U.S. patent number 7,758,876 [Application Number 10/533,634] was granted by the patent office on 2010-07-20 for method of preventing infections from bioterrorism agents with immunostimulatory cpg oligonucleotides.
This patent grant is currently assigned to N/A, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Bruce Ivins, Dennis M. Klinman, Daniela Verthelyi.
United States Patent |
7,758,876 |
Klinman , et al. |
July 20, 2010 |
Method of preventing infections from bioterrorism agents with
immunostimulatory CpG oligonucleotides
Abstract
The present disclosure relates to a method of preventing or
treating an infection caused by a bioterrorism agent, specifically
to a method of increasing an immune response to a bioterrorism
agent using an oligodeoxynucleotide including a CpG motif, and a
method of enhancing the immunogenicity of a vaccine against a
bioterrorism agent using an oligodeoxynucleotide including a CpG
motif.
Inventors: |
Klinman; Dennis M. (Potomac,
MD), Ivins; Bruce (Frederick, MD), Verthelyi; Daniela
(Potomac, MD) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
N/A (N/A)
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Family
ID: |
33434787 |
Appl.
No.: |
10/533,634 |
Filed: |
October 31, 2003 |
PCT
Filed: |
October 31, 2003 |
PCT No.: |
PCT/US03/34523 |
371(c)(1),(2),(4) Date: |
April 29, 2005 |
PCT
Pub. No.: |
WO2004/098491 |
PCT
Pub. Date: |
November 18, 2004 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060019239 A1 |
Jan 26, 2006 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60422964 |
Nov 1, 2002 |
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Current U.S.
Class: |
424/278.1 |
Current CPC
Class: |
A61P
31/20 (20180101); A61K 39/39 (20130101); A61P
43/00 (20180101); A61P 37/04 (20180101); A61K
39/07 (20130101); A61K 31/711 (20130101); A61P
31/14 (20180101); A61P 31/04 (20180101); A61P
31/10 (20180101); A61K 48/00 (20130101); A61K
31/7125 (20130101); A61K 2039/57 (20130101); A61K
2039/55561 (20130101); Y02A 50/30 (20180101); Y02A
50/407 (20180101); A61K 9/0019 (20130101); Y02A
50/396 (20180101) |
Current International
Class: |
A61K
45/00 (20060101) |
References Cited
[Referenced By]
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WO 97/28259 |
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WO 2004/005476 |
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Primary Examiner: Le; Emily M.
Attorney, Agent or Firm: Klarquist Sparkman, LLP
Parent Case Text
PRIORITY CLAIM
This application is a .sctn.371 U.S. National Stage of
International Application No. PCT/US2003/034523, filed Oct. 31,
2003, which was published in English under PCT Article 21(2), which
in turn claims the benefit of U.S. Provisional Application No.
60/422,964, filed Nov. 1, 2002, which is incorporated herein by
reference in its entirety.
Claims
The invention claimed is:
1. A method of enhancing the immunogenicity of a vaccine against
Bacillus anthracis in a subject, comprising administering to the
subject a therapeutically effective amount of an
oligodeoxynucleotide consisting of the nucleic acid sequence set
forth as SEQ ID NO: 200 in combination with the vaccine against
Bacillus anthracis, thereby enhancing the immunogenicity of the
vaccine.
2. The method of claim 1, wherein the vaccine is an antigen
vaccine, a DNA vaccine, a protein subunit vaccine, a peptide
vaccine, an attenuated vaccine, or a heat-killed vaccine.
3. The method of claim 1, wherein the vaccine is an antigen from
Bacillus anthracis.
4. The method of claim 3, wherein the antigen is recombinant
Protective Antigen or Protective Antigen.
5. The method of claim 1, wherein the oligodeoxynucleotide is
administered before the vaccine is administered to the subject.
6. The method of claim 5, wherein the oligodeoxynucleotide is
administered from about two weeks to about one day before the
vaccine is administered to the subject.
7. The method of claim 1, wherein the oligodeoxynucleotide is
administered to the subject concurrently with the vaccine.
8. The method of claim 1, wherein the oligodeoxynucleotide is
administered after the vaccine is administered to the subject.
9. The method of claim 8, wherein the oligodeoxynucleotide is
administered from about two weeks to about one day after the
vaccine is administered to the subject.
10. A method of enhancing the immunogenicity of Anthrax Vaccine
Adsorbed (AVA) vaccine, comprising administering to a subject a
therapeutically effective amount of an oligodeoxynucleotide
comprising the nucleotide sequence set forth as SEQ ID NO: 200 and
a therapeutically effective amount of Anthrax Vaccine Adsorbed
(AVA) vaccine, thereby enhancing the immunogenicity of Anthrax
Vaccine Adsorbed (AVA) vaccine.
11. A method of enhancing the immunogenicity of a vaccine
comprising anthrax protective antigen, comprising administering to
a subject a therapeutically effective amount of an
oligodeoxynucleotide comprising the nucleotide sequence set forth
as SEQ ID NO: 200 and a therapeutically effective amount of anthrax
protective antigen, thereby enhancing the immunogenicity of the
vaccine.
12. The method of claim 10, wherein enhancing the immunogenicity of
AVA comprises increasing the IgG or IgM titer.
13. The method of claim 10, wherein enhancing the immunogenicity of
AVA comprises increasing survival of the subject upon subsequent
exposure to anthrax.
14. The method of claim 1, wherein the vaccine is Anthrax Vaccine
Adsorbed (AVA).
15. The method of claim 11, wherein the subject is human.
16. The method of claim 14, comprising administering to the subject
a therapeutically effective amount of the oligodeoxynucleotide and
a therapeutically effective amount of anthrax protective antigen at
an initial time point and at two and four weeks following the
initial time point, thereby enhancing the immunogenicity of the
vaccine.
17. The method of claim 1, wherein the subject is human.
18. The method of claim 1, comprising administering to the subject
a therapeutically effective amount of the oligodeoxynucleotide and
a therapeutically effective amount of anthrax protective antigen at
an initial time point and at two and four weeks following the
initial time point, thereby enhancing the immunogenicity of the
vaccine.
19. A method of enhancing the immunogenicity of a vaccine against
Bacillus anthracis in a subject, comprising administering to the
subject a therapeutically effective amount of an
oligodeoxynucleotide comprising the nucleic acid sequence set forth
as SEQ ID NO: 200 in combination with the vaccine against Bacillus
anthracis, thereby enhancing the immunogenicity of the vaccine.
20. The method of claim 19, wherein the vaccine is an antigen
vaccine, a DNA vaccine, a protein subunit vaccine, a peptide
vaccine, an attenuated vaccine, or a heat-killed vaccine.
21. The method of claim 19, wherein the vaccine is an antigen from
Bacillus anthracis.
22. The method of claim 19, wherein the antigen is recombinant
Protective Antigen or Protective Antigen.
23. The method of claim 19, wherein the oligodeoxynucleotide is
administered before the vaccine is administered to the subject.
24. The method of claim 19, wherein the oligodeoxynucleotide is
administered from about two weeks to about one day before the
vaccine is administered to the subject.
25. The method of claim 19, wherein the oligodeoxynucleotide is
administered to the subject concurrently with the vaccine.
26. The method of claim 19, wherein the oligodeoxynucleotide is
administered after the vaccine is administered to the subject.
27. The method of claim 19, wherein the oligodeoxynucleotide is
administered from about two weeks to about one day after the
vaccine is administered to the subject.
Description
FIELD
The present disclosure relates to a method of inhibiting or
treating an infection caused by a bioterrorism agent, specifically
to a method of increasing an immune response to a bioterrorism
agent using an oligodeoxynucleotide including a CpG motif.
BACKGROUND
Bioterrorism agents are bacteria, viruses, and toxins that are
dispersed deliberately in an environment to cause disease or death
in humans or animals. Bioterrorism agents include, but are not
limited to, Bacillus anthracis (anthrax), Yersinia pestis (plague),
Variola major (smallpox), tick-borne encephalitis virus (TBEV)
(tick-borne encephalitis), and Ebola virus (Ebola). Bioterrorism
agents can also include biotoxins, which are toxins produced by
certain biological organisms. Exemplary biotoxins are botulinum
toxin, which is produced by the bacterium Clostridium botulinum,
and ricin, which is isolated from castor oil seeds.
The immune system has evolved two general strategies for combating
infections from bioterrorism agents such as anthrax. A rapid
"innate" immune response is induced when Toll-like receptors (TLR)
on host cells interact with highly conserved pathogen associated
molecular patterns (PAMPs) expressed by infectious microorganisms
(Marrack and Kappler, Cell 76:323, 1994; Medzhitov and Janeway,
Cur. Op. Immunol. 9:4, 1997). The resultant production of
polyreactive antibodies and immunostimulatory cytokines check the
pathogen's early proliferation and spread (Marrack and Kappler,
Cell 76:323, 1994; Medzhitov and Janeway, Cur. Op. Immunol. 9:4,
1997; Medzhitov and Janeway, Cell 91:295, 1998). A subsequent
antigen-specific immune response is then generated against
determinants unique to the pathogen that helps to eradicate the
remaining organisms and provide long-lasting protective memory.
Vaccination can be used to protect against the effects of some
bioterrorism agents. For example, in the case of anthrax,
"protective antigen" (PA) is necessary for vaccine immunogenicity
(Ivins et al., Infect. Immun. 60:662, 1992; Welkos and Friedlander,
Microb. Pathog. 5:127, 1998). Antibodies against PA prevent anthrax
toxin from binding to host cells, thus abrogating toxicity (Little
and Ivins, Microbes. Infect. 1:131, 1999). Additionally, antibodies
to PA can inhibit the germination of spores while improving their
phagocytosis and killing by macrophages (Welkos et al.,
Microbiology 147:1677, 2001). Unfortunately, the currently licensed
human anthrax vaccine (AVA) requires six vaccinations over eighteen
months followed by yearly boosters to induce and maintain
protective anti-PA titers (Pittman et al., Vaccine 20:1412, 2002;
Pittman et al., Vaccine 20:972, 2001). In some vaccinees, this
regimen is associated with undesirable local reactogenicity
(Pittman et al., Vaccine 20:972, 2001).
Thus, there exists a need for agents that prevent or treat
infections caused by bioterrorism agents, or that increase the
immunogenicity of a vaccine against a bioterrorism agent, in order
to treat or prevent infections in individuals exposed to or at risk
of exposure to bioterrorism agents.
SUMMARY
Described herein are methods of treating or preventing an infection
in a subject who has been exposed to or is at risk for exposure to
a bioterrorism agent. In some embodiments, the method is a method
of increasing an immune response to a bioterrorism agent using an
oligodeoxynucleotide including a CpG motif. Other methods are
methods of increasing an immune response to a bioterrorism agent
using an oligodeoxynucleotide including a CpG motif and an
additional anti-infective agent. Still other methods include
enhancing the immunogenicity of a vaccine against a bioterrorism
agent using an oligodeoxynucleotide including a CpG motif.
In some embodiments, a therapeutically effective amount of an
immunostimulatory D oligodeoxynucleotide or an immunostimulatory K
oligodeoxynucleotide is administered to the subject, thereby
treating or preventing the infection.
Also described herein are methods of treating or preventing an
infection in a subject who has been exposed to or is at risk for
exposure to Bacillus anthracis. In some embodiments, the method
includes administering a therapeutically effective amount of an
immunostimulatory D oligodeoxynucleotide or an immunostimulatory K
oligodeoxynucleotide to a subject.
Other methods described herein are methods of treating or
preventing an infection in a subject who has been exposed to or is
at risk for exposure to a bioterrorism agent by administering a
therapeutically effective amount of an immunostimulatory D
oligodeoxynucleotide or an immunostimulatory K oligodeoxynucleotide
to the subject in combination with an anti-infective agent, thereby
treating or preventing the infection.
Further embodiments are methods of treating or preventing an
infection in a subject who has been exposed to or is at risk for
exposure to Bacillus anthracis. In some embodiments, the method
includes administering a therapeutically effective amount of an
immunostimulatory D oligodeoxynucleotide or an immunostimulatory K
oligodeoxynucleotide in combination with an anti-infective agent to
a subject.
Also described herein are methods of enhancing the immunogenicity
of a vaccine against a bioterrorism agent in a subject. In some
embodiments, a therapeutically effective amount of an
immunostimulatory D oligodeoxynucleotide or an immunostimulatory K
oligodeoxynucleotide is administered to a subject in combination
with a vaccine against a bioterrorism agent, thereby enhancing the
immunogenicity of the vaccine against a bioterrorism agent.
Still further embodiments are methods of enhancing the
immunogenicity of an antigen from Bacillus anthracis, comprising
administering to the subject a therapeutically effective amount of
an immunostimulatory D oligodeoxynucleotide or an immunostimulatory
K oligodeoxynucleotide in combination with an antigen from Bacillus
anthracis, thereby enhancing the immunogenicity of the antigen.
The features and advantages will become more apparent from the
following detailed description of several embodiments, which
proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a graph showing that K ODN significantly increases the
maximum, average, and long-term IgG anti-PA response in Rhesus
macaques when co-administered with AVA or rPA. Rhesus macaques
(5-6/group) were immunized SQ at 0 and 4 weeks with 0.5 ml of AVA
or 50 .mu.g of rPA in alum. In some cases, these vaccines were
co-administered with 250 .mu.g of an equimolar mixture of K3, K23
and K123 (K) or D19, D29 and D35 (D) ODN. Animals were "challenged"
IM with the live attenuated veterinary vaccine strain of anthrax on
week 27. Results show the geometric mean (+SEM) IgG anti-PA titer
calculated by analyzing serum from each animal independently at the
time points shown. The time-averaged magnitude of the response
induced by K ODN plus AVA or rPA significantly exceeded that of
either vaccine alone, p<0.05.
FIG. 2 is a graph showing that K ODN increase serum IgG anti-PA
titers and avidity. Six rhesus macaques were immunized SQ at 0 and
4 weeks with 0.5 ml of AVA and then "challenged" IM with the live
attenuated veterinary vaccine strain of anthrax on week 27. Serum
IgG anti-PA titers and avidity (% of Ab remaining bound after
elution with 6 M urea) are shown. Results reflect the geometric
mean (+SEM) IgG anti-PA titer derived by analyzing serum from each
animal independently at the time points shown.
FIG. 3 is a graph showing that dose and timing of CpG
administration influences CpG-mediated protection in mice exposed
to Ebola virus. In FIG. 3a, mice were treated with 25-150 .mu.g of
CpG ODN on day 0, and then challenged with 300 LD50 of
mouse-adapted Ebola Zaire. In FIG. 3b, mice were treated with 100
.mu.g of CpG ODN on the day shown, and then challenged with 300
LD50 of mouse-adapted Ebola Zaire.
N=10 mice/group.
FIG. 4 is a graph showing that CpG ODNs increase survival times in
mice exposed to anthrax spores. Mice were treated at the times
shown with 100 .mu.g of CpG ODN, and then challenged with 11 LD 50
anthrax spores. Survival is shown (N=10/group).
FIG. 5 is a graph that shows the effect of K ODN on the avidity of
the anti-PA response. N=6 per group.
FIG. 6 is a line graph of the geometric mean anti-PA IgG titer
following treatment with the anthrax vaccine AVA alone, AVA plus K
ODN, AVA plus ODN 10103, and AVA plus ODN 7909.
FIG. 7 is a graph of the geometric mean anti-PA IgM titers
following treatment with the anthrax vaccine AVA alone, AVA plus K
ODN, AVA plus ODN 10103, and AVA plus ODN 7909.
FIG. 8 is line graph of the geometric mean TNA titers over the
entire study period following treatment with the anthrax vaccine
AVA alone, AVA plus K ODN, AVA plus ODN 10103, and AVA plus ODN
7909.
FIG. 9 is a graphical representation of the correlation between
anti-PA titer and overall survival percentages.
SEQUENCE LISTING
The nucleic acid sequences listed in the accompanying sequence
listing are shown using standard letter abbreviations for
nucleotide bases, as defined in 37 C.F.R. 1.822. It will be clear
to one of skill in the art that whereas the letter X is used in the
specification to refer to any unspecified nucleotide, the letter N
is used in the Sequence Listing to refer to any unspecified
nucleotide. In the accompanying sequence listing:
SEQ ID NOs 1-16, 17, 18, and 21-25 are immunostimulatory CpG D
oligonucleotide sequences.
SEQ ID NOs 19, 20, and 26-28 are control D oligonucleotide
sequences.
SEQ ID NOs 29-43 are K oligonucleotide sequences.
DETAILED DESCRIPTION
I. Abbreviations
A: adenine Ab: antibody AVA: anthrax vaccine adsorbed C: cytosine
CpG ODN: an oligodexoynucleotide (either a D or a K type) including
a CpG motif DC: dendritic cell EU: Endotoxin units FCS: fetal calf
serum G: guanine h: hour IFN-.alpha.: interferon alpha i.m.:
intramuscular i.p.: intraperitoneal IFN-.gamma.: interferon gamma
.mu.g: microgram mRNA: messenger ribonucleic acid NK: natural
killer cells ODN: oligodeoxynucleotide PA: protective antigen
PAMPs: pathogen-associated molecular patterns Pu: purine Py:
pyrimidine rPA: recombinant PA antigen SQ: subcutaneous T: thymine
TLR: Toll-like receptor
II. Terms
Unless otherwise noted, technical terms are used according to
conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
In order to facilitate review of the various embodiments of this
disclosure, the following explanations of specific terms are
provided:
Animal: Living multi-cellular vertebrate organisms, a category that
includes, for example, mammals and birds. The term mammal includes
both human and non-human mammals. Similarly, the term "subject"
includes both human and veterinary subjects, for example, humans,
non-human primates, dogs, cats, horses, and cows.
Antigen: A compound, composition, or substance that can stimulate
the production of antibodies or a T cell response in an animal,
including compositions that are injected or absorbed into an
animal. An antigen reacts with the products of specific humoral or
cellular immunity, including those induced by heterologous
immunogens. The term "antigen" includes all related antigenic
epitopes. In one embodiment, an antigen is a bioterrorism agent
antigen. In some embodiments, an antigen is a component of a
vaccine against a bioterrorism agent, which is an antigen
associated with or expressed by any bacterium, virus, fungus, or
biotoxin that can be dispersed to cause disease or death in animals
or humans.
Anti-infectious agent: A substance (such as a chemical compound,
protein, antisense oligonucleotide, or other molecule) of use in
treating infection of a subject. Anti-infectious agents include,
but are not limited to, anti-fungal compounds, anti-viral
compounds, and antibiotics. Antibiotics include, but are not
limited to, amoxicillin, clarithromycin, cefuroxime, cephalexin
ciprofloxacin, doxycycline, metronidazole, terbinafine,
levofloxacin, nitrofurantoin, tetracycline, and azithromycin.
Anti-fungal compounds, include, but are not limited to,
clotrimazole, butenafine, butoconazole, ciclopirox, clioquinol,
clioquinol, clotrimazole, econazole, fluconazole, flucytosine,
griseofulvin, haloprogin, itraconazole, ketoconazole, miconazole,
naftifine, nystatin, oxiconazole, sulconazole, terbinafine,
terconazole, fluconazole, and tolnaftate. Anti-viral compounds,
include, but are not limited to, zidovudine, didanosine,
zalcitabine, stavudine, lamivudine, abacavir, tenofovir,
nevirapine, delavirdine, efavirenz, saquinavir, ritonavir,
indinavir, nelfinavir, saquinavir, amprenavir, and lopinavir.
Anti-infectious agents also include hyper-immune globulin.
Hyper-immune globulin is gamma globulin isolated from a donor, or
from a pool of donors, that has been immunized with a substance of
interest. Specifically, hyper-immune globulin is antibody purified
from a donor who was repeatedly vaccinated against a pathogen. In
several embodiments, hyper-immune globulin is gamma globulin
isolated from a donor, or from a pool of donors, repeatedly
immunized with an antigen, a micro-organism (including a
heat-killed micro-organism), or a virus. In one specific,
non-limiting example, hyper-immune globulin against anthrax is
produced using serum from a donor repeatedly immunized with the
anthrax vaccine (AVA).
Antisense, Sense and Antigene: Double-stranded DNA (dsDNA) has two
strands, a 5'.fwdarw.3' strand, referred to as the plus strand, and
a 3'.fwdarw.5' strand (the reverse compliment), referred to as the
minus strand. Because RNA polymerase adds nucleic acids in a
5'.fwdarw.3' direction, the minus strand of the DNA serves as the
template for the RNA during transcription. Thus, the RNA formed
will have a sequence complementary to the minus strand and
identical to the plus strand (except that U is substituted for
T).
Antisense molecules are molecules that are specifically
hybridizable or specifically complementary to either RNA or the
plus strand of DNA. Sense molecules are molecules that are
specifically hybridizable or specifically complementary to the
minus strand of DNA. Antigene molecules are either antisense or
sense molecules directed to a dsDNA target. In one embodiment, an
antisense molecule specifically hybridizes to a target mRNA and
inhibits transcription of the target mRNA.
AVA: The only licensed human anthrax vaccine in the United States,
AVA, is produced by BioPort Corporation in Lansing, Mich., and is
prepared from a cell-free filtrate of B. anthracis culture that
contains no dead or live bacteria. The strain used to prepare the
vaccine is a toxigenic, nonencapsulated strain known as V770-NP1-R.
The filtrate contains a mix of cellular products including PA
(protective antigen) and is adsorbed to aluminum hydroxide
(Amphogel, Wyeth Laboratories) as adjuvant. The amount of PA and
other proteins per 0.5 mL dose is unknown, and all three toxin
components, LF (lethal factor), EF (edema factor), and PA, are
present in the product.
Generally, primary vaccination generally consists of three
subcutaneous injections at 0, 2, and 4 weeks, and three booster
vaccinations at 6, 12, and 18 months. To maintain immunity, the
manufacturer recommends an annual booster injection. However,
schedules with a reduced number of doses and with intramuscular
(IM) administration have been proposed. Following a suspected
exposure to B. anthracis, AVA may be given concurrently with
antibiotic prophylaxis.
Bacillus Anthracis: The etiologic agent of anthrax, Bacillus
anthracis is a large, gram-positive, nonmotile, spore-forming
bacterial rod. The three virulence factors of B. anthracis are
edema toxin, lethal toxin and a capsular antigen. Infection with B.
anthracis is the cause of human anthrax disease. Human anthrax has
three major clinical forms: cutaneous, inhalation, and
gastrointestinal. Cutaneous anthrax is a result of introduction of
the spore through the skin; inhalation anthrax, through the
respiratory tract; and gastrointestinal anthrax, by ingestion. If
untreated, anthrax in all forms can lead to septicemia and death.
Early treatment of cutaneous anthrax is usually curative, and early
treatment of all forms is important for recovery. Patients with
gastrointestinal anthrax have reported case-fatality rates ranging
from 25% to 75%. Case-fatality rates for inhalational anthrax are
thought to approach 90 to 100%.
Bacillus anthracis secretes a toxin made up of three proteins:
protective antigen (PA), oedema factor (OF) and lethal factor (LF)
(Stanley et al., J. Gen. Microbiol. 26:49-66, 1961; Beall et al.,
J. Bacteriol. 83:1274-1280, 1962). PA binds to cell-surface
receptors on the host's cell membranes. After being cleaved by a
protease (Bradley et al., Nature 414:225, 2001), PA binds to the
two toxic enzymes, OF and LA, and mediates their transportation
into the cytosol where they exert their pathogenic effects. Thus,
the smaller cleaved 63 kD PA remnant PA.sub.63) oligomerizes
features a newly exposed, second binding domain and binds to either
EF, an 89 kD protein, to form edema toxin, or LF, a 90 kD protein,
to form lethal toxin (LeTx) (Leppla et al., Salisbury Med. Bull.
Suppl. 68:41-43, 1990), and the complex is internalized into the
cell (Singh et al., Infect. Immun. 67:1853, 1999; Friedlander, J.
Biol. Chem. 261:7123, 1986). From these endosomes, the PA.sub.63
channel enables translocation of LF and EF to the cytosol by a pH-
and voltage-dependent mechanism (Zhao et al., J. Biol. Chem,
270:18626, 1995).
Bioterrorism agents: Any of various bacteria, viruses, and toxins
that can be dispersed deliberately to cause disease or death to
humans or animals. Examples of bioterrorism agents include Bacillus
anthracis, which causes anthrax, Yersinia pestis, which causes
plague, and Variola major, which causes smallpox, tick-borne
encephalitis virus (TBEV), which causes tick-borne encephalitis,
and Ebola virus, which causes Ebola. Bioterrorism agents also
include biotoxins, which are toxins produced by certain biological
organisms. Exemplary biotoxins are botulinum toxin, produced by the
bacterium Clostridium botulinum, and ricin isolated from castor oil
seeds. Western counter-proliferation agencies currently recognize
23 types of bacteria, 43 types of viruses, and 14 types of
biotoxins as potential bioterrorism agents.
Other examples of bioterrorism agents include, but are not limited
to, Escherichia coli, Haemophilus influenzae, cobra venom,
shellfish toxin, botulinum toxin, saxitoxin, ricin toxin, Shigella
flexneri, S. dysenteriae (Shigella bacillus), Salmonella,
Staphylococcus enterotoxin B, Histoplasma capsulatum, tricothecene
mycotoxin, aflatoxin. Bioterrorism agents can also result in
cryptococcosis, brucellosis (undulant fever), coccidioidomycosis
(San Joaquin Valley or desert fever), psittacosis (parrot fever),
bubonic plague, tularemia (rabbit fever), malaria, cholera,
typhoid, hemorrhagic fever, tick-borne encephalitis, Venezuelan
equine encephalitis, pneumonic plague, Rocky Mountain spotted
fever, dengue fever, Rift Valley fever, diphtheria, melioidosis,
glanders, tuberculosis, infectious hepatitis, encephalitides,
blastomycosis, nocardiosis, yellow fever, typhus, and Q fever.
CpG or CpG motif: A nucleic acid having a cytosine followed by a
guanine linked by a phosphate bond in which the pyrimidine ring of
the cytosine is unmethylated. The term "methylated CpG" refers to
the methylation of the cytosine on the pyrimidine ring, usually
occurring at the 5-position of the pyrimidine ring. A CpG motif is
a pattern of bases that include an unmethylated central CpG
surrounded by at least one base flanking (on the 3' and the 5' side
of) the central CpG. Without being bound by theory, the bases
flanking the CpG confer part of the activity to the CpG
oligodeoxynucleotide. A CpG oligonucleotide is an oligonucleotide
that is at least about ten nucleotides in length and includes an
unmethylated CpG. CpG oligonucleotides include both D and K
oligodeoxynucleotides (see below). CpG oligodeoxynucleotides are
single-stranded. The entire CpG oligodeoxynucleotide can be
unmethylated or portions may be unmethylated. In one embodiment, at
least the C of the 5' CG 3' is unmethylated.
Cytokine: Proteins made by cells that affect the behavior of other
cells, such as lymphocytes. In one embodiment, a cytokine is a
chemokine, a molecule that affects cellular trafficking. Specific
non-limiting examples of cytokines are IFN-.gamma., IL-6, and
IL-10.
D Type Oligodeoxynucleotide (D ODN): An oligodeoxynucleotide
including an unmethylated CpG motif that has a sequence represented
by the formula: 5'RY-CpG-RY3' wherein the central CpG motif is
unmethylated, R is A or G (a purine), and Y is C or T (a
pyrimidine). D-type oligodeoxynucleotides include an unmethylated
CpG dinucleotide. Inversion, replacement or methylation of the CpG
reduces or abrogates the activity of the D
oligodeoxynucleotide.
In one embodiment, a D type ODN is at least about 16 nucleotides in
length and includes a sequence represented by Formula III:
5'X.sub.1X.sub.2X.sub.3Pu.sub.1Py.sub.2CpGPu.sub.3Py.sub.4X.sub.4X.sub.5X-
.sub.6CV).sub.M(G).sub.N-3' wherein the central CpG motif is
unmethylated, Pu is a purine nucleotide, Py is a pyrimidine
nucleotide, X and W are any nucleotide, M is any integer from 0 to
10, and N is any integer from 4 to 10. Generally D ODNs can
stimulate a cellular response. For example, D ODNs stimulate
natural killer cells and the maturation of dendritic cells.
Epitope: An antigenic determinant. These are particular chemical
groups or peptide sequences on a molecule that are antigenic, i.e.,
that elicit a specific immune response. An antibody binds a
particular antigenic epitope.
Functionally Equivalent: Sequence alterations, for example in an
immunostimulatory ODN, that yield the same results as described
herein. Such sequence alterations can include, but are not limited
to, deletions, base modifications, mutations, labeling, and
insertions.
Immune response: A response of a cell of the immune system, such as
a B cell or a T cell, to a stimulus. In one embodiment, the
response is specific for a particular antigen (an "antigen-specific
response"). A "parameter of an immune response" is any particular
measurable aspect of an immune response, including, but not limited
to, cytokine secretion (IL-6, IL-10, IFN-.gamma., etc.),
immunoglobulin production, dendritic cell maturation, and
proliferation of a cell of the immune system. One of skill in the
art can readily determine an increase in any one of these
parameters, using known laboratory assays. In one specific
non-limiting example, to assess cell proliferation, incorporation
of .sup.3H-thymidine can be assessed. A "substantial" increase in a
parameter of the immune response is a significant increase in this
parameter as compared to a control. Specific, non-limiting examples
of a substantial increase are at least about a 50% increase, at
least about a 75% increase, at least about a 90% increase, at least
about a 100% increase, at least about a 200% increase, at least
about a 300% increase, and at least about a 500% increase. One of
skill in the art can readily identify a significant increase using
known statistical methods. One specific, non-limiting example of a
statistical test used to assess a substantial increase is the use
of a Z test to compare the percent of samples that respond to a
vaccine against a bioterrorism agent alone as compared to the
percent of samples that respond using a vaccine against a
bioterrorism agent administered in conjunction with an
immunostimulatory ODN. A non-parametric ANOVA can be used to
compare differences in the magnitude of the response induced by
vaccine alone as compared to the percent of samples that respond
using vaccine administered in conjunction with an immunostimulatory
ODN. In this example, p.ltoreq.0.05 is significant, and indicates a
substantial increase in the parameter of the immune response. One
of skill in the art can readily identify other statistical assays
of use.
An "immunoprotective response" is an immune response that results
in a decrease of symptoms upon infection with a bioterrorism agent
or results in a delay or prevention of a disease associated with
infection. "Enhancing the immunogenicity of a vaccine" is an
example of an increase in an immune response.
Inhibiting or treating a disease: "Inhibiting" a disease refers to
reducing the full development of a disease, for example in a person
who is known to have a predisposition to a disease such as a person
who has been or is at risk for being exposed to a bioterrorism
agent. Examples of persons at risk for being exposed to a
bioterrorism agent include, but are not limited to, military
personnel, mail handlers, medical personnel, and governmental
officials, as well as those with weakened immune systems, for
example, the elderly, people on immunosuppressive drugs, subjects
with cancer, and subjects infected with HIV. "Treatment" refers to
a therapeutic intervention that ameliorates a sign or symptom of a
disease or pathological condition after it has begun to
develop.
Isolated: An "isolated" biological component (such as a nucleic
acid, peptide or protein) has been substantially separated,
produced apart from, or purified away from other biological
components in the cell of the organism in which the component
naturally occurs, i.e., other chromosomal and extrachromosomal DNA
and RNA, and proteins. Nucleic acids, peptides and proteins which
have been "isolated" thus include nucleic acids and proteins
purified by standard purification methods. The term also embraces
nucleic acids, peptides and proteins prepared by recombinant
expression in a host cell as well as chemically synthesized nucleic
acids.
K Type Oligodeoxynucleotide (K ODN): An oligodeoxynucleotide
including an unmethylated CpG motif that has a sequence represented
by the formula:
5'N.sub.1N.sub.2N.sub.3Q-CpG-WN.sub.4N.sub.5N.sub.63' wherein the
central CpG motif is unmethylated, Q is T, G or A, W is A or T, and
N.sub.1, N.sub.2, N.sub.3, N.sub.4, N.sub.5, and N.sub.6 are any
nucleotides. In one embodiment, Q is a T. K type CpG ODNs have been
previously described (see U.S. Pat. No. 6,194,388; U.S. Pat. No.
6,207,646; U.S. Pat. No. 6,214,806; U.S. Pat. No. 6,218,371; U.S.
Pat. No. 6,239,116, U.S. Pat. No. 6,339,068; U.S. Pat. No.
6,406,705 and U.S. Pat. No. 6,429,199, which are herein
incorporated by reference). Generally K ODNs can stimulate a
humoral response. For example, K ODNs stimulate the production of
IgM.
Leukocyte: Cells in the blood, also termed "white cells," that are
involved in defending the body against infective organisms and
foreign substances. Leukocytes are produced in the bone marrow.
There are 5 main types of white blood cell, subdivided between 2
main groups: polymorphomnuclear leukocytes (neutrophils,
eosinophils, basophils) and mononuclear leukocytes (monocytes and
lymphocytes). When an infection is present, the production of
leukocytes increases.
Mammal: This term includes both human and non-human mammals.
Similarly, the term "subject" includes both human and veterinary
subjects.
Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in
either single or double stranded form, and unless otherwise
limited, encompasses known analogues of natural nucleotides that
hybridize to nucleic acids in a manner similar to naturally
occurring nucleotides.
Oligonucleotide or "oligo": Multiple nucleotides (i.e. molecules
comprising a sugar (e.g. ribose or deoxyribose) linked to a
phosphate group and to an exchangeable organic base, which is
either a substituted pyrimidine (Py) (e.g. cytosine (C), thymine
(T) or uracil (U)) or a substituted purine (Pu) (e.g. adenine (A)
or guanine (G)). The term "oligonucleotide" as used herein refers
to both oligoribonucleotides (ORNs) and oligodeoxyribonucleotides
(ODNs). The term "oligonucleotide" also includes oligonucleosides
(i.e. an oligonucleotide minus the phosphate) and any other organic
base polymer. Oligonucleotides can be obtained from existing
nucleic acid sources (e.g. genomic or cDNA), but are preferably
synthetic (e.g. produced by oligonucleotide synthesis).
A "stabilized oligonucleotide" is an oligonucleotide that is
relatively resistant to in vivo degradation (for example via an
exo- or endo-nuclease). In one embodiment, a stabilized
oligonucleotide has a modified phosphate backbone. One specific,
non-limiting example of a stabilized oligonucleotide has a
phosphorothioate modified phosphate backbone (wherein at least one
of the phosphate oxygens is replaced by sulfur). Other stabilized
oligonucleotides include: nonionic DNA analogs, such as alkyl- and
aryl-phosphonates (in which the charged phosphonate oxygen is
replaced by an alkyl or aryl group), phosphodiester and
alkylphosphotriesters, in which the charged oxygen moiety is
alkylated. Oligonucleotides which contain a diol, such as
tetraethyleneglycol or hexaethyleneglycol, at either or both
termini have also been shown to be substantially resistant to
nuclease degradation.
An "immunostimulatory oligonucleotide," "immunostimulatory CpG
containing oligodeoxynucleotide," "CpG ODN," refers to an
oligodeoxynucleotide, which contains a cytosine, guanine
dinucleotide sequence and stimulates (e.g. has a mitogenic effect)
vertebrate immune cells. The cytosine, guanine is unmethylated. The
term "immunostimulatory ODN" includes both D and K type ODNs.
An "oligonucleotide delivery complex" is an oligonucleotide
associated with (e.g. ionically or covalently bound to; or
encapsulated within) a targeting means (e.g. a molecule that
results in a higher affinity binding to a target cell (e.g. B cell
or natural killer (NK) cell) surface and/or increased cellular
uptake by target cells). Examples of oligonucleotide delivery
complexes include oligonucleotides associated with: a sterol (e.g.
cholesterol), a lipid (e.g. cationic lipid, virosome or liposome),
or a target cell specific binding agent (e.g. a ligand recognized
by a target cell specific receptor). Preferred complexes must be
sufficiently stable in vivo to prevent significant uncoupling prior
to internalization by the target cell. However, the complex should
be cleavable or otherwise accessible under appropriate conditions
within the cell so that the oligonucleotide is functional. (Gursel,
J. Immunol. 167: 3324, 2001)
Protective Antigen (PA): Bacillus anthracis secretes a toxin made
up of three proteins: protective antigen (PA), edema factor (EF)
and lethal factor (LF). PA binds to cell-surface receptors on the
host's cell membranes. After being cleaved by a protease, PA binds
to the two toxic enzymes, EF and LA, and mediates their
transportation into the cytosol where they exert their pathogenic
effects.
The only licensed human anthrax vaccine in the United States, AVA,
contains a mix of cellular products including PA (protective
antigen). The sequence of the protection antigen is known, as is
set forth as GenBank Accession No. 13423, which is incorporated
herein by reference. Vaccine preparations including PA are
described, for example, in U.S. Pat. No. 5,591,631, which is
incorporated herein by reference. Recombinant Protective Antigen
(rPA) is an anthrax vaccine that is currently under development.
rPA is a recombinant version of the PA vaccine.
Pharmaceutical agent or drug: A chemical compound or composition
capable of inducing a desired therapeutic or prophylactic effect
when properly administered to a subject. Pharmaceutical agents
include, but are not limited to, anti-infective agents, such as
antibiotics, anti-fungal compounds, anti-viral compounds, and
hyper-immune globulin.
Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this disclosure are conventional.
Remington's Pharmaceutical Sciences, by E. W. Martin, Mack
Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of the fusion proteins herein disclosed.
In general, the nature of the carrier will depend on the particular
mode of administration being employed. For instance, parenteral
formulations usually comprise injectable fluids that include
pharmaceutically and physiologically acceptable fluids such as
water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
Self-complementary nucleic acid sequence: A nucleic acid sequence
that can form Watson-Crick base pairs. The four bases
characteristic of deoxyribonucleic unit of DNA are the purines
(adenine and guanine) and the pyrimidines (cytosine and thymine).
Adenine pairs with thymine via two hydrogen bonds, while guanine
pairs with cytosine via three hydrogen bonds. If a nucleic acid
sequence includes two or more bases in sequence that can form
hydrogen bonds with two or more other bases in the same nucleic
acid sequence, then the nucleic acid includes a self-complementary
sequence.
Therapeutically effective dose: A dose sufficient to prevent
advancement, or to cause regression of the disease, or which is
capable of relieving symptoms caused by the disease, such as fever,
respiratory symptoms, pain or swelling.
Vaccine: A preparation of antigen, DNA, protein subunit, peptide,
attenuated microorganisms (including but not limited to bacteria
and viruses), living microorganisms, or killed microorganisms,
administered for the inhibition, prevention, amelioration or
treatment of infectious disease. In some embodiments, the vaccine
against a bioterrorism agent includes an antigen from a
bioterrorism agent, for example AVA or PA.
Generally, the first step in making a vaccine is to isolate or
create an organism, or part of one, that is unable to cause the
disease, but that still retains the antigens responsible for
inducing the host's immune response. This can be done in many ways.
One way is to kill the organism using heat or formalin; vaccines
produced in this way are called "inactivated" or "killed" vaccines.
Examples of killed vaccines in common use today are the typhoid
vaccine and the Salk poliomyelitis vaccine.
Another way to produce a vaccine is to use only the antigenic part
of the disease-causing organism, for example the capsule, the
flagella, or part of the protein cell wall; these types of vaccines
are called "acellular vaccines." An example of an acellular vaccine
is the Haemophilus influenzae B (HIB) vaccine. Acellular vaccines
exhibit some similarities to killed vaccines: neither killed nor
acellular vaccines generally induce the strongest immune responses
and may therefore require a "booster" every few years to insure
their continued effectiveness. In addition, neither killed nor
acellular vaccines can cause disease and are therefore considered
to be safe for use in immunocompromised patients.
A third way of making a vaccine is to "attenuate" or weaken a live
microorganism by mutating the organism to alter its growth
capabilities. In one embodiment, an attenuated vaccine is not
replication competent or lacks essential proteins. Examples of
attenuated vaccines are those that protect against measles, mumps,
and rubella. Immunity is often life-long with attenuated vaccines
and does not require booster shots.
Vaccines can also be produced from a toxin. In these cases, the
toxin is often treated with aluminum or adsorbed onto aluminum
salts to form a "toxoid." Examples of toxoids are the diphtheria
and the tetanus vaccines. Vaccines made from toxoids often induce
low-level immune responses and are therefore sometimes administered
with an "adjuvant"--an agent which increases the immune response.
For example, the diphtheria and tetanus vaccines are often combined
with the pertussis vaccine and administered together as a DPT
immunization. The pertussis acts as an adjuvant in this vaccine.
When more than one vaccine is administered together it is called a
"conjugated vaccine."
Another way of making a vaccine is to use an organism which is
similar to the virulent organism but that does not cause serious
disease, such as using the cowpox virus to protect against
infection with smallpox virus, or BCG vaccine, an attenuated strain
of Mycobacterium bovis, used to protect against Mycobacterium
tuberculosis.
"Subunit vaccines" are vaccines which use a polypeptide from an
infectious organism to stimulate a strong immune response. An
"antigen vaccine" uses an immunogenic epitope of a polypeptide to
induce a protective immune response. A "DNA vaccine" uses a nucleic
acid encoding an antigen to induce a protective immune
response.
A "vaccine against a bioterrorism agent" can be, but is not limited
to, a heat or formalin-killed vaccine, attenuated vaccine, subunit
vaccine, antigen vaccine, DNA vaccine, acellular vaccine, or toxoid
vaccine directed against Bacillus anthracis, Yersinia pestis,
Variola major, tick-borne encephalitis virus (TBEV), Ebola virus,
Escherichia coli, Haemophilus influenzae, cobra venom, shellfish
toxin, botulinum toxin, saxitoxin, ricin toxin, Shigella flexneri,
S. dysenteriae (Shigella bacillus), Salmonella, Staphylococcus
enterotoxin B, Histoplasma capsulatum, tricothecene mycotoxin,
aflatoxin. A "vaccine against a bioterrorism agent" can also be
used to induce a protective immune response against cryptococcosis,
brucellosis (undulant fever), coccidioidomycosis (San Joaquin
Valley or desert fever), psittacosis (parrot fever), bubonic
plague, tularemia (rabbit fever), malaria, cholera, typhoid,
hemorrhagic fever, tick-borne encephalitis, Venezuelan equine
encephalitis, pneumonic plague, Rocky Mountain spotted fever,
dengue fever, Rift Valley fever, diphtheria, melioidosis, glanders,
tuberculosis, infectious hepatitis, encephalitides, blastomycosis,
nocardiosis, yellow fever, typhus, and Q fever.
Virus: A microscopic infectious organism that reproduces inside
living cells. A virus consists essentially of a core of a single
nucleic acid surrounded by a protein coat, and has the ability to
replicate only inside a living cell. "Viral replication" is the
production of additional virus by the occurrence of at least one
viral life cycle. A virus may subvert the host cells' normal
functions, causing the cell to behave in a manner determined by the
virus. For example, a viral infection may result in a cell
producing a cytokine, or responding to a cytokine, when the
uninfected cell does not normally do so.
Unless otherwise explained, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs. The
singular terms "a," "an," and "the" include plural referents unless
context clearly indicates otherwise. Similarly, the word "or" is
intended to include "and" unless the context clearly indicates
otherwise. It is further to be understood that all base sizes or
amino acid sizes, and all molecular weight or molecular mass
values, given for nucleic acids or polypeptides are approximate,
and are provided for description. Although methods and materials
similar or equivalent to those described herein can be used in the
practice or testing of this disclosure, suitable methods and
materials are described below. The term "comprises" means
"includes." All publications, patent applications, patents, and
other references mentioned herein are incorporated by reference in
their entirety. In case of conflict, the present specification,
including explanations of terms, will control. In addition, the
materials, methods, and examples are illustrative only and not
intended to be limiting.
III. Description of Several Embodiments
A. D and K-Type ODNs
The present disclosure relates to a class of DNA motifs that
stimulates immune activation, for example the innate immune
response or the adaptive immune response by B cells, monocytes,
dendritic cells, and natural killer (NK) cells. K type CpG ODNs
have been previously described (see U.S. Pat. No. 6,194,388; U.S.
Pat. No. 6,207,646; U.S. Pat. No. 6,214,806; U.S. Pat. No.
6,218,371; U.S. Pat. No. 6,239,116, U.S. Pat. No. 6,339,068; U.S.
Pat. No. 6,406,705 and U.S. Pat. No. 6,429,199, which are herein
incorporated by reference). K ODNs that exhibit the greatest
immunostimulatory activity share specific characteristics. These
characteristics differ from those of the Formula II or D ODN (see
below). In addition, K ODNs have specific effects on the cells of
the immune system, which differ from the effects of D ODN. For
example, K ODNs stimulate proliferation of B cells and stimulate
the production of IL-6.
The K ODNs include at least about 10 nucleotides and include a
sequence represented by Formula I:
5'N.sub.1N.sub.2N.sub.3T-CpG-WN.sub.4N.sub.5N.sub.63' wherein the
central CpG motif is unmethylated, W is A or T, and N.sub.1,
N.sub.2, N.sub.3, N.sub.4, N.sub.5, and N.sub.6 are any
nucleotides.
These Formula I or K ODNs stimulate B cell proliferation and the
secretion of IgM and IL-6, processes involved in the body's humoral
immunity, such as the production of antibodies against foreign
antigens. In one embodiment, the K ODNs induce a humoral immune
response.
Certain K oligonucleotides are of the formula:
5'N.sub.1N.sub.2N.sub.3T-CpG-WN.sub.4N.sub.5N.sub.63' contain a
phosphate backbone modification. In one specific, non-limiting
example, the phosphate backbone modification is a phosphorothioate
backbone modification (i.e., one of the non-bridging oxygens is
replaced with sulfur, as set forth in International Patent
Application WO 95/26204, herein incorporated by reference). In one
embodiment, K ODNs halve a phosphorothioate backbone, and at least
one unmethylated CpG dinucleotide. Eliminating the CpG dinucleotide
motif from the K ODN significantly reduces immune activation.
Incorporating multiple CpGs in a single K ODN increases immune
stimulation. In some embodiments, the K ODNs are at least 12 bases
long. In addition, K ODNs containing CpG motifs at the 5' end are
the most stimulatory, although at least one base upstream of the
CpG is required. More particularly, the most active K ODNs contain
a thymidine immediately 5' from the CpG dinucleotide, and a TpT or
a TpA in a position 3' from the CpG motif. Modifications which are
greater than 2 base pairs from the CpG dinucleotide motif appear to
have little effect on K ODN activity.
Examples of a K ODN include, but are not limited to:
TABLE-US-00001 TCCATGTCGCTCCTGATGCT (SEQ ID NO: 29)
TCCATGTCGTTCCTGATGCT (SEQ ID NO: 30) TCGTCGTTTTGTCGTTTTGTCGT (SEQ
ID NO: 31) TCGTCGTTGTCGTTGTCGTT (SEQ ID NO: 32)
TCGTCGTTTTGTCGTTTGTCGTT (SEQ ID NO: 33) TCGTCGTTGTCGTTTTGTCGTT (SEQ
ID NO: 34) GCGTGCGTTGTCGTTGTCGTT (SEQ ID NO: 35)
TGTCGTTTGTCGTTTGTCGTT (SEQ ID NO: 36) TGTCGTTGTCGTTGTCGTT (SEQ ID
NO: 37) TCGTCGTCGTCGTT (SEQ ID NO: 38) TCCTGTCGTTCCTTGTCGTT (SEQ ID
NO: 39) TCCTGTCGTTTTTTGTCGTT (SEQ ID NO: 40) TCGTCGCTGTCTGCCCTTCTT
(SEQ ID NO: 41) TCGTCGCTGTTGTCGTTTCTT (SEQ ID NO: 42)
TCCATGACGTTCCTGACGTT (SEQ ID NO: 43)
In particular, non-limiting examples, the K oligodeoxynucleotide
includes a sequence selected from the group consisting of SEQ ID
NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33,
SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID
NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID) NO:
42, and SEQ ID NO: 43.
D ODNs differ both in structure and activity from K ODNs. The
unique activities of D ODNs are disclosed below. For example, as
disclosed herein, D oligodeoxynucleotides stimulate the release of
cytokines from cells of the immune system. In specific,
non-limiting examples, D oligonucleotides stimulate the release or
production of IP-10 and IFN-.alpha. by monocytes and/or
plasmacytoid dendritic cells and the release or production of
IFN-.gamma. by NK cells. The stimulation of NK cells by D
oligodeoxynucleotides can be either direct or indirect.
With regard to structure, a CpG motif in D oligonucleotides can be
described by Formula II: 5'RY-CpG-RY3' wherein the central CpG
motif is unmethylated, R is A or G (a purine), and Y is C or T (a
pyrimidine). D oligonucleotides include an unmethylated CpG
dinucleotide. Inversion, replacement or methylation of the CpG
reduces or abrogates the activity of the D oligonucleotide.
Certain D ODNs are at least about 16 nucleotides in length and
includes a sequence represented by Formula III:
5'X.sub.1X.sub.2X.sub.3Pu.sub.1Py.sub.2CpG
Pu.sub.3Py.sub.4X.sub.4X.sub.5X.sub.6(W).sub.M(G).sub.N-3' wherein
the central CpG motif is unmethylated, Pu is a purine nucleotide,
Py is a pyrimidine nucleotide, X and W are any nucleotide, M is any
integer from 0 to 10, and N is any integer from 4 to 10.
The region Pu.sub.1 Py.sub.2 CpG Pu.sub.3 Py.sub.4 is termed the
CpG motif. The region X.sub.1X.sub.2X.sub.3 is termed the 5'
flanking region, and the region X.sub.4X.sub.5X.sub.6 is termed the
3' flanking region. If nucleotides are included 5' of
X.sub.1X.sub.2X.sub.3 in the D ODN, these nucleotides are termed
the 5' far flanking region. Nucleotides 3' of X.sub.4X.sub.5X.sub.6
in the D ODN are termed the 3' far flanking region.
In one specific, non-limiting example, Py.sub.2 is a cytosine. In
another specific, non-limiting example, Pu.sub.3 is a guanidine. In
yet another specific, non-limiting example, Py.sub.2 is a thymidine
and Pu.sub.3 is an adenine. In a further specific, non-limiting
example, Pu.sub.1 is an adenine and Py.sub.2 is a tyrosine. In
another specific, non-limiting example, Pu.sub.3 is an adenine and
Py.sub.4 is a tyrosine.
In one specific, not limiting example, N is from about 4 to about
8. In another specific, non-limiting example, N is about 6.
D CpG oligonucleotides can include modified nucleotides. Without
being bound by theory, modified nucleotides can be included to
increase the stability of a D oligonucleotide. Without being bound
by theory, because phosphorothioate-modified nucleotides confer
resistance to exonuclease digestion, the D ODN are "stabilized" by
incorporating phosphorothioate-modified nucleotides. In one
embodiment, the CpG dinucleotide motif and its immediate flanking
regions include phosphodiester rather than phosphorothioate
nucleotides. In one specific non-limiting example, the sequence
Pu.sub.1 Py.sub.2 CpG Pu.sub.3 Py.sub.4 includes phosphodiester
bases. In another specific, non-limiting example, all of the bases
in the sequence Pu.sub.1 Py.sub.2 CpG Pu.sub.3 Py.sub.4 are
phosphodiester bases. In yet another specific, non-limiting
example, X.sub.1X.sub.2X.sub.3 and X.sub.4X.sub.5X.sub.6(W).sub.M
(G).sub.N include phosphodiester bases. In yet another specific,
non-limiting example, X.sub.1X.sub.2X.sub.3 Pu.sub.1 Py.sub.2 CpG
Pu.sub.3 Py.sub.4 X.sub.4X.sub.5X.sub.6(W).sub.M (G).sub.N include
phosphodiester bases. In further non-limiting examples, the
sequence X.sub.1X.sub.2X.sub.3 includes at most one or at most two
phosphothioate bases and/or the sequence X.sub.4X.sub.5X.sub.6
includes at most one or at most two phosphotioate bases. In
additional non-limiting examples, X.sub.4X.sub.5X.sub.6(W).sub.M
(G).sub.N includes at least 1, at least 2, at least 3, at least 4,
or at least 5 phosphothioate bases. Thus, a D oligodeoxynucleotide
can be a phosphorothioate/phosphodiester chimera.
As disclosed herein, any suitable modification can be used in the
present disclosure to render the D oligodeoxynucleotide resistant
to degradation in vivo (e.g., via an exo- or endo-nuclease). In one
specific, non-limiting example, a modification that renders the
oligodeoxynucleotide less susceptible to degradation is the
inclusion of nontraditional bases such as inosine and quesine, as
well as acetyl-, thio- and similarly modified forms of adenine,
cytidine, guanine, thymine, and uridine. Other modified nucleotides
include nonionic DNA analogs, such as alkyl or aryl phosphonates
(i.e., the charged phosphonate oxygen is replaced with an alkyl or
aryl group, as set forth in U.S. Pat. No. 4,469,863),
phosphodiesters and alkylphosphotriesters (i.e., the charged oxygen
moiety is alkylated, as set forth in U.S. Pat. No. 5,023,243 and
European Patent No. 0 092 574). Oligonucleotides containing a diol,
such as tetraethyleneglycol or hexaethyleneglycol, at either or
both termini, have also been shown to be more resistant to
degradation. The D oligodeoxynucleotides can also be modified to
contain a secondary structure (e.g., stem loop structure). Without
being bound by theory, it is believed that incorporation of a stem
loop structure renders and oligodeoxynucleotide more effective.
In a further embodiment, Pu.sub.1 Py.sub.2 and Pu.sub.3 Py.sub.4
are self-complementary. In another embodiment,
X.sub.1X.sub.2X.sub.3 and X.sub.4X.sub.5X.sub.6 are self
complementary. In yet another embodiment X.sub.1X.sub.2X.sub.3
Pu.sub.1 Py.sub.2 and Pu.sub.3 Py.sub.4 X.sub.4X.sub.5X.sub.6 are
self complementary.
Specific non-limiting examples of a D oligonucleotide wherein
Pu.sub.1 Py.sub.2 and Pu.sub.3 Py.sub.4 are self-complementary
include, but are not limited to, ATCGAT (SEQ ID NO: 9), ACCGGT (SEQ
ID NO: 10), ATCGAC (SEQ ID NO: 11), ACCGAT (SEQ ID NO: 12), GTCGAC
(SEQ ID NO: 13), or GCCGGC (SEQ ID NO: 14). Without being bound by
theory, the self-complementary base sequences can help to form a
stem-loop structure with the CpG dinucleotide at the apex to
facilitate immunostimulatory functions. Thus, in one specific,
non-limiting example, D oligonucleotides wherein Pu.sub.1 Py.sub.2
and Pu.sub.3 Py.sub.4 are self-complementary induce higher levels
of IFN-.gamma. production from a cell of the immune system (see
below). The self-complementary need not be limited to Pu.sub.1
Py.sub.2 and Pu.sub.3 Py.sub.4. Thus, in another embodiment,
additional bases on each side of the three bases on each side of
the CpG-containing hexamer form a self-complementary sequence (see
above).
One specific non-limiting example of a sequence wherein Pu.sub.1
Py.sub.2 and Pu.sub.3 Py.sub.4 are self-complementary but wherein
the far-flank-ing sequences are not self-complementary is:
TABLE-US-00002 GGTGCATCGATACAGGGGGG. (ODN D 113, SEQ ID NO: 15)
This oligodeoxynucleotide has a far flanking region that is not
self-complementary and induces high levels of IFN-.gamma. and
IFN-.alpha..
Another specific, non-limiting example of a D oligodeoxynucleotides
is:
TABLE-US-00003 GGTGCGTCGATGCAGGGGGG. (D28, SEQ ID NO: 16)
This oligodeoxynucleotide is of use for inducing production and/or
release of cytokines from immune cells, although it lacks a
self-complementary motif.
In one embodiment, the D oligodeoxynucleotides disclosed herein are
at least about 16 nucleotides in length. In a second embodiment, a
D oligodeoxynucleotide is at least about 18 nucleotides in length.
In another embodiment, a D oligodeoxynucleotide is from about 16
nucleotides in length to about 100 nucleotides in length. In yet
another embodiment, a D oligodexoynucleotide is from about 16
nucleotides in length to about 50 nucleotides in length. In a
further embodiment, a D oligodeoxynucleotide is from about 18
nucleotides in length to about 30 nucleotides in length.
In another embodiment, the oligodeoxynucleotide is at least 18
nucleotides in length, and at least two G's are included at the 5'
end of the molecule, such that the oligodeoxynucleotide includes a
sequence represented by Formula IV:
5'GGX.sub.1X.sub.2X.sub.3Pu.sub.1Py.sub.2CpGPu.sub.3Py.sub.4X.sub.4X.sub.-
5X.sub.6(W).sub.M(G).sub.N-3'. The D oligodeoxynucleotide can
include additional G's at the 5' end of the oligodeoxynucleotide.
In one specific example, about 1 or about 2 G's are included at the
5' end of an olgiodeoxynucleotide including a sequence as set forth
as Formula IV.
Examples of a D oligodeoxynucleotide include, but are not limited
to:
TABLE-US-00004 5'XXTGCATCGATGCAGGGGGG3', (SEQ ID NO: 1)
5'XXTGCACCGGTGCAGGGGGG3', (SEQ ID NO: 2) 5'XXTGCGTCGACGCAGGGGGG3',
(SEQ ID NO: 3) 5'XXTGCGTCGATGCAGGGGGG3', (SEQ ID NO: 4)
5'XXTGCGCCGGCGCAGGGGGG3', (SEQ ID NO: 5) 5'XXTGCGCCGATGCAGGGGGG3',
(SEQ ID NO: 6) 5'XXTGCATCGACGCAGGGGGG3', (SEQ ID NO: 7)
5'XXTGCGTCGGTGCAGGGGGG3', (SEQ ID NO: 8)
wherein X is any base, or is no base at all. In one specific,
non-limiting example, X is a G. In particular, non-limiting
examples, the oligodeoxynucleotide includes a sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,
SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID
NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,
SEQ ID NO: 24, and SEQ ID NO: 25.
The D and K oligodeoxynucleotides disclosed herein can be
synthesized de novo using any of a number of procedures well known
in the art. For example, the oligodeoxynucleotides can be
synthesized as set forth in U.S. Pat. No. 6,194,388, which is
herein incorporated by reference in its entirety. A D
oligodeoxynucleotide may be synthesized using, for example, by the
B-cyanoethyl phosphoramidite method or nucleoside H-phosphonate
method. These chemistries can be performed by a variety of
automated oligonucleotide synthesizers available in the market.
Alternatively, oligodeoxynucleotides can be prepared from existing
nucleic acid sequences (e.g. genomic or cDNA) using known
techniques, such as employing restriction enzymes, exonucleases or
endonucleases, although this method is less efficient than direct
synthesis.
B. Pharmaceutical Compositions
The immunostimulatory ODNs described herein may be formulated in a
variety of ways depending on the location and type of disease to be
treated or prevented. Pharmaceutical compositions are thus provided
for both local (e.g. topical or inhalational) use and for systemic
use. Therefore, the disclosure includes within its scope
pharmaceutical compositions comprising at least one
immunostimulatory ODN formulated for use in human or veterinary
medicine. While the immunostimulatory ODNs will typically be used
to treat human subjects, they may also be used to treat similar or
identical diseases in other vertebrates, such other primates, dogs,
cats, horses, and cows.
Pharmaceutical compositions that include at least one
immunostimulatory K or D ODN as described herein as an active
ingredient, or that include both an immunostimulatory ODN and an
additional anti-infective agent as active ingredients, may be
formulated with an appropriate solid or liquid carrier, depending
upon the particular mode of administration chosen. Additional
active ingredients include, for example, anti-infective agents,
such as antibiotics, anti-fungal compounds, anti-viral compounds,
and hyper-immune globulin. A suitable administration format may
best be determined by a medical practitioner for each subject
individually. Various pharmaceutically acceptable carriers and
their formulation are described in standard formulation treatises,
e.g., Remington's Pharmaceutical Sciences by E. W. Martin. See also
Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and
Technology, Technical Report No. 10, Supp. 42: 2S, 1988.
The dosage form of the pharmaceutical composition will be
determined by the mode of administration chosen. For instance, in
addition to injectable fluids, inhalational and oral formulations
can be employed. Inhalational preparations can include aerosols,
particulates, and the like. In general, the goal for particle size
for inhalation is about 1 .mu.m or less in order that the
pharmaceutical reach the alveolar region of the lung for
absorption. Oral formulations may be liquid (e.g., syrups,
solutions, or suspensions), or solid (e.g., powders, pills,
tablets, or capsules). For solid compositions, conventional
non-toxic solid carriers can include pharmaceutical grades of
mannitol, lactose, starch, or magnesium stearate. Actual methods of
preparing such dosage forms are known, or will be apparent, to
those of ordinary skill in the art.
In some embodiments, the bioavailability and duration of action of
CpG ODN may improve their therapeutic efficacy. One potential
method for protecting CpG ODN from degradation while increasing
their uptake by cells of the immune system involves liposome
encapsulation (MacDonald et al., Biochim. Biophys. Acta 1061:297,
1991; Takeshita et al., Eur. J. Immunol. 30:108, 2000). Sterically
stabilized cationic liposomes (SSCL) compositions efficiently
incorporate and deliver K type CpG ODNs to cells in vitro and in
vivo. The SSCLs are liposomes that include a cationic lipid, a
colipid, and a stabilizing additive, as described below.
Cationic lipids include, but are not limited to
spermidine-cholesterol, spermine-cholesterol, is
dimethylaminoethae-carbomol-chlesteroc (DC-CHOL), and
dioctadecylamidoglycylspermine (DOGS). In one embodiment, the
cationic lipid is dimethylaminoethane-carbomol-cholesterol
(DC-CHOL). Colipids include, but are not limited to, neutral,
zwitterionic, and anionic lipids. In one embodiment, the colipid is
dioleoylphosphatidylethanolamine (DOPE). The colipid can be a
moiety that allows the stabilizing additive (see below) to be
incorporated into the complex. Without being bound by theory,
derivatization of the lipid with an additive allows the moiety to
anchor the stabilizing additive to the cationic lipid complex. The
colipid can be conjugated to additives which prevent aggregation
and precipitation of cationic lipid-nucleic acid complexes.
Colipids which may be used to incorporate such additives to
compositions disclosed herein include, but are not limited to,
zwitterionic or other phospholipids. Preferably, the colipid is
inert and biocompatible.
The ratio of cationic lipid to colipid (as a molar ratio) is from
about 3:7 to about 7:3. In one embodiment, the ratio of cationic
lipid to colipid (molar ratio) is about 4:6 to about 6:4. In a
further embodiment, the lipid to colipid (molar ratio) is about
4:6. Thus, in one specific, non-limiting example DC-CHOL and DOPE
are included in the sterically stabilized cationic liposome at a
molar ratio of about 4:6.
Stabilizing agents are also included in the sterically stabilized
cationic liposomes. Without being bound by theory, it is believed
that the stabilizing agent maintains the integrity of the complex,
maintains stability during sizing procedures, and increases shelf
life. In one embodiment, the additives are bound to a moiety
capable of being incorporated into or binding to the complex, for
example, a colipid. Such additives generally are selected from
among hydrophilic polymers, which include, but are not limited to,
polyethylene glycol, polyvinylpyrrolidine, polymethyloxazoline,
polyethyl-oxazoline, polyhydroxypropyl methacrylamide, polylactic
acid, polyglycolic acid, and derivatized celluloses such as
hydroxymethylcellulose or hydroxyethylcellulose (see published PCT
Application No. WO 94/22429). Other stabilizing agents include, but
are not limited to perfluorinated or partially fluorinated alkyl
chains, fluorinated phospholipids, fatty acids and
perfluoroalkylated phospholipids and polyglucoronic acids (Oku et
al., Critical Reviews in Therapeutic Drug Carrier System,
11:231-270, 1994).
A variety of methods are available for preparing liposomes as
described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467,
1980; U.S. Pat. No. 4,186,183; U.S. Pat. No. 4,217,344; U.S. Pat.
No. 4,235,871; U.S. Pat. No. 4,261,975; U.S. Pat. No. 4,485,054;
U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,774,085; U.S. Pat. No.
4,837,028; U.S. Pat. No. 4,946,787; PCT Publication No. WO
91/17424; Szoka & Papahadjopoulos, Proc. Natl. Acad. Sci. USA
75:4194-4198, 1978; Deamer & Bangham, Biochim. Biophys. Acta
443:629-634, 1976; Fraley et al., Proc. Natl. Acad. Sci. USA
76:3348-3352, 1979; Hope et al., Biochim. Biophys. Acta 812:55-65,
1985; Mayer et al., Biochim. Biophys. Acta 858:161-168, 1986;
Williams et al., Proc. Natl. Acad. Sci. USA 85:242-246, 1988,
Liposomes, ch. 1 (Ostro, ed., 1983); and Hope et al., Chem. Phys.
Lip. 40:89, 1986; U.S. Pat. No. 6,410,049. Suitable methods
include, e.g., sonication, extrusion, high pressure/homogenization,
microfluidization, detergent dialysis, calcium-induced fusion of
small liposome vesicles, and ether-infusion methods, all well known
in the art.
In one embodiment, a pharmacological composition is provided that
includes a D or K oligonucleotide and a pharmacologically
acceptable carrier. Pharmacologically acceptable carriers (e.g.,
physiologically or pharmaceutically acceptable carriers) are well
known in the art. A suitable pharmacological composition can be
formulated to facilitate the use of K or D type ODN in vivo. Such a
composition can be suitable for delivery of the active ingredient
to any suitable host, such as a patient for medical application,
and can be manufactured in a manner that is itself known, e.g., by
means of conventional mixing dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
The compositions or pharmaceutical compositions can be administered
by any route, including parenteral administration, for example,
intravenous, intraperitoneal, intramuscular, intraperitoneal,
intrasternal, or intra-articular injection or infusion, or by
sublingual, oral, topical, intra-nasal, or transmucosal
administration, or by pulmonary inhalation. When immunostimulatory
ODNs are provided as parenteral compositions, e.g. for injection or
infusion, they are generally suspended in an aqueous carrier, for
example, in an isotonic buffer solution at a pH of about 3.0 to
about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to
6.0, or 3.5 to about 5.0. Useful buffers include sodium
citrate-citric acid and sodium phosphate-phosphoric acid, and
sodium acetate/acetic acid buffers.
For oral administration, the pharmaceutical compositions can take
the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets can be
coated by methods well known in the art. Liquid preparations for
oral administration can take the form of, for example, solutions,
syrups or suspensions, or they can be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations can
also contain buffer salts, flavoring, coloring, and sweetening
agents as appropriate.
For administration by inhalation, the compounds for use according
to the present invention are conveniently delivered in the form of
an aerosol spray presentation from pressurized packs or a
nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol, the dosage unit can be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges for use in an inhaler or insufflator can be
formulated containing a powder mix of the compound and a suitable
powder base such as lactose or starch.
Pharmaceutical compositions that comprise an immunostimulatory ODN
as described herein as an active ingredient will normally be
formulated with an appropriate solid or liquid carrier, depending
upon the particular mode of administration chosen. The
pharmaceutically acceptable carriers and excipients of use are
conventional. For instance, parenteral formulations usually
comprise injectable fluids that are pharmaceutically and
physiologically acceptable fluid vehicles such as water,
physiological saline, other balanced salt solutions, aqueous
dextrose, glycerol or the like. Excipients that can be included
are, for instance, proteins, such as human serum albumin or plasma
preparations. If desired, the pharmaceutical composition to be
administered may also contain minor amounts of non-toxic auxiliary
substances, such as wetting or emulsifying agents, preservatives,
and pH buffering agents and the like, for example sodium acetate or
sorbitan monolaurate. Actual methods of preparing such dosage forms
are known, or will be apparent, to those skilled in the art.
For example, for parenteral administration, immunostimulatory ODNs
can be formulated generally by mixing them at the desired degree of
purity, in a unit dosage injectable form (solution, suspension, or
emulsion), with a pharmaceutically acceptable carrier, i.e., one
that is non-toxic to recipients at the dosages and concentrations
employed and is compatible with other ingredients of the
formulation. A pharmaceutically acceptable carrier is a non-toxic
solid, semisolid or liquid filler, diluent, encapsulating material
or formulation auxiliary of any type.
Generally, the formulations are prepared by contacting the
immunostimulatory ODNs each uniformly and intimately with liquid
carriers or finely divided solid carriers or both. Then, if
necessary, the product is shaped into the desired formulation.
Optionally, the carrier is a parenteral carrier, and in some
embodiments it is a solution that is isotonic with the blood of the
recipient. Examples of such carrier vehicles include water, saline,
Ringer's solution, and dextrose solution. Non-aqueous vehicles such
as fixed oils and ethyl oleate are also useful herein, as well as
liposomes.
The pharmaceutical compositions that comprise an immunostimulatory
ODN, in some embodiments, will be formulated in unit dosage form,
suitable for individual administration of precise dosages. The
amount of active compound(s) administered will be dependent on the
subject being treated, the severity of the affliction, and the
manner of administration, and is best left to the judgment of the
prescribing clinician. Within these bounds, the formulation to be
administered will contain a quantity of the active component(s) in
amounts effective to achieve the desired effect in the subject
being treated.
The therapeutically effective amount of immunostimulatory ODN will
be dependent on the ODN utilized, the subject being treated, the
severity and type of the affliction, and the manner of
administration. For example, a therapeutically effective amount of
immunostimulatory ODN can vary from about 0.01 .mu.g per kilogram
(kg) body weight to about 1 g per kg body weight, such as about 1
.mu.g to about 5 mg per kg body weight, or about 5 .mu.g to about 1
mg per kg body weight. The exact dose is readily determined by one
of skill in the art based on the potency of the specific compound
(such as the immunostimulatory ODN utilized), the age, weight, sex
and physiological condition of the subject.
C. Therapeutic Uses
Methods are disclosed herein for treating or preventing an
infection in a subject who has been exposed to or is at risk for
exposure to a bioterrorism agent. These methods include: 1.
administering a therapeutically effective amount of the
immunostimulatory ODN to a subject who has been exposed to or is at
risk for exposure to a bioterrorism agent, 2. administering a
therapeutically effective amount of the immunostimulatory ODN in
combination with an anti-infective agent to a subject who has been
exposed to or is at risk for exposure to a bioterrorism agent,
thereby treating or preventing the infection in a subject, and 3.
administering to the subject a therapeutically effective amount of
an immunostimulatory D oligodeoxynucleotide or an immunostimulatory
K oligodeoxynucleotide in combination with a vaccine against a
bioterrorism agent.
Bioterrorism agents include, but are not limited to Bacillus
anthracis, Yersinia pestis, Variola major, Histoplasma capsulatum,
Haemophilus influenzae, Ebola virus, tick-borne encephalitis virus
(TBEV), Escherichia coli, Shigella flexneri, S. dysenteriae
(Shigella bacillus), Salmonella, Staphylococcus enterotoxin B,
botulinum toxin, ricin toxin, cobra venom, shellfish toxin,
botulinum toxin, saxitoxin, ricin toxin, tricothecene mycotoxin, or
aflatoxin. Exposure to bioterrorism agents can result in
infections, such as, but not limited to, anthrax, cryptococcosis,
brucellosis, coccidioidomycosis, psittacosis, bubonic plague,
tularemia, malaria, cholera, typhoid, hemorrhagic fever, tick-borne
encephalitis, Venezuelan equine encephalitis, pneumonic plague,
Rocky Mountain spotted fever, dengue fever, Rift Valley fever,
diphtheria, melioidosis, glanders, tuberculosis, infectious
hepatitis, encephalitides, blastomycosis, nocardiosis, yellow
fever, typhus, or Q fever.
Some embodiments of the methods include administering a
therapeutically effective amount of the immunostimulatory ODN to a
subject who has been exposed to or is at risk for exposure to a
bioterrorism agent, thereby treating or preventing the infection in
a subject. Without being bound by theory, administration of the
immunostimulatory ODN increases the general immune activation,
leading to an increase in the immune response to the bioterrorism
agent or vaccine. In this manner, a subject can be at reduced risk
of infection or susceptible to fewer symptoms, and can obtain
further treatment if necessary. In one embodiment, the
immunostimulatory ODN can be administered locally, such as by
inhalation. In another embodiment, the immunostimulatory ODN is
administered systemically, such as by intravenous injection,
intramuscular injection, or subcutaneous injection.
In some embodiments, the immunostimulatory ODN is administered to
the subject prior to exposure to a bioterrorism agent, for example,
two weeks, one week, one day, six hours, or one hour prior to
exposure. The immunostimulatory ODN can also be administered to the
subject after exposure to a bioterrorism agent, for example, two
weeks, one week, one day, six hours, or one hour after exposure. In
some embodiments, the immunostimulatory ODN is administered to a
subject who is at risk for exposure to a bioterrorism agent, for
example, a member of the military, a police officer, a mail
handler, a government official, or any other individual who is at
risk for exposure to a bioterrorism agent. For example, in one
specific, non-limiting example, an immunostimulatory ODN is
administered to a member of the military three days, six days, or
two weeks before deployment. Suitable subjects also include those
who are more prone to illness following bioterrorism agent
exposure, such as subjects with weakened immune systems, for
example, the elderly, people on immunosuppressive drugs, subjects
with cancer, and subjects infected with HIV
Combinations of these immunostimulatory ODNs are also of use. Thus,
in one embodiment, more than one immunostimulatory D or K ODN, or
both D and K ODNs, each with a different nucleic acid sequence, are
administered to the subject. In several specific, non-limiting
examples, at least two, at least three, or at least four
immunostimulatory D ODNs are administered to the subject. In other
specific, non-limiting examples, at least two, at least three, or
at least four immunostimulatory K ODNs are administered to the
subject. In still further specific, non-limiting examples, at least
two, at least three, or at least four immunostimulatory D ODNs are
administered to the subject in combination with at least two, at
least three, or at least four immunostimulatory K ODNs.
An effective amount of an immunostimulatory ODN can be administered
in a single dose, or in multiple doses, for example weekly, during
a course of treatment. In one embodiment, a therapeutically
effective amount of an immunostimulatory ODN is administered as a
single pulse dose, as a bolus dose, or as pulse doses administered
over time. Thus, in pulse doses, a bolus administration of an
immunostimulatory ODN is provided, followed by a time period
wherein no immunostimulatory ODN is administered to the subject,
followed by a second bolus administration. In specific,
non-limiting examples, pulse doses of an immunostimulatory ODN are
administered during the course of a day, during the course of a
week, or during the course of a month.
Thus, the immunostimulatory ODNs disclosed herein may be
administered to a subject for the treatment of a bioterrorism
agent-induced infection in that individual. ODN administration can
be systemic or local. Local administration of the ODN is performed
by methods well known to those skilled in the art. By way of
example, one method of administration to the lungs of an individual
is by inhalation through the use of a nebulizer or inhaler. For
example, the ODN is formulated in an aerosol or particulate and
drawn into the lungs using a standard nebulizer well known to those
skilled in the art.
In other embodiments, the administration of the immunostimulatory
ODN is systemic. Oral, intravenous, intra-arterial, subcutaneous,
intra-peritoneal, intra-muscular, and even rectal administration is
contemplated. Prevention of an infection includes both prevention
of symptoms and delaying the onset of symptoms. In specific,
non-limiting examples, administration of an immunostimulatory ODN
delays symptoms of an infection until further treatment is
sought.
The effectiveness of treatment with an immunostimulatory ODN can be
measured by monitoring symptoms of infection, for example, fever.
For example, a decrease in fever over time is an indicator of
efficacy of ODN treatment.
In some embodiments, the method includes administering to the
subject an anti-infective agent, such as an antibiotic, anti-viral
compound, anti-fungal compound, or hyper-immune globulin, in
conjunction with an immunostimulatory ODN. The administration of
the additional anti-infective agent and the immunostimulatory ODN
can be sequential or simultaneous.
Anti-infectious agents include, but are not limited to, anti-fungal
compounds, anti-viral compounds, and antibiotics. Antibiotics
include, but are not limited to, amoxicillin, clarithromycin,
cefuroxime, cephalexin ciprofloxacin, doxycycline, metronidazole,
terbinafine, levofloxacin, nitrofurantoin, tetracycline, and
azithromycin. Anti-fungal compounds, include, but are not limited
to, clotrimazole, butenafine, butoconazole, ciclopirox, clioquinol,
clioquinol, clotrimazole, econazole, fluconazole, flucytosine,
griseofalvin, haloprogin, itraconazole, ketoconazole, miconazole,
naftifine, nystatin, oxiconazole, sulconazole, terbinafine,
terconazole, tioconazole, and tolnaftate. Anti-viral compounds,
include, but are not limited to, zidovudine, didanosine,
zalcitabine, stavudine, lamivudine, abacavir, tenofovir,
nevirapine, delavirdine, efavirenz, saquinavir, ritonavir,
indinavir, nelfinavir, saquinavir, amprenavir, and lopinavir.
Anti-infectious agents also include hyper-immune globulin.
Other methods described herein are methods of enhancing the
immunogenicity of a vaccine against a bioterrorism agent in a
subject. In some embodiments, the vaccine against a bioterrorism
agent is an antigen. The method includes administering to the
subject a therapeutically effective amount of an immunostimulatory
D oligodeoxynucleotide or an immunostimulatory K
oligodeoxynucleotide in combination with a vaccine against a
bioterrorism agent, thereby enhancing the efficacy of the vaccine.
The vaccine can be a preparation of antigen, DNA, protein subunit,
peptide, attenuated microorganisms (including but not limited to
bacteria and viruses), living microorganisms, or killed
microorganisms, administered for the prevention, amelioration or
treatment of a disease caused by a bioterrorism agent.
In some embodiments, the vaccine is a heat or formalin-killed
vaccine. Examples of heat-killed vaccines in common use today are
the typhoid vaccine and the Salk poliomyelitis vaccine.
In other embodiments, the vaccine is an acellular vaccine.
Acellular vaccines are made using only the antigenic part of the
disease-causing organism, for example the capsule, the flagella, or
part of the protein cell wall. In still other embodiments, the
vaccine is an attenuated vaccine. Attenuated vaccines are made by
"attenuating" or weakening a live microorganism by aging it or
altering its growth conditions. In still further embodiments, the
vaccine is a toxoid.
In other embodiments, the vaccine is made from a related, less
virulent pathogen. The related pathogen does not cause serious
disease, but provides protection from the more virulent pathogen.
For example, the relatively mild cowpox virus is used to protect
against the similar, but often lethal, smallpox virus. In still
further embodiments, the vaccine is a subunit vaccine or a DNA
vaccine.
Thus, a CpG oligonucleotide can be used in conjunction with a wide
variety of vaccines against a bioterrorism agent, including but not
limited to, a heat or formalin-killed vaccine, attenuated vaccine,
protein subunit vaccine, antigen vaccine, DNA vaccine, acellular
vaccine, or toxoid vaccine directed against Bacillus anthracis,
Yersinia pestis, Variola major, tick-borne encephalitis virus
(TBEV), Ebola virus, Escherichia coli, Haemophilus influenzae,
cobra venom, shellfish toxin, botulinum toxin, saxitoxin, ricin
toxin, Shigella flexneri, S. dysenteriae (Shigella bacillus),
Salmonella, Staphylococcus enterotoxin B, Histoplasma capsulatum,
tricothecene mycotoxin, aflatoxin. The vaccine can also be directed
against cryptococcosis, brucellosis (undulant fever),
coccidioidomycosis (San Joaquin Valley or desert fever),
psittacosis (parrot fever), bubonic plague, tularemia (rabbit
fever), malaria, cholera, typhoid, hemorrhagic fever, tick-borne
encephalitis, Venezuelan equine encephalitis, pneumonic plague,
Rocky Mountain spotted fever, dengue fever, Rift Valley fever,
diphtheria, melioidosis, glanders, tuberculosis, infectious
hepatitis, encephalitides, blastomycosis, nocardiosis, yellow
fever, typhus, and Q fever. In some embodiments, the vaccine is an
antigen from Bacillus anthracis, Ebola virus, tick-borne
encephalitis virus (TBEV), Yersinia pestis, Variola major,
Histoplasma capsulatum, Haemophilus influenzae, Escherichia coli,
Shigella flexneri, S. dysenteriae (Shigella bacillus), Salmonella,
or Staphylococcus.
In particular examples of certain embodiments, the vaccine is an
anthrax vaccine, such as, but not limited to AVA, or an anthrax
antigen, such as, but not limited to Protective Antigen (PA) or
recombinant Protective Antigen (rPA).
Primary vaccination with AVA generally consists of three
subcutaneous injections at 0, 2, and 4 weeks, and three booster
vaccinations at 6, 12, and 18 months. To maintain immunity, the
manufacturer recommends an annual booster injection. Because of the
complexity of a six-dose primary vaccination schedule and frequency
of local injection-site reactions, schedules with a reduced number
of doses would be desirable. Administration of AVA in conjunction
with an immunostimulatory D or K ODN provides a better immune
response to the vaccine than use of the vaccine alone, and can
result in a decreased frequency of immunizations required to attain
an immune protective response.
In particular, non-limiting examples, the vaccine is a DNA sequence
encoding the non-toxic protective antigen (PA) from B. anthracis or
an immunogenic fragment thereof. The sequence for PA has been
determined and has been deposited in GenBank at Accession No.
M22589. Other antigens of use include, but are not limited to, B.
anthracis lethal factor (LF) or an immunogenic fragment thereof,
disclosed in U.S. Publication No. U.S. 2002/0051791A1, hantavirus
antigens, for example those disclosed in U.S. Pat. No. 5,614,193,
smallpox antigens, for example those disclosed in U.S. Pat. No.
4,567,147, plague antigens, for example those disclosed in WO
98/24912 A2, Ebola virus antigens, for example those disclosed in
WO 00/00617A2, tick-borne encephalitis antigens, for example those
disclosed in U.S. Pat. No. 6,372,221 and EP 0691404 B1, Histoplasma
capsulatum antigens, for example those disclosed in WO 99/55874A2
and U.S. Pat. No. 6,391,313, Haemophilus influenzae antigens, for
example those disclosed in U.S. Pat. No. 6,342,232, EP 0432220 B1,
and U.S. Pat. No. RE 37741, E. coli antigens, for example those
disclosed in U.S. Pat. No. 5,370,872, U.S. Pat. No. 6,077,516, and
U.S. Pat. No. 3,975,517, Shigella antigens, for example those
disclosed in U.S. Pat. No. 5,077,044, U.S. Pat. No. 5,686,580, and
U.S. Pat. No. 5,681,736, Salmonella antigens, for example those
disclosed in WO 01/70247 A2, U.S. Publication No. 2001/0021386A1
and EP 1112747A1, and Staphylococcus antigens, for example those
disclosed in EP 0694309A3 and U.S. Pat. No. 6,391,315.
The method includes administering a therapeutically effective
amount of the immunostimulatory D and/or K ODN to a subject in
conjunction with a vaccine against a bioterrorism agent, thereby
enhancing the immunogenicity of the vaccine. In one embodiment, the
immunostimulatory ODN can be administered locally, such as
topically or by inhalation. In another embodiment, the
immunostimulatory ODN is administered systemically, such as by
intravenous injection, intramuscular injection, or subcutaneous
injection.
Combinations of immunostimulatory ODNs are also of use in enhancing
the immunogenicity of a vaccine against a bioterrorism agent. Thus,
in one embodiment, more than one immunostimulatory ODN, each with a
different nucleic acid sequence, are administered to the subject in
combination with the vaccine. In several specific, non-limiting
examples, at least two, at least three, or at least four
immunostimulatory ODNs are administered to the subject in
combination with the vaccine.
An effective amount of an immunostimulatory ODN can be administered
in combination with a vaccine against a bioterrorism agent in a
single dose, or in multiple doses. For example, in some
embodiments, boosters of the vaccine and immunostimulatory ODN can
be administered periodically after the initial administration, for
example, at one month, two months, or three months after the
initial administration. In specific, non-limiting examples, pulse
doses of an immunostimulatory ODN, in combination with a vaccine
against a bioterrorism agent, are administered at 2 weeks, 4 weeks,
6 months, 12 months, 18 months, or yearly after the initial bolus
administration.
In other embodiments, a subject who likely has been exposed to a
bioterrorism agent can receive a vaccine against a bioterrorism
agent in conjunction with an immunostimulatory D or K ODN and an
anti-infective agent. For example, during a course of treatment of
a suspect who has been, or is likely to have been exposed to a
bioterrorism agent, the vaccine and ODN can be administered daily,
weekly, or every two weeks.
The immunostimulatory ODNs can be administered before vaccine
administration, concurrently with vaccine administration or after
vaccine administration. For example, the immunostimulatory ODN can
be administered before the vaccine is administered, for instance,
two weeks, one week, one day, or one hour before the vaccine is
administered to the subject. Alternatively, the immunostimulatory
ODN can be administered concurrently with vaccine administration,
or, for instance, two weeks, one week, one day, or one hour after
the vaccine is administered to the subject.
Thus, the immunostimulatory ODNs described herein can be
administered to a subject in combination with a vaccine against a
bioterrorism agent in order to enhance the immunogenicity of the
vaccine. The effectiveness of the ODN administration can be
measured by monitoring vaccine against a bioterrorism agent titer
or avidity of antibody response, or cytotoxic T cell response, by
methods known to one of skill in the art. For example, an increase
in vaccine against a bioterrorism agent titer or avidity of
antibody response over time is an indicator of efficacy of ODN
treatment.
The disclosure is illustrated by the following non-limiting
Examples.
EXAMPLES
Example 1
Materials and Methods
Reagents
Phosphorothioate ODN (Table 1) were synthesized or obtained from a
commercial source. Specifically, CpG 7909 (SEQ ID NO: 200) and CpG
10103 are both K type ODN that were obtained from Coley
Pharmaceuticals (Wellesley, Mass.). All synthesized ODN had less
than <0.1 EU of endotoxin per mg of ODN as assessed by a Limulus
amebocyte lysate assay (QCL-1000, BioWhittaker).
AVA was obtained from BioPort Corporation (East Lansing, Md.).
Recombinant PA (rPA) was produced as previously described (Farchaus
et al., Appl. Environ. Microbiol. 64:982, 1998). For vaccinations,
50 .mu.g of rPA was dissolved in 0.5 ml of PBS plus 0.5 mg of
aluminum. (Alhydrogel, SuperFos/BioSector, Denmark).
The Bacillus anthracis Ames and Vollum 1B strains were obtained
from the culture collection of the United States Army Medical
Research Institute of Infectious Diseases, Fort Detrick, Md. Spores
were prepared and stored as previously described (Ivins et al.,
Infect. Immun. 58:303, 1990).
TABLE-US-00005 TABLE 1 Sequence and backbone of murine and human
ODN Desig- Species nation SEQ ID NO: Sequence Mouse CpG ODN SEQ ID
NO: 17 GCTAGACGTTAGCGT 1555 Mouse CpG ODN SEQ ID NO: 18 TCAACGTTGA
1466 Mouse Control SEQ ID NO: 19 GCTAGAGCTTAGGCT ODN Mouse Control
SEQ ID NO: 20 TCAAGCTTGA ODN Human CpG ODN SEQ ID NO: 21
GGTGCATCGATGCAGGGGGG D19 Human CpG ODN SEQ ID NO: 22
GGTGCACCGGTGCAGGGGGG D29 Human CpG ODN SEQ ID NO: 21
GGTGCATCGATGCAGGGGGG D35 Human CpG ODN SEQ ID NO: 23
ATCGACTCTCGAGCGTTCTC K3 Human CpG ODN SEQ ID NO: 24 TCGTTCGTTCTC
K123 Human CpG ODN SEQ ID NO: 25 TCGAGCGTTCTC K23 Human Control SEQ
ID NO: 26 GGTGCATTGATGCAGGGGGG ODN Human Control SEQ ID NO: 27
TTGAGTGTTCTC ODN Human Control SEQ ID NO: 28 GGGCATGCATGGGGGG
ODN
Bases shown in italics are phosphodiester while all others are
phosphorothioate. CpG dinucleotides are underlined.
Animals
All animal studies were ACUC approved and were conducted in AAALAC
accredited facilities. Animals were monitored daily by
veterinarians. Specific pathogen-free BALB/c mice were obtained
from the Jackson Laboratories (Bar Harbor, Me.) and housed in
sterile micro-isolator cages in a barrier environment. Mice were
injected i.p. at 6-8 weeks of age with 50 .mu.g of CpG ODN and then
challenged SQ with 11-70 LD50 B. anthracis Vollum 1B spores.
Hartley guinea pigs, 325-375 gm (Charles River) were immunized IM
with 0.5 ml-doses of AVA plus 100-300 .mu.g of CpG ODN, and boosted
with the same material 4 weeks later. Animals were challenged IM at
week 10 with 5,000 (50 LD50) Ames spores.
Healthy 3 year old female rhesus macaques were obtained from the
FDA colony in South Carolina. Five to six animals/group were
immunized subcutaneously at 0 and 6 weeks with the normal human
dose of AVA (0.5 ml) or rPA (50 .mu.g) plus 250 .mu.g of "K" or "D"
CpG ODN. Animals were "challenged" IM with the live veterinary
vaccine strain of anthrax (Sterne) on week 27. Treatments were
administered and peripheral blood samples obtained from ketamine
anesthetized animals (10 mg/kg, Ketaject, Phoenix Pharmaceuticals,
St. Joseph, Md.).
For several of the experiments, five groups of 5 male and female
rhesus macaques/group were immunized subcutaneously (SQ) or
intramuscularly (1) on study days 0 and 42 with 0.5 mL of AVA plus
0 or 250 .mu.g of CpG ODN. All animals were monitored daily by
veterinarians. Treatments were administered under appropriate
anesthesia. A baseline blood sample was collected from each
non-human primate (NHP) 10 days before the first injection (study
day-10). Blood was collected from each NHP on days 1, 4, 11, 16,
21, 28, 35, 42, 49, 56, and 63 after the first injection.
Anti-PA ELISA and Avidity Assays
The titer of IgG against the anthrax PA was monitored by enzyme
linked immunosorbent assay (ELISA). Anti-PA IgG is considered a
marker of vaccine efficacy as anti-PA antibodies confer protection
to host cells by blocking the binding of anthrax toxin (see Pittman
et al., Vaccine 20:1412-1420, 2002). Anti-PA antibodies also
inhibit spore germination and increase the uptake and elimination
of spores by macrophages (Welkos et al., Microbiology
147:1677-1685, 2001). The titer of anti-PA IgM was also evaluated
by ELISA to evaluate any differences in the immunogenicity of the
vaccine and adjuvant combinations.
Microtiter plates (96-well Immulon 2; Dynex Technologies Inc.,
Chantilly, Va.) were coated with 1 .mu.g/ml of rPA in PBS and then
blocked with PBS-5% non-fat milk and dried overnight. Serum samples
diluted in blocking buffer were incubated on rPA-coated microtiter
plates for 2 hours. After coating, plates were blocked with 0.1%
Tween 20 with 2% non-fat dry milk in PBS for one hour at room
temperature. Plates were then overlaid with serially diluted serum
for 1 hour at 37.degree. C. as described (Ivins et al., Infect
Immun 60, 662-668, 1992). The plates were washed, and bound
antibody detected using peroxidase-conjugated goat anti-monkey IgG
or IgM (Kirkegaard & Perry, Gaithersburg, Md.) followed by ABTS
substrate (Kirkegaard & Perry). Antibody titers were determined
by comparison to a standard curve generated using high-titered
anti-PA serum. All samples were analyzed in triplicate. All assays
were performed the same day using the same reagents for all
plates.
For avidity studies, the plates were washed, and were treated for
15 minutes with 200 .mu.l of 6M urea. Bound antibodies were
detected by adding peroxidase-labeled goat anti-monkey IgG
(Kirkegaard & Perry Laboratories, Gaithersburg, Md.) followed
by ABTS (Kirkegaard & Perry Laboratories). Titers were
determined by comparison to a standard curve generated using
high-titered anti-serum. All samples were analyzed in
triplicate.
TNA Assay
The TNA assay evaluates the ability of the test serum to neutralize
the cytotoxic effects of anthrax lethal toxin (a mixture of PA and
anthrax lethal factor (LF)) in an in vitro assay. A murine target
cell line susceptible to anthrax lethal toxin is incubated with
mixtures of lethal toxin with control sera or lethal toxin with
test sera. Percent cytotoxicity is determined by the addition of a
colorimetric substrate, MTT, that in turn is hydrolyzed by the
remaining viable cells into a purple formazan precipitate, which is
solubilized and quantitated on a plate reader. The effective dose
50 (ED.sub.50) is calculated as the reciprocal of the dilution of
test serum at which 50% neutralization of the lethal toxin is
achieved. The concentration of neutralizing antibody is calculated
using the ratio of the ED.sub.50 of the test serum compared to the
ED.sub.50 of a standard reference serum.
Mouse Seroprotection Assay
Serum samples collected for the immunogenicity assays described
above were stored frozen at -80.degree. C. until use. Equal volumes
of serum from all animals in each treatment group from study day 11
and separately from study day 16 were pooled. 100 .mu.l of this
serum pool was injected IV into 6 week old, male A/JCr mice (10
recipients/group). Twenty-four hours later, blood was collected
from the tail vein from a subset of these animals (4-total) to
assess serum IgG anti-PA titers. All mice were then challenged i.p.
with 30-60 LD.sub.50 of Sterne strain anthrax spores diluted in 500
.mu.l of PBS. Survival of these mice was monitored daily for 3
weeks, and the time to death recorded. No mortality was observed in
any group after day 10. The experiment was conducted twice.
Statistical Analysis
Challenge experiments were performed using a minimum of 5-10
mice/group. Survival differences were evaluated using Student's t
test, while differences in serum anti-PA Ab titers (or avidity)
were evaluated by multiple regression ANOVA.
Example 2
Immunoprotective Activity of CpG ODN
The ability of CpG ODN to improve the survival of normal BALB/c
mice challenged with B. anthracis Vollum 1B spores was
demonstrated. The fraction of BALB/c mice surviving infection,
and/or their mean time to death (MTD), was significantly improved
by CpG ODN treatment 3-6 days prior to challenge (Table II). The
earlier treatment was initiated, the greater the beneficial effect,
consistent with the hypothesis that CpG motifs stimulate an immune
cascade that matures over several days and then persists for
several weeks (Elkins et al., J. Immunol. 162:2291, 1999; Klinman
et al., Immunity 11:123, 1999). A modest prolongation in mean time
to death (MTD) was observed in mice treated with CpG ODN one day
prior to challenge, while treatment at the same time or after
pathogen exposure had no effect on survival (Table II).
TABLE-US-00006 TABLE II Effect of CpG ODN on the survival of mice
challenged with anthrax Experiment 1 Experiment 2 Treatment Day %
survival MTD % survival MTD CpG ODN -6 50* 120 CpG ODN -3 0 110 20
114 CpG ODN 0 0 98 CpG ODN 1 0 89 No Rx 0 98 0 96 BALB/c mice (N =
10/group) were treated with 50-100 .mu.g of CpG D ODN on the day
shown, and infected with 70 (Exp 1) or 11 (Exp 2) B. anthracis 1B
spores on Day 0. Survival and mean time to death (MTD in hours) is
shown. *Significantly improved survival compared to untreated mice,
p < .05.
Thus, these CpG ODN can significantly reduce the mortality of mice
infected with anthrax. Although 100% survival was not achieved, the
mean time to death (MTD) was consistently prolonged, thus providing
a "window of opportunity" to initiate life-saving antibiotic
therapy. The activity of the ODN was unambiguously attributed to
their CpG content, since control ODN in which the CpG dinucleotide
was inverted to a GpC provided no protection.
Example 3
CpG ODN as Vaccine Adjuvants
Since exposure to anthrax is rarely predictable, the possibility
that CpG ODN could be used to enhance the immunogenicity of
protective vaccines was demonstrated. Due to evolutionary
divergence in CpG recognition between species, CpG motifs that are
highly active in rodents are poorly immunostimulatory in primates,
and vice versa (Bauer et al., Immunology 97:699, 1999; Hartmann and
Krieg, J. Immunology 164:944, 2000; Verthelyi et al., J. Immunol.
166:2372, 2001). Thus, primate models are optimal for examining the
activity of CpG ODN being developed for human use. Immune cells
from rhesus macaques respond to the same two classes of ODN that
stimulate human cells (Streilein et al., Immunol. Today 18:443,
1997). "D" type ODN trigger primate cells to secrete IFN-.alpha.
and IFNg and promote the functional maturation of APC, whereas "K"
type ODN induce immune cells to proliferate and secrete IL-6 and/or
IgM (Verthelyi et al., J. Immunol. 166:2372, 2001).
Mixtures of K and D ODN that strongly stimulate PBMC from both
humans and macaques (Streilein et al., Immunol. Today 18:443, 1997)
were co-administered as adjuvants with the licensed AVA vaccine and
with the rPA vaccine currently undergoing clinical evaluation. As
seen in FIG. 1, K ODN significantly increased the maximum, average,
and long-term IgG anti-PA responses. Over the duration of study, K
ODN increased the GMT of vaccinated macaques by 2.1+0.3 fold
(p<0.03).
The quality of a vaccine-adjuvant combination is reflected by both
the avidity and titer of the resultant Ab response. The avidity of
the IgG anti-PA Abs elicited by AVA or rPA vaccination was
monitored by incubating bound serum Abs with 6 M urea. This
treatment selectively elutes low avidity Abs (Eggers et al., J.
Med. Virol. 60:324, 2000; Cozon et al., Eur. J. Clin. Microbiol.
Infect. Dis. 17:32, 1998). As seen in FIG. 2, nearly 90% of the
serum Abs generated by primary vaccination with AVA were eluted by
urea treatment. By comparison, <50% of the serum anti-PA Abs
present in animals boosted with AVA and then challenged with
attenuated anthrax could be eluted, consistent with affinity
maturation of the memory response.
There was little difference in the average avidity of the serum
anti-PA response of macaques initially immunized with AVA or rPA
plus CpG ODN. However, the use of K and D ODN as adjuvants
generated Abs of significantly higher avidity post boost when
compared to animals immunized and boosted with vaccine alone
(p<0.02).
Since CpG motifs that activate human immune cells tend to be weakly
immunostimulatory in mice, their adjuvant effects are best assessed
in non-human primates. Fortunately, rhesus macaques respond to the
same CpG motifs that stimulate human PBMC (Verthelyi et al., J.
Immunol. 168:1659, 2002). When co-administered with the AVA
vaccine, K ODN (which support B cell activation) significantly
increased both the titer and avidity of the IgG anti-PA antibody
response (FIGS. 1 and 3). When "challenged" with a non-lethal
strain of anthrax, macaques immunized with AVA or rPA plus K ODN
mounted stronger and longer lasting immune responses (FIG. 1).
Additional studies were performed using two K type ODNs, ODN 7909
(SEQ ID NO: 200) and ODN 10103. For these studies, five groups of 5
male and female rhesus macaques/group were immunized subcutaneously
(SQ) or intramuscularly (IM) on study days 0 and 42 with 0.5 mL of
AVA plus 0 or 250 .mu.g of CpG ODN. All animals were monitored
daily by veterinarians. Treatments were administered under
appropriate anesthesia. A baseline blood sample was collected from
each non-human primate (NHP) 10 days before the first injection
(study day--10). Blood was collected from each NHP on days 1, 4,
11, 16, 21, 28, 35, 42, 49, 56, and 63 after the first
injection.
The geometric means of the anti-PA IgG titer of the five
vaccination groups for each day were computed. Anti-PA IgG titers
for each animal on each serum collection day are shown in Appendix
III. The area under the curve (AUC), Tmax and Cmax, for 3 different
intervals were also computed as follows: 1. The entire study period
up through Day 63 after the first vaccination. 2. The study period
up through Day 42, which includes only data prior to the second
vaccination. 3. The study period up through Day 21, to more closely
assess the early titer response.
AUC was calculated using the trapezoidal rule. Tmax is the time
(days after first vaccination) that the animal had the maximum
anti-PA IgG titer during the given time period, and Cmax is the
anti-PA IgG on that day.
Table III shows the median and mean values for the AUC, Tmax, and
Cmax for each of the 3 time periods defined (0-63 days, 042 days,
0-21 days). Numbers with an asterisk indicate that that group was
significantly different at the 0.05 significance level than the AVA
SQ group, based on the Wilcoxon Rank Sum test.
TABLE-US-00007 TABLE III Median (Mean) Area Under the Curve (AUC),
Time to Maximum Concentration (Tmax), and Maximum Concentration
(Cmax) by Treatment Group AVA + 10103 AVA + 7909 p- AVA SQ AVA + K
SQ SQ SQ AVA + 7909 IM value** Through Day 63 AUC 27,247,740
30,215,702 122,771,755 43,564,150 25,522,306 0.32 (72,440,976)
(34,771,333) (213,490,038) (49,416,690) (35,012,286) Tmax 49 49 49
*56 *56 0.35 (49) (51.8) (53.2) (53.2) (53.2) Cmax 2,350,000
2,080,000 10,867,300 2,205,000 1,600,000 0.25 (8,491,400)
(2,164,000) (25,783,660) (4,013,800) (2,929,600) Through Day 42 AUC
2,163,950 4,419,240 *11,657,355 *5,479,006 5,969,975 0.036
(2,761,226) (7,507,173) (10,965,198) (6,094,460) (4,922,086) Tmax
35 35 16 16 16 0.28 (27.4) (27.4) (18.4) (23.6) (17) Cmax 125,000
246,000 *548,000 *284,500 384,250 0.048 (155,932) (356,222)
(605,512) (310,522) (285,132) Through Day 21 AUC 492,292 1,108,240
*4,283,805 *2,052,506 2,985,975 0.035 (620,416) (2,402,633)
(4,289,102) (2,216,740) (2,196,426) Tmax 16 16 16 16 16 0.42 (16)
(15) (17) (17) (17) Cmax 67,600 164,300 *539,300 284,500 384,250
0.034 (86,322) (319,814) (600,312) (258,672) (285,132) *p < 0.05
in comparison to AVA SQ, using the Wilcoxon Rank Sum test **p-value
based on Kruskal-Wallis test comparing all 5 treatment groups
Statistical comparisons were made using nonparametric tests. For
each outcome, the 5 treatment groups were compared overall, using
the Kruskal-Wallis test. In addition, for each outcome, the
Wilcoxon Rank Sum test was performed for the AVA Alone group versus
each of the 4 other treatment groups separately. Outcomes that were
significantly different from AVA Alone group by the Wilcoxon Rank
Sum test at p<0.05 are indicated by an asterisk in the
tables.
All analyses included all 25 NHPs in the experiment. No adjustments
for the multiple significance tests were made. The significance
tests are provided to guide interpretation of the data only. The
fact that there are only 5 animals in each group means that
differences that may be clinically important will not necessarily
be statistically significant. On the other hand, because of the
multiple significance tests performed, some of the differences
noted may be due to chance alone.
The geometric means of the anti-PA IgG titers on each day are shown
in Table IV, along with the p-values comparing all 5 treatment
groups. Numbers with an asterisk indicate that that group was
significantly different at the 0.05 significance level from the AVA
Alone group on that day. Some significant increases in geometric
mean titers were noted when comparing groups receiving AVA in
combination with a CpG ODN on Days 11, 16, 21, 28, 35, 42, and 49
compared to AVA Alone. The geometric means of the anti-PA IgG for
all groups and all time points are plotted in FIG. 6.
TABLE-US-00008 TABLE IV Anti-PA IgG Geometric Means Titer p- Study
Day AVA SQ AVA + K SQ AVA + 10103 SQ AVA + 7909 SQ AVA + 7909 IM
value** 10 days pre 224 1,935 282 538 75 0.47 1 136 72 1,420 247 55
0.77 4 381 633 318 243 18 0.84 11 10,850 35,558 *69,896 *70,334
28,685 0.059 16 72,922 119,545 *538,025 183,630 212,095 0.059 21
35,370 61,983 *310,753 *127,623 *165,425 0.029 28 94,204 114,936
*325,414 119,436 93,301 0.101 35 119,719 182,093 *319,666 227,795
59,609 0.018 42 45,397 63,868 97,958 *184,346 61,636 0.24 49
4,323,503 1,541,858 8,143,388 1,563,387 *926,026 0.058 56 808,048
1,324,476 1,340,915 2,698,954 2,014,763 0.26 63 859,924 508,446
1,867,685 653,155 670,124 0.21 *p < 0.05 in comparison to AVA
sc, using the Wilcoxon Rank Sum test **p-value based on
Kruskal-Wallis test comparing all 5 treatment groups
The earliest time a meaningful antibody titer was detected in
animals was 11 days after the initial injection. Four of 5 and 5 of
5 animals receiving AVA+CpG 10103 SQ and AVA+CpG 7909 IM,
respectively, had peak IgG concentrations at day 16. Whereas, 3 of
5, in the group receiving AVA+CpG 7909 SQ, and 2 of 5 in the groups
receiving AVA alone or AVA+K type ODN had peak IgG concentrations
at day 16. The remaining animals in these groups had peak
concentrations at week 5. There was variability in responses
between animals consistent with other vaccine preparations. As
there were significant differences at some time points in each
group receiving AVA with a CpG ODN, it appears that CpG ODN are
increasing anti-PA antibody titers earlier during the immunization
process.
Anti-PA IgM titers were determined by ELISA at baseline and up to
16 days after the first injection. FIG. 7 shows the geometric mean
anti-PA IgM titers in all 5 study groups. The geometric mean TNA
titer on each day is shown in Table V, along with the p-values
comparing all 5 treatment groups. Numbers with an asterisk indicate
that that group was significantly different at the 0.05
significance level from the AVA Alone group on that day.
Significant differences were noted on days 4, 11, 16, 35, 42, and
56 (see also FIG. 8).
TABLE-US-00009 TABLE V Geometric Mean TNA Titer AVA + 10103 AVA +
7909 AVA + 7909 Day AVA SQ AVA + K SQ SQ SQ IM p-value** 4 750 564
818 *278 674 0.10 11 709 3,990 *5,880 *6,659 2,295 0.04 16 9,923
*40,130 *36,453 *84,737 13,095 0.003 21 30,566 49,148 38,805 67,262
24,579 0.13 28 58,216 46,442 102,761 137,442 31,483 0.07 35 32,348
39,984 59,652 *168,815 24,991 0.04 42 20,409 29,759 *68,738 *80,926
17,593 0.02 49 1,170,338 403,990 1,492,207 885,196 1,135,368 0.12
56 227,357 195,412 *914,143 262,595 *472,711 0.01 63 464,681
246,756 598,617 313,282 414,113 0.20 *p < 0.05 in comparison to
AVA sc, using the Wilcoxon Rank Sum test **p-value based on
Kruskal-Wallis test comparing all 5 treatment groups
The correlation of TNA titers with anti-PA IgG titers was examined.
Specifically, using linear regression models, TNA titer was plotted
versus anti-PA IgG titer and correlation coefficients and
probabilities were calculated. Correlations were statistically
significant (P<0.05) between the titers of the two analytes on
days 11, 21, 28, 35, 42, and 63.
Example 4
Mouse Seroprotection Assay
Mice were passively immunized with pooled serum collected from NHPs
on days 11 and 16 post their first immunization. The four vaccine
groups for which serum was pooled included AVA alone, AVA+CpG10103
SQ, AVA+CpG7909 SQ, and AVA+CpG7909 IM. Serum from a control group
of untreated NHPs and from a pool of pre-treatment serum was also
tested. Two experiments were performed (10 mice/group). Twenty-four
hours after injecting mice IV with 0.1 mL of a pooled antiserum,
serum was collected from 4 mice in each group and anti-PA IgG
titers determined.
Mice were followed for survival after an i.p. challenge with 30-60
LD.sub.50 of Sterne strain anthrax spores. Statistical analysis on
survival data was performed using the log-rank test within the PROC
LIFETEST of SAS software for Windows, Version 8.2. Table VI
summarizes the percentage survival of treatment and control groups
of mice in both experiments.
TABLE-US-00010 TABLE VI Percentage Survival of Mice Post Injection
with Pooled NHP Serum and Challenge with Anthrax Spores (N = 10
mice per group) Experiment 1 Experiment 2 Percentage Survival
Vaccination Day 11 Day 16 Day 11 Day 16 Group serum serum serum
serum AVA 10 50 10 20 AVA + 10103 SQ 40 40 0 60 AVA + 7909 SQ 60 70
40 40 AVA + 7909 IM 50 30 10 20 Control 0 0 Pre-Treatment Bleed 10
10
For NHP Serum Collected on Day 11 (two experiments combined), the
survival curves were significantly different by the log-rank test
when all 6 groups were included (p=0.0002), but were not
significantly different when the 2 control groups were excluded
(p=0.12). The pairwise analyses comparing the AVA-CpG combinations
to AVA alone gave one significant result, for AVA+CpG7909 SQ (p
0.02). For NHP Serum Collected on Day 16 (two experiments
combined), the survival curves were significantly different by the
log-rank test when all 6 groups were included (p<0.0001), but
were not significantly different when the 2 control groups were
excluded (p=0.12). The pairwise analyses comparing the AVA-CpG
combinations to AVA alone did not give any significant results.
Overall survival percentages for each treatment group were plotted
versus the arithmetic mean anti-PA IgG titer of mouse sera
collected for each treatment group 24 hours after injection of NHP
serum just before anthrax spore challenge (FIG. 9). Linear
regression was used to fit a line to the data points to be used as
a predictor of the serum titer needed to be circulating in mice at
the time of challenge to achieve protection against an injection of
30 to 60 LD.sub.50 of anthrax spores. The two data points with
titers above 6,000 were excluded from the calculation of the
regression line. Interpolating from the regression line, this data
suggest that it takes a circulating anti-PA IgG titer of 2,500 to
protect 50% of mice from a lethal challenge of 30 to 60 LD.sub.50
of anthrax spores.
Thus, CpG ODN can be safely administered in amounts of 250 .mu.g in
combination with the approved human dose of AVA to rhesus macaques.
The combination of CpG ODN with AVA increased the total anti-PA IgG
concentration after a single priming immunization compared to the
group receiving AVA alone. The peak response was also accelerated
in time for more of the animals receiving AVA+CpG 10103 and CpG
7909 compared to AVA alone. Although antibody titers were
statistically significantly increased at some time points in some
of the groups immunized with AVA in combination with CpG ODN
compared to AVA alone, the numbers of animals per group were
relatively small, and therefore, the statistical significance of
these results should be viewed with caution. However, the results
overall are highly encouraging that this combination may potentiate
the development of protective antibodies when B type CpG are
combined with the licensed anthrax vaccine.
Example 5
Effect of CpG ODN on the Protective Efficacy of AVA
The critical measure of an antigen-adjuvant combination is its
ability to induce protective immunity. Based on results indicating
that the immune response of macaques to AVA was improved by the
co-administration of CpG ODN, studies to demonstrate the protective
efficacy of CpG ODN with AVA were undertaken. Due to restrictions
associated with the use of macaques in trials involving lethal
anthrax challenge, the widely accepted guinea pig model was
employed. Normal guinea pigs succumb rapidly to challenge by 50
LD50 Ames strain anthrax spores (Table VII). Immunization and
boosting with AVA alone improved survival, although most animals
still died from infection (Table III). By comparison, nearly 75% of
the animals immunized and boosted with CpG-adjuvanted vaccine
survived (p=0.05). Co-administering CpG ODN with AVA also resulted
in a modest increase in the MID of the challenged animals, although
this effect did not reach statistical significance.
TABLE-US-00011 TABLE VII Effect of CpG ODN plus AVA on the survival
of guinea pigs challenged with anthrax MTD # Surviving/total %
Surviving (days) Untreated 1/28 3.6 2.1 AVA 15/32 46.9 6.1 AVA +
CpG ODN 23/31* 74.2 6.9 Guinea pigs were immunized on day 0 and
boosted on week 4 with AVA plus 100-300 .mu.g of an equimolar
mixture of CpG D ODNs 1555, 1466 and K3 (all known to be active in
this species). Six weeks later they were challenged IM with 50 LD50
Ames spores. Survival and mean time to death (MTD in days) is
shown. *Significantly improved survival compared to animals
immunized with AVA alone, p = 0.05.
The fraction of guinea pigs surviving otherwise lethal anthrax
challenge was significantly improved by co-administration of CpG
ODN with AVA. Without being bound by theory, several
characteristics of CpG ODN contribute to their utility as
immunotherapeutic agents. First, a single dose can provide
protection for several weeks, allowing ODN to be administered to
"at risk" populations (e.g., medical and/or military personnel) in
advance of potential pathogen exposure (Elkins et al., J. Immunol.
162:2291, 1999). Repeated doses can extend the duration of
protection for many months (Klinman et al., Infect. Immun. 67:5658,
1999). Second, CpG ODN are effective against a wide range of
pathogens, and thus may be of use before the causative agent has
been identified. Third, CpG ODN improve the innate immune response
of individuals whose adaptive immune response is impaired (such as
newborns and the elderly), and thus can provide broad
population-based protection against infection (Klinman et al.,
Immunity 11:123, 1999).
Furthermore, repeated doses of CpG ODN can be safely administered
to rodents and primates without adverse consequences (Verthelyi et
al., J. Immunol. 168:1659, 2002; Klinman et al., Infect. Immun.
67:565, 1999). In the present example, animals treated with CpG ODN
(alone or in combination with anthrax vaccine) remained healthy and
active prior to pathogen challenge.
Example 6
Dose and Timing of CpG Administration Influences CpG-Mediated
Protection in Mice Exposed to Ebola Virus
In order to demonstrate the effect of CpG dosage on protection from
Ebola virus, mice were treated with 25-150 .mu.g of CpG ODN on day
0, and then challenged with 300 LD50 of mouse-adapted Ebola Zaire.
As shown in FIG. 3a, mice treated with 100-150 .mu.g of CpG ODN
attained maximal protection from the virus.
In order to determine the effect of timing of CpG administration on
protection from Ebola, mice were treated with 100 .mu.g of CpG ODN
on the day shown, and then challenged with 300 LD50 of
mouse-adapted Ebola Zaire. As shown in FIG. 3b, mice showed maximal
protection from Ebola infection when CpG ODN were administered
concurrently with Ebola exposure. However, partial protection was
attained when CpG ODN were administered up to three days prior to
exposure or up to three days after exposure.
Example 7
CpG ODNs Increase Survival Times in Mice Exposed to Anthrax
Spores
In order to demonstrate that CpG ODNs increase survival times in
mice exposed to anthrax spores, mice were treated at the times
shown with 100 .mu.g of CpG ODN, and then challenged with 11 LD 50
anthrax spores. As shown in FIG. 4, CpG administration increased
survival times in mice exposed to anthrax spores.
Example 8
Adjuvant Effect of CpG ODN with AVA/rPA in Mice
This example demonstrates the adjuvant effect of CpG ODN with
AVA/rPA in mice. Mice were immunized with 2.5 .mu.g of rPA or 5
.mu.g of AVA plus 50 .mu.g of CpG ODN. The magnitude of the IgG
anti-PA response and IFN.gamma. response 10 days after the second
immunization is shown below (N=4 mice/group).
TABLE-US-00012 TABLE VIII Effect of CpG ODN on the immune response
of mice to AVA and rPA Group IgG anti-PA titer (.times.1000)
IFN.gamma. production Naive 0 0 AVA 13 + 8 250 + 125 AVA + CpG 15 +
12 757~87 rPA 45 + 3 254 + 163 rPA + CpG 182 + 61 343 + 148 CpG 0
0
Example 9
Effect of CpG ODN Alone (No Antigen) to Prevent Infection by
TBEV
This example demonstrates the efficacy of CpG ODN alone in
preventing infection by tick-borne encephalitis virus. Mice were
injection on the day shown with 100 .mu.g of CpG ODN. They were
challenged with TBEC, and survival monitored. N=10/group.
TABLE-US-00013 TABLE IX Effect of CpG ODN on the immune response of
mice to tick borne encephalitis virus Group Percent surviving (day
12) Control ODN 0 CpG ODN day 0 80 CpG ODN day 2 30 CpG ODN day 4
20
Example 10
Effect of K ODN on the Avidity of the Anti-PA Response
This example demonstrates the effect of K ODN on the avidity of the
anti-PA response. Animals were treated with ODN as described in
Elkins et al., J. Immunol 162:2291, 1999. FIG. 5 shows that K ODN
increase the avidity of the anti-PA response.
Example 11
Effect of Poly (Lactide-Co-gGycolide) (PLG) Microparticle
Formulation of DNA onto cationic poly(lactide-coglycolide) (PLG)
microparticles, has been developed as a means to better target DNA
to antigen-presenting cells (APCs). PLG microparticles are an
attractive approach for vaccine delivery, since the polymer is
biodegradable and biocompatible and has been used to develop
several drug delivery systems (Okada et al, Adv. Drug Deliv. Rev.
28:43-70, 1997). In addition, PLG microparticles have also been
used for a number of years as delivery systems for entrapped
vaccine antigens (Singh and O'Hanagan, Nat. Biotechnol.
17:1075-1081, 1999). More recently, PLG microparticles have been
described as a delivery system for vaccines, such as entrapped DNA
vaccines (Hedley et al., Nat. Med. 4:365-368, 1998; Jones et al.,
Vaccine 15:814-817, 1997; U.S. Pat. No. 6,309,569; U.S. Pat. No.
6,565,777; U.S. Pat. No. 6,548,302).
Encapsulating a bioactive agent in a polymer microparticle, such as
a PLG microparticle generally includes (1) dissolving polymer in a
solvent to form a polymer solution; (2) preparing an aqueous
solution of the bioactive agent, such as the CpG ODN; (3) combining
the polymer and bioactive agent solutions with agitation to form a
water-in-oil emulsion; (4) adding the water-in-oil emulsion to a
further aqueous phase containing a stabilizer or surfactant with
agitation to form a (water-in-oil)-in-water emulsion; (5) adding
the (water-in-oil)-in-water emulsion to excess of an aqueous phase
to extract the solvent, thereby forming polymer microparticles of a
size up to 10 microns in diameter. The microparticles contain the
bioactive agent. Generally, the polymer includes or consists of PLG
of molecular weight of 40 kD or lower (see U.S. Pat. No.
6,309,569). In one example, the molecular weight of the PLG is 30
kD or lower. In other examples, the microparticles include PLG of 3
kD, 6 kD, 9 kD, 22 kD and mixtures thereof. It has been proposed
that the molecular weight range of suitable polymer is 1.5 kD-250
kD, and commercial preparations of 3, 6, 9, 12, 18, 22, 60, 65
& 90 kD PLG have been utilized (see U.S. Pat. No. 5,309,569).
It is believed that the hydrolysis rate of the polymer is related
to the molecular weight. Thus, lower molecular weight polymers
degrade more rapidly.
The effectiveness of cationic microparticles with adsorbed DNA at
inducing immune responses was investigated in mice. The PLG polymer
(RG505) can be obtained from Boehringer Ingelheim.
Several exemplary protocol for the preparation of cationic
microparticles using a modified solvent evaporation process
follows. 1. Briefly, the microparticles were prepared by
emulsifying 10 ml of a 5% (wt/vol) polymer solution in methylene
chloride with 1 ml of phosphate-buffered saline (PBS) at high speed
using an IKA homogenizer. The primary emulsion was then added to 50
ml of distilled water containing cetyltrimethylammonium bromide
(CTAB) (0.5% wt/vol), resulting in the formation of a
water-in-oil-in-water emulsion, which was stirred at 6,000 rpm for
12 hours at room temperature, allowing the methylene chloride to
evaporate. The resulting microparticles were washed twice in
distilled water by centrifugation at 10,000.times.g and
freeze-dried. DNA was adsorbed onto the microparticles by
incubating 100 mg of cationic microparticles in a I-mg/ml solution
of DNA at 4.degree. C. for 6 hours. The microparticles were then
separated by centrifugation, the pellet was washed with TE
(Tris-EDTA) buffer, and the microparticles were freeze-dried.
Physical characteristics were monitored as previously described
(see Singh et al., Proc. Natl. Acad. Sci. USA 97:811-816; O'Hagan
et al., J. Virol. 75 (19):9037-9043, 2001). 2. Cationic
microparticles were prepared by using a modified solvent
evaporation process. Briefly, the microparticles were prepared by
emulsifying 10 ml of a 5% (wt/vol) polymer solution in methylene
chloride with 1 ml of PBS at high speed using an Ika homogenizer
(Ika-Werk Instruments, Cincinnati, Ohio). The primary emulsion then
was added to 50 ml of distilled water containing
cetyltrimethylammonium bromide (CTAB) (0.5% wt/vol). This resulted
in the formation of a water/oil/water emulsion that was stirred at
6,000 rpm for 12 hours at room temperature, allowing the methylene
chloride to evaporate. The resulting microparticles were washed
twice in distilled water by centrifugation at 10,000 g and
freeze-dried. For preparing PLG-dimethyl dioctadecyl ammonium
bromide (DDA) and PLG-1,2-dioleoyl-1,3-trimethylammoniopropane
(DOTAP) microparticles, DDA or DOTAP was dissolved in the polymer
solution along with PLG polymer, and the primary emulsion then was
added to 0.5% polyvinyl alcohol solution to form the
water/oil/water emulsion (see Singh et al., Proc. Natl. Acad. Sci.
97:811-816, 2000).
After preparation, washing, and collection, DNA was adsorbed onto
the microparticles by incubating 100 mg of cationic microparticles
in a 1 mg/ml solution of DNA at 4.degree. C. for 6 hours. The
microparticles then were separated by centrifugation, the pellet
was washed with Tris-EDTA buffer, and the microparticles were
freeze-dried (see Singh et al., Proc. Natl. Acad. Sci. 97:811-816,
2000).
For the studies described below, the CpG ODN was conjugated to the
PLG, whereas the rPA was free (but was co-administered after mixing
with the PLG-ODN).
Mice were immunized with either 2.5, 8 or 25 .mu.l of AVA, AVA plus
CpG ODN, AVA plus PLG-CpG, AVA plus GpC ODN (a control), AVA plus
PLG-GpG ODN (and additional control), or AVA plus PLG (a further
control). The IgM anti-PA titer, IgG anti-PA titer, IgG1 anti-PA
titer, IgG2a anti-PA titer, and survival of the animals were
monitored.
The results indicated that mice immunized with a single dose of AVA
plus CpG or poly (lactide-co-glycolide) microparticle
(PLG)-encapsulated CpG through cross-linking developed specific
antibody responses as early as day 11 post-immunization. The
antibody response elicited by the AVA-CpG-ODN immunogen was five to
one hundred times greater than that of mice immunized with AVA
alone or AVA plus control ODN or PLG-control ODN. Immunization of
AVA plus CpG or PLG-CpG also elicited a stronger TH1 response with
higher ratios of IgG2a/IgG1 than control groups. Whereas most of
control mice died after a challenge with 300-9000 LD50 of Sterne
strain spores (STI) of Bacillus anthracis at week 1 and 2
post-immunization, a 70-100% survival rate was observed in mice
immunized with AVA plus CpG or PLG-CpG. These results suggested
that using CpG or PLG-CpG as adjuvants not only improved the
immunogenicity and protection of AVA against STI spore challenge,
but also accelerated specific immune responses with great potential
for counter-bioterrorism usage.
It will be apparent that the precise details of the methods or
compositions described may be varied or modified without departing
from the spirit of the described disclosure. We claim all such
modifications and variations that fall within the scope and spirit
of the claims below.
SEQUENCE LISTINGS
1
200120DNAArtificial sequenceCpG D oligonucleotide 1nntgcatcga
tgcagggggg 20220DNAArtificial sequenceCpG D oligonucleotide
2nntgcaccgg tgcagggggg 20320DNAArtificial sequenceCpG D
oligonucleotide 3nntgcgtcga cgcagggggg 20420DNAArtificial
sequenceCpG D oligonucleotide 4nntgcgtcga tgcagggggg
20520DNAArtificial sequenceCpG D oligonucleotide 5nntgcgccgg
cgcagggggg 20620DNAArtificial sequenceCpG D oligonucleotide
6nntgcgccga tgcagggggg 20720DNAArtificial sequenceCpG D
oligonucleotide 7nntgcatcga cgcagggggg 20820DNAArtificial
sequenceCpG D oligonucleotide 8nntgcgtcgg tgcagggggg
2096DNAArtificial sequenceCpG D oligonucleotide 9atcgat
6106DNAArtificial sequenceCpG D oligonucleotide 10accggt
6116DNAArtificial sequenceCpG D oligonucleotide 11atcgac
6126DNAArtificial sequenceCpG D oligonucleotide 12accgat
6136DNAArtificial sequenceCpG D oligonucleotide 13gtcgac
6146DNAArtificial sequenceCpG D oligonucleotide 14gccggc
61520DNAArtificial sequenceCpG D oligonucleotide 15ggtgcatcga
tacagggggg 201620DNAArtificial sequenceCpG D oligonucleotide
16ggtgcgtcga tgcagggggg 201715DNAArtificial sequenceCpG D
oligonucleotide 17gctagacgtt agcgt 151810DNAArtificial sequenceCpG
D oligonucleotide 18tcaacgttga 101915DNAArtificial sequenceControl
D oligonucleotide 19gctagagctt aggct 152010DNAArtificial
sequenceControl D oligonucleotide 20tcaagcttga 102120DNAArtificial
sequenceCpG D oligonucleotide 21ggtgcatcga tgcagggggg
202220DNAArtificial sequenceCpG D oligonucleotide 22ggtgcaccgg
tgcagggggg 202320DNAArtificial sequenceCpG D oligonucleotide
23atcgactctc gagcgttctc 202412DNAArtificial sequenceCpG D
oligonucleotide 24tcgttcgttc tc 122512DNAArtificial sequenceCpG D
oligonucleotide 25tcgagcgttc tc 122620DNAArtificial sequenceControl
D oligonucleotide 26ggtgcattga tgcagggggg 202712DNAArtificial
sequenceControl D oligonucleotide 27ttgagtgttc tc
122816DNAArtificial sequenceControl D oligonucleotide 28gggcatgcat
gggggg 162920DNAArtificial sequenceK oligonucleotide 29tccatgtcgc
tcctgatgct 203020DNAArtificial sequenceK oligonucleotide
30tccatgtcgt tcctgatgct 203123DNAArtificial sequenceK
oligonucleotide 31tcgtcgtttt gtcgttttgt cgt 233220DNAArtificial
sequenceK oligonucleotide 32tcgtcgttgt cgttgtcgtt
203323DNAArtificial sequenceK oligonucleotide 33tcgtcgtttt
gtcgtttgtc gtt 233422DNAArtificial sequenceK oligonucleotide
34tcgtcgttgt cgttttgtcg tt 223521DNAArtificial sequenceK
oligonucleotide 35gcgtgcgttg tcgttgtcgt t 213621DNAArtificial
sequenceK oligonucleotide 36tgtcgtttgt cgtttgtcgt t
213719DNAArtificial sequenceK oligonucleotide 37tgtcgttgtc
gttgtcgtt 193814DNAArtificial sequenceK oligonucleotide
38tcgtcgtcgt cgtt 143920DNAArtificial sequenceK oligonucleotide
39tcctgtcgtt ccttgtcgtt 204020DNAArtificial sequenceK
oligonucleotide 40tcctgtcgtt ttttgtcgtt 204121DNAArtificial
sequenceK oligonucleotide 41tcgtcgctgt ctgcccttct t
214221DNAArtificial sequenceK oligonucleotide 42tcgtcgctgt
tgtcgtttct t 214320DNAArtificial sequenceK oligonucleotide
43tccatgacgt tcctgacgtt 204416DNAArtificial sequencesynthetic
44nnnrycgryn nngggg 164517DNAArtificial sequencesynthetic
45nnnrycgryn nnngggg 174618DNAArtificial sequencesynthetic
46nnnrycgryn nnnngggg 184719DNAArtificial sequencesynthetic
47nnnrycgryn nnnnngggg 194820DNAArtificial sequencesynthetic
48nnnrycgryn nnnnnngggg 204921DNAArtificial sequencesynthetic
49nnnrycgryn nnnnnnnggg g 215022DNAArtificial sequencesynthetic
50nnnrycgryn nnnnnnnngg gg 225123DNAArtificial sequencesynthetic
51nnnrycgryn nnnnnnnnng ggg 235224DNAArtificial sequencesynthetic
52nnnrycgryn nnnnnnnnnn gggg 245325DNAArtificial sequencesynthetic
53nnnrycgryn nnnnnnnnnn ngggg 255426DNAArtificial sequencesynthetic
54nnnrycgryn nnnnnnnnnn nngggg 265517DNAArtificial
sequencesynthetic 55nnnrycgryn nnggggg 175618DNAArtificial
sequencesynthetic 56nnnrycgryn nnnggggg 185719DNAArtificial
sequencesynthetic 57nnnrycgryn nnnnggggg 195820DNAArtificial
sequencesynthetic 58nnnrycgryn nnnnnggggg 205921DNAArtificial
sequencesynthetic 59nnnrycgryn nnnnnngggg g 216022DNAArtificial
sequencesynthetic 60nnnrycgryn nnnnnnnggg gg 226123DNAArtificial
sequencesynthetic 61nnnrycgryn nnnnnnnngg ggg 236224DNAArtificial
sequencesynthetic 62nnnrycgryn nnnnnnnnng gggg 246325DNAArtificial
sequencesynthetic 63nnnrycgryn nnnnnnnnnn ggggg 256426DNAArtificial
sequencesynthetic 64nnnrycgryn nnnnnnnnnn nggggg
266527DNAArtificial sequencesynthetic 65nnnrycgryn nnnnnnnnnn
nnggggg 276618DNAArtificial sequencesynthetic 66nnnrycgryn nngggggg
186719DNAArtificial sequencesynthetic 67nnnrycgryn nnngggggg
196820DNAArtificial sequencesynthetic 68nnnrycgryn nnnngggggg
206921DNAArtificial sequencesynthetic 69nnnrycgryn nnnnnggggg g
217022DNAArtificial sequencesynthetic 70nnnrycgryn nnnnnngggg gg
227123DNAArtificial sequencesynthetic 71nnnrycgryn nnnnnnnggg ggg
237224DNAArtificial sequencesynthetic 72nnnrycgryn nnnnnnnngg gggg
247325DNAArtificial sequencesynthetic 73nnnrycgryn nnnnnnnnng ggggg
257426DNAArtificial sequencesynthetic 74nnnrycgryn nnnnnnnnnn
gggggg 267527DNAArtificial sequencesynthetic 75nnnrycgryn
nnnnnnnnnn ngggggg 277628DNAArtificial sequencesynthetic
76nnnrycgryn nnnnnnnnnn nngggggg 287719DNAArtificial
sequencesynthetic 77nnnrycgryn nnggggggg 197820DNAArtificial
sequencesynthetic 78nnnrycgryn nnnggggggg 207921DNAArtificial
sequencesynthetic 79nnnrycgryn nnnngggggg g 218022DNAArtificial
sequencesynthetic 80nnnrycgryn nnnnnggggg gg 228123DNAArtificial
sequencesynthetic 81nnnrycgryn nnnnnngggg ggg 238224DNAArtificial
sequencesynthetic 82nnnrycgryn nnnnnnnggg gggg 248325DNAArtificial
sequencesynthetic 83nnnrycgryn nnnnnnnngg ggggg 258426DNAArtificial
sequencesynthetic 84nnnrycgryn nnnnnnnnng gggggg
268527DNAArtificial sequencesynthetic 85nnnrycgryn nnnnnnnnnn
ggggggg 278628DNAArtificial sequencesynthetic 86nnnrycgryn
nnnnnnnnnn nggggggg 288729DNAArtificial sequencesynthetic
87nnnrycgryn nnnnnnnnnn nnggggggg 298820DNAArtificial
sequencesynthetic 88nnnrycgryn nngggggggg 208921DNAArtificial
sequencesynthetic 89nnnrycgryn nnnggggggg g 219022DNAArtificial
sequencesynthetic 90nnnrycgryn nnnngggggg gg 229123DNAArtificial
sequencesynthetic 91nnnrycgryn nnnnnggggg ggg 239224DNAArtificial
sequencesynthetic 92nnnrycgryn nnnnnngggg gggg 249325DNAArtificial
sequencesynthetic 93nnnrycgryn nnnnnnnggg ggggg 259426DNAArtificial
sequencesynthetic 94nnnrycgryn nnnnnnnngg gggggg
269527DNAArtificial sequencesynthetic 95nnnrycgryn nnnnnnnnng
ggggggg 279628DNAArtificial sequencesynthetic 96nnnrycgryn
nnnnnnnnnn gggggggg 289729DNAArtificial sequencesynthetic
97nnnrycgryn nnnnnnnnnn ngggggggg 299830DNAArtificial
sequencesynthetic 98nnnrycgryn nnnnnnnnnn nngggggggg
309921DNAArtificial sequencesynthetic 99nnnrycgryn nngggggggg g
2110022DNAArtificial sequencesynthetic 100nnnrycgryn nnnggggggg gg
2210123DNAArtificial sequencesynthetic 101nnnrycgryn nnnngggggg ggg
2310224DNAArtificial sequencesynthetic 102nnnrycgryn nnnnnggggg
gggg 2410325DNAArtificial sequencesynthetic 103nnnrycgryn
nnnnnngggg ggggg 2510426DNAArtificial sequencesynthetic
104nnnrycgryn nnnnnnnggg gggggg 2610527DNAArtificial
sequencesynthetic 105nnnrycgryn nnnnnnnngg ggggggg
2710628DNAArtificial sequencesynthetic 106nnnrycgryn nnnnnnnnng
gggggggg 2810729DNAArtificial sequencesynthetic 107nnnrycgryn
nnnnnnnnnn ggggggggg 2910830DNAArtificial sequencesynthetic
108nnnrycgryn nnnnnnnnnn nggggggggg 3010931DNAArtificial
sequencesynthetic 109nnnrycgryn nnnnnnnnnn nngggggggg g
3111022DNAArtificial sequencesynthetic 110nnnrycgryn nngggggggg gg
2211123DNAArtificial sequencesynthetic 111nnnrycgryn nnnggggggg ggg
2311224DNAArtificial sequencesynthetic 112nnnrycgryn nnnngggggg
gggg 2411325DNAArtificial sequencesynthetic 113nnnrycgryn
nnnnnggggg ggggg 2511426DNAArtificial sequencesynthetic
114nnnrycgryn nnnnnngggg gggggg 2611527DNAArtificial
sequencesynthetic 115nnnrycgryn nnnnnnnggg ggggggg
2711628DNAArtificial sequencesynthetic 116nnnrycgryn nnnnnnnngg
gggggggg 2811729DNAArtificial sequencesynthetic 117nnnrycgryn
nnnnnnnnng ggggggggg 2911830DNAArtificial sequencesynthetic
118nnnrycgryn nnnnnnnnnn gggggggggg 3011931DNAArtificial
sequencesynthetic 119nnnrycgryn nnnnnnnnnn nggggggggg g
3112032DNAArtificial sequencesynthetic 120nnnrycgryn nnnnnnnnnn
nngggggggg gg 3212110DNAArtificial sequencesynthetic 121nnndcgwnnn
1012210DNAArtificial sequencesynthetic 122nnntcgwnnn
1012318DNAArtificial sequencesynthetic 123ggnnnrycgr ynnngggg
1812419DNAArtificial sequencesynthetic 124ggnnnrycgr ynnnngggg
1912520DNAArtificial sequencesynthetic 125ggnnnrycgr ynnnnngggg
2012621DNAArtificial sequencesynthetic 126ggnnnrycgr ynnnnnnggg g
2112722DNAArtificial sequencesynthetic 127ggnnnrycgr ynnnnnnngg gg
2212823DNAArtificial sequencesynthetic 128ggnnnrycgr ynnnnnnnng ggg
2312924DNAArtificial sequencesynthetic 129ggnnnrycgr ynnnnnnnnn
gggg 2413025DNAArtificial sequencesynthetic 130ggnnnrycgr
ynnnnnnnnn ngggg 2513126DNAArtificial sequencesynthetic
131ggnnnrycgr ynnnnnnnnn nngggg 2613227DNAArtificial
sequencesynthetic 132ggnnnrycgr ynnnnnnnnn nnngggg
2713328DNAArtificial sequencesynthetic 133ggnnnrycgr ynnnnnnnnn
nnnngggg 2813419DNAArtificial sequencesynthetic 134ggnnnrycgr
ynnnggggg 1913520DNAArtificial sequencesynthetic 135ggnnnrycgr
ynnnnggggg 2013621DNAArtificial sequencesynthetic 136ggnnnrycgr
ynnnnngggg g 2113722DNAArtificial sequencesynthetic 137ggnnnrycgr
ynnnnnnggg gg 2213823DNAArtificial sequencesynthetic 138ggnnnrycgr
ynnnnnnngg ggg 2313924DNAArtificial sequencesynthetic 139ggnnnrycgr
ynnnnnnnng gggg 2414025DNAArtificial sequencesynthetic
140ggnnnrycgr ynnnnnnnnn ggggg 2514126DNAArtificial
sequencesynthetic 141ggnnnrycgr ynnnnnnnnn nggggg
2614227DNAArtificial sequencesynthetic 142ggnnnrycgr ynnnnnnnnn
nnggggg 2714328DNAArtificial sequencesynthetic 143ggnnnrycgr
ynnnnnnnnn nnnggggg 2814429DNAArtificial sequencesynthetic
144ggnnnrycgr ynnnnnnnnn nnnnggggg 2914520DNAArtificial
sequencesynthetic 145ggnnnrycgr ynnngggggg 2014621DNAArtificial
sequencesynthetic 146ggnnnrycgr ynnnnggggg g 2114722DNAArtificial
sequencesynthetic 147ggnnnrycgr ynnnnngggg gg 2214823DNAArtificial
sequencesynthetic 148ggnnnrycgr ynnnnnnggg ggg 2314924DNAArtificial
sequencesynthetic 149ggnnnrycgr ynnnnnnngg gggg
2415025DNAArtificial sequencesynthetic 150ggnnnrycgr ynnnnnnnng
ggggg 2515126DNAArtificial sequencesynthetic 151ggnnnrycgr
ynnnnnnnnn gggggg 2615227DNAArtificial sequencesynthetic
152ggnnnrycgr ynnnnnnnnn ngggggg 2715328DNAArtificial
sequencesynthetic 153ggnnnrycgr ynnnnnnnnn nngggggg
2815429DNAArtificial sequencesynthetic 154ggnnnrycgr ynnnnnnnnn
nnngggggg 2915530DNAArtificial sequencesynthetic 155ggnnnrycgr
ynnnnnnnnn nnnngggggg
3015621DNAArtificial sequencesynthetic 156ggnnnrycgr ynnngggggg g
2115722DNAArtificial sequencesynthetic 157ggnnnrycgr ynnnnggggg gg
2215823DNAArtificial sequencesynthetic 158ggnnnrycgr ynnnnngggg ggg
2315924DNAArtificial sequencesynthetic 159ggnnnrycgr ynnnnnnggg
gggg 2416025DNAArtificial sequencesynthetic 160ggnnnrycgr
ynnnnnnngg ggggg 2516126DNAArtificial sequencesynthetic
161ggnnnrycgr ynnnnnnnng gggggg 2616227DNAArtificial
sequencesynthetic 162ggnnnrycgr ynnnnnnnnn ggggggg
2716328DNAArtificial sequencesynthetic 163ggnnnrycgr ynnnnnnnnn
nggggggg 2816429DNAArtificial sequencesynthetic 164ggnnnrycgr
ynnnnnnnnn nnggggggg 2916530DNAArtificial sequencesynthetic
165ggnnnrycgr ynnnnnnnnn nnnggggggg 3016631DNAArtificial
sequencesynthetic 166ggnnnrycgr ynnnnnnnnn nnnngggggg g
3116722DNAArtificial sequencesynthetic 167ggnnnrycgr ynnngggggg gg
2216823DNAArtificial sequencesynthetic 168ggnnnrycgr ynnnnggggg ggg
2316924DNAArtificial sequencesynthetic 169ggnnnrycgr ynnnnngggg
gggg 2417025DNAArtificial sequencesynthetic 170ggnnnrycgr
ynnnnnnggg ggggg 2517126DNAArtificial sequencesynthetic
171ggnnnrycgr ynnnnnnngg gggggg 2617227DNAArtificial
sequencesynthetic 172ggnnnrycgr ynnnnnnnng ggggggg
2717328DNAArtificial sequencesynthetic 173ggnnnrycgr ynnnnnnnnn
gggggggg 2817429DNAArtificial sequencesynthetic 174ggnnnrycgr
ynnnnnnnnn ngggggggg 2917530DNAArtificial sequencesynthetic
175ggnnnrycgr ynnnnnnnnn nngggggggg 3017631DNAArtificial
sequencesynthetic 176ggnnnrycgr ynnnnnnnnn nnnggggggg g
3117732DNAArtificial sequencesynthetic 177ggnnnrycgr ynnnnnnnnn
nnnngggggg gg 3217823DNAArtificial sequencesynthetic 178ggnnnrycgr
ynnngggggg ggg 2317924DNAArtificial sequencesynthetic 179ggnnnrycgr
ynnnnggggg gggg 2418025DNAArtificial sequencesynthetic
180ggnnnrycgr ynnnnngggg ggggg 2518126DNAArtificial
sequencesynthetic 181ggnnnrycgr ynnnnnnggg gggggg
2618227DNAArtificial sequencesynthetic 182ggnnnrycgr ynnnnnnngg
ggggggg 2718328DNAArtificial sequencesynthetic 183ggnnnrycgr
ynnnnnnnng gggggggg 2818429DNAArtificial sequencesynthetic
184ggnnnrycgr ynnnnnnnnn ggggggggg 2918530DNAArtificial
sequencesynthetic 185ggnnnrycgr ynnnnnnnnn nggggggggg
3018631DNAArtificial sequencesynthetic 186ggnnnrycgr ynnnnnnnnn
nngggggggg g 3118732DNAArtificial sequencesynthetic 187ggnnnrycgr
ynnnnnnnnn nnnggggggg gg 3218833DNAArtificial sequencesynthetic
188ggnnnrycgr ynnnnnnnnn nnnngggggg ggg 3318924DNAArtificial
sequencesynthetic 189ggnnnrycgr ynnngggggg gggg
2419025DNAArtificial sequencesynthetic 190ggnnnrycgr ynnnnggggg
ggggg 2519126DNAArtificial sequencesynthetic 191ggnnnrycgr
ynnnnngggg gggggg 2619227DNAArtificial sequencesynthetic
192ggnnnrycgr ynnnnnnggg ggggggg 2719328DNAArtificial
sequencesynthetic 193ggnnnrycgr ynnnnnnngg gggggggg
2819429DNAArtificial sequencesynthetic 194ggnnnrycgr ynnnnnnnng
ggggggggg 2919530DNAArtificial sequencesynthetic 195ggnnnrycgr
ynnnnnnnnn gggggggggg 3019631DNAArtificial sequencesynthetic
196ggnnnrycgr ynnnnnnnnn nggggggggg g 3119732DNAArtificial
sequencesynthetic 197ggnnnrycgr ynnnnnnnnn nngggggggg gg
3219833DNAArtificial sequencesynthetic 198ggnnnrycgr ynnnnnnnnn
nnnggggggg ggg 3319934DNAArtificial sequencesynthetic 199ggnnnrycgr
ynnnnnnnnn nnnngggggg gggg 3420024DNAArtificial sequencesynthetic
200tcgtcgtttt gtcgttttgt cgtt 24
* * * * *