25; 10. In some embodiments,
an ISS-containing polynucleotide is greater than about any of the
following lengths (in bases or base pairs): 8; 10; 15; 20; 25; 30;
40; 50; 60; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500;
750; 1000; 2000; 5000; 7500; 10000; 20000; 50000. Alternately, the
ISS can be any of a range of sizes having an upper limit of 10,000;
5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300; 250; 200; 175;
150; 125; 100; 75; 50; 25; or 10 and an independently selected
lower limit of 8; 10; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125;
150; 175; 200; 250; 300; 350; 400; 500; 750; 1000; 2000; 5000;
7500, wherein the lower limit is less than the upper limit.
[0061] In some embodiments, the ISS comprises any of the following
sequences: GACGCTCC; GACGTCCC; GACGTTCC; GACGCCCC; AGCGTTCC;
AGCGCTCC; AGCGTCCC; AGCGCCCC; AACGTCCC; AACGCCCC; AACGTTCC;
AACGCTCC; GGCGTTCC; GGCGCTCC; GGCGTCCC; GGCGCCCC; GACGCTCG;
GACGTCCG; GACGCCCG; GACGTTCG; AGCGCTCG; AGCGTTCG; AGCGTCCG;
AGCGCCCG; AACGTCCG; AACGCCCG; AACGTTCG; AACGCTCG; GGCGTTCG;
GGCGCTCG; GGCGTCCG; GGCGCCCG. In some embodiments, the
immunomodulatory polynucleotide comprises the sequence
5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1).
[0062] In some embodiments, the ISS comprises any of the following
sequences: GACGCU; GACGUC; GACGUU; GACGUT; GACGTU; AGCGUU; AGCGCU;
AGCGUC; AGCGUT; AGCGTU; AACGUC; AACGUU; AACGCU; AACGUT; AACGTU;
GGCGUU; GGCGCU; GGCGUC; GGCGUT; GGCGTU.
[0063] In some embodiments, the ISS comprises any of the following
sequences: GABGCTCC; GABGTCCC; GABGTTCC; GABGCCCC; AGBGTTCC;
AGBGCTCC; AGBGTCCC; AGBGCCCC; AABGTCCC; AABGCCCC; AABGTTCC;
AABGCTCC; GGBGTTCC; GGBGCTCC; GGBGTCCC; GGBGCCCC; GABGCTCG;
GABGTCCG; GABGCCCG; GABGTTCG; AGBGCTCG; AGBGTTCG; AGBGTCCG;
AGBGCCCG; AABGTCCG; AABGCCCG; AABGTTCG; AABGCTCG; GGBGTTCG;
GGBGCTCG; GGBGTCCG; GGBGCCCG; GABGCTBG; GABGTCBG; GABGCCBG;
GABGTTBG; AGBGCTBG; AGBGTTBG; AGBGTCBG; AGBGCCBG; AABGTCBG;
AABGCCBG; AABGTTBG; AABGCTBG; GGBGTTBG; GGBGCTBG; GGBGTCBG;
GGBGCCBG, where B is 5-bromocytosine.
[0064] In some embodiments, the ISS comprises any of the following
sequences: GABGCUCC; GABGUCCC; GABGUTCC; GABGTUCC; GABGUUCC;
AGBGUUCC; AGBGTUCC; AGBGUTCC; AGBGCUCC; AGBGUCCC; AABGUCCC;
AABGUUCC; AABGUTCC; AABGTUCC; AABGCUCC; GGBGUUCC; GGBGUTCC;
GGBGTUCC; GGBGCUCC; GGBGUCCC; GABGCUCG; GABGUCCG; GABGUUCG;
GABGUTCG; GABGTUCG; AGBGCUCG; AGBGUUCG; AGBGUTCG; AGBGTUCG;
AGBGUCCG; AABGUCCG; AABGUUCG; AABGUTCG; AABGTUCG; AABGCUCG;
GGBGUUCG; GGBGUTCG; GGBGTUCG; GGBGCUCG; GGBGUCCG; GABGCUBG;
GABGUCBG; GABGUUBG; GABGUTBG; GABGTUBG; AGBGCUBG; AGBGUUBG;
AGBGUCBG; AGBGUTBG; AGBGTUBG; AABGUCBG; AABGUUBG; AABGUTBG;
AABGTUBG; AABGCUBG; GGBGUUBG; GGBGUTBG; GGBGTUBG; GGBGCUBG;
GGBGUCBG, where B is 5-bromocytosine.
[0065] In other embodiments, the ISS comprises any of the
sequences: 5'-TGACCGTGAACGTTCGAGATGA-3' (SEQ ID NO:2);
5'-TCATCTCGAACGTTCCACAGTCA-3' (SEQ ID NO:3);
5'-TGACTGTGAACGTTCCAGATGA-3' (SEQ ID NO:4);
5'-TCCATAACGTTCGCCTAACGTTCGTC-3' (SEQ ID NO:5);
5'-TGACTGTGAABGTTCCAGATGA- -3' (SEQ ID NO:6), where B is
5-bromocytosine; 5'-TGACTGTGAABGTTCGAGATGA-3- ' (SEQ ID NO:7),
where B is 5-bromocytosine and 5'-TGACTGTGAABGTTBGAGATGA-- 3' (SEQ
ID NO:8), where B is 5-bromocytosine.
[0066] An ISS and/or ISS-containing polynucleotide may contain
modifications. Modifications of ISS include any known in the art,
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. Various such
modifications are described below.
[0067] An ISS may be single stranded or double stranded DNA, as
well as single or double-stranded RNA or other modified
polynucleotides. An ISS may or may not include one or more
palindromic regions, which may be present in the motifs described
above or may extend beyond the motif. An ISS may comprise
additional flanking sequences, some of which are described herein.
An ISS may contain naturally-occurring or modified, non-naturally
occurring bases, and may contain modified sugar, phosphate, and/or
termini. For example, phosphate modifications include, but are not
limited to, methyl phosphonate, phosphorothioate, phosphoramidate
(bridging or non-bridging), phosphotriester and phosphorodithioate
and may be used in any combination. Other non-phosphate linkages
may also be used. Preferably, oligonucleotides of the present
invention comprise phosphorothioate backbones. Sugar modifications
known in the field, such as 2'-alkoxy-RNA analogs, 2'-amino-RNA
analogs and 2'-alkoxy- or amino-RNA/DNA chimeras and others
described herein, may also be made and combined with any phosphate
modification. Examples of base modifications include, but are not
limited to, addition of an electron-withdrawing moiety to C-5
and/or C-6 of a cytosine of the ISS (e.g., 5-bromocytosine,
5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine).
[0068] The ISS can be synthesized using techniques and nucleic acid
synthesis equipment which are well known in the art including, but
not limited to, enzymatic methods, chemical methods, and the
degradation of larger oligonucleotide sequences. See, for example,
Ausubel et al. (1987); and Sambrook et al. (1989). When assembled
enzymatically, the individual units can be ligated, for example,
with a ligase such as T4 DNA or RNA ligase. U.S. Pat. No.
5,124,246. Oligonucleotide degradation can be accomplished through
the exposure of an oligonucleotide to a nuclease, as exemplified in
U.S. Pat. No. 4,650,675.
[0069] The ISS can also be isolated using conventional
polynucleotide isolation procedures. Such procedures include, but
are not limited to, hybridization of probes to genomic or cDNA
libraries to detect shared nucleotide sequences, antibody screening
of expression libraries to detect shared structural features and
synthesis of particular native sequences by the polymerase chain
reaction.
[0070] Circular ISS can be isolated, synthesized through
recombinant methods, or chemically synthesized. Where the circular
ISS is obtained through isolation or through recombinant methods,
the ISS will preferably be a plasmid. The chemical synthesis of
smaller circular oligonucleotides can be performed using any method
described in the literature. See, for instance, Gao et al. (1995)
Nucleic Acids Res. 23:2025-2029; and Wang et al. (1994) Nucleic
Acids Res. 22:2326-2333.
[0071] The techniques for making oligonucleotides and modified
oligonucleotides are known in the art. Naturally occurring DNA or
RNA, containing phosphodiester linkages, is generally synthesized
by sequentially coupling the appropriate nucleoside phosphoramidite
to the 5'-hydroxy group of the growing oligonucleotide attached to
a solid support at the 3'-end, followed by oxidation of the
intermediate phosphite triester to a phosphate triester. Once the
desired oligonucleotide sequence has been synthesized, the
oligonucleotide is removed from the support, the phosphate triester
groups are deprotected to phosphate diesters and the nucleoside
bases are deprotected using aqueous ammonia or other bases. See,
for example, Beaucage (1993) "Oligodeoxyribonucleotide Synthesis"
in Protocols for Oligonucleotides and Analogs, Synthesis and
Properties (Agrawal, ed.) Humana Press, Totowa, N.J.; Warner et al.
(1984) DNA 3:401 and U.S. Pat. No. 4,458,066.
[0072] The ISS can also contain phosphate-modified
oligonucleotides. Synthesis of polynucleotides containing modified
phosphate linkages or non-phosphate linkages is also know in the
art. For a review, see Matteucci (1997) "Oligonucleotide Analogs:
an Overview" in Oligonucleotides as Therapeutic Agents, (D. J.
Chadwick and G. Cardew, ed.) John Wiley and Sons, New York, N.Y.
The phosphorous derivative (or modified phosphate group) which can
be attached to the sugar or sugar analog moiety in the
oligonucleotides of the present invention can be a monophosphate,
diphosphate, triphosphate, alkylphosphonate, phosphorothioate,
phosphorodithioate or the like. The preparation of the above-noted
phosphate analogs, and their incorporation into nucleotides,
modified nucleotides and oligonucleotides, per se, is also known
and need not be described here in detail. Peyrottes et al. (1996)
Nucleic Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucleic
Acids Res. 24:2318-2323; and Schultz et al. (1996) Nucleic Acids
Res. 24:2966-2973. For example, synthesis of phosphorothioate
oligonucleotides is similar to that described above for naturally
occurring oligonucleotides except that the oxidation step is
replaced by a sulfurization step (Zon (1993) "Oligonueleoside
Phosphorothioates" in Protocols for Oligonucleotides and Analogs,
Synthesis and Properties (Agrawal, ed.) Humana Press, pp. 165-190).
Similarly the synthesis of other phosphate analogs, such as
phosphotriester (Miller et al. (1971) JACS 93:6657-6665),
non-bridging phosphoramidates (Jager et al. (1988) Biochem.
27:7247-7246), N3' to P5' phosphoramidates (Nelson et al. (1997)
JOC 62:7278-7287) and phosphorodithioates (U.S. Pat. No. 5,453,496)
has also been described. Other non-phosphorous based modified
oligonucleotides can also be used (Stirchak et al. (1989) Nucleic
Acids Res. 17:6129-6141). Oligonucleotides with phosphorothioate
backbones can be more immunogenic than those with phosphodiester
backbones and appear to be more resistant to degradation after
injection into the host. Braun et al. (1988) J. Immunol.
141:2084-2089; and Latimer et al. (1995) Mol. Immunol.
32:1057-1064.
[0073] ISS-containing polynucleotides used in the invention can
comprise ribonucleotides (containing ribose as the only or
principal sugar component), deoxyribonucleotides (containing
deoxyribose as the principal sugar component), or, as is known in
the art, modified sugars or sugar analogs can be incorporated in
the ISS. Thus, in addition to ribose and deoxyribose, the sugar
moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose,
arabinose, xylose, lyxose, and a sugar "analog" cyclopentyl group.
The sugar can be in pyranosyl or in a furanosyl form. In the ISS,
the sugar moiety is preferably the furanoside of ribose,
deoxyribose, arabinose or 2'-O-alkylribose, and the sugar can be
attached to the respective heterocyclic bases either in a or .beta.
anomeric configuration. Sugar modifications include, but are not
limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs and
2'-alkoxy- or amino-RNA/DNA chimeras. The preparation of these
sugars or sugar analogs and the respective "nucleosides" wherein
such sugars or analogs are attached to a heterocyclic base (nucleic
acid base) per se is known, and need not be described here, except
to the extent such preparation can pertain to any specific example.
Sugar modifications may also be made and combined with any
phosphate modification in the preparation of an ISS.
[0074] The heterocyclic bases, or nucleic acid bases, which are
incorporated in 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.
[0075] 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 in the art, and that as long as other criteria of the
present invention are satisfied, 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 includes, but is not limited to,
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-yl,
2-amino-4-oxopyrolo [2,3-d] pyrimidin-5-yl, 2-amino-4-oxopyrrolo
[2.3-d] pyrimidin-3-yl groups, where the purines are attached to
the sugar moiety of the ISS via the 9-position, the pyrimidines via
the 1-position, the pyrrolopyrimidines via the 7-position and the
pyrazolopyrimidines via the 1-position.
[0076] The ISS may comprise at least one modified base as
described, for example, in the commonly owned international
application WO 99/62923. As used herein, the term "modified base"
is synonymous with "base analog", for example, "modified cytosine"
is synonymous with "cytosine analog." Similarly, "modified"
nucleosides or nucleotides are herein defined as being synonymous
with nucleoside or nucleotide "analogs." Examples of base
modifications include, but are not limited to, addition of an
electron-withdrawing moiety to C-5 and/or C-6 of a cytosine of the
ISS. Preferably, the electron-withdrawing moiety is a halogen. Such
modified cytosines can include, but are not limited to,
azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine,
chlorinated cytosine, cyclocytosine, cytosine arabinoside,
5-fluorocytosine, fluoropyrimidine, fluorouracil,
5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil,
5-nitrocytosine, uracil, and any other pyrimidine analog or
modified pyrimidine.
[0077] The preparation of base-modified nucleosides, and the
synthesis of modified oligonucleotides using said base-modified
nucleosides as precursors, has been described, for example, in U.S.
Pat. Nos. 4,910,300, 4,948,882, and 5,093,232. These base-modified
nucleosides have been designed so that they can be incorporated by
chemical synthesis into either terminal or internal positions of an
oligonucleotide. Such base-modified nucleosides, present at either
terminal or internal positions of an oligonucleotide, can serve as
sites for attachment of a peptide or other antigen. Nucleosides
modified in their sugar moiety have also been described (including,
but not limited to, e.g., U.S. Pat. Nos. 4,849,513, 5,015,733,
5,118,800, 5,118,802) and can be used similarly.
[0078] The ISS used in the methods of the invention may be produced
as ISS-microcarrier complexes. ISS-microcarrier complexes comprise
an ISS-containing polynucleotide bound to a microcarrier (MC).
ISS-MC complexes comprise an ISS bound to the surface of a
microcarrier (i.e., the ISS is not encapsulated in the MC),
adsorbed within a microcarrier (e.g., adsorbed to PLGA beads), or
encapsulated within a MC (e.g., incorporated within liposomes).
[0079] ISS-containing oligonucleotides bound to microparticles
(SEPHAROSE.RTM. beads) have previously been shown to have
immunostimulatory activity in vitro (Liang et al., (1996), J. Clin.
Invest. 98:1119-1129). However, recent results show that
ISS-containing oligonucleotides bound to gold, latex and magnetic
particles are not active in stimulating proliferation of 7TD1
cells, which proliferate in response to ISS-containing
oligonucleotides (Manzel et al., (1999), Antisense Nucl. Acid Drug
Dev. 9:459-464).
[0080] Microcarriers are not soluble in pure water, and are less
than about 50-60 .mu.m in size, preferably less than about 10 .mu.m
in size, more preferably from about 10 nm to about 10 .mu.m, 25 nm
to about 5 .mu.m, 50 nm to about 4.5 .mu.m or 1.0 .mu.m to about
2.0 .mu.m in size. Microcarrers may be any shape, such as
spherical, ellipsoidal, rod-shaped, and the like, although
spherical microcarriers are normally preferred. Preferred
microcarriers have sizes of or about 50 nm, 200 nm, 1 .mu.m, 1.2
.mu.m, 1.4 .mu.m, 1.5 .mu.m, 1.6 .mu.m, 1.8 .mu.m, 2.0 .mu.m, 2.5
.mu.m or 4.5 .mu.m. The "size" of a microcarier is generally the
"design size" or intended size of the particles stated by the
manufacturer. Size may be a directly measured dimension, such as
average or maximum diameter, or may be determined by an indirect
assay such as a filtration screening assay. Direct measurement of
microcarrier size is typically carried out by microscopy, generally
light microscopy or scanning electron microscopy (SEM), in
comparison with particles of known size or by reference to a
micrometer. As minor variations in size arise during the
manufacturing process, microcarriers are considered to be of a
stated size if measurements show the microcarriers are .+-. about
5-10% of the stated measurement. Size characteristics may also be
determined by dynamic light scattering. Alternately, microcarrier
size may be determined by filtration screening assays. A
microcarrier is less than a stated size if at least 97% of the
particles pass through a "screen-type" filter (i.e., a filter in
which retained particles are on the surface of the filter, such as
polycarbonate or polyethersulfone filters, as opposed to a "depth
filter" in which retained particles lodge within the filter) of the
stated size. A microcarrier is larger than a stated size if at
least about 97% of the microcarrier particles are retained by a
screen-type filter of the stated size. Thus, at least about 97%
microcarriers of about 10 .mu.m to about 10 nm in size pass through
a 10 .mu.m pore screen filter and are retained by a 10 nm screen
filter.
[0081] As above discussion indicates, reference to a size or size
range for a microcarrier implicitly includes approximate variations
and approximations of the stated size and/or size range. This is
reflected by use of the term "about" when referring to a size
and/or size range, and reference to a size or size range without
reference to "about" does not mean that the size and/or size range
is exact.
[0082] Microcarriers may be solid phase (e.g., polystyrene beads)
or liquid phase (e.g., liposomes, micelles, or oil droplets in an
oil and water emulsion). Liquid phase microcarriers include
liposomes, micelles, oil droplets and other lipid or oil-based
particles. One preferred liquid phase microcarrier is oil droplets
within an oil-in-water emulsion. Preferably, oil-in-water emulsions
used as microcarriers comprise biocompatible substituents such as
squalene. Liquid phase microcarriers are normally considered
nonbiodegradable, but may be biodegradable liquid phase
microcarriers may be produced by incorporation of one or more
biodegradable polymers in the liquid microcarrier formulation. In
one preferred embodiment, the microcarrier is oil droplets in an
oil-in-water emulsion prepared by emulsification of squalene,
sorbitan trioleate, TWEEN 80.RTM. in an aqueous pH buffer.
[0083] Solid phase microcarriers for use in ISS-microcarrier
complexes may be made from biodegradable materials or
nonbiodegradable materials, and may include or exclude agarose or
modified agarose microcarriers. Useful solid phase biodegradable
microcarriers include, but are not limited to: biodegradable
polyesters, such as poly(lactic acid), poly(glycolic acid), and
copolymers (including block copolymers) thereof, as well as block
copolymers of poly(lactic acid) and poly(ethylene glycol);
polyorthoesters such as polymers based on
3,9-diethylidene-2,4,8,10-tetra- oxaspiro[5.5]undecane (DETOSU);
polyanhydrides such as poly(anhydride) polymers based on sebacic
acid, p-(carboxyphenoxy)propane, or p-(carboxyphenoxy)hexane;
polyanhydride imides, such as polyanhydride polymers based on
sebacic acid-derived monomers incorporating amino acids (i.e.,
linked to sebacic acid by imide bonds through the amino-terminal
nitrogen) such as glycine or alanine; polyanhydride esters;
polyphosphazenes, especially poly(phosphazenes) which contain
hydrolysis-sensitive ester groups which can catalyze degradation of
the polymer backbone through generation of carboxylic acid groups
(Schacht et al. (1996) Biotechnol. Bioeng. 1996:102); and
polyamides such as poly(lactic acid-co-lysine). A wide variety of
nonbiodegradable materials suitable for manufacturing microcarriers
are also known, including, but not limited to polystyrene,
polyethylene, latex, gold, and ferromagnetic or paramagnetic
materials. Solid phase microcarriers may be covalently modified to
incorporate one or more moieties for use in linking the ISS, for
example by addition of amine groups for covalent linking using
amine-reactive crosslinkers.
[0084] The ISS-microcarrier complexes of the invention may be
covalently or non-covalently linked. Covalently linked ISS-MC
complexes may be directly linked or be linked by a crosslinking
moiety of one or more atoms (typically the residue of a
crosslinking agent). The ISS may be modified to allow or augment
binding to the MC (e.g., by incorporation of a free sulfhydryl for
covalent crosslinking or addition of a hydrophobic moieties such as
lipids, steroids, sterols such as cholesterol, and terpenes, for
hydrophobic bonding), although unmodified ISS may be used for
formation of non-covalent ISS-MC complex formation by electrostatic
interaction or by base pairing (e.g., by base pairing at least one
portion of the ISS with a complementary oligonucleotide bound to
the microcarrier). ISS-containing polynucleotides may be linked to
solid phase microcarriers or other chemical moieties to facilitate
ISS-MC complex formation using conventional technology known in the
art, such as use of available heterobifunctional crosslinkers
(e.g., succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
or its sulfo-derivatives for covalently linking an
amine-derivatized microcarrier and an ISS modified to contain a
free sulfhydryl) or by addition of compounds such as cholesterol
(e.g., by the method of Godard et al. (1995) Eur. J. Biochem.
232:404-410) to facilitate binding to hydrophobic microcarriers
such as oil droplets in oil-in-water emulsions. Alternatively,
modified nucleosides or nucleotides, such as known in the art, can
be incorporated at either terminus, or at internal positions in the
ISS. These can contain blocked functional groups which, when
deblocked, are reactive with a variety of functional groups which
can be present on, or attached to, the microcarrier or a moiety
which would facilitate binding to a microcarrier. Certain
embodiments of noncovalently linked ISS-MC complexes utilize a
binding pair (e.g., an antibody and its cognate antigen or biotin
and streptavidin or avidin), where one member of the binding pair
is bound to the ISS and the microcarrier is derivatized with the
other member of the binding pair (e.g., a biotinylated ISS and a
streptavidin-derivatized microcarrier may be combined to form a
noncovalently linked ISS-MC complex).
[0085] Non-covalent ISS-MC complexes bound by electrostatic binding
typically exploit the highly negative charge of the polynucleotide
backbone. Accordingly, microcarriers for use in non-covalently
bound ISS-MC complexes are generally positively charged at
physiological pH (e.g., about pH 6.8-7.4). The microcarrier may
intrinsically possess a positive charge, but microcarriers made
from compounds not normally possessing a positive charge may be
derivatized or otherwise modified to become positively charged. For
example, the polymer used to make the microcarrier may be
derivatized to add positively charged groups, such as primary
amines. Alternately, positively charged compounds may be
incorporated in the formulation of the microcarrier during
manufacture (e.g., positively charged surfactants may be used
during the manufacture of poly(lactic acid)/poly(glycolic acid)
copolymers to confer a positive charge on the resulting
microcarrier particles.
[0086] Solid phase microspheres are prepared using techniques known
in the art. For example, they can be prepared by emulsion-solvent
extraction/evaporation technique. Generally, in this technique,
biodegradable polymers such as polyanhydrates,
poly(alkyl-.alpha.-cyanoac- rylates) and poly(.alpha.-hydroxy
esters), for example, poly(lactic acid), poly(glycolic acid),
poly(D,L-lactic-co-glycolic acid) and poly(caprolactone), are
dissolved in a suitable organic solvent, such as methylene
chloride, to constitute the dispersed phase (DP) of emulsion. DP is
emulsified by high-speed homogenization into excess volume of
aqueous continuous phase (CP) that contains a dissolved surfactant,
for example, polyvinylalcohol (PVA) or polyvinylpirrolidone (PVP).
Surfactant in CP is to ensure the formation of discrete and
suitably-sized emulsion droplet. The organic solvent is then
extracted into the CP and subsequently evaporated by raising the
system temperature. The solid microparticles are then separated by
centrifugation or filtration, and dried, for example, by
lyophilization or application of vaccum, before storing at
4.degree. C.
[0087] Generally, to prepare cationic microspheres, cationic lipids
or polymers, for example,
1,2-dioleoyl-1,2,3-trimethylammoniopropane (DOTAP),
cetyltrimethylammonium bromide (CTAB) or polylysine, are added
either to DP or CP, as per their solubility in these phases.
[0088] Physico-chemical characteristics such as mean size, size
distribution and surface charge of dried microspheres may be
determined. Size characteristics are determined, for example, by
dynamic light scattering technique and the surface charge was
determined by measuring the zeta potential.
[0089] Generally, ISS-containing polynucleotides can be adsorbed
onto the cationic microspheres by overnight aqueous incubation of
ISS and the particles at 4.degree. C. Microspheres are
characterized for size and surface charge before and after ISS
association. Selected batches may then evaluated for activity as
described herein.
[0090] Administration
[0091] An ISS-containing polynucleotide may be administered before,
during and/or after exposure to a respiratory virus. An ISS
polynucleotide may also be administered before, during and/or after
infection by a respiratory virus. An ISS polynucleotide may also be
administered before or after onset of symptoms of respiratory virus
infection. Accordingly, administration of ISS-containing
polynucleotide may be at various times with respect to exposure to,
infection by and/or onset of symptoms by infection by virus.
Further, there may be one or more administrations. If the
ISS-containing polynucleotide is administered on multiple
occasions, the ISS may be administered on any schedule selected by
the clinician, such as daily, every other day, every three days,
every four days, every five days, every six days, weekly, biweekly,
monthly or at ever longer intervals (which may or may not remain
the same during the course of treatment). Where multiple
administrations are given, the ISS-containing polynucleotide may be
given in 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separate
administrations. Generally, but not necessarily, an interval of at
least about three days is necessary to allow effect of
ISS-containing polynucleotides.
[0092] When ISS-containing polynucleotide is administered to an
individual at risk of exposure to virus (i.e., before infection),
ISS-containing polynucleotide is preferably administered less than
about 14 days before exposure to virus, preferably less than about
10 days before exposure to virus, more preferably less than about 7
days before exposure to virus, even more preferably less than about
5 days before exposure to virus. In some embodiments,
ISS-containing polynucleotide is administered about 3 days before
exposure to virus.
[0093] In a further embodiment, the ISS-containing polynucleotide
is administered after exposure to a respiratory virus, but prior to
appearance of symptoms. Preferably, the ISS-containing
polynucleotide is administered less than about three days after
exposure, more preferably less than about one day, 12 hours, six
hours or two hours after exposure, if the time of exposure is known
or suspected.
[0094] In another embodiment, the ISS-containing polynucleotide is
administered after appearance of at least one symptom of
respiratory virus infection. Preferably, ISS-containing
polynucleotide is administered within about 28, 21, 14, 7, 5 or 3
days following appearance of a symptom of respiratory virus
infection. However, some infected individuals exhibiting symptoms
will already have undertaken one or more courses of treatment with
another therapy. In such individuals, or in individuals who failed
to appreciate the import of their symptoms, the ISS-containing
polynucleotide may be administered at any point following
infection.
[0095] Additionally, treatments employing an ISS-containing
polynucleotide may also be employed in conjunction with other
treatments or as `second line` treatments employed after failure of
a `first line` treatment. Treatments for respiratory virus
infection are known in the art.
[0096] ISS polynucleotides may be formulated in any form known in
the art, such as dry powder, semi-solid or liquid formulations. For
parenteral administration ISS polynucleotides preferably
administered in a liquid formulation, although solid or semi-solid
formulations may also be acceptable, particularly where the ISS
polynucleotide is formulated in a slow release depot form. ISS
polynucleotides are generally formulated in liquid or dry powder
form for topical administration, although semi-solid formulations
may occasionally be useful.
[0097] ISS polynucleotide formulations may contain additional
components such as salts, buffers, bulking agents, osmolytes,
antioxidants, detergents, surfactants and other
pharmaceutically-acceptable excipients as are known in the art.
Generally, liquid ISS polynucleotide formulations made in USP water
for injection and are sterile, isotonic and pH buffered to a
physiologically-acceptable pH, such as about pH 6.8 to 7.5.
[0098] ISS-containing polynucleotides may be formulated in delivery
vehicles such as liposomes, oil/water emulsion or slow release
depot formulations. Methods of formulating polynucleotides in such
forms are well known in the art.
[0099] ISS-containing polynucleotide formulations may also include
or exclude immunomodulatory agents such as adjuvants and
immunostimulatory cytokines, which are well known in the art.
[0100] A suitable dosage range or effective amount is one that
provides the desired reduction of symptoms and/or suppression of
viral infection and depends on a number of factors, including the
particular respiratory virus, ISS sequence of the polynucleotide,
molecular weight of the polynucleotide and route of administration.
Dosages are generally selected by the physician or other health
care professional in accordance with a variety of parameters known
in the art, such as severity of symptoms, history of the patient
and the like. Generally, for an ISS-containing polynucleotide of
about 20 bases, a dosage range may be selected from, for example,
an independently selected lower limit such as about 0.1, 0.25, 0.5,
1, 2, 5, 10, 20, 30, 40, 50, 60, 80, 100, 200, 300, 400 or 500
.mu.g/kg up to an independently selected upper limit, greater than
the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750,
1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or
10,000 .mu.g/kg. For example, a dose may be about any of the
following: 0.1 to 100 .mu.g/kg, 0.1 to 50 .mu.g/kg, 0.1 to 25
.mu.g/kg, 0.1 to 10 .mu.g/kg, 1 to 500 .mu.g/kg, 100 to 400
.mu.g/kg, 200 to 300 .mu.g/kg, 1 to 100 .mu.g/kg, 100 to 200
.mu.g/kg, 300 to 400 .mu.g/kg, 400 to 500 .mu.g/kg, 500 to 1000
.mu.g/kg, 500 to 5000 .mu.g/kg, or 500 to 10,000 .mu.g/kg.
Generally, parenteral routes of administration may require higher
doses of ISS compared to more direct application to infected
tissue, as do ISS-containing polynucleotides of increasing
length.
[0101] Polynucleotides comprising an ISS may be administered by
systemic (e.g., parenteral) or local (e.g., topical)
administration.
[0102] In one embodiment, the ISS-containing polynucleotide(s) is
topically administered, at a site of infection, such as respiratory
mucosa (such as nasal passages or lung). Nasopharyngeal and
pulmonary routes of administration include, but are not limited to,
intranasal, inhalation, transbronchial and transalveolar routes.
The ISS-containing polynucleotide may thus be administered by
inhalation of aerosols, atomized liquids or powders. Devices
suitable for administration by inhalation of ISS-containing
compositions include, but are not limited to, nebulizers,
atomizers, vaporizers, and metered-dose inhalers. Nebulizers,
atomizers, vaporizers and metered-dose inhalers filled with or
employing reservoirs containing formulations comprising the
ISS-containing polynucleotide(s) are among a variety of devices
suitable for use in inhalation delivery of the ISS-containing
polynucleotide(s). Other methods of delivering to respiratory
mucosa include delivery of liquid formulations, such as by nose
drops.
[0103] In other embodiments, the ISS-containing polynucleotide is
administered parenterally. Parenteral routes of administration
include, but are not limited to, transdermal, transmucosal and
direct injection. Parenteral administration by injection may be by
any parenteral injection route, including, but not limited to,
intravenous (IV), intraperitoneal (IP), intramuscular (IM),
subcutaneous (SC) and intradermal (ID) routes. Transdermal and
transmucosal administration may be accomplished by, for example,
inclusion of a carrier (e.g., dimethylsulfoxide, DMSO), by
application of electrical impulses (e.g., iontophoresis) or a
combination thereof. A variety of devices are available for
transdermal administration which may be used in accordance with the
invention.
[0104] Because respiratory viruses infect cells of the respiratory
tract, routes which deliver ISS polynucleotides to the respiratory
tract, such as inhalation and intranasal delivery (discussed
above), are considered local routes of administration rather than
systemic routes of administration, even though delivery through
such routes are normally considered parenteral, systemic routes of
administration.
[0105] IV, IP, IM and ID administration may be by bolus or infusion
administration. For SC administration, administration may be by
bolus, infusion or by implantable device, such as an implantable
minipump (e.g., osmotic or mechanical minipump) or slow release
implant. The ISS polynucleotide(s) may also be delivered in a slow
release formulation adapted for IV, IP, IM, ID or SC
administration. Administration by inhalation is preferably
accomplished in discrete doses (e.g., via a metered dose inhaler),
although delivery similar to an infusion may be accomplished
through use of a nebulizer. Administration via the transdermal and
transmucosal routes may be continuous or pulsatile.
[0106] Respiratory Syncytial Virus (RSV)
[0107] If the respiratory virus is RSV, administration (at least a
first administration) preferably occurs less than about 10 days,
preferably less than about 7 days, preferably about 3 days prior to
exposure to virus. Even more preferably, administration(s) is
local, such as aerosol administration to nasal and/or bronchial
passages. The ISS containing polynucleotide may comprise the
sequence 5'-T, C, G-3'. The ISS containing polynucleotide used
preferably comprises the sequence 5'-purine, purine, C, G,
pyrimidine, pyrimidine C, G-3', more preferably comprises
5'-AACGTTCG-3', and more preferably comprises (or, alternatively,
consists of) the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID
NO:1).
[0108] Assessment
[0109] In some embodiments, administration of an ISS-containing
polynucleotide results in prevention, palliation, and/or
improvement in one or more symptoms of respiratory virus infection,
such as RSV infection. The exact form of prevention, palliation or
improvement will depend on the particular respiratory virus. In
some embodiments, administration of an ISS-containing
polynucleotide results in a reduction in viral titer (a reduction
of which indicates suppression of viral infection). In some
embodiments, duration of respiratory viral infection is reduced. In
other embodiments, viral infection is suppressed, which may be
indicated by any one or more of a number of parameters, including,
but not limited to, extent of one or more symptoms and viral
titer.
[0110] Symptoms of infection may be assessed before and/or after
administration of ISS-containing polynucleotide by the individual
or the clinician. Rhinitis, nasal mucous production, severity of
cough, myalgia, elevated body temperature, and other symptoms of
respiratory virus infection may be easily measured using simple
tests and/or scales as are known in the art.
[0111] Biological samples can be examined for evidence of virus
and/or pathological effects of virus before, during and/or after
administration of an ISS-containing polynucleotide. Biological
samples for examination include, but are not limited to, lung
tissue specimens, bronchoalveolar lavage specimens, sputum, and
upper respiratory tract swab, aspirate and wash specimens. See
Ksiazek et al. (N. Engl. J. Med., published at www.nejm.org on Apr.
10, 2003) for examples of specimens in which SARS-associated
coronavirus was identified. Viral titer may be assessed in
biological samples using standard methods of the art. Levels of
viral nucleic acid may be assessed by isolating nucleic acid from
the sample and blot analysis using a viral polynucleotide sequence
as a probe, or PCR analysis. Another assay is to test for virus
particles in the sample. Another assay is for plaque forming units
(PFU). Another assay measures virus induced cytopathic effects
(CPE), such as formation of syncytia, as is described in the
Examples. Extent or amount of viral particles may be measured from
any infected area, such as infected tissue or mucosal discharge.
When the sample is a liquid, viral titer is calculated in some
indication of number or amount of virus or virus particles (e.g.,
infectious particles, plaque forming units, infectious doses, or
median tissue culture infectious doses (TCID 50)) per unit volume.
In solid samples, such as a tissue sample, viral titer is
calculated in virus particles per unit weight. Reduction is
indicated by comparing an estimated titer (based, for example, on
animal or clinical studies) that represents untreated infection,
and/or a titer measured at an earlier timepoint, with the measured
viral titer after treatment.
[0112] Kits of the Invention
[0113] The invention provides kits for carrying out the methods of
the invention. Accordingly, a variety of kits are provided. The
kits may be used for any one or more of the following (and,
accordingly, may contain instructions for any one or more of the
following uses): reducing severity of a symptom of a respiratory
virus infection in an individual at risk of being exposed to,
exposed to or infected by a respiratory virus; suppressing
infection in an individual at risk of being exposed to, exposed to
or infected by a respiratory virus; preventing a symptom of a
respiratory virus infection in an individual at risk of being
exposed to, exposed to or infected by a respiratory virus; delaying
development of a symptom of a respiratory virus infection in an
individual at risk of being exposed to, exposed to or infected by a
respiratory virus; reducing duration of a respiratory virus
infection in an individual at risk of being exposed to, exposed to
or infected by a respiratory virus. As is understood in the art,
any one or more of these uses would be included in instructions
directed to treating or preventing a respiratory virus
infection.
[0114] The kits of the invention comprise one or more containers
comprising an ISS-containing polynucleotide and a set of
instructions, generally written instructions although electronic
storage media (e.g., magnetic diskette or optical disk) containing
instructions are also acceptable, relating to the use and dosage of
the ISS-containing polynucleotide for the intended treatment (e.g.,
reducing severity of a symptoms of a respiratory virus infection in
an individual at risk of being exposed to, exposed to, or infected
by a respiratory virus, suppressing infection in an individual at
risk of being exposed to, exposed to, or infected by a respiratory
virus, preventing a symptom of a respiratory virus infection in an
individual at risk of being exposed to, exposed to, or infected by
a respiratory virus, delaying development of a symptom of a
respiratory virus infection in an individual at risk of being
exposed to, exposed to, or infected by a respiratory virus, and/or
reducing duration of a respiratory virus infection in an individual
at risk of being exposed to, exposed to, or infected by a
respiratory virus). The instructions included with the kit
generally include information as to dosage, dosing schedule, and
route of administration for the intended treatment. The containers
of ISS-containing polynucleotide may be unit doses, bulk packages
(e.g., multi-dose vials) or sub-unit doses.
[0115] The kits of the invention do not include any packages or
containers which contain viral antigens from the respiratory
virus(es) the kit is intended to be used to treat. For example, in
a kit intended for the prevention, suppression, amelioration or
treatment of RSV, neither the container(s) comprising the
ISS-containing polynucleotide nor any other containers in the kit
contain viral antigens from the RSV.
[0116] The ISS-containing polynucleotide component of the kit may
be packaged in any convenient, appropriate packaging. For example,
if the ISS is a freeze-dried formulation, a vial with a resilient
stopper is normally used, so that the drug may be easily
reconstituted by injecting fluid through the resilient stopper.
Ampoules with non-resilient, removable closures (e.g., sealed
glass) or with resilient stoppers are most conveniently used for
injectable forms of ISS. Also, prefilled syringes may be used when
the kit is supplied with a liquid formulation of the ISS-containing
polynucleotide. The kit may contain the ISS in an ointment for
topical formulation in appropriate packaging. Also contemplated are
packages for use in combination with a specific device, such as an
inhaler, nasal administration device (e.g., an atomizer) or an
infusion device such as a minipump or transdermal administration
device.
[0117] As stated above, any ISS-containing polynucleotide described
herein may be used, such as, for example, any polynucleotide
comprising any of the following ISS: the sequence 5'-cytosine,
guanine-3', the sequence 5'-T, C, G-3', the sequence 5'-C, G,
pyrimidine, pyrimidine, C, G-3', the sequence 5'-purine, purine, C,
G, pyrimidine, pyrimidine, C, G-3', the sequence 5'-purine, purine,
C, G, pyrimidine, pyrimidine, C, C-3'; the sequence SEQ ID NO:
1018; the sequence 5'-purine, purine, B, G, pyrimidine,
pyrimidine-3' wherein B is 5-bromocytosine or the sequence
5'-purine, purine, B, G, pyrimidine, pyrimidine, C, G-3' wherein B
is 5-bromocytosine.
[0118] The following Examples are provided to illustrate, but not
limit, the invention.
EXAMPLES
Example 1
Animal Model and Experimental Methods for Respiratory Viruses
[0119] Rat Model for RSV Infection and ISS Administration
[0120] Cotton rats, 50-100 g and 4-12 weeks old (Sigmoden hispidis)
of either sex were used in these studies. All of the animals were
descendants of two pair of cotton rats obtained in 1984 from the
Small Animal Section of the Veterinary Research Branch, Division of
Research Services, National Institutes of Health.
[0121] RSV strain A2 was purchased from the ATCC (ATCC VR26).
Working stocks of this virus were prepared as described in detail
by Wyde et al. (1995) Pediatr. Res. 38:543-550. ISS sequence tested
for RSV experiments was 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1)
(phosphorothioate). Control, non-ISS sequences used were
5'-TGACTGTGAAGGTTAGAGATGA-3' (SEQ ID NO:9) (phosphorothioate) and
5'-TCACTCTCTTCCTTACTCTTCT-3' (SEQ ID NO:10) (phosphorothioate), as
well as PBS.
[0122] Assay for RSV Viral Titer
[0123] RSV levels in virus pools and lung lavage fluids (L.F.) were
determined using sterile 96-well, flat bottom tissue culture plates
(Falcon 3072), serial 3-fold dilutions and 2% FCS-MEM as described
in detail previously (Wyde et al., 1995). The wells in these assay
plates were observed for virus-induced cytopathic effects (CPE)
including formation of synctia. After the dilutions in the last
wells of replicate rows exhibiting virus-induced CPE were
determined, mean virus titers were calculated using the method of
Karber, Rhodes and Van Rhodes and Van Rooyen (1953) Textbook of
Virology (2nd ed. Williams and Wilkins pp 66-69). The amount of
virus in virus pools was expressed as a median tissue culture
infectious doses (TCID.sub.5O/ml, log.sub.10). Titers of virus in
L.F. were expressed as TCID.sub.50/g lung tissue (log.sub.10). The
minimum detectable virus concentration in these assays was 1.3
log.sub.10 TCID.sub.50/ml (virus pools) or 1.6 log.sub.10
TCID.sub.50/g lung.
Example 2
Local Administration of ISS Reduces RSV Viral Titer
[0124] These experiments were performed to test the effect of local
administration of ISS in terms of antiviral activity against
respiratory syncytial virus (RSV) in cotton rats.
[0125] On day -3 (i.e., 3 days before infection with virus), 20
cotton rats (CRs) were selected and divided into five groups of
four animals. The animals in Group 1 were lightly anesthetized and
50 .mu.L of phosphate buffered saline (PBS) was administered
intranasally (IN). The CRs in Group 2 were similarly administered
150 .mu.g of ISS (5'-TGACTGTGAACGTTCGAGATGA-3') (SEQ ID NO:1),
while the animals in Group 3 were similarly administered 150 .mu.g
of control non-ISS sequence 5'-TGACTGTGAAGGTTAGAGATGA-3' (SEQ ID
NO:9). Three days later, on Day 0, each of CRs in Group 4 were
anesthetized and 150 .mu.g of ISS was administered IN, and the
animals in Group 5 were administered, in a like manner, 150 .mu.g
of control non-ISS sequence 5'-TGACTGTGAAGGTTAGAGATGA-3- ' (SEQ ID
NO:9).
[0126] Thirty minutes later, all of the CRs were inoculated IN with
100 median tissue culture infectious doses (TCID.sub.50) of RSV A2.
Four days later (Day 4), all of the animals were sacrificed and the
lungs of each animal were removed, lavaged, and assessed for RSV
levels. A summary of the protocol is shown in Table 1. The results
are shown in FIG. 1 and Table 2.
1TABLE 1 Protocol Dose ISS Day Day ISS given ISS RSV Day CRs Group
admin. (.mu.g/CR) given given harvested End-point 1 PBS 0 Day -3
Day 0 Day 4 RSV in lung 2 ISS 150 Day -3 Day 0 Day 4 RSV in lung 3
non-ISS 150 Day -3 Day 0 Day 4 RSV in lung 4 ISS 150 Day 0 Day 0
Day 4 RSV in lung 5 non-ISS 150 Day 0 Day 0 Day 4 RSV in lung
[0127]
2TABLE 2 RSV Titers RSV titer (log.sub.10/g lung) in CR No. Group
Treatment Day given 1 2 3 4 Mean Std. Dev. 1 PBS -3 4.5 4.5 3.5 4
4.1 0.5 2 ISS -3 3 3 2.5 2.5 2.8 0.3 3 non-ISS -3 4.5 4.5 3.5 4 4.1
0.5 4 ISS 0 4 4 4.5 3 3.9 0.6 5 non-ISS 0 4.5 4 4.5 3 4.0 0.7 Using
the Kruskall-Wallis nonparametric ANOVA p = 0.061, not quite
statistically significant.
[0128] These results indicate that administration of ISS reduced
viral titer in infected tissue compared to PBS or non-ISS
administration. The results also indicate that a first
administration of ISS on the day of infection was not effective,
while administration before infection (in this experiment, 3 days)
was effective at reducing viral titers.
Example 3
Non-Local Administration of ISS and RSV Viral Titer
[0129] These experiments were performed to test the effect of
non-local administration of ISS in terms of antiviral activity
against RSV in cotton rats.
[0130] Twenty cotton rats were divided into 5 groups of 4 animals.
Administered to these animals, either intraperitoneally (IP) or
subcutaneously (SC), was PBS, immunostimulatory sequence (ISS)
5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1) or non-ISS sequence
5'-TCACTCTCTTCCTTACTCTTCT-3' (SEQ ID NO:10), each sequence at 150
.mu.g/injection. On Day 0 each of these animals was inoculated IN
with 100 TCID.sub.50 of RSV A2. Four days later each cotton rat was
sacrificed. The lungs of each animal were removed, lavaged and
assessed for RSV. The protocol is summarized in Table 3. The
results from IP administration are shown in Table 4. The results
from SC administration are shown in Table 5.
3TABLE 3 Protocol Dose ISS Day Day ISS given ISS RSV Day CRs Group
admin. (.mu.g/CR) given given Sacrificed End-point 1 PBS 0 -3, -1 0
Day 4 RSV in lung 2 ISS 150 -1 0 Day 4 RSV in lung 3 ISS 150 -3 0
Day 4 RSV in lung 4 non-ISS 150 -1 0 Day 4 RSV in lung 5 non-ISS
150 -3 0 Day 4 RSV in lung
[0131]
4TABLE 4 RSV Titers RSV titer (log.sub.10/g lung) in cotton rat no.
Treatment Day (s) Std. Group (IP) given 1 2 3 4 Mean Dev. 1 PBS -1,
-3 4.3 3.8 3.8 3.3 3.8 0.3 2 ISS -1 3.8 3.3 3.3 3.8 3.6 0.3 3 ISS
-3 3.3 3.8 3.8 3.8 3.7 0.3 4 Non-ISS -1 1.8 3.3 3.8 3.3 3.1 0.9 5
Non-ISS -3 3.3 4.3 3.3 3.3 3.6 0.5
[0132]
5TABLE 5 RSV titers RSV titer (log.sub.10/g Treatment Days lung) in
CR no. Std. Group (SC) given 1 2 3 4 Mean Dev. 1 PBS -1, -3 4 4 3.5
4 3.9 0.3 2 ISS -1 4 4.5 3.5 4 4.0 0.4 3 ISS -3 4 4.5 4 4 4.1 0.3 4
Non-ISS -1 4.5 4.5 3.5 4 4.1 0.5 5 Non-ISS -3 3.5 4 4 3.5 3.8
0.3
[0133] In each experiment, IP and SC administration of 150 .mu.g of
ISS-containing polynucleotide failed to cause a statistically
significant reduction in viral titers compared to PBS
administration.
Example 4
Local Administration of ISS and Influenza Viral Titer
[0134] These experiments were performed to test the effect of local
administration of ISS in terms of antiviral activity against
influenza virus in mice.
[0135] Thirty-five mice were divided into 5 groups of 7 animals
each. On Day -3 (relative to virus inoculation), PBS (50 .mu.l) was
administered intranasally (IN) to the animals in Group 1, while ISS
5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1) was administered IN (50
.mu.g in 50 .mu.l/mouse) to the animals in Group 2 and non-ISS
control sequence 5'-TGACTGTGAAGGTTAGAGATGA-3' (SEQ ID NO:9) was
administered IN (50 .mu.g in 50 .mu.l/mouse) to the animals in
Group 3. Three days later (Day 0), ISS (50 .mu.g/mouse) or non-ISS
control of sequence (50 .mu.g/mouse) were administered IN to the
animals in Groups 4 and 5, respectively. On day 0, 50 .mu.l of PBS
was administered IN to the animals in Group 1. Shortly after these
administrations on day 0, all of the mice were inoculated IN with
approximately 100 median tissue culture infectious doses
(TCID.sub.50) of influenza A/Mississippi (H3N2) virus. Four days
later, all of the mice were sacrificed and the lungs of each were
tested for influenza virus titer. The protocol is summarized in
Table 6. The results are summarized in Table 7. The results show
that IN administration of this dose of ISS before viral infection
fails to cause a satisfactory significant reduction in virus titer
compared to PBS administration.
6TABLE 6 Protocol Day Virus Day Test Group Treatment given inoc.
Sacrifice parameter 1 PBS -3, 0 Day 0 Day 4 Pulmonary virus 2 ISS
-3 Day 0 Day 4 titer 3 non-ISS -3 Day 0 Day 4 4 ISS 0 Day 0 Day 4 5
non-ISS 0 Day 0 Day 4
[0136]
7TABLE 7 Influenza Virus Titers Day Pulmonary virus titer
(log.sub.10/ Treat- ISS lung) in mouse no. Std. Group ment given 1
2 3 4 5 6 7 Mean Dev. 1 PBS -3, 0 3.5 4 4.5 6 4.5 4.5 4 4.4 0.8 2
ISS -3 5.5 4 6 5.5 5 4 3 4.7 1.1 3 non-ISS -3 3.5 3.5 4 3 5 5 4 4.0
0.8 4 ISS 0 4 5.5 5 4.5 4.5 4.5 4.5 4.6 0.5 5 non-ISS 0 5.5 4 4.5
4.5 6 5.5 4.5 4.9 0.7
Example 5
Non-Local Administration of ISS and Influenza Viral Titer
[0137] These experiments were performed to test the effect of
non-local administration of ISS in terms of antiviral activity
against influenza virus in mice.
[0138] Twenty-five mice were divided into 5 groups of 5 animals
each. On Day -3 (relative to virus inoculation), PBS was
administered intraperitoneally (IP) to the animals in Group 1,
while ISS 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO:1) was
administered IP (50 .mu.g/mouse) to the animals in Group 3 and
non-ISS control sequence 5'-TCACTCTCTTCCTTACTCTTCT-3' (SEQ ID
NO:10) was administered IP (50 .mu.g/mouse) to the animals in Group
5. On Day -1, ISS (50 .mu.g/mouse) or non-ISS control of sequence
(50 .mu.g/mouse) were administered IP to the animals in Groups 2
and 4, respectively. The next day (Day 0), all of the mice were
inoculated intranasally (IN) with approximately 100 median
TCID.sub.50 of influenza A/Mississippi (H3N2) virus. Four days
later, all of the mice were sacrificed and the lungs of each were
tested for influenza virus titer. The protocol is summarized in
Table 8. The results are summarized in Table 9. The results show
that IP administration of this dose of ISS before viral infection
fails to cause a satisfactory significant reduction in virus titer
compared to PBS administration.
8TABLE 8 Protocol Day Virus Day Test Group Treatment given inoc.
Sacrifice parameter 1 PBS -3, -1 Day 0 Day 4 Pulmonary virus 2 ISS
-1 Day 0 Day 4 titer 3 ISS -3 Day 0 Day 4 4 non-ISS -1 Day 0 Day 4
5 non-ISS -3 Day 0 Day 4
[0139]
9TABLE 9 Influenza Virus Titers Pulmonary virus titer Day ISS
(log.sub.10/lung) in mouse no. Group Treatment given 1 2 3 4 5 Mean
Dev. 1 None -3, -1 5.8 7.3 5.8 6.3 6.3 6.3 0.6 2 ISS -1 6.3 6.8 7.3
6.8 6.8 6.8 0.4 3 ISS -3 7.3 5.8 7.3 6.8 7.3 6.9 0.7 4 non-ISS -1
6.8 6.3 5.8 5.8 5.8 6.1 0.4 5 non-ISS -3 5.8 5.8 6.3 7.3 7.3 6.5
0.8
[0140] The present invention has been detailed both by direct
description and by example. Equivalents and modifications of the
present invention will be apparent to those skilled in the art, and
are encompassed within the scope of the invention.
* * * * *
References