U.S. patent application number 13/675897 was filed with the patent office on 2013-11-28 for administration of interferon for prophylaxis against or treatment of pathogenic infection.
This patent application is currently assigned to Defyrus, Inc.. The applicant listed for this patent is Defyrus, Inc.. Invention is credited to Jane E. Ennis, Jeffrey D. Turner.
Application Number | 20130315952 13/675897 |
Document ID | / |
Family ID | 43308334 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130315952 |
Kind Code |
A1 |
Turner; Jeffrey D. ; et
al. |
November 28, 2013 |
ADMINISTRATION OF INTERFERON FOR PROPHYLAXIS AGAINST OR TREATMENT
OF PATHOGENIC INFECTION
Abstract
The invention provides compositions and methods for the
prophylaxis or treatment of diseases or disorders in a subject
(e.g., a mammal, such as a human) including, e.g., diseases or
disorders caused by biological agents, autoimmune diseases, and
cancer. The compositions include a delivery vector (e.g., a viral
vector, such as an Ad5 vector) encoding an interferon (e.g.,
IFN-.alpha.), and are provided to the subject by, e.g., intranasal
or pulmonary administration.
Inventors: |
Turner; Jeffrey D.;
(Toronto, CA) ; Ennis; Jane E.; (Toronto,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Defyrus, Inc. |
Toronto |
|
CA |
|
|
Assignee: |
Defyrus, Inc.
Toronto
CA
|
Family ID: |
43308334 |
Appl. No.: |
13/675897 |
Filed: |
November 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12797575 |
Jun 9, 2010 |
8309531 |
|
|
13675897 |
|
|
|
|
61185261 |
Jun 9, 2009 |
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Current U.S.
Class: |
424/204.1 ;
128/200.21; 128/203.12; 128/203.15; 424/93.2; 435/320.1;
514/44R |
Current CPC
Class: |
A61K 39/12 20130101;
A61P 31/18 20180101; A61P 33/02 20180101; Y02A 50/463 20180101;
A61K 9/0073 20130101; A61P 31/00 20180101; A61P 31/14 20180101;
Y02A 50/30 20180101; A61M 11/02 20130101; C12N 2799/022 20130101;
A61P 33/00 20180101; C12N 15/86 20130101; A61K 9/007 20130101; A61P
37/00 20180101; A61P 31/16 20180101; A61K 9/0043 20130101; A61P
31/12 20180101; A61P 31/10 20180101; A61P 31/04 20180101; A61M
15/00 20130101; A61P 37/04 20180101; A61P 31/22 20180101; A61P
35/00 20180101; A61M 16/14 20130101; A61P 33/06 20180101; A61P
31/20 20180101; A61K 38/212 20130101; Y02A 50/387 20180101; A61K
48/005 20130101; A61P 37/02 20180101 |
Class at
Publication: |
424/204.1 ;
435/320.1; 514/44.R; 424/93.2; 128/203.12; 128/200.21;
128/203.15 |
International
Class: |
A61K 38/21 20060101
A61K038/21; A61K 39/12 20060101 A61K039/12; A61M 15/00 20060101
A61M015/00; A61M 16/14 20060101 A61M016/14; A61M 11/02 20060101
A61M011/02; C12N 15/86 20060101 C12N015/86; A61K 9/00 20060101
A61K009/00 |
Claims
1. A composition comprising a vector comprising a nucleic acid
molecule encoding an interferon (IFN), wherein said composition is
formulated as: a) a dry, lyophilized powder, gel, or liquid,
wherein said composition is stable at room temperature for at least
one week; or b) a frozen, non-stabilized liquid, wherein said
composition, once thawed, is stable at room temperature for at
least 24 hours.
2. The composition of claim 1, wherein said interferon is IFN-alpha
(IFN-.alpha.).
3. The composition of claim 2, wherein said IFN-.alpha. is
consensus IFN-.alpha. (conIFN-.alpha.).
4. The composition of claim 1, wherein said vector is a viral or
non-viral vector.
5. The composition of claim 4, wherein said viral vector is an
adenoviral vector.
6. The composition of claim 5, wherein said adenoviral vector is an
adenoviral 5 (Ad5) vector.
7. The composition of claim 6, wherein said Ad5 vector is a
replication deficient vector that comprises deletions of the E1 and
E3 genes.
8. The composition of claim 1, wherein said nucleic acid molecule
of said vector is operably linked to a promoter selected from an
SV40 promoter, CMV promoter, adenovirus early and late promoter,
metallothioneine gene (MT-1) promoter, Rous sarcoma virus (RSV)
promoter, and human Ubiquitine C (UbC) promoter.
9. The composition of claim 1, wherein said vector further
comprises one or more of a signal sequence, a polyadenylation
sequence, and enhancer, an upstream activation sequence, and a
transcription termination factor that facilitates expression of
said nucleic acid molecule encoding said interferon.
10. The composition of claim 3, wherein said conIFN-.alpha. encoded
by said nucleic acid molecule has a polypeptide sequence comprising
the sequence set forth in SEQ ID NO: 11.
11. The composition of claim 1, wherein said composition further
comprises a pharmaceutically acceptable excipient selected from one
or more of fructose, maltose, galactose, glucose, D-mannose,
sorbose, lactose, sucrose, trehalose, cellobiose, raffinose,
melezitose, maltodextrins, dextrans, starches, mannitol, xylitol,
xylose, maltitol, lactitol, xylitol sorbitol, sorbitose, pyranosyl
sorbitol, myoinositol, glycine, CaCl.sub.2, hydroxyectoine,
ectoine, gelatin, di-myo-inositol phosphate (DIP), cyclic 2,3
diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate (DGP),
.beta.-mannosylglycerate (firoin), .beta.-mannosylglyceramide
(firoin A), and proline betaine.
12. The composition of claim 1, wherein said composition is
formulated for aerosolized delivery.
13. The composition of claim 1, wherein said composition is stable
at room temperature for at least one month to at least 1 year.
14. The composition of claim 1, wherein said composition is admixed
with a pharmaceutically acceptable liquid to form said liquid or
gel.
15. The composition of claim 1, further comprising an additional
therapeutic agent selected from an anti-viral agent, an
anti-bacterial agent, an anti-fungal agent, an anti-parasitic
agent, an immunostimulatory agent, a vaccine, and a
chemotherapeutic agent.
16. The composition of claim 15, wherein said vaccine is an Ebola
virus vaccine.
17. A method for treating or reducing the effects of an infection,
autoimmune disease, or cancer in a subject in need thereof
comprising administering an amount of the composition of claim 1 to
the pulmonary or nasal mucosa of the subject one or more times.
18. The method of claim 17, wherein administration of said
composition results in expression of said interferon (IFN) in
pulmonary or nasal epithelial cells of said mucosa.
19. The method of claim 17, wherein said composition comprises an
adenoviral 5 (Ad5) vector encoding said IFN and said method
comprises administering said Ad5 vector in an amount in the range
of at least about 1.times.10.sup.3 to about 1.times.10.sup.14 viral
particles per dose.
20. The method of claim 17, wherein said IFN is IFN-.alpha..
21. The method of claim 20, wherein said IFN-.alpha. is consensus
IFN-.alpha. (conIFN-.alpha.).
22. The method of claim 17, wherein said subject receives said
composition prior to or after exposure to said pathogen or
diagnosis of said autoimmune disease or cancer.
23. The method of claim 22, wherein said subject receives said
composition at least 15 minutes to at least 1 week prior to
exposure to said pathogen.
24. The method of claim 22, wherein said subject receives said
composition immediately after exposure to said pathogen or
diagnosis of said autoimmune disease or cancer or at least 15
minutes to at least 48 hours after exposure to said pathogen or
diagnosis of said autoimmune disease or cancer.
25. The method of claim 17, wherein said pathogen is a bacterium,
virus, fungus, or parasite.
26. The method of claim 25, wherein: i) said bacterium is selected
from Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia
coli, Klebsiella pneumoniae, Bruscella, Burkholderia mallei,
Yersinia pestis, and Bacillus anthraci; ii) said virus is selected
from a member of the Flaviviridae, Arenaviridae, Bunyaviridae,
Filoviridae, Togaviridae, Poxyiridae, Herpesviridae,
Orthomyxoviridae, Coronaviridae, Rhabdoviridae, Paramyxoviridae,
Picornaviridae, Hepadnaviridae, Papillamoviridae, Parvoviridae,
Astroviridae, Polyomaviridae, Calciviridae, Reoviridae, and the
Retroviridae family; iii) said fungus is selected from Aspergillus,
Blastomyces dermatitidis, Candida, Coccidioides immitis,
Cryptococcus neoformans, Histoplasma capsulatum var. capsulatum,
Paracoccidioides brasiliensis, Sporothrix schenckii, Zygomycetes
spp., Absidia corymbifera, Rhizomucor pusillus, and Rhizopus
arrhizus; or iv) said parasite is selected from Toxoplasma gondii,
Plasmodium falciparum, P. vivax, P. ovale, P. malariae, Trypanosoma
spp., and Legionella spp.
27. The method of claim 26, wherein said virus is selected from
hepatitis C virus, Yellow fever virus, Gadgets Gully virus, Kadam
virus, Kyasanur Forest disease virus, Langat virus, Omsk
hemorrhagic fever virus, Powassan virus, Royal Farm virus, Karshi
virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin
virus, Louping ill virus, Negishi virus, Meaban virus, Saumarez
Reef virus, Tyuleniy virus, Aroa virus, dengue virus, Kedougou
virus, Cacipacore virus, Koutango virus, Japanese encephalitis
virus, Murray Valley encephalitis virus, St. Louis encephalitis
virus, Usutu virus, West Nile virus, Yaounde virus, Kokobera virus,
Bagaza virus, Ilheus virus, Israel turkey meningoencephalo-myelitis
virus, Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui
virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik virus,
Uganda S virus, Wesselsbron virus, yellow fever virus, Entebbe bat
virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa
virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasa
bat virus, Carey Island virus, Dakar bat virus, Montana myotis
leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus,
Tamana bat virus, Cell fusing agent virus, Ippy virus, Lassa virus,
lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia
virus, Amapari virus, Flexal virus, Guanarito virus, Junin virus,
Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde
virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus,
Whitewater Arroyo virus, Chapare virus, Lujo virus, Hantaan virus,
Sin Nombre virus, Dugbe virus, Bunyamwera virus, Rift Valley fever
virus, La Crosse virus, Punta Toro virus (PTV), California
encephalitis virus, Crimean-Congo hemorrhagic fever (CCHF) virus,
Ebola virus, Marburg virus, Venezuelan equine encephalitis virus
(VEE), Eastern equine encephalitis virus (EEE), Western equine
encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki
Forest virus, Ross River virus, Barmah Forest virus, O'nyong'nyong
virus, chikungunya virus, smallpox virus, monkeypox virus, vaccinia
virus, herpes simplex virus (HSV), human herpes virus,
cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella-Zoster
virus, Kaposi's sarcoma associated-herpesvirus (KSHV), influenza
virus, severe acute respiratory syndrome (SARS) virus, rabies
virus, vesicular stomatitis virus (VSV), human respiratory
syncytial virus (RSV), Newcastle disease virus, hendravirus,
nipahvirus, measles virus, rinderpest virus, canine distemper
virus, Sendai virus, human parainfluenza virus, rhinovirus, mumps
virus, coxsackievirus, hepatitis B virus, human papilloma virus,
adeno-associated virus, astrovirus, JC virus, BK virus, SV40 virus,
Norwalk virus, rotavirus, human immunodeficiency virus (HIV), and
human T-lymphotropic virus (HTLV).
28. The method of claim 17, wherein said composition is inhaled as
a lyophilized powder.
29. The method of claim 17, wherein said composition is admixed
with a pharmaceutically acceptable liquid and inhaled as an
aerosolized mist.
30. The method of claim 29, wherein said pharmaceutically
acceptable liquid is water or saline.
31. The method of claim 17, wherein said subject is a human.
32. The method of claim 17, wherein said subject is administered at
least 2 doses of said composition.
33. The method of claim 17, wherein said composition is
administered as a gel.
34. The method of claim 17, wherein said method further comprises
administering an additional therapeutic agent selected from an
anti-viral agent, an anti-bacterial agent, an anti-fungal agent, an
anti-parasitic agent, an immunostimulatory agent, a vaccine, and a
chemotherapeutic agent.
35. The method of claim 34, wherein said vaccine is an Ebola virus
vaccine.
36. The method of claim 34, wherein said therapeutic agent is
administered separately or concurrently with said composition.
37. The method of claim 34, wherein said therapeutic agent is
admixed with said composition.
38. A device comprising the composition of claim 1, wherein said
device comprises: a) a container comprising said composition; b) a
nozzle for directing said composition to the pulmonary or nasal
mucosa of a subject; c) a mechanical delivery pump for delivering
the composition to the nozzle, wherein activation of said pump
results in a fluid connection between said nozzle and said
container; and d) an actuation mechanism for activating said
mechanical delivery pump.
39. The device of claim 38, wherein the actuation mechanism
comprises a trigger for actuating the delivery pump at a
predeterminable pressure or flow rate.
40. The device of claim 38, wherein the delivery pump comprises a
liquid delivery pump for delivering a metered volume of said
composition in liquid or powder form.
41. The device of claim 38, wherein the nozzle is configured to
deliver an aerosol or a jet.
42. A kit comprising i) a first container comprising a composition
comprising a vector comprising a nucleic acid molecule encoding an
interferon (IFN), wherein said composition is formulated as: a) a
dry, lyophilized powder, gel, or liquid, wherein said composition
is stable at room temperature for at least one week; or b) a
frozen, non-stabilized liquid, wherein said composition, once
thawed, is stable at room temperature for at least 24 hours; ii) a
second container comprising a pharmaceutically acceptable liquid;
and iii) the device of claim 38 and, optionally, instructions for
using the device to deliver the contents of said first container,
or for combining the contents of said first and second containers
to form a combined composition and then using the device to deliver
the combined composition, to a subject for treating or inhibiting
infection by a pathogen.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
12/797,575, filed on Jun. 9, 2010, now U.S. Pat. No. 8,309,531,
which claims priority to U.S. Provisional Application No.
61/185,261, filed on Jun. 9, 2009.
FIELD OF THE INVENTION
[0002] The invention is directed to the treatment of or prophylaxis
against diseases or disorders caused by biological or chemical
agents in a subject (e.g., a mammal, such as a human).
BACKGROUND OF THE INVENTION
[0003] There is a suite of emerging viruses that are endemic,
pandemic, engineered, or weaponized. To date, there is no
broad-spectrum antiviral therapy that can effectively prevent
infection or treat illness resulting from these viruses. According
to the U.S. Centers for Disease Control and Prevention (CDC; Rotz
et al, CDC Emerging Infectious Diseases Vol. 8, No. 2, 2002) there
are six Category A threats, which includes smallpox, which is
caused by, e.g., variola virus (Smallpox), and viral hemorrhagic
fever, which is caused by, e.g., filoviruses, such as Ebola virus,
bunyaviruses, such as hantavirus, and arenaviruses, such as Lassa
virus. Category A agents have the greatest potential for adverse
public health impact with mass casualties. Biological agents that
have potential for large-scale dissemination with resultant illness
but generally fewer fatalities are classified as Category B
threats. Several viral threats are identified as Category B
threats; these include viral encephalitis, such as, e.g.,
Venezuelan equine encephalitis virus (VEEV), eastern equine
encephalitis virus (EEEV), and western equine encephalitis virus
(WEEV), which are all alphaviruses. There are also many emerging
Category C threats, which include diseases caused by Nipah virus
and hantavirus.
[0004] In addition to the CDC list, the U.S. Department of Health
and Human Services (HHS) has released a list of viruses under their
Public Health Emergency Medical Countermeasures Enterprise (PHEMCE)
program that lists Arenaviridae (e.g., Junin and Lassa viruses),
Filoviridae (e.g., Ebola and Marburg viruses), Poxyiridae (Smallpox
and monkey pox viruses), and Orthomyxoviridae (e.g., Influenzavirus
A, such as H5N1 and H1N1 viruses). Clearly it is not feasible to
vaccinate an entire population against all viral strains of all of
these viral agents. Indeed, the large-scale vaccination of the
public against bioterrorist threats, e.g., anthrax, was a
failure.
[0005] Interferon-alpha (IFN-.alpha.) has been used clinically and
commercially (e.g., RoferonA.RTM., IntronA.RTM., Pegasys.RTM.,
Peglntron.RTM. etc) to successfully treat various cancers,
including, e.g., malignant melanoma, hairy cell leukemia,
non-Hodgkin's lymphoma, AIDS-related Kaposi's sarcoma, as well as
infectious diseases, such as severe acute respiratory syndrome
(SARS), chronic Hepatitis B, and chronic Hepatitis C. IFN-.alpha.
is a type I interferon, which binds to the IFN-.alpha.
receptor.
[0006] IFN-.alpha. is one of the earliest cytokines released by
antigen presenting cells as part of the innate immune response. It
is directly responsible for NK and T cell responsiveness, which
drives the subsequent immune response. Because of the early
response of IFN-.alpha. in the immune cascade, its primary role is
suggested to be to induce a priming state during the initial
response to infection, and it has been shown that low dose
IFN-.alpha. results in increased protection from a viral
challenge.
[0007] IFN-.alpha., as a recombinant human therapeutic agent, is
expensive to manufacture by cGMP, is hindered by its short
half-life in vivo, and is produced in a non-glycosylated form.
IFN-.alpha. has an initial distributive half-life of 7 minutes and
a beta half-life of 2 to 5 hours. This rapid decay requires
multiple injections, usually three times weekly, to maintain
therapeutic levels. Thus, at $2,500 per dose retail, the cost of
using recombinant human IFN-.alpha. as a broad-spectrum antiviral
in counter bioterrorism or military operations is prohibitive.
[0008] In order to mitigate this rapid in vivo degradation,
PEGylated forms of IFN-.alpha. have been developed that have
half-lives that are on the order of days instead of hours, thus
reducing the number of injections to once per week. Nonetheless,
the PEGylation process has been shown to reduce the activity of the
IFN-.alpha., and PEG-IFN-.alpha. is even more expensive to
manufacture than IFN-.alpha..
[0009] Currently, there is a need for a broad-spectrum antiviral
that could be administered for pre- or post-exposure prophylaxis to
guard against or in response to, respectively, infectious diseases,
such as viral threats (e.g., a viral bioweapon used during a
terrorist event or in the event of pandemic disease).
SUMMARY OF THE INVENTION
[0010] In a first aspect, the invention features a composition that
includes a vector having a nucleic acid molecule encoding an
interferon (IFN) and a pharmaceutically acceptable excipient, in
which the composition is formulated as a dry, lyophilized powder,
gel, or liquid, and in which the composition is stable at room
temperature for at least one week. In an embodiment, the interferon
is IFN-alpha (IFN-.alpha.; e.g., consensus IFN-.alpha.
(conIFN-.alpha.; set forth in, e.g., SEQ ID NO: 11) or that is
substantially identical (e.g., at least about 75%, 80%, 85%, 90%,
95%, 97%, or 99% or more identical) to the sequence set forth in
SEQ ID NO: 11). In another embodiment, the vector is a viral vector
(e.g., an adenoviral vector (e.g., an adenoviral strain 5 (Ad5)
vector)). In another embodiment, the adenoviral vector (e.g., the
Ad5 vector) includes a deletion of all or part of the E1 and E3
genes, which makes it replication deficient. In yet another
embodiment, the vector is a non-viral vector.
[0011] In another embodiment of the first aspect of the invention,
in vivo expression of the IFN upon administration of the
composition of the first aspect of the invention produces a
protective immune response against a pathogen (e.g., a bacterium,
virus, fungus, or parasite) in a mammal (e.g., a human) to which
the composition is administered or treats infection by the pathogen
in the mammal. In another embodiment, in vivo expression of the IFN
upon administration of the composition of the first aspect of the
invention produces a protective response against an autoimmune
disease in a mammal (e.g., a human) to which the composition is
administered.
[0012] In other embodiments of the first aspect of the invention,
the nucleic acid molecule of the vector is operably linked to a
promoter selected from an SV40 promoter, CMV promoter, adenovirus
early and late promoter, metallothioneine gene (MT-1) promoter,
Rous sarcoma virus (RSV) promoter, and human Ubiquitine C (UbC)
promoter, or the vector further includes one or more of a signal
sequence, a polyadenylation sequence, and enhancer, an upstream
activation sequence, and a transcription termination factor that
facilitates expression of the nucleic acid molecule encoding the
interferon. In yet other embodiments, the excipient, which is
present in the composition in an amount in the range of from 1% to
90% by weight (e.g., in an amount in the range of from 5% to 30% by
weight), is selected from one or more of fructose, maltose,
galactose, glucose, D-mannose, sorbose, lactose, sucrose,
trehalose, cellobiose, raffinose, melezitose, maltodextrins,
dextrans, starches, mannitol, xylitol, xylose, maltitol, lactitol,
xylitol sorbitol, sorbitose, pyranosyl sorbitol, myoinositol,
glycine, CaCl.sub.2, hydroxyectoine, ectoine, gelatin,
di-myo-inositol phosphate (DIP), cyclic 2,3 diphosphoglycerate
(cDPG), 1,1-di-glycerol phosphate (DGP), .beta.-mannosylglycerate
(firoin), .beta.-mannosylglyceramide (firoin A), and proline
betaine.
[0013] In a preferred embodiment, the excipient is one that is
capable of stabilizing the IFN-encoding delivery vehicle (e.g., the
Ad5-IFN delivery vehicle) for an extended period of time (e.g.,
greater than 1 week, and preferably greater than 1 year or more) at
room temperature with a loss of less than 20% of the viral titer or
biological activity (e.g., if the delivery vehicle is non-viral).
Non-limiting examples of such excipients include, e.g., trehalose,
sorbitol, sucrose, mannitol, glycine, CaCl.sub.2, hydroxiectoin,
ectoin, firoin and gelatine.
[0014] In still other embodiments, the composition can be
formulated for aerosolized delivery; is stable at room temperature
for at least one month (e.g., 1 year or more); and can be admixed
with a pharmaceutically acceptable liquid to form the liquid or
gel.
[0015] In a second aspect, the invention features a method for
prophylaxis or treatment of infection by a biological agent (e.g.,
an infectious pathogen, such as a bacteria, virus, fungus, or
parasite), autoimmune disease, or cancer in a subject in need
thereof (e.g., a mammal, such as a primate, dog, cat, cow, horse,
pig, goat, rat, mouse, or human, or a bird) by administering an
amount of the composition of the first aspect of the invention to
the pulmonary or nasal mucosa of a subject (e.g., a mammal, such as
a primate, dog, cat, cow, horse, pig, goat, rat, mouse, or human,
or a bird) one or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10
times, e.g., within the course of one or more months or one or more
years, or as needed). In an embodiment, the vector targets
pulmonary or nasal epithelial cells upon said administration. In
yet another embodiments, transfection of the vector into the
targeted cells results in expression of the interferon (IFN; e.g.,
IFN-.alpha., such as consensus IFN-.alpha. (conIFN-.alpha.; set
forth in, e.g., SEQ ID NO: 11)) in the cells of the subject and the
IFN acts locally and/or is secreted by the cells into the subject's
bloodstream. In other embodiments, the composition includes an
adenovirus strain 5 (Ad5) vector encoding the IFN and the
composition includes the Ad5 vector in an amount in the range of at
least about 1.times.10.sup.3 to about 1.times.10.sup.14 viral
particles per dose.
[0016] In still other embodiments of the second aspect of the
invention, the subject receives the composition prior to exposure
to the pathogen (e.g., at least about 15 to 30 minutes prior to
exposure to the pathogen, preferably at least about 1, 2, 4, 6, 8,
10, 15, 20, or 24 hours prior to exposure to the pathogen, and more
preferably at least about 1-2 weeks prior to exposure to the
pathogen) or the subject receives the composition following
exposure to the pathogen (e.g., immediately after exposure to the
pathogen or at least about 15-30 minutes following exposure to the
pathogen or at least about 1, 2, 4, 6, 8, 10, 15, 20, 24, 48, or 72
hours, or more, after exposure to the pathogen. In other
embodiments, the pathogen is a bacterium, virus, fungus, or
parasite.
[0017] In other embodiments, the subject receives the composition
of the first aspect of the invention prior to or after development
of autoimmune disease or cancer, or symptoms thereof.
In still other embodiments of the second aspect of the invention,
the composition can be inhaled as a lyophilized powder (e.g., as an
unreconstituted powder) or admixed with a pharmaceutically
acceptable liquid (e.g., water or saline) and inhaled as an
aerosolized mist. In other embodiments, the aerosolized mist
includes droplets having a diameter of greater than 2 .mu.m. In yet
another embodiment, prior to administration of the composition of
the first aspect of the invention, the subject is tested to
determine whether the subject has been exposed to the pathogen,
exhibits symptoms of autoimmune disease, or has cancer. In another
embodiment, following administration of the composition of the
first aspect of the invention, the method further includes
determining the level of IFN in the subject's serum and
administering a subsequent dose of the composition if the level of
IFN in the serum is less than about 1000 IU/ml, preferably less
than about 500 IU/ml, more preferably less than 100 IU/ml, e.g., in
the range of about 0.0001 to about 250 IU/ml. In other embodiments,
the level of IFN in the serum, following administration of a
composition of the invention is in the range of about 100 IU/ml to
about 5.0.times.10.sup.5 IU/ml, preferably in the range of about
200 to 10,000 IU/ml, more preferably in the range of about 250 to
5,000 IU/ml. In other embodiments, the subject is administered at
least 2 doses (e.g., 3, 4, 5, 6, 7, 8, 9, and 10 doses) of the
composition. Preferably, the composition protects the subject from
infection by the pathogen for at least about 24 hours, 36 hours, 48
hours, or 72 hours, preferably for at least about 1, 2, 3, 4, or 5
weeks, and more preferably for at least about 2, 6, 12, 18 or 24
months or more. In other embodiments, administration of the
composition of the first aspect of the invention reduces or
dimishes symptoms associated with autoimmune disease or results in
a decrease of 20, 40, 60, 80, or 100% in the size of a tumor or in
the number of cancerous cells, as determined using standard methods
(for example, at least 20, 40, 60, 80, 90, or 95% of the treated
subjects have a complete remission in which all evidence of the
tumor or cancer disappears). Desirably, the tumor or cancer does
not reappear or reappears after at least 5, 10, 15, or 20
years.
[0018] In other embodiments, the composition is administered as a
liquid or a gel. The composition may be administered by the subject
or by another person, such as an attending physician.
[0019] In other embodiments of the second aspect of the invention,
following administration of the composition of the first aspect of
the invention, the method further includes determining the level of
an IFN-induced response as a correlate for the activity of IFN in
the subject. For example, the method can include determining or
measuring the upregulation or activity of the double-stranded RNA
(dsRNA)-dependent protein kinase R (PKR), the 2'-5'-oligoadenylate
synthetase (2'-5'-OAS), IFN-inducible Mx proteins, a
tryptophan-degrading enzyme (see, e.g., Pfefferkorn, Proc. Natl.
Acad. Sci. USA 81:908-912, 1984), adenosine deaminase (ADAR1),
IFN-stimulated gene 20 (ISG20), p 56, ISG15, mGBP2, GBP-1, the
APOBEC proteins, viperin, or other factors (see, e.g., Zhang et
al., J. Virol., 81:11246-11255, 2007, and U.S. Pat. No. 7,442,527,
which is incorporated by reference herein in its entirety).
[0020] A third aspect of the invention features a device that
contains the composition of any embodiments of the first aspect of
the invention. Preferably, the device includes a) a container that
includes the composition; b) a nozzle for directing the composition
to the pulmonary or nasal mucosa of a subject; c) a mechanical
delivery pump for delivering the composition to the nozzle, such
that activation of the pump results in a fluid connection between
the nozzle and the container; and d) an actuation mechanism for
activating the mechanical delivery pump (e.g., a trigger capable of
actuating the delivery pump at a predeterminable pressure or flow
rate). The delivery pump can also include a liquid delivery pump
for delivering a metered volume of the composition in liquid form
or a powder delivery pump for delivering a metered amount of the
composition in powder form. In an embodiment, the nozzle can be
configured to deliver an aerosol (e.g., a mist) or a jet. Devices
for use in the third aspect of the invention are described
herein.
[0021] A fourth aspect of the invention features a kit that
includes a first container having the composition of any
embodiments of the first aspect of the invention, a second
container having a pharmaceutically acceptable liquid, and the
device of any embodiments of the third aspect of the invention,
and, optionally, instructions for using the device to deliver the
contents of the first container, or for combining the contents of
the first and second containers to form a combined composition and
then using the device to deliver the combined composition, e.g., to
a subject for treating or inhibiting infection by a pathogen,
autoimmune disease or symptoms thereof, or cancer. In an embodiment
of all aspects of the invention, the vector is a recombinant viral
vector (e.g., an adenoviral vector, such as Ad5) that includes a
nucleic acid molecule encoding a cytokine (e.g., interferon-alpha
(IFN-.alpha.), such as consensus IFN-.alpha.); the composition can
be administered to a subject (e.g., a mammal, such as a primate,
dog, cat, cow, horse, pig, goat, rat, mouse, or human, or a bird)
to protect against challenge from, or to treat infection by, a
biological agent. The biological agent can be an infectious
pathogen, such as a bacterium, virus, fungus, or parasite.
[0022] In an embodiment of all aspects of the invention, the
bacterium is selected from Pseudomonas aeruginosa, Salmonella
typhimurium, Escherichia coli, Klebsiella pneumoniae, Bruscella,
Burkholderia mallei, Yersinia pestis, and Bacillus anthracis.
[0023] In an embodiment of all aspects of the invention, the virus
is selected from a member of the Flaviviridae family (e.g., a
member of the Flavivirus, Pestivirus, and Hepacivirus genera),
which includes the hepatitis C virus, Yellow fever virus;
Tick-borne viruses, such as the Gadgets Gully virus, Kadam virus,
Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fever
virus, Powassan virus, Royal Farm virus, Karshi virus, tick-borne
encephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill
virus and the Negishi virus; seabird tick-borne viruses, such as
the Meaban virus, Saumarez Reef virus, and the Tyuleniy virus;
mosquito-borne viruses, such as the Aroa virus, dengue virus,
Kedougou virus, Cacipacore virus, Koutango virus, Japanese
encephalitis virus, Murray Valley encephalitis virus, St. Louis
encephalitis virus, Usutu virus, West Nile virus, Yaounde virus,
Kokobera virus, Bagaza virus, Ilheus virus, Israel turkey
meningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika
virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus,
Saboya virus, Sepik virus, Uganda S virus, Wesselsbron virus,
yellow fever virus; and viruses with no known arthropod vector,
such as the Entebbe bat virus, Yokose virus, Apoi virus, Cowbone
Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San
Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar bat
virus, Montana myotis leukoencephalitis virus, Phnom Penh bat
virus, Rio Bravo virus, Tamana bat virus, and the Cell fusing agent
virus.
[0024] In another embodiment of all aspects of the invention, the
virus is selected from a member of the Arenaviridae family, which
includes the Ippy virus, Lassa virus (e.g., the Josiah, LP, or
GA391 strain), lymphocytic choriomeningitis virus (LCMV), Mobala
virus, Mopeia virus, Amapari virus, Flexal virus, Guanarito virus,
Junin virus, Latino virus, Machupo virus, Oliveros virus, Parana
virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe virus,
Tamiami virus, Whitewater Arroyo virus, Chapare virus, and Lujo
virus.
[0025] In yet other embodiments of all aspects of the invention,
the virus is selected from a member of the Bunyaviridae family
(e.g., a member of the Hantavirus, Nairovirus, Orthobunyavirus, and
Phlebovirus genera), which includes the Hantaan virus, Sin Nombre
virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La
Crosse virus, Punta Toro virus (PTV), California encephalitis
virus, and Crimean-Congo hemorrhagic fever (CCHF) virus.
[0026] In still other embodiments of all aspects of the invention,
the virus is selected from a member of the Filoviridae family,
which includes the Ebola virus (e.g., the Zaire, Sudan, Ivory
Coast, Reston, and Uganda strains) and the Marburg virus (e.g., the
Angola, Ci67, Musoke, Popp, Ravn and Lake Victoria strains); a
member of the Togaviridae family (e.g., a member of the Alphavirus
genus), which includes the Venezuelan equine encephalitis virus
(VEE), Eastern equine encephalitis virus (EEE), Western equine
encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki
Forest virus, Ross River virus, Barmah Forest virus, O'nyong'nyong
virus, and the chikungunya virus; a member of the Poxyiridae family
(e.g., a member of the Orthopoxvirus genus), which includes the
smallpox virus, monkeypox virus, and vaccinia virus; a member of
the Herpesviridae family, which includes the herpes simplex virus
(HSV; types 1, 2, and 6), human herpes virus (e.g., types 7 and 8),
cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella-Zoster
virus, and Kaposi's sarcoma associated-herpesvirus (KSHV); a member
of the Orthomyxoviridae family, which includes the influenza virus
(A, B, and C), such as the H5N1 avian influenza virus or H1N1 swine
flu; a member of the Coronaviridae family, which includes the
severe acute respiratory syndrome (SARS) virus; a member of the
Rhabdoviridae family, which includes the rabies virus and vesicular
stomatitis virus (VSV); a member of the Paramyxoviridae family,
which includes the human respiratory syncytial virus (RSV),
Newcastle disease virus, hendravirus, nipahvirus, measles virus,
rinderpest virus, canine distemper virus, Sendai virus, human
parainfluenza virus (e.g., 1, 2, 3, and 4), rhinovirus, and mumps
virus; a member of the Picornaviridae family, which includes the
poliovirus, human enterovirus (A, B, C, and D), hepatitis A virus,
and the coxsackievirus; a member of the Hepadnaviridae family,
which includes the hepatitis B virus; a member of the
Papillamoviridae family, which includes the human papilloma virus;
a member of the Parvoviridae family, which includes the
adeno-associated virus; a member of the Astroviridae family, which
includes the astrovirus; a member of the Polyomaviridae family,
which includes the JC virus, BK virus, and SV40 virus; a member of
the Calciviridae family, which includes the Norwalk virus; a member
of the Reoviridae family, which includes the rotavirus; and a
member of the Retroviridae family, which includes the human
immunodeficiency virus (HIV; e.g., types 1 and 2), and human
T-lymphotropic virus Types I and II (HTLV-1 and HTLV-2,
respectively).
[0027] In still other embodiments of all aspects of the invention,
the fungus can be Aspergillus, Blastomyces dermatitidis, Candida,
Coccidioides immitis, Cryptococcus neoformans, Histoplasma
capsulatum var. capsulatum, Paracoccidioides brasiliensis,
Sporothrix schenckii, Zygomycetes spp., Absidia corymbifera,
Rhizomucor pusillus, or Rhizopus arrhizus.
[0028] In another embodiment of all aspects of the invention, the
parasite is selected from Toxoplasma gondii, Plasmodium falciparum,
P. vivax, P. ovale, P. malariae, Trypanosoma spp., and Legionella
spp.
[0029] In another embodiment of all aspects of the invention, the
autoimmune disease includes systemic autoimmune diseases and
organ-specific autoimmune diseases. Typical examples of autoimmune
diseases include insulin-dependent diabetes (also known as type 1
diabetes), systemic lupus erythematosus, chronic rheumatoid
arthritis, Hashimoto's disease, alopecia greata, ankylosing
spondylitis, antiphospholipid syndrome, autoimmune Addison's
disease, autoimmune hemolytic anemia, autoimmune hepatitis,
Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac
sprue-dermatitis, chronic fatigue immune dysfunction syndrome
(CFIDS), chronic inflammatory demyelinating polyneuropathy,
Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome,
cold agglutinin disease, Crohn's disease, discoid lupus, ulcerative
colitis, psoriatic arthritis, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre,
hypothyroidism, idiopathic pulmonary fibrosis, idiopathic
thrombocytopenia purpura (ITP), IgA nephropathy, juvenile
arthritis, lichen planus, lupus, Meniere's disease, mixed
connective tissue disease, multiple sclerosis, myasthenia gravis,
pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,
polychondritis, polyglandular syndromes, polymyalgia rheumatica,
polymyositis and dermatomyositis, primary agammaglobulinemia,
primary biliary cirrhosis, psoriasis, Raynaud's phenomenon,
Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma,
Sjogren's syndrome, Stiff-Man syndrome, Devic's disease, Takayasu
arteritis, temporal arteritis/giant cell arteritis, ulcerative
colitis, uveitis, vasculitis, vitiligo, and Wegener's
granulomatosis.
[0030] In another embodiment of all aspects of the invention, the
cancer include such cancers as melanoma, clear cell sarcoma, head
and neck cancer, bladder cancer, breast cancer, colon cancer,
ovarian cancer, endometrial cancer, gastric cancer, pancreatic
cancer, renal cancer, prostate cancer, salivary gland cancer, lung
cancer, liver cancer, skin cancer, and brain cancer.
[0031] In yet another embodiment of all aspects of the invention,
the compositions and methods of the first, second, third, and
fourth aspects of the invention further include administering with,
or expressing in, the vector (e.g., viral vector), a supplemental
therapeutic agent or regimen, e.g., a polypeptide, such as an
antibody or antibody fragment (e.g., recombinant, humanized,
chimeric, or monoclonal antibody or fragment), a microbial antigen,
a cytokine or growth factor, a hormone, a clotting factor, a drug
resistance or anti-viral resistance polypeptide, an anti-venom
agent, an antioxidant, a receptor or ligand, an immunomodulatory
factor, a detectable label, a cellular factor, or a vaccine. In
other embodiments, the antibody or antibody fragment can be a
single chain antibody (scFv), Fab, Fab'2, scFv, SMIP, diabody,
nanobody, aptamer, or domain antibody. In yet other embodiments,
the cytokine or growth factor can be tumor necrosis factor alpha
(TNF-.alpha.), TNF-.beta., IFN-.beta., IFN-.gamma., interleukin 1
(IL-1), IL-1.beta., interleukin 2-14, granulocyte macrophage
colony-stimulating factor (GM-CSF), granulocyte colony-stimulating
factor (G-CSF), RANTES, MIP-1.alpha.), transforming growth
factor-beta (TGF-.beta.), platelet derived growth factor (PGDF),
insulin-like growth factor (IGF), epidermal growth factor (EGF),
vascular endothelial growth factor (VEGF), keratinocyte growth
factor (KGF), erythropoietin (EPO), or thrombopoietin (TPO). The
hormone can be angiotensinogen, angiotensin, parathyroid hormone
(PTH), basic fibroblast growth factor-2, luteinizing hormone,
follicle-stimulating hormone, adrenocorticotrophic hormone (ACTH),
vasopressin, oxytocin, somatostatin, gastrin, cholecystokinin,
leptin, atrial-natriuretic peptide, epinephrine, norephinephrine,
dopamine, calcitonin, or insulin. The clotting factor can be factor
VII, factor VIII, factor IX, or fibrinogen. The enzyme can be can
be butyrylcholinesterase (BChE), adenosine deaminase,
glucocerebrosidase, alpha-1 antitrypsin, a viral thymidine kinase,
hypoxanthine phosphoribosyl transferase, manganese superoxide
dismutase (Mn-SOD), catalase, copper-zinc-superoxide dismutase
(CuZn-SOD), extracellular superoxide dismutase (EC-SOD),
glutathione reductase, phenylalanine hydroxylase, nitric oxide
synthetase, or paraoxinase. The receptor or ligand can be a T-cell
receptor (TCR), LDL receptor, surface-bound immunoglobulin, soluble
CD4, cystic fibrosis transmembrane conductance receptor (CFTR), or
a F.sub.c receptor. The immunomodulatory factor can be CTLA-4, VCP,
PLIF, LSF-1, Nip, CD200, uromodulin, CD40L (CD154), FasL, CD27L,
CD30L, 4-1BBL, CD28, CD25, B7.1, B7.2, or OX40L. The detectable
label can be green fluorescent protein (GFP). The cellular factor
can be cytochrome b, ApoE, ApoC, ApoAI, MDR, tissue plasminogen
activator (tPA), urokinase, hirudin, .beta.-globin, .alpha.-globin,
HbA, ras, src, or bcl. The polypeptide can be a cellular protein
that acts as an antigen, thereby generating an immune response in
the subject against a biological or chemical agent. The vaccine can
be, e.g., a bacterial, viral, fungal, or parasite vaccine known in
the art for treating one or more of the bacterial, viral, fungal,
or parasitic agents described herein. For example, the vaccine may
be directed against a bacterium selected from Pseudomonas
aeruginosa, Salmonella typhimurium, Escherichia coli, Klebsiella
pneumoniae, Bruscella, Burkholderia mallei, Yersinia pestis, and
Bacillus anthracis; a virus selected from a member of the
Flaviviridae family (e.g., a member of the Flavivirus, Pestivirus,
and Hepacivirus genera), which includes the hepatitis C virus,
Yellow fever virus; Tick-borne viruses, such as the Gadgets Gully
virus, Kadam virus, Kyasanur Forest disease virus, Langat virus,
Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus,
Karshi virus, tick-borne encephalitis virus, Neudoerfl virus,
Sofjin virus, Louping ill virus and the Negishi virus; seabird
tick-borne viruses, such as the Meaban virus, Saumarez Reef virus,
and the Tyuleniy virus; mosquito-borne viruses, such as the Aroa
virus, dengue virus, Kedougou virus, Cacipacore virus, Koutango
virus, Japanese encephalitis virus, Murray Valley encephalitis
virus, St. Louis encephalitis virus, Usutu virus, West Nile virus,
Yaounde virus, Kokobera virus, Bagaza virus, Ilheus virus, Israel
turkey meningoencephalo-myelitis virus, Ntaya virus, Tembusu virus,
Zika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra
virus, Saboya virus, Sepik virus, Uganda S virus, Wesselsbron
virus, yellow fever virus; and viruses with no known arthropod
vector, such as the Entebbe bat virus, Yokose virus, Apoi virus,
Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus,
San Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar
bat virus, Montana myotis leukoencephalitis virus, Phnom Penh bat
virus, Rio Bravo virus, Tamana bat virus, and the Cell fusing agent
virus; a virus selected from a member of the Arenaviridae family,
which includes the Ippy virus, Lassa virus (e.g., the Josiah, LP,
or GA391 strain), lymphocytic choriomeningitis virus (LCMV), Mobala
virus, Mopeia virus, Amapari virus, Flexal virus, Guanarito virus,
Junin virus, Latino virus, Machupo virus, Oliveros virus, Parana
virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe virus,
Tamiami virus, Whitewater Arroyo virus, Chapare virus, and Lujo
virus; a virus selected from a member of the Bunyaviridae family
(e.g., a member of the Hantavirus, Nairovirus, Orthobunyavirus, and
Phlebovirus genera), which includes the Hantaan virus, Sin Nombre
virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La
Crosse virus, Punta Toro virus (PTV), California encephalitis
virus, and Crimean-Congo hemorrhagic fever (CCHF) virus; a virus
selected from a member of the Filoviridae family, which includes
the Ebola virus (e.g., the Zaire, Sudan, Ivory Coast, Reston, and
Uganda strains) and the Marburg virus (e.g., the Angola, Ci67,
Musoke, Popp, Ravn and Lake Victoria strains); a member of the
Togaviridae family (e.g., a member of the Alphavirus genus), which
includes the Venezuelan equine encephalitis virus (VEE), Eastern
equine encephalitis virus (EEE), Western equine encephalitis virus
(WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross
River virus, Barmah Forest virus, O'nyong'nyong virus, and the
chikungunya virus; a member of the Poxyiridae family (e.g., a
member of the Orthopoxvirus genus), which includes the smallpox
virus, monkeypox virus, and vaccinia virus; a member of the
Herpesviridae family, which includes the herpes simplex virus (HSV;
types 1, 2, and 6), human herpes virus (e.g., types 7 and 8),
cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella-Zoster
virus, and Kaposi's sarcoma associated-herpesvirus (KSHV); a member
of the Orthomyxoviridae family, which includes the influenza virus
(A, B, and C), such as the H5N1 avian influenza virus or H1N1 swine
flu; a member of the Coronaviridae family, which includes the
severe acute respiratory syndrome (SARS) virus; a member of the
Rhabdoviridae family, which includes the rabies virus and vesicular
stomatitis virus (VSV); a member of the Paramyxoviridae family,
which includes the human respiratory syncytial virus (RSV),
Newcastle disease virus, hendravirus, nipahvirus, measles virus,
rinderpest virus, canine distemper virus, Sendai virus, human
parainfluenza virus (e.g., 1, 2, 3, and 4), rhinovirus, and mumps
virus; a member of the Picornaviridae family, which includes the
poliovirus, human enterovirus (A, B, C, and D), hepatitis A virus,
and the coxsackievirus; a member of the Hepadnaviridae family,
which includes the hepatitis B virus; a member of the
Papillamoviridae family, which includes the human papilloma virus;
a member of the Parvoviridae family, which includes the
adeno-associated virus; a member of the Astroviridae family, which
includes the astrovirus; a member of the Polyomaviridae family,
which includes the JC virus, BK virus, and SV40 virus; a member of
the Calciviridae family, which includes the Norwalk virus; a member
of the Reoviridae family, which includes the rotavirus; and a
member of the Retroviridae family, which includes the human
immunodeficiency virus (HIV; e.g., types 1 and 2), and human
T-lymphotropic virus Types I and II (HTLV-1 and HTLV-2,
respectively); or a fungus selected from Aspergillus, Blastomyces
dermatitidis, Candida, Coccidioides immitis, Cryptococcus
neoformans, Histoplasma capsulatum var. capsulatum,
Paracoccidioides brasiliensis, Sporothrix schenckii, Zygomycetes
spp., Absidia corymbifera, Rhizomucor pusillus, and Rhizopus
arrhizus; or parasite selected from Toxoplasma gondii, Plasmodium
falciparum, P. vivax, P. ovale, P. malariae, Trypanosoma spp., and
Legionella spp.
[0032] In yet other embodiments of all aspects of the invention,
the vector (e.g., viral vector) can be modified to express one or
more oligonucleotides, e.g., an RNA interference (RNAi) molecule
capable of inhibiting viral replication or infection. The RNAi
molecule can be a small inhibitory RNA (siRNA) or short hairpin RNA
(shRNA) molecule.
[0033] In another embodiment of all aspects of the invention, the
subject has been or is suspected to have been exposed to a
biological or chemical agent prior to receiving a pharmaceutical
composition of the invention. In another embodiment of all aspects
of the invention, the subject has been diagnosed with or exhibits
symptoms of autoimmune disease or cancer prior to receiving a
pharmaceutical composition of the invention. The subject can be
administered single or multiple doses of the pharmaceutical
composition of the invention. In another embodiment of all aspects
of the invention, the pharmaceutical composition of the invention
can be administered to a subject (e.g., a mammal, such as a human)
as a prophylactic, e.g., as a vaccine-type preventative, prior to
exposure to a biological or chemical agent to protect the subject
(e.g., immediately prior to exposure, e.g., at least about 5, 10,
or 30 minutes prior to exposure, or, preferably, at least about 1,
2, 3, 4, or 5 hours prior to exposure, more preferably at least
about 6, 24, 36, 48, or 72 hours prior to exposure, and more
preferably at least about 1, 2, 3, or 4 weeks or more prior to
exposure) or prior to the diagnosis of, or development of symptoms
of, autoimmune disease or cancer. The pharmaceutical composition of
the invention can be administered to a subject intravenously,
intramuscularly, orally, by inhalation, parenterally,
intraperitoneally, intraarterially, transdermally, sublingually,
nasally, transbuccally, liposomally, adiposally, opthalmically,
intraocularly, subcutaneously, intrathecally, topically, or
locally. In a preferred embodiment, the pharmaceutical composition
is administered to the pulmonary or intranasal mucosa of a subject.
If the IFN-encoding delivery vehicle composition is a viral vector,
the subject can be administered at least about 1.times.10.sup.3
viral particles (vp)/dose or between 1.times.10.sup.1 and
1.times.10.sup.14 vp/dose, preferably between 1.times.10.sup.3 and
1.times.10.sup.12 vp/dose, and more preferably between
1.times.10.sup.5 and 1.times.10.sup.10 vp/dose. If the IFN-encoding
delivery vehicle composition is a non-viral vector, the subject can
be administered at least about 1.times.10.sup.1 molecules/dose,
e.g., between 1.times.10.sup.1 and 1.times.10.sup.15
molecules/dose, preferably between 1.times.10.sup.3 and
1.times.10.sup.10 molecules/dose, and more preferably between
1.times.10.sup.4 and 1.times.10.sup.8 molecules/dose, of the
non-viral delivery vector.
[0034] In other embodiments of all aspects of the invention,
expression of the heterologous protein (e.g., IFN, such as a
consensus IFN-.alpha.) in a subject (as determined by measuring
serum levels) occurs for greater than one week, one month, two
months, or six months. In yet other embodiments, the effects of
expression of interferon (e.g., IFN-.alpha., such as a consensus
IFN-.alpha.) occurs for greater than one week, one month, two
months, six months or 1-2 years (as determined by using surrogate
markers for interferon expression, as is discussed herein).
[0035] In another embodiment of all aspects of the invention, the
pharmaceutical composition of the invention can be administered to
a subject in combination with one or more supplemental agents that
enhance or prolong the prophylactic or therapeutic effect of the
interferon (e.g., consensus IFN-.alpha.) treatment. The
supplemental agent can be, e.g., a cytokine, antiviral agent,
anti-bacterial agent, anti-fungal agent, anti-parasitic agent,
immunostimulant, or immunization vaccine. In another embodiment,
the pharmaceutical composition of the invention includes an IFN
expression vector (e.g., an Ad5 vector that encodes IFN-.alpha.), a
vaccine, and a pharmaceutically acceptable carrier, in which the
composition is fast-acting (e.g., exhibiting >80% (e.g., 85%,
90%, 95%, or 99% or more (e.g., 100%)) treatment efficacy (e.g., as
measured by survival) when administered within at least 24 hours
(e.g., 1, 2, 4, 6, 8, 10, 12, 15, or 18 hours) post-exposure or
even within as little as 15-30 minutes post-exposure. In another
embodiment, the vaccine is a viral vaccine (e.g., an Ebola vaccine
(e.g., the Ebola Zaire vaccine Ad-CAGoptZGP; see Richardson et al.
(PloS 4:e5308, 2009)). In another embodiment, the pharmaceutical
composition of the invention includes an IFN expression vector
(e.g., an Ad5 vector that encodes IFN-.alpha.) and a
pharmaceutically acceptable carrier, which is administered
separately or in combination with a vaccine (e.g., a viral vaccine,
such as an Ebola vaccine (e.g., the Ebola Zaire vaccine
Ad-CAGoptZGP; see Richardson et al. (PLoS 4:e5308, 2009)). For
example, the pharmaceutical composition of the invention is
administered within 15-30 minutes of the vaccine or within 1, 2, 4,
8, 10, 12, 24, 48, or 72 hours of the vaccine or within 1-2 weeks
after the vaccine.
[0036] In yet another embodiment of all aspects of the invention,
the vector (e.g., viral vector, such as Ad5 vector) is administered
with a pharmaceutically acceptable carrier or excipient.
DEFINITIONS
[0037] The term "about" is used herein to mean a value that is
.+-.10% of the recited value.
[0038] As used herein, by "administering" is meant a method of
giving a dosage of a pharmaceutical composition to a subject. The
compositions utilized in the methods described herein can be
administered by a route selected from, e.g., parenteral, dermal,
transdermal, ocular, inhalation, buccal, sublingual, perilingual,
nasal, rectal, topical, and oral. Parenteral administration
includes intra-arterial, intravenous, intraperitoneal,
subcutaneous, and intramuscular administration. The preferred
method of administration can vary depending on various factors
(e.g., the components of the composition being administered and the
severity of the condition being treated).
[0039] By "an amount sufficient to treat" is meant the amount of a
composition administered to improve, inhibit, or ameliorate a
condition of a subject, or a symptom of a disorder, in a clinically
relevant manner (e.g., improve, inhibit, or ameliorate infection,
e.g., by one or more viruses or viral strains, or one or more
symptoms that occur following infection, or to improve, treat, or
ameliorate autoimmune disease or cancer, or one or more symptoms
thereof). Any improvement in the subject is considered sufficient
to achieve treatment. Preferably, an amount sufficient to treat is
an amount that reduces, inhibits, or prevents the occurrence or one
or more symptoms of a viral infection (e.g., symptoms that result
from infection by at least one and preferably two or more viruses
or viral strains) or is an amount that reduces the severity of, or
the length of time during which a subject suffers from, one or more
symptoms of the infection (e.g., by at least 10%, 20%, or 30%, more
preferably by at least 50%, 60%, or 70%, and most preferably by at
least 80%, 90%, 95%, 99%, or more, relative to a control subject
that is not treated with a composition of the invention). A
sufficient amount of the pharmaceutical composition used to
practice the methods described herein (e.g., the treatment of viral
infection(s)) varies depending upon the manner of administration
and the age, body weight, and general health of the subject being
treated. A physician or researcher can decide the appropriate
amount and dosage regimen.
[0040] By "host, "subject" or "patient" is meant any organism, such
as a mammal (e.g., a primate, dog, cat, cow, horse, pig, goat, rat,
and mouse) or a bird; preferably the organism is a human. A host
may also be a domestic animal (e.g., a farm animal) or a companion
animal (e.g., a pet).
[0041] By "inducing an immune response" is meant eliciting a
humoral response (e.g., the production of antibodies) or a cellular
response (e.g., the activation of T cells, macrophages,
neutrophils, and natural killer cells) directed against one or more
viruses or viral strains (e.g., two, three, four, or more viruses
or viral strains) in a subject to which the pharmaceutical
composition (e.g., a vaccine) has been administered.
[0042] As used here, "interferon" or "IFN" refers to a peptide or
protein having an amino acid sequence substantially identical
(e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,
or even 100% identical) to all or a portion of the sequence of an
interferon (e.g., a human interferon), such as IFN-.alpha. (e.g.,
IFN-.alpha.-1a; see U.S. Patent Application No. 20070274950,
incorporated herein by reference in its entirety), IFN-.alpha.-1b
(SEQ ID NOs: 1 and 2), IFN-.alpha.-2a (see PCT Application No. WO
07/044,083, herein incorporated by reference in its entirety) and
IFN-.alpha.-2b (SEQ ID NOs: 3 and 4)), consensus IFN-.alpha. (SEQ
ID NO: 11), IFN-.beta. (e.g., described in U.S. Pat. No. 7,238,344,
incorporated by reference in its entirety; IFN-.beta.-1a, as
described in U.S. Pat. No. 6,962,978; incorporated by reference in
its entirety) and IFN-.beta.-1b (as described in U.S. Pat. Nos.
4,588,585; 4,959,314; 4,737,462; and 4,450,103; incorporated by
reference in their entirety; see also SEQ ID NOs: 5 and 6),
IFN-.gamma. (see, e.g., SEQ ID NOs: 7 and 8), and IFN-.tau. (as
described in U.S. Pat. No. 5,738,845 and U.S. Patent Application
Publication Nos. 20040247565 and 20070243163; incorporated by
reference in their entirety; see also SEQ ID NOs: 9 and 10).
[0043] The term "interferon alpha" or "IFN-.alpha." as used herein
means the family of highly homologous species-specific proteins
that inhibit viral replication and cellular proliferation and
modulate immune response. Typical suitable interferon-alphas
include, but are not limited to, recombinant interferon alpha-2a,
recombinant interferon alpha-2b, recombinant interferon alpha-2c,
alpha 2 interferon, and a consensus alpha interferon, such as those
described in U.S. Pat. Nos. 4,897,471 and 4,695,623 (especially
Examples 7, 8 or 9 thereof), which are incorporated herein by
reference.
[0044] By "pharmaceutical composition" is meant any composition
that contains a therapeutically or biologically active agent (e.g.,
at least one nucleic acid molecule that encodes all or part of a
cytokine (e.g., an interferon, such as IFN-.alpha. (e.g., consensus
IFN-.alpha.) either incorporated into a viral vector or independent
of a viral vector (e.g., incorporated into a liposome,
microparticle, or nanoparticle)) that is suitable for
administration to a subject and that is capable of inducing an
immune response against at least one virus (e.g., at least two,
three, four, or more different viruses or viral strains) or that
treats autoimmune disease or cancer or reduces or ameliorates one
or more symptoms of autoimmune disease or cancer. For the purposes
of this invention, pharmaceutical compositions suitable for
delivering a therapeutic or biologically active agent can include,
e.g., tablets, gelcaps, capsules, pills, powders, granulates,
suspensions, emulsions, solutions, gels, hydrogels, oral gels,
pastes, eye drops, ointments, creams, plasters, drenches, delivery
devices, suppositories, enemas, injectables, implants, sprays, or
aerosols. Any of these formulations can be prepared by well-known
and accepted methods of art. See, for example, Remington: The
Science and Practice of Pharmacy (21.sup.st ed.), ed. A. R.
Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia
of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare,
2006, each of which is hereby incorporated by reference.
[0045] By "pharmaceutically acceptable diluent, excipient, carrier,
or adjuvant" is meant a diluent, excipient, carrier, or adjuvant
which is physiologically acceptable to the subject while retaining
the therapeutic properties of the pharmaceutical composition with
which it is administered. One exemplary pharmaceutically acceptable
carrier is physiological saline. Other physiologically acceptable
diluents, excipients, carriers, or adjuvants and their formulations
are known to one skilled in the art.
[0046] By "recombinant," with respect to a vector, such as a viral
vector, is meant a vector (e.g., a viral genome that has been
incorporated into one or more delivery vehicles (e.g., a plasmid,
cosmid, etc.)) that has been manipulated in vitro, e.g., using
recombinant nucleic acid techniques, to introduce changes to the
vector (e.g., to include heterologous nucleic acid sequences (such
as IFN (e.g., conIFN-.alpha.) in a viral genome (e.g., a
replication deficient Ad5 genome)). An example of a recombinant
viral vector of the invention is a vector that includes all or part
of the adenovirus (e.g., adenovirus strain 5 (Ad5)) genome and that
includes the nucleic acid sequence for all or part of, e.g., a
cytokine gene sequence, such as an interferon-.alpha. gene (e.g.,
the consensus IFN-.alpha. sequence).
[0047] By "room temperature" is meant a temperature of about
5.degree. C. to about 30.degree. C., in particular from about
10.degree. C. to about 27.degree. C. (e.g., about 23-27.degree.
C.).
[0048] The term "substantial identity" or "substantially
identical," when used in the context of comparing a polynucleotide
or polypeptide sequence to a reference sequence, means that the
polynucleotide or polypeptide sequence has the same sequence as the
reference sequence or has a specified percentage of nucleotides or
amino acid residues that are the same at the corresponding
locations within the reference sequence when the two sequences are
optimally aligned. For instance, an amino acid sequence that is
"substantially identical" to a reference sequence has at least
about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher percentage
identity (up to 100%) to the reference sequence when compared and
aligned for maximum correspondence over the full length of the
reference sequence as measured using a BLAST or BLAST 2.0 sequence
comparison algorithms with default parameters, or by manual
alignment and visual inspection (see, e.g., NCBI web site).
[0049] By "treating" is meant administering a pharmaceutical
composition of the invention for prophylactic and/or therapeutic
purposes. Prophylactic treatment may be administered, for example,
to a subject who is not yet ill, but who is susceptible to, or
otherwise at risk of, a particular biological condition, e.g.,
infection by a bacteria, virus, fungus, or parasite (e.g., the
subject may already have been exposed to the infectious agent but
is asymptomatic or the level of exposure to the infectious agent is
unknown), or the development of autoimmune disease or cancer.
Therapeutic treatment may be administered, for example, to a
subject already suffering from contact with a biological agent in
order to improve or stabilize the subject's condition (e.g., a
patient already infected with a pathogenic virus) or a subject
already suffering from an autoimmune disease or cancer. Thus, in
the claims and embodiments described herein, treating is the
administration to a subject either for therapeutic or prophylactic
purposes. In some instances, as compared with an equivalent
untreated control, treatment may ameliorate a disorder (e.g.,
infection by a pathogen, such as a virus, autoimmune disease, and
cancer) or a symptom of the disorder, or reduce the progression,
severity, or frequency of one or more symptoms of the disorder by,
e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%
as measured by any standard technique. For example, for measuring
symptoms of infection, one may use, e.g., blood tests to check for
antibodies directed against the pathogen or for the antigens
themselves; cultures for samples of blood, bodily fluid, or other
material taken from the infected area; spinal tap to examine
cerebrospinal fluid; polymerase chain reaction (PCR) techniques to
amplify nucleic acid material from the pathogen; magnetic and
resonance imaging (MRI) to detect increased swelling in the
temporal lobes). Symptoms of pathogenic infection, which may vary
from mild to severe and may depend on what part of the body is
affected, the type of pathogen, and the age and overall health of
the affected person, include, e.g., fever, muscle aches, coughing,
sneezing, runny nose, sore throat, headache, chills, diarrhea,
vomiting, rash, weakness, dizziness, bleeding under the skin, in
internal organs, or from body orifices like the mouth, eyes, or
ears, shock, nervous system malfunction, delirium, seizures, renal
(kidney) failure, personality changes, neck stiffness, dehydration,
seizures, lethargy, paralysis of the limbs, confusion, back pain,
loss of sensation, impaired bladder and bowel function, and
sleepiness that can progress into coma or death. In some instances,
treating can result in the inhibition of the pathogenic infection,
the treatment of the infection, and/or the amelioration of symptoms
of the infection (e.g., hemorrhagic fever). Detecting an
improvement in, or the absence of, one or more symptoms of the
infection, indicates successful treatment. Treatment can also be
confirmed by the absence of, or the inability to detect the
presence of, the pathogen (e.g., a virus) in the treated
subject.
[0050] For the treatment or prophylaxis of autoimmune disease, one
can measure, e.g., decreased levels of autoantibodies, decreased
levels of autoreactive T cells, increase of targeted cells (e.g.,
pancreatic .beta.-islet cells), and improvements in fatigue,
depression, sensitivity to cold, weight gain, muscle weakness,
constipation, insomnia, irritability, weight loss, bulging eyes,
muscle tremors, skin rashes, painful or swollen joints, sensitivity
to the sun, loss of coordination, and paralysis.
[0051] For the treatment or reduction of cancer, one can measure
reductions in the size of a tumor or in the number of cancer cells,
the slowing or prevention of an increase in the size of a tumor or
cancer cell proliferation, an increase in the disease-free survival
time between the disappearance of a tumor or other cancer and its
reappearance, the prevention of an initial or subsequent occurrence
of a tumor or other cancer, or the reduction of an adverse symptom
associated with a tumor or other cancer. In a desired embodiment,
the percent of tumor or cancerous cells surviving the treatment is
at least 20, 40, 60, 80, or 100% lower than the initial number of
tumor or cancerous cells, as measured using any standard assay
(e.g., caspase assays, TUNEL and DNA fragmentation assays, cell
permeability assays, and Annexin V assays). Desirably, the decrease
in the number of tumor or cancerous cells induced by administration
of an agent of the invention is at least 2, 5, 10, 20, or 50-fold
greater than the decrease in the number of non-tumor or
non-cancerous cells. Desirably, the methods of the present
invention result in a decrease of 20, 40, 60, 80, or 100% in the
size of a tumor or in the number of cancerous cells, as determined
using standard methods. Desirably, at least 20, 40, 60, 80, 90, or
95% of the treated subjects have a complete remission in which all
evidence of the tumor or cancer disappears. Desirably, the tumor or
cancer does not reappear or reappears after at least 5, 10, 15, or
20 years.
[0052] A subject to be treated according to the methods described
herein (e.g., a subject infected with, or at risk of being infected
with, a bacterium, virus, fungus, or parasite) may be one who has
been diagnosed by a medical practitioner as having such a
condition. Diagnosis may be performed by any suitable means. A
subject in whom the development of an infection is being prevented
may or may not have received such a diagnosis. One skilled in the
art will understand that a subject to be treated according to the
present invention may have been subjected to standard tests or may
have been identified, without examination, as one at high risk due
to the presence of one or more risk factors (e.g., exposure to a
biological agent, such as a virus).
[0053] By "viral vector" is meant a composition that includes one
or more genes from a viral species, such as an adenoviral species
(e.g., Ad5), that is able to transmit one or more heterologous
genes from a viral or non-viral source to a host or subject. The
nucleic acid material of the viral vector may be encapsulated,
e.g., in a lipid membrane or by structural proteins (e.g., capsid
proteins), that may include one or more viral polypeptides (e.g., a
glycoprotein). The viral vector can be used to infect cells of a
subject (e.g., nasal epithelium), which, in turn, promotes the
translation of the heterologous gene(s) of the viral vector into a
protein product (e.g., IFN-.alpha.).
[0054] Alternatively, the viral vector can be administered to a
subject so that it infects one or more cells of the subject, which
then promotes expression of the one or more heterologous genes of
the viral vector and stimulates an immune response (directly or
indirectly) that is protective against infection by a pathogen
(e.g., bacteria, virus, fungus, or parasite) or that treats
infection by the pathogen.
[0055] The term "vaccine," as used herein, is defined as material
used to provoke an immune response and confer immunity after
administration of the vaccine to a subject.
[0056] The term "virus," as used herein, is defined as an
infectious agent that is unable to grow or reproduce outside a host
cell and that infects mammals (e.g., humans) or birds.
[0057] Other features and advantages of the invention will be
apparent from the detailed description and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0058] FIG. 1 is a table providing comparative amino acid sequences
of human leukocyte interferon subtypes and a consensus human
leukocyte interferon.
[0059] FIG. 2 is a schematic showing insertion of the nucleic acid
molecule encoding consensus interferon-alpha (conINF-.alpha.) into
an adenoviral vector.
[0060] FIG. 3 is a schematic showing delivery of an
Ad5-conIFN-.alpha. construct of the invention to the nasal
epithelial cells of a patient, expression of the conIFN-.alpha.
nucleic acid molecule in the cells, and release of IFN polypeptide
into the bloodstream of the patient.
[0061] FIG. 4 is a diagram showing the benefits of an
Ad5-conIFN-.alpha. construct of the invention.
[0062] FIG. 5 is a table summarizing the results of experiments (in
the indicated animal model) using compositions of the invention to
treat or prevent infection by the indicated virus.
[0063] FIG. 6 is a graph showing the effect of intranasal (IN)
Ad5-IFN.alpha. treatment on survival outcome in hamsters challenged
with Punta Toro virus (PTV). Animals in each group were treated
once 24 hours prior to IN instillation with PTV with the indicated
amount of Ad5-IFN.alpha. or empty vector virus particles. Ribavirin
treatment was i.p. once daily for 6 days starting 4 hours prior to
PTV infection. *P<0.05, **P<0.01 compared to PBS vehicle
placebo-treated animals. .sup.a<0.001 as compared to EV-treated
animals.
[0064] FIGS. 7A and 7B are graphs showing the effect of IN
Ad5-IFN.alpha. treatment on survival outcome in mice challenged
with WEE virus. Animals in each group were treated with 10.sup.7
PFU Ad5-IFN.alpha., as per the groups outlined in Example 9 below,
and challenged with WEE virus via IN instillation. IFN.alpha. B/D
was given daily as a positive control group.
[0065] FIGS. 8A and 8B are graphs showing the effect of IN
Ad5-IFN.alpha. treatment on survival outcome in mice challenged
with SARS virus. FIG. 8A shows the results of prophylyaxis: Animals
in each group were treated with 10.sup.6 PFU Ad5-IFN.alpha., as per
the groups outlined in Example 10 below, and challenged with SARS
virus via IN instillation. FIG. 8B shows the results of treatment:
Animals in each group were treated with 10.sup.6 or 10.sup.5 PFU
Ad5-IFN.alpha. as per the groups outlined in Example 10 below, and
challenged with SARS virus via IN instillation Poly IC/LC was used
as a positive control group, with saline as negative control.
[0066] FIGS. 9A and 9B are graphs showing the effect of IN
Ad5-IFN.alpha. treatment on survival outcome in mice challenged
with YF virus. FIG. 9A shows the results of dose range
prophylyaxis: Animals were treated with Ad5-IFN.alpha. as per the
groups outlined in Example 11 below, and challenged with YF virus
via IN instillation. Complete protection was observed at the two
highest doses, with a dose response curve for the lower doses. FIG.
9B shows the results of treatment: Animals in each group were
treated with 5.times.10.sup.7 PFU Ad5-IFN.alpha., as per the groups
outlined in Example 11 below, and challenged with SARS virus via IN
instillation Complete survival was observed for the -4 hr and +1
dpi groups with a drop in survival correlated with delayed
treatment in other groups.
[0067] FIGS. 10A and 10B are graphs showing the effect of IN
Ad5-IFN.alpha. treatment on survival outcome in mice challenged
with ZEBOV. FIG. 10A shows the results of mouse treatment: Animals
were challenged with 100 LD50 EBOV and 30 minutes later treated
with Ad5-IFN.alpha. by either the 1M or 1N route. Complete
protection was observed with 10.sup.7 PFU with both routes of
administration. FIG. 10B shows the results of guinea pig treatment:
Animals were challenged with 100 LD50 EBOV and 30 minutes later
treated with Ad5-IFN.alpha. IN. Complete protection was observed
with 2.times.10.sup.8 PFU.
[0068] FIG. 11 is a graph showing the effect of IN Ad5-IFN.alpha.
treatment on survival outcome in mice challenged with Pichinde
virus. Animals were treated with Ad5-IFN.alpha., as per the groups
outlined in Example 13 below, and challenged with PCV via IN
instillation. Complete protection was observed at the highest dose,
with a dose response curve at lower doses.
[0069] FIG. 12 is a graph showing the effect of IN Ad5-IFN.alpha.
treatment in conjunction with Ad-EBOV vaccine on survival outcome
in mice challenged with EBOV. Animals were treated with
Ad5-IFN.alpha., as per the groups outlined in Example 14 below, and
challenged with PCV via IN instillation. Complete protection was
observed at the highest dose, with a dose response curve at lower
doses.
DETAILED DESCRIPTION OF THE INVENTION
[0070] The invention features compositions and methods for the
prophylaxis (pre- or post-exposure) and treatment of diseases or
disorders caused by an infectious pathogen (e.g., infectious
agents, such as viruses, bacteria, fungi, and parasites) in a
subject (e.g., a mammal, such as a human). The infectious pathogen
may be naturally occurring or it may be formulated for, or adapted
to, use as a biological agent. The invention also features the use
of the compositions of the invention to treat or reduce one or more
symptoms of autoimmune disease and cancer in a subject (e.g., a
mammal, such as a human).
[0071] The compositions of the invention can be used as, e.g., a
broad-spectrum prophylaxis or treatment to guard against or treat
infection by several different infectious pathogens, in particular,
viral agents. Of particular note, the compositions of the invention
can be administered for pre-exposure prophylaxis (e.g., 1-30
minutes (e.g., 15-30 minutes) before exposure, preferably 1, 2, 3,
4, 5, 6-12, 24-72 hours before exposure, or 1-6 weeks or more
(e.g., at least 2 weeks) before exposure to an infectious agent),
as well as for post-exposure prophylaxis or treatment (e.g.,
immediately after exposure, e.g., 1-30 minutes (e.g., 15-30
minutes) after exposure, or within 1, 2, 3, 4, 5, 6-12, 24, 48, or
72 hours or 1-2 weeks after exposure to an infectious agent). Thus,
the compositions of the invention provide benefits in the
prophylaxis or treatment, respectively, of a subject in
anticipation of, or following, e.g., exposure to an infectious
pathogen (e.g., a virus, such as during a bioterrorist attack). The
benefits include both long-lasting protection as well as rapid
protection, as needed.
[0072] In order to circumvent the fast decay of traditional
IFN-.alpha. protein-based drugs in vivo, the compositions of the
invention utilize a delivery vector (e.g., a viral vector, such as
an adenoviral vector (e.g., an adenovirus 5 (Ad5) delivery
platform)) that is capable of delivering a nucleic acid molecule
encoding IFN, which drives the continuous in situ production of IFN
(e.g., human IFN-.alpha., such as consensus IFN-.alpha. (con
IFN-.alpha.)) by cells transduced or transfected with the delivery
vector. The production of IFN continues in the transduced or
transfected cell (e.g., for the life of the cell).
[0073] For example, a nucleic acid molecule encoding IFN-.alpha. is
inserted into the replication defective Ad5 virus, and the
Ad5-IFN-.alpha. vector is then delivered to a subject (e.g., a
mammal, such as a human). In an embodiment, delivery of the viral
vector is intranasal. Intranasal administration of the compositions
of the invention prevents the host immune system from recognizing
the Ad5 vector, thereby bypassing any pre-existing immunity the
subject might typically present against the delivery vector itself.
In addition, intranasal administration avoids the use of needles,
which allows for easier, less invasive administration in the event
mass administration to the public is needed in response to, e.g., a
bioterrorist attack, or in the absence of ready access to a medical
facility. Compositions of the invention can also be delivered to
the pulmonary system (e.g., the upper and/or lower respiratory
tract) by delivery to the lungs through the mouth.
[0074] The compositions of the invention also provide benefit due
to their long-term storage potential and extended shelf life. The
compositions of the invention can be stored at room temperature for
significant periods of time (e.g., for at least 1 week and up to 1
year or more). Alternatively, the compositions of the invention can
be stored at temperatures in the range of 30.degree.-55.degree. C.
(e.g., at 45.degree. C.) for significant periods of time (e.g., for
at least 2-3 days, 1-3 week, 1-6 months, and up to 1 year or more).
In an embodiment, the compositions of the invention are in powder
form when stored at temperatures in the range of
30.degree.-55.degree. C. In yet other embodiments, the compositions
of the invention can be stored frozen (e.g., at temperatures below
at least 4.degree. C. (e.g., in the range of 0.degree. to
-20.degree. C.)), either in a powder or liquid form. For example,
the compositions can be stored frozen as a non-stabilized, liquid
formulation (e.g., without any or with only one or a few
stabilizing agents, such as, e.g., trehalose, sorbitol, sucrose,
mannitol, glycine, CaCl.sub.2, hydroxiectoin, ectoin, firoin and
gelatin).
[0075] In an embodiment, the compositions of the invention are
stored as a stable lyophilized powder. The powder can be used
directly (e.g., in powder form without reconstitution of any kind)
or reconstituted just before use (e.g., using a hydration medium,
such as saline or water, preferably sterilized, or any other
pharmaceutically acceptable hydration medium) and administered as,
e.g., an aqueous mist. Reconstitution of powder forms of the
compositions of the invention is possible where clean water is
available, such as a medical facility or rear echelons in the
military. Alternatively, the powder compositions of the invention
can be reconstituted in a gel form. Nasal gels are high-viscosity
thickened solutions or suspensions. The advantages of a nasal gel
includes the reduction of post-nasal drip due to high viscosity,
reduction of taste impact due to reduced swallowing, reduction of
anterior leakage of the formulation, reduction of irritation by
using soothing/emollient excipients, and target to mucosa for
better absorption.
[0076] The powder form of compositions of the invention can be
provided in a kit with a vial of sterile hydrating medium (e.g.,
water or saline) that can be used to reconstitute the powder (e.g.,
to form a liquid or gel). If water is to be used as the hydrating
medium, the composition of the invention can but need not be
formulated to include reagents (e.g., buffers) that adjust the
conditions of the composition in its final form (e.g., the pH,
osmolarity, or ionic concentration) so that it is suitable for, or
tolerable to, a subject administered the composition.
[0077] Administration of the compositions of the invention in
powder form is more likely in, e.g., emerging economies,
expeditionary military operations, and in rapid response
situations. For those compositions of the invention that are not
formulated to exhibit an extended shelf life at room temperature or
at higher temperatures (e.g., those compositions of the invention
that exhibit a shelf life of less than 1 week when stored at room
temperature), it is preferable that the compositions be stored at a
temperature in the range of about -20.degree. C. to about
20.degree. C. to extend shelf life. These compositions may be
formulated with an excipient that does not stabilize the Ad5-IFN
delivery vehicle such that it can only remain at room temperature
for periods of less than, e.g., 1 week to 1 month, unless
refrigerated.
[0078] Compositions of the invention (e.g., an Ad5-IFN.alpha.
construct) have been tested successfully to date in animal models
of human disease, such as mouse, Guinea pig, and hamster models,
against challenges from representative viruses from important viral
families, e.g., Filoviridae (Ebola virus, Zaire strain),
Flaviviridae (Yellow Fever), Arenaviridae (Pichinde), Bunyaviridae
(Punta Toro), Coronaviridae (SARS), Togaviridae (VEEV and WEEV);
see FIG. 5. The compositions of the invention have an excellent
treatment profile and a good prophylactic window with data
indicating full protection to 21 days with partial protection at
further time points. Compositions of the invention are fast acting,
and impart both therapeutic and prophylactic benefits to the
recipient within minutes to hours; the benefits of the compositions
of the invention remain effective for days and even months after
administration.
Compositions of the Invention
[0079] The compositions of the invention include a delivery vector
containing a nucleic acid molecule encoding a cytokine (e.g., an
IFN, such as conIFN-.alpha.). The compositions of the invention may
be formulated for any route of administration (e.g., the
administration routes described herein, such as by nasal inhalation
and/or inhalation through the mouth for delivery to the upper
and/or lower respiratory tract). The compositions may be
administered in a single dose or in multiple doses to a subject in
need thereof, either pre- or post-exposure to an infectious
pathogen or prior to the diagnosis of, or after development of
symptoms of, autoimmune disease or cancer. The compositions of the
invention may also further include secondary agents (either as a
nucleic acid molecule to be expressed by a cell of the subject or
as a polypeptide or drug) or they may be administered in
combination with one or more additional therapeutic regimens (e.g.,
vaccines), as is discussed below.
Interferons
[0080] The compositions of the invention for use in the pre- or
post-exposure prophylaxis or treatment, respectively, of a
pathogenic infection (e.g., a viral, bacterial, fungal, or
parasitic infection) or for use in the treatment of autoimmune
disease or cancer (or one or more symptoms thereof) include a
delivery vector containing a nucleic acid molecule encoding an IFN.
The nucleic acid molecule encodes an interferon having an amino
acid sequence substantially identical (e.g., at least 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% identical) to
the sequence of a human IFN-.alpha. (e.g., IFN-.alpha.-1a,
IFN-.alpha.-1b, IFN-.alpha.-2a, IFN-.alpha.-2b, and consensus
IFN-.alpha. (conIFN-.alpha.); FIG. 1), a human IFN-.beta. (e.g.,
IFN-.beta.-1a and IFN-.beta.-1b), a human IFN-.gamma.), or an
IFN-.tau. or a polypeptide that demonstrates the same or similar
biological activity to an interferon (e.g., at least 50%, 60%, 70%,
75%, 80%, 85%, 90%, 95%, or 100% of the activity of a human
IFN-.alpha., a human IFN-.beta., a human IFN-.gamma., an IFN-.tau.,
or a conIFN-.alpha. (SEQ ID NOs: 2, 4, 6, 8, 10, and 11,
respectively). The nucleic acid molecule may have the sequence set
forth in any one of SEQ ID NOs: 1, 3, 5, 7, or 9 corresponding to a
human IFN-.alpha., a human IFN-.beta., a human IFN-.gamma., or an
IFN-.tau., respectively, or the nucleic acid molecule may have a
sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or even 100% identity to one of SEQ ID NOs: 1, 3, 5, 7,
or 9.
[0081] The biological activity of an interferon of the invention
can be confirmed using, e.g., a virus-plaque-reduction assay,
assays that measure the inhibition of cell proliferation, the
regulation of functional cellular activities, the regulation of
cellular differentiation, and immunomodulation mediated by IFN, as
well as a reporter gene assay, in which the promoter region of IFN
responsive genes is linked with a heterologous reporter gene, for
example, firefly luciferase or alkaline phosphatase, and
transfected into an IFN-sensitive cell line such that stably
transfected cell lines exposed to IFN increase expression of the
reporter gene product in direct relation to the dose of IFN (see,
e.g., Balducci et al., Appl. Microbiol. 11:310-314, 1963; McNeil,
J. Immunol. Methods 46:121-127, 1981; and Meager et al., J.
Immunol. Methods 261:21-36, 2002). Other assays for measuring the
activity of IFN include measuring the upregulation or activity of
the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR),
the 2'-5'-oligoadenylate synthetase (2'-5'-OAS), IFN-inducible Mx
proteins, a tryptophan-degrading enzyme (see, e.g., Pfefferkorn,
Proc. Natl. Acad. Sci. USA 81:908-912, 1984), adenosine deaminase
(ADAR1), IFN-stimulated gene 20 (ISG20), p 56, ISG15, mGBP2, GBP-1,
the APOBEC proteins, viperin, or other factors (see, e.g., Zhang et
al., J. Virol., 81:11246-11255, 2007, and U.S. Pat. No. 7,442,527,
which is incorporated by reference herein in its entirety).
[0082] Interferon alpha (IFN-.alpha.), as used herein, refers to a
cytokine with multiple biological activities that include antiviral
activity, regulation of cell proliferation and differentiation and
immunomodulation, as exemplified in, e.g., Pfeffer et al. (Cancer
Res. 58:2489-2499, 1998). In an embodiment of the invention, the
IFN-.alpha. may be selected from, e.g., IFN-.alpha.2a,
IFN-.alpha.2b, IFN-.alpha.2c, and consensus IFN-.alpha.
(conIFN-.alpha.) (see FIG. 4 and, e.g., U.S. Pat. No. 4,695,623,
incorporated herein by reference). In an embodiment, the
IFN-.alpha. is conIFN-.alpha..
[0083] Unlike the compositions of the invention, recombinant human
IFN, in particular rhconIFN-.alpha., which is fully approved and
marketed as Infergen.RTM. for the treatment of chronic Hepatitis C,
is made via prokaryotic fermentation, and thus lacks glycosylation.
Moreover, Infergen.RTM. is formulated for administration via
injection into patients.
Viral Vectors
[0084] In the invention described herein, the interferon (e.g.,
IFN-.alpha., such as conIFN-.alpha.) can be formulated for delivery
using a viral vector that includes a nucleic acid molecule encoding
the interferon. Any suitable viral vector system can be used
including, e.g., adenoviruses (e.g., Ad2, Ad5, Ad9, Ad15, Ad17,
Ad19, Ad20, Ad22, Ad26, Ad27, Ad28, Ad30, or Ad39; see, e.g., FIG.
2), rhabdoviruses (e.g., vesicular stomatitis virus), retroviruses
(see, e.g., Miller, Curr. Top. Microbiol. Immunol. 158:1-24, 1992;
Salmons and Gunzburg, Human Gene Therapy 4:129-141, 1993; and
Miller et al., Methods in Enzymology 217:581-599, 1994),
adeno-associated vectors (reviewed in Carter, Curr. Opinion
Biotech. 3:533-539, 1992; and Muzcyzka, Curr. Top. Microbiol.
Immunol. 158:97-129, 1992), poxviruses, herpes viral vectors, and
Sindbis viral vectors (see viral vectors discussed generally in,
e.g., Jolly, Cancer Gene Therapy 1:51-64, 1994; Latchman, Molec.
Biotechnol. 2:179-195, 1994; Johanning et al., Nucl. Acids Res.
23:1495-1501, 1995; Berencsi et al., J. Infect. Dis. 183:1171-1179,
2001; Rosenwirth et al., Vaccine 19:1661-1670, 2001; Kittlesen et
al., J. Immunol. 164:4204-4211, 2000; Brown et al., Gene Ther.
7:1680-1689, 2000; Kanesa-thasan et al., Vaccine 19:483-491, 2000;
and Sten Drug 60:249-271, 2000. Compositions comprising such
vectors and an acceptable excipient are also a feature of the
invention.
[0085] Ad5 is a virus of the family Adenoviridae, species C,
subtype 5. This virus is naturally occurring and causes mild upper
respiratory infections, usually in children. Ad5 can be used as a
delivery platform to deliver the genetic information to make human
interferon in situ. Typically, the Ad5 is rendered replication
defective (by specific gene deletion; e.g., all or a portion of the
E1 or E3 genes). Ad5 vectored vaccines have been approved for
clinical studies widely in the past. Ad5 is widely used in clinical
trials as a vector delivery system. As of June 2010, there are
currently 29 clinical trials that are currently active using Ad5
vectored delivery of biologics/drugs. Adenovirus 5 based vectors
exhibit an excellent safety profile. The Ad5 vector has additional
benefits over conventional vaccines such as live-attenuated
vaccines, a type of vaccine where pathogenic viruses are partially
crippled via chemical or heat treatment prior to injection, in that
there is no risk the Ad5 system could revert and cause illness.
Further, Ad5 is a live vaccine which has been shown to provide
prompt immunologic protection. Ad5-based vectors for delivery of
cytokine genes for providing protection against biological weapons
is described in, e.g., U.S. Pat. Nos. 6,565,853 and 6,936,257, both
of which are incorporated herein by reference.
[0086] Intravenous or intramuscular administration of agents for
biodefense medical counter measure indications using the Ad5 system
have previously failed because the body's immune system recognizes
this viral vector and destroys the vector before the gene has been
delivered to a host cell. This occurred most recently with Merck's
HIV-1 vaccine clinical trial, which resulted in the study being
halted early on the grounds of futility (see Robb, Lancet 372,
2008). Intranasal administration of compositions of the invention
(e.g., an Ad5-vector encoding IFN) circumvents this problem by
avoiding the body's immune targeting of the Ad5 vector, as is
discussed herein.
[0087] The viral vector may be constructed using conventional
techniques known to one of skill in the art. For example, the viral
vector may contain at least one sequence encoding a heterologous
gene (e.g., consensus IFN-.alpha.), which is under the control of
regulatory sequences that direct its expression in a cell (e.g., an
epithelial cells, such as a nasal or pulmonary epithelial cell).
Appropriate amounts for vector-mediated delivery of the
heterologous gene can be readily determined by one of skill in the
art based on the information provided herein.
[0088] The delivery of IFN-.alpha. using an adenoviral vector is
described in, e.g., Ahmed et al. (J. Interferon Cytokine Res. 21:
399408, 2001), Zhang et al. (Proc. Natl. Acad. Sci. USA
93:4513-4518, 1996), Ahmed (Hum. Gene Ther. 10:77-84, 1999), and
Santodonato et al. (Cancer Gene Ther. 8:63-72, 2001). The delivery
of IFN-.alpha. using a retroviral vector is described in, e.g.,
Tuting et al. (Gene Ther. 4:1053-1060, 1997) and Mecchia et al.
(Gene Ther. 7:167-179, 2000).
[0089] In an embodiment, the Ad5 vector contains a nucleic acid
molecule encoding human interferon alpha consensus sequence under
the transcriptional regulation of the intermediate-early promoter
of CMV and Simian virus 40 (SV40) polyadenylation sequence. In
another embodiment, the human Ad5 vector includes E1 and E3
deletions to render it replication deficient. The Ad5-IFN-.alpha.
vector can be further stabilized with an excipient of
polysaccharides and electrolytes during lyophilization and storage,
as is described herein. As adenoviruses are fragile to thermal
stress and maintenance of the cold chain in the field is onerous,
the temperature stability of the compositions of the invention
impart a significant advantage. We have developed a systematic
process for the stabilization of viral-based vaccines, including
adenoviruses, based on a novel eigenvector approach (see, e.g.,
Kueltzo et al., J. Pharm. Sci. 92:1805-1820, 2003; Fan et al., J.
Pharm. Sci. 94:1893-1911, 2005; Ausar et al., Mol. Pharm.
2:491-499, 2005; and Rexroad et al., J. Pharm. Sci. 95:237-247,
2005). Multiple assays are then used to identify a number of
potential excipients that are tested for their ability to stabilize
the virus against physical and chemical degradation pathways that
result in loss of activity (e.g. physicochemical integrity,
biological activity, etc.).
[0090] An increase in the expression level of a transfected nucleic
acid molecule (e.g., the con IFN-.alpha. sequence) in a host cell
(e.g., an epithelial cell, such as a nasal or pulmonary epithelial
cell) can be promoted by operably linking the nucleic acid molecule
to an open frame expression control sequence, which can work in the
selected expression host. Expression control sequences useful for
eukaryotic host cells can be a native or foreign to the nucleic
acid molecule to be expressed, as well as to the delivery vector.
Examples of expression control sequences include, but are not
limited to, leader sequences, polyadenylation sequences, propeptide
sequences, promoters, enhancers, upstream activation sequences,
signal peptide sequences, and transcription termination factors.
Expression control sequences include those derived from, e.g., SV40
(e.g., early and late promoters of SV40), bovine papilloma virus,
adenovirus (e.g., early and late promoters of adenovirus),
cytomegalovirus (CMV; e.g., the human cytomegalovirus early gene
promoter), MT-1 (metallothioneine gene) promoter, Rous sarcoma
virus (RSV) promoter, and human Ubiquitine C (UbC) promoter. In
order to further improve expression in mammalian cells, synthetic
intron sequences can be inserted into a non-transcription region of
a nucleotide sequence encoding the IFN-.alpha. polypeptide.
[0091] Other vector components that can be used in practicing the
present invention include a signal peptide. This sequence is
typically located at the 5' of a gene encoding a protein and is
thus added to the amino terminus of the protein during expression.
The presence or absence of a signal peptide varies depending on the
expression host cell to be used in production of the IFN-.alpha.
polypeptide and the preference of producing a secreted product
(i.e., according to whether the IFN-.alpha. polypeptide is to be
expressed intra-cellularly or extra-cellularly). In an embodiment,
the IFN-.alpha. (e.g., the conIFN-.alpha.) is secreted from the
host cell during expression. The signal peptide can be homologous
or heterologous to either the IFN-.alpha. polypeptide or the host
cell.
[0092] A nucleic acid molecule is "operably linked" to another
nucleic acid molecule when they are arranged in a functional
relationship. This means that an appropriate molecule (for example,
a transcription activator) binds to a regulatory sequence(s), a
gene, or a regulatory sequence(s) linked in such a way that the
expression of the nucleic acid molecule is modulated. For example,
when a pre-sequence or secretory leader participates in secretion
of a mature protein, they are operably linked to the promoter. When
a promoter affects transcription of a coding sequence, the promoter
is operably linked to the coding sequence. When a ribosomal binding
site is located at a place capable of being read as a coding
sequence, the ribosomal binding site is operably linked to the
coding sequence. Generally "operably linked" means in contact with
a linked nucleic acid molecule and a secretory leader and to be in
a reading frame.
Non-Viral Vectors
[0093] Non-viral approaches can also be employed to introduce a
therapeutic nucleic acid molecule (e.g., an IFN-.alpha.-encoding
nucleic acid molecule) into cells to treat or prevent pathogenic
infection (e.g., viral infection) or to treat or reduce the
symptoms of autoimmune disease or cancer. For example, a
heterologous gene (e.g., an interferon, such as IFN-.alpha. (e.g.,
consensus IFN-.alpha.) can be introduced into a cell (e.g., an
epithelial cell, such as a nasal or pulmonary epithelial cell) by
lipofection (see, e.g., Felgner et al., Proc. Natl. Acad. Sci. USA
84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990;
Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al.,
Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine
conjugation (see, e.g., Wu et al., Journal of Biological Chemistry
263:14621, 1988; Wu et al., Journal of Biological Chemistry
264:16985, 1989), or, less preferably, micro-injection under
surgical conditions (see, e.g., Wolff et al., Science 247:1465,
1990). Gene transfer can also be achieved by the use of calcium
phosphate, DEAE dextran, electroporation, and protoplast fusion.
Liposomes, microparticles, or nanoparticles can also be potentially
beneficial for delivery of a nucleic acid molecule (e.g., an
IFN-.alpha.-encoding nucleic acid molecule) or a protein into a
cell or into a patient in order to stimulate an immune response
against a pathogen (e.g., a virus). Other non-viral methods of
delivering IFN-.alpha. are described in, e.g., Coleman et al., Hum.
Gene Ther. 9:2223-2230, 1998, and Horton et al., Proc. Natl. Acad.
Sci. USA 96:1553-1558, 1999).
Methods of Prophylaxis or Treatment of Pathogenic
Infection Using Compositions of the Invention
[0094] The pharmaceutical compositions of the invention can be used
as gene therapy and/or genetic vaccines for treating or inhibiting
infection by pathogens, such as bacteria, viruses, fungus, and
parasites. In particular, the compositions of the invention can be
used to treat (pre- or post-exposure) infection by viruses (e.g., a
member of the Flaviviridae family (e.g., a member of the
Flavivirus, Pestivirus, and Hepacivirus genera), which includes the
hepatitis C virus, Yellow fever virus; Tick-borne viruses, such as
the Gadgets Gully virus, Kadam virus, Kyasanur Forest disease
virus, Langat virus, Omsk hemorrhagic fever virus, Powassan virus,
Royal Farm virus, Karshi virus, tick-borne encephalitis virus,
Neudoerfl virus, Sofjin virus, Louping ill virus and the Negishi
virus; seabird tick-borne viruses, such as the Meaban virus,
Saumarez Reef virus, and the Tyuleniy virus; mosquito-borne
viruses, such as the Aroa virus, dengue virus, Kedougou virus,
Cacipacore virus, Koutango virus, Japanese encephalitis virus,
Murray Valley encephalitis virus, St. Louis encephalitis virus,
Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagaza
virus, Ilheus virus, Israel turkey meningoencephalo-myelitis virus,
Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus,
Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S
virus, Wesselsbron virus, yellow fever virus; and viruses with no
known arthropod vector, such as the Entebbe bat virus, Yokose
virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus,
Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Carey
Island virus, Dakar bat virus, Montana myotis leukoencephalitis
virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, and
the Cell fusing agent virus; a member of the Arenaviridae family,
which includes the Ippy virus, Lassa virus (e.g., the Josiah, LP,
or GA391 strain), lymphocytic choriomeningitis virus (LCMV), Mobala
virus, Mopeia virus, Amapari virus, Flexal virus, Guanarito virus,
Junin virus, Latino virus, Machupo virus, Oliveros virus, Parana
virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe virus,
Tamiami virus, Whitewater Arroyo virus, Chapare virus, and Lujo
virus; a member of the Bunyaviridae family (e.g., a member of the
Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus genera),
which includes the Hantaan virus, Sin Nombre virus, Dugbe virus,
Bunyamwera virus, Rift Valley fever virus, La Crosse virus, Punta
Toro virus (PTV), California encephalitis virus, and Crimean-Congo
hemorrhagic fever (CCHF) virus; a member of the Filoviridae family,
which includes the Ebola virus (e.g., the Zaire, Sudan, Ivory
Coast, Reston, and Uganda strains) and the Marburg virus (e.g., the
Angola, Ci67, Musoke, Popp, Ravn and Lake Victoria strains); a
member of the Togaviridae family (e.g., a member of the Alphavirus
genus), which includes the Venezuelan equine encephalitis virus
(VEE), Eastern equine encephalitis virus (EEE), Western equine
encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki
Forest virus, Ross River virus, Barmah Forest virus, O'nyong'nyong
virus, and the chikungunya virus; a member of the Poxyiridae family
(e.g., a member of the Orthopoxvirus genus), which includes the
smallpox virus, monkeypox virus, and vaccinia virus; a member of
the Herpesviridae family, which includes the herpes simplex virus
(HSV; types 1, 2, and 6), human herpes virus (e.g., types 7 and 8),
cytomegalovirus (CMV), Epstein-Ban virus (EBV), Varicella-Zoster
virus, and Kaposi's sarcoma associated-herpesvirus (KSHV); a member
of the Orthomyxoviridae family, which includes the influenza virus
(A, B, and C), such as the H5N1 avian influenza virus or H1N1 swine
flu; a member of the Coronaviridae family, which includes the
severe acute respiratory syndrome (SARS) virus; a member of the
Rhabdoviridae family, which includes the rabies virus and vesicular
stomatitis virus (VSV); a member of the Paramyxoviridae family,
which includes the human respiratory syncytial virus (RSV),
Newcastle disease virus, hendravirus, nipahvirus, measles virus,
rinderpest virus, canine distemper virus, Sendai virus, human
parainfluenza virus (e.g., 1, 2, 3, and 4), rhinovirus, and mumps
virus; a member of the Picornaviridae family, which includes the
poliovirus, human enterovirus (A, B, C, and D), hepatitis A virus,
and the coxsackievirus; a member of the Hepadnaviridae family,
which includes the hepatitis B virus; a member of the
Papillamoviridae family, which includes the human papilloma virus;
a member of the Parvoviridae family, which includes the
adeno-associated virus; a member of the Astroviridae family, which
includes the astrovirus; a member of the Polyomaviridae family,
which includes the JC virus, BK virus, and SV40 virus; a member of
the Calciviridae family, which includes the Norwalk virus; a member
of the Reoviridae family, which includes the rotavirus; and a
member of the Retroviridae family, which includes the human
immunodeficiency virus (HIV; e.g., types 1 and 2), and human
T-lymphotropic virus Types I and II (HTLV-1 and HTLV-2,
respectively)).
[0095] The pharmaceutical compositions include vectors encoding IFN
(e.g., IFN-.alpha., such as conIFN-.alpha.) that can be
administered in vivo or ex vivo.
[0096] IFN-.alpha. is one of the earliest cytokines released by the
antigen-presenting cell as part of the innate immune response and
is directly responsible for NK and T cell responsiveness, which
drives the subsequent immune response. NK cells are one of the
first professional killing cells to arrive in the early antiviral
immune response. In addition, IFN-.alpha. appears to be the
principle cytokine mediating expansion of CD8+ T cells. Because of
the early response of IFN-.alpha. in the immune cascade, its
primary role is suggested to be to induce a priming state during
the initial response to infection, and it has been shown that low
dose IFN-.alpha. results in increased protection from a viral
challenge (see, e.g., Brassard et al., J. Leuk. Biol. 71:565-581,
2002).
[0097] In addition, interferon induces the expression of MX
proteins, which are 7-80 kDa proteins with GTPase activity that
affect viral replication by interfering with transcription (i.e.,
they inhibit viral RNA polymerases) of influenza and other negative
strand RNA viruses (Acheson, In "Fundamentals of Molecular
Virology," J. Wiley and Sons, Hoboken N.J., 2007).
[0098] Interferon also induces the expression of ribonuclease L,
which degrades viral (and host) mRNA, and thus leads to an
inhibition of viral replication by suppression of viral protein
synthesis. (Acheson, 2007). Thus, the expression of IFN-.alpha. in
the transduced/transfected cells (e.g., epithelial cells) of a
subject provides prophylaxis and/or treatment of pathogenic
infection by, in part, activating these and other pathways that
stimulate the subject's immune response and protect the subject,
pre- and post-exposure, against pathogenic (e.g., viral)
infection.
[0099] The pharmaceutical compositions of the invention act via a
two-step process: administration and expression. For example, after
intranasal administration, the Ad5 virus enters the epithelial
cells of the upper and/or lower respiratory tract and transports
the IFN-.alpha. nucleic acid molecule to the nucleus. Next, the
IFN-.alpha. nucleic acid molecule is transcribed and the resulting
mRNA is translated, post-translationally modified with
glycosylation, expressed as a mature IFN-.alpha. cytokine on the
cell surface. The adenovirus itself does not replicate as it has
been rendered replication deficient. Once the IFN-.alpha. is
expressed on the cell surface, it functions in the same manner as
naturally in situ-produced IFN-.alpha..
[0100] Accordingly, the vectors can be used to transduce or
transfect a subject's cells in vivo (e.g., epithelial cells, such
as nasal or pulmonary epithelial cells) by administering the vector
in a dosage and form discussed herein (e.g., as an aerosolized
powder, liquid mist, or gel) to the subject (e.g., via intranasal
or pulmonary administration) to provide prophylaxis and/or
treatment of pathogenic infection. Alternatively, cells can be
removed from the subject and transduced or transfected ex vivo with
the vector encoding IFN and those cells can be returned to the
subject to provide prophylaxis and/or treatment of pathogenic
infection. In an embodiment, cells of the subject are removed and
treated ex vivo with the Ad5-IFN-.alpha. vector of the invention.
The cells are then administered to the patient, pre- or
post-exposure, to treat or inhibit pathogenic infection. Preferably
at least about 1.times.10.sup.4 to about 10.times.10.sup.6 cells
are treated and reintroduced to the subject.
[0101] In an embodiment, a sufficient amount of the pharmaceutical
composition is administered to a subject to achieve a peak blood
level of IFN-.alpha. due to expression from the
transfected/transduced cells of at least between about 0.0001 to
5.0.times.10.sup.5 IU/ml, preferably between about 0.0002 to
2.0.times.10.sup.5 IU/mL, and most preferably between about 0.0005
to 1.0.times.10.sup.5 IU/mL (see, e.g., NIBSC code: 94/784 and
94/786; WHO International Standard for INTERFERON ALPHA, (Human
leukocyte-derived); dated 14 Feb. 2008; Meager et al., J. Immunol.
Methods 257:17-33, 2001; and Mire-Sluis et al., J. Interferon
Cytokine Res. 16:637-643, 1996). In another embodiment, the amount
of circulating IFN-.alpha. is between about 100 IU/ml and 1,000
IU/ml (e.g., about 250 IU/ml). Preferably, the circulating levels
of IFN-.alpha. remain within this range for at least 1 to 15 days,
or at least 1, 2, 3, or 4 weeks, or at least 2-6 months. The
expression levels of IFN-.alpha. can be determined by measuring the
amount of IFN-.alpha. in, e.g., the subject's serum (see, e.g.,
Forti et al., J. Clin. Microbiol. 21:689-693, 1985). In other
embodiments, the anti-viral effects of IFN-.alpha. remain evident
in the subject for at least 1, 2, 3, or 4 weeks, more preferably
for at least 2, 4, or 6 months, and most preferably for 1 year or
more. The anti-viral effects of IFN-.alpha. can be determined by
measuring the upregulation or activity of the double-stranded RNA
(dsRNA)-dependent protein kinase R (PKR), the 2'-5'-oligoadenylate
synthetase (2'-5'-OAS), IFN-inducible Mx proteins, a
tryptophan-degrading enzyme (see, e.g., Pfefferkorn, Proc. Natl.
Acad. Sci. USA 81:908-912, 1984), adenosine deaminase (ADAR1),
IFN-stimulated gene 20 (ISG20), p 56, ISG15, mGBP2, GBP-1, the
APOBEC proteins, viperin, or other factors (see, e.g., Zhang et
al., J. Virol., 81:11246-11255, 2007). Assays for measuring the
anti-viral effects of IFN-.alpha. can be found in, e.g., U.S. Pat.
No. 7,442,527, which is incorporated by reference herein in its
entirety.
[0102] Upon administration of the pharmaceutical composition
including the IFN-.alpha. delivery vector (e.g., an Ad5 delivery
vector), e.g., to nasal or pulmonary epithelial cells, the nucleic
acid molecule encoding IFN-.alpha. incorporates into the cells.
These cells then produce IFN-.alpha. during the course of their
lifespan until death or apoptosis, thereby allowing for expression
of human IFN-.alpha. lasting for several hours, days, or weeks or
more (e.g., about 1-15 days, 1-4 weeks, or 2-6 months) compared to
hours for exogenously administered rhIFN-.alpha.. Furthermore, the
IFN produced from, e.g., an Ad5-hIFN vector will be fully
glycosylated unlike the rhIFN-.alpha. currently being commercially
prepared by eukaryotic fermentation (i.e., Infergen.RTM. (Alfacon;
DIN 2239832)). In addition, the therapeutic effects (e.g.,
anti-viral effects) of IFN-.alpha. can extend for at least 1, 2, 3,
or 4 weeks, more preferably for at least 2, 4, or 6 months, and
most preferably for 1 year or more.
[0103] Naturally occurring IFN-.alpha. is glycosylated. Most rhIFN
products are not glycosylated as they are made via prokaryotic
fermentation. Due to the location of the glycosylation sites, there
is no risk of impeding receptor binding with the addition of
glycosylation. However, the pharmacokinetics of glycosylated and
unglycosylated IFN-.alpha. may well be different, and the stability
of the protein may be influenced by glycosylation, as is the case
for human granulocyte-macrophage colony-stimulating factor (GM-CSF;
see Adolf et al. (Biochem. J. 276:511-518, 1991). Further, the
immunogenicity of rhIFN-.alpha. might be affected by the lack of
glycosylation. Gribben et al. have reported that four out of 16
patients receiving rhGM-CSF produced in yeast developed antibodies
to this protein; these antibodies reacted with epitopes that were
exposed in the recombinant factor, but would have been protected by
glycosylation (see Gribben et al., Lancet 335:434-437, 1990).
Induction of antibodies to non-glycosylated rhIFN-.alpha. after
prolonged treatment of patients has been described, and it has been
speculated that natural IFN-.alpha. may be less immunogenic than
the recombinant proteins (see Figlin and Itri, Semin. Hematol.
25:9-15, 1988, and Galton et al., Lancet 2:572-573, 1989).
[0104] Although there is evidence using all forms of IFN (e.g.,
.alpha., .beta., .omega., .gamma.) that glycosylation does not
appear to affect the specific antiviral/biological activity of the
protein (see Bocci, Trends Biochem Sci 8:432-434, 1983, and Adolf
et al., Biochem J. 276:511-518, 1991), it is believed that
glycosylation of IFN may be important for other reasons. There are
studies specifically working on different translational methods to
manufacture fully glycosylated hIFN.alpha. ex vivo (see, e.g.,
Rossmann et al., Prot. Exp. Purif. 7:335-342, 1996), and patents
filed protecting these methods (see, e.g., U.S. Pat. Nos.
7,445,774; 7,338,654; 7,311,903; and 7,129,390). Thus,
glycosylation is clearly a desirable factor in IFN. The
pharmaceutical compositions of the invention, which deliver a
vector that promotes expression of a fully glycosylated hIFN in
situ, will likely result in a protein with more stability and less
immunogenic effects than currently administered rhIFN polypeptides
lacking glycosylation, while maintaining the same level of
therapeutic (e.g., antiviral) activity.
[0105] Expression of IFN-.alpha. (e.g., conIFN-.alpha.) in the
cells of a subject transfected/transduced with the delivery vector
of the invention provides fast acting protection to the subject
against pathogenic infection (e.g., viral infection). The
IFN-.alpha. delivery vector of the invention is fast acting because
the Ad5 vector incorporates into epithelial cells (e.g., nasal or
pulmonary epithelial cells), journeying from the cell surface to
the nucleus within 30 minutes. The IFN-.alpha. delivery vector of
the invention is particularly effective when administered, e.g.,
intranasally, because the nasal cavity has a large surface area
(100-200 cm square), which allows the Ad5 delivery vector to
penetrate into millions of upper and/or lower respiratory
epithelial cells. Once incorporated, the epithelial cells begin to
generate the IFN-.alpha. (e.g., conIFN-.alpha.) as if it was
endogenous to the cell; the IFN-.alpha. is expressed on the cell
surface and it is secreted into the host circulation.
[0106] Expression of IFN-.alpha. typically occurs within 24 hours
or less (e.g., as early as 3 hours) after administration of the
delivery vector. This result is beneficial, especially in cases
where rapid treatment response is preferable (e.g., viral outbreaks
in the public arena or in situations where a pathogen has been
intentionally released (e.g., against military personnel deployed
on the frontline)). The IFN-.alpha. delivery vector of the
invention provides medical personnel in the public sector, as well
as military planners and others with the ability to act quickly
when responding to various operational threat situations where
there may be uncertainty as to the presence of an infectious
pathogen. For example, today, military planners will not deploy
into areas with endemic pathogenic risks without the proper
vaccinations. This delays greatly the ability of the military, law
enforcement agents, or local emergency coordinator (LEC) to respond
promptly to global threats. The pharmaceutical compositions of the
invention can be used to mitigate those risks and speed the
response time against pathogenic exposure or outbreaks.
[0107] The compositions of the invention may be administered in a
single dose or in multiple doses separately from or coextensively
with other therapies for pathogenic infection (e.g., vaccines), or
as a stand-alone therapy. The compositions of the invention may,
but need not, also include additional therapeutic agents. These
additional therapeutic agents can also be encoded as nucleic acid
molecules in the same or a different delivery vector (e.g., a viral
vector) and expressed as a polypeptide with the IFN or they can be
administered as polypeptides or drugs with the compositions of the
invention, e.g., as a single pharmaceutical composition or in
separate pharmaceutical compositions.
[0108] The compositions of the invention can be administered to a
subject (e.g., a human), pre- or post-exposure to a pathogenic
infection (e.g., a viral infection), to treat, prevent, ameliorate,
inhibit the progression of, or reduce the severity of one or more
symptoms of the pathogenic infection in the subject. Examples of
the symptoms of pathogenic infection, in particular, viral
infection, that can be treated using the compositions of the
invention include, e.g., fever, muscle aches, coughing, sneezing,
runny nose, sore throat, headache, chills, diarrhea, vomiting,
rash, weakness, dizziness, bleeding under the skin, in internal
organs, or from body orifices like the mouth, eyes, or ears, shock,
nervous system malfunction, delirium, seizures, renal (kidney)
failure, personality changes, neck stiffness, dehydration,
seizures, lethargy, paralysis of the limbs, confusion, back pain,
loss of sensation, impaired bladder and bowel function, and
sleepiness that can progress into coma or death. These symptoms,
and their resolution during treatment, may be measured by, e.g., a
physician during a physical examination or by other tests and
methods known in the art.
[0109] The dose of the compositions of the invention (e.g., the
number of IFN-encoding delivery vectors, viral or otherwise) or the
number of treatments using the compositions of the invention may be
increased or decreased based on the severity of, occurrence of, or
progression of, the pathogenic infection in the patient (e.g.,
based on the severity of one or more symptoms of, e.g., viral
infection).
Uses
[0110] IFN is known to be effective against a broad range of
pathogens, in particular, viruses. Hence the pharmaceutical
compositions of this invention are referred to as a "Broad Spectrum
Antiviral." Viruses against which the compositions of the invention
can be used include the following: a member of the Flaviviridae
family (e.g., a member of the Flavivirus, Pestivirus, and
Hepacivirus genera), which includes the hepatitis C virus, Yellow
fever virus; Tick-borne viruses, such as the Gadgets Gully virus,
Kadam virus, Kyasanur Forest disease virus, Langat virus, Omsk
hemorrhagic fever virus, Powassan virus, Royal Farm virus, Karshi
virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin
virus, Louping ill virus and the Negishi virus; seabird tick-borne
viruses, such as the Meaban virus, Saumarez Reef virus, and the
Tyuleniy virus; mosquito-borne viruses, such as the Aroa virus,
dengue virus, Kedougou virus, Cacipacore virus, Koutango virus,
Japanese encephalitis virus, Murray Valley encephalitis virus, St.
Louis encephalitis virus, Usutu virus, West Nile virus, Yaounde
virus, Kokobera virus, Bagaza virus, Ilheus virus, Israel turkey
meningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika
virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus,
Saboya virus, Sepik virus, Uganda S virus, Wesselsbron virus,
yellow fever virus; and viruses with no known arthropod vector,
such as the Entebbe bat virus, Yokose virus, Apoi virus, Cowbone
Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San
Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar bat
virus, Montana myotis leukoencephalitis virus, Phnom Penh bat
virus, Rio Bravo virus, Tamana bat virus, and the Cell fusing agent
virus; a member of the Arenaviridae family, which includes the Ippy
virus, Lassa virus (e.g., the Josiah, LP, or GA391 strain),
lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia
virus, Amapari virus, Flexal virus, Guanarito virus, Junin virus,
Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde
virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus,
Whitewater Arroyo virus, Chapare virus, and Lujo virus; a member of
the Bunyaviridae family (e.g., a member of the Hantavirus,
Nairovirus, Orthobunyavirus, and Phlebovirus genera), which
includes the Hantaan virus, Sin Nombre virus, Dugbe virus,
Bunyamwera virus, Rift Valley fever virus, La Crosse virus, Punta
Toro virus (PTV), California encephalitis virus, and Crimean-Congo
hemorrhagic fever (CCHF) virus; a member of the Filoviridae family,
which includes the Ebola virus (e.g., the Zaire, Sudan, Ivory
Coast, Reston, and Uganda strains) and the Marburg virus (e.g., the
Angola, Ci67, Musoke, Popp, Ravn and Lake Victoria strains); a
member of the Togaviridae family (e.g., a member of the Alphavirus
genus), which includes the Venezuelan equine encephalitis virus
(VEE), Eastern equine encephalitis virus (EEE), Western equine
encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki
Forest virus, Ross River virus, Barmah Forest virus, O'nyong'nyong
virus, and the chikungunya virus; a member of the Poxyiridae family
(e.g., a member of the Orthopoxvirus genus), which includes the
smallpox virus, monkeypox virus, and vaccinia virus; a member of
the Herpesviridae family, which includes the herpes simplex virus
(HSV; types 1, 2, and 6), human herpes virus (e.g., types 7 and 8),
cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella-Zoster
virus, and Kaposi's sarcoma associated-herpesvirus (KSHV); a member
of the Orthomyxoviridae family, which includes the influenza virus
(A, B, and C), such as the H5N1 avian influenza virus or H1N1 swine
flu; a member of the Coronaviridae family, which includes the
severe acute respiratory syndrome (SARS) virus; a member of the
Rhabdoviridae family, which includes the rabies virus and vesicular
stomatitis virus (VSV); a member of the Paramyxoviridae family,
which includes the human respiratory syncytial virus (RSV),
Newcastle disease virus, hendravirus, nipahvirus, measles virus,
rinderpest virus, canine distemper virus, Sendai virus, human
parainfluenza virus (e.g., 1, 2, 3, and 4), rhinovirus, and mumps
virus; a member of the Picornaviridae family, which includes the
poliovirus, human enterovirus (A, B, C, and D), hepatitis A virus,
and the coxsackievirus; a member of the Hepadnaviridae family,
which includes the hepatitis B virus; a member of the
Papillamoviridae family, which includes the human papilloma virus;
a member of the Parvoviridae family, which includes the
adeno-associated virus; a member of the Astroviridae family, which
includes the astrovirus; a member of the Polyomaviridae family,
which includes the JC virus, BK virus, and SV40 virus; a member of
the Calciviridae family, which includes the Norwalk virus; a member
of the Reoviridae family, which includes the rotavirus; and a
member of the Retroviridae family, which includes the human
immunodeficiency virus (HIV; e.g., types 1 and 2), and human
T-lymphotropic virus Types I and II (HTLV-1 and HTLV-2,
respectively).
[0111] Particular indications that are contemplated for the
pharmaceutical compositions of the invention, and which are
currently being or have been evaluated in conjunction with the
Division of Microbiology and Infectious Disease (DMID), part of the
National Institute of Allergy and Infectious Disease (NIAID),
include: Dengue, Punta Toro (a BSL-2 surrogate for Rift Valley
Fever), monkeypox, Flu A (H5N1 and H1N1), SARS, Yellow Fever,
Pichinde (a BSL-2 surrogate for Lassa Fever), Western Equine
Encephalitis, Venezuelan Equine Encephalitis, and West Nile Virus.
In broader terms, the IFN-.alpha. delivery vector and
pharmaceutical compositions containing it will be effective
against, at least, the following viral families: Alphaviridae,
Filoviridae, Flaviviridae, Orthomyxoviridae, Bunyaviridae,
Arenaviridae, Herpesviridae, Hepadnaviridae, Coronaviridae, and
Poxyiridae (see Examples).
[0112] A significant proportion of the human population has been
exposed to many adenoviral strains, including Ad5. Thus, there is a
good probability the immune system of any potential recipient of
the pharmaceutical compositions of the invention has "seen" Ad5
before and would be able to quickly mount an immune response to it.
This was the case with the MRKAd5 HIV-1 gag/pol/nef HIV vaccine,
which was tested on HIV negative patients in a phase II clinical
trial in 2008. This trial, which utilized injections, resulted in
"futility," meaning there was no protection seen: the levels of
infection in inoculated subjects was the same as non-inoculated
ones (Buchbinder et al., Lancet 372:1881-1893, 2008). Positive
serostatus for Ad5 was significantly associated with acquisition
(Robb, Lancet 372:1857-1858, 2008), and the design of the vaccine
is "at the centre of the study's failure" (White, Lancet 373:805,
2009). Thus, Ad5 vectored vaccines were thought to be useless due
to the high probability of pre-existing immunity. Indeed, all
military personnel are actively vaccinated with Ad4 and Ad7
vaccines during basic training medical preparation following
enlistment.
[0113] To circumvent pre-existing immunity to the delivery vector,
the IFN-.alpha. delivery vector of the invention, and
pharmaceutical compositions containing it, can be administered via,
e.g., a pulmonary or intranasal route, which avoids problems with
pre-existing immunity to the delivery vector. This is believed to
be due to the lack of contact between the vector (e.g., the
adenoviral vector (e.g., Ad5)) and the immune system (e.g., the
immune components in blood), as the vector incorporates into, e.g.,
epithelial cells directly upon administration. These epithelial
cells act as a functional barrier to the cells and antibodies of
the immune system. Thus, the delivery vector is not exposed to the
circulation; only the IFN is released into the bloodstream with no
traces of the vector remaining (see FIG. 3).
Methods of Prophylaxis or Treatment of Autoimmune
Disease or Cancer Using the Compositions of the Invention
[0114] The pharmaceutical compositions of the invention can also be
used as gene therapy and/or genetic vaccines for treating or
reducing one or more symptoms of autoimmune disease and cancer. The
mechanism of action of the compositions of the invention described
above applies equally to their use in this context.
[0115] Interferons exhibit both antiviral and antiproliferative
activity. IFN-.alpha. is currently approved in the United States
and other countries for the treatment of hairy cell leukemia,
venereal warts, Kaposi's Sarcoma, and chronic non-A, non-B
hepatitis. Two variants of IFN-.alpha. have received approval for
therapeutic use: Interferon alfa-2a, marketed under the trade name
ROFERON.TM.-A, and Interferon alfa-2b, marketed under the trade
name INTRON.TM. A. The amino acid sequences of ROFERON.TM.-A and
INTRON.TM. A differ at a single position but otherwise are
identical to the amino acid sequence of alpha-interferon subtype 2
(subtype A).
[0116] In addition to the labeled indications, IFN-.alpha. is being
used or evaluated alone or in conjunction with chemotherapeutic
agents in a variety of other cellular proliferation disorders,
including chronic myelogenous leukemia, multiple myeloma,
superficial bladder cancer, skin cancers (basal cell carcinoma and
malignant melanoma), renal cell carcinoma, ovarian cancer, low
grade lymphocytic and cutaneous T cell lymphoma, and glioma.
IFN-.alpha. may be effective in combination with other chemotherapy
agents for the treatment of solid tumors that arise from lung,
colorectal and breast cancer (see Rosenberg et al. "Principles and
Applications of Biologic Therapy" in Cancer: Principles and
Practices of Oncology, 3rd ed., Devita et al., eds. pp. 301-547
(1989), Balmer DICP, Ann Pharmacother 24, 761-768 (1990)).
[0117] BETASERON.TM. (Schering Corp's recombinant interferon
beta-1b) was the first drug indicated specifically for the
treatment of MS. In a major clinical trial, BETASERON.TM. was found
to be effective in reducing the number and severity of
exacerbations, or relapses, suffered by MS patients, as well as
decreasing magnetic resonance imaging (MRI) evidence of MS activity
in the brain. Importantly, the results of the trial pertained only
to the relapsing-remitting patient group, since other forms of MS
were not represented in the trial. Moreover, the trial demonstrated
no beneficial effect of the drug on ultimate disability of MS over
the 2 to 3 years of the study, and the effectiveness of the drug is
significantly impaired by its side effects. U.S. Pat. Nos.
7,105,154; 5,372,808; 5,846,526; 6,204,022; 6,060,450; and
6,361,769 also describe the use of IFN therapy for treating
autoimmune diseases and cancer; each of these publications is
incorporated herein by reference). U.S. Pat. No. 7,442,380
describes the treatment of autoimmune diseases caused by viral
infection using interferons.
[0118] Thus, the compositions of the invention (e.g., an
Ad5-IFN.alpha.) can be administered to a subject (e.g., a human) to
treat or reduce one or more symptoms of autoimmune disease (e.g.,
multiple sclerosis, type I diabetes, lupus, Addison's disease,
myasthenia gravis, and amyotrophic lateral sclerosis) or cancer in
the subject. Examples of the symptoms of autoimmune disease that
can be treated or reduced using the compositions of the invention
include, e.g., increased levels of autoantibodies, increased levels
of autoreactive T cells, loss of targeted cells (e.g., pancreatic
.beta.-islet cells), fatigue, depression, sensitivity to cold,
weight gain, muscle weakness, constipation, insomnia, irritability,
weight loss, bulging eyes, muscle tremors, skin rashes, painful or
swollen joints, sensitivity to the sun, loss of coordination, and
paralysis. These symptoms, and their resolution during treatment,
may be measured by, e.g., a physician during a physical examination
or by other tests and methods known in the art.
[0119] The dose of the compositions of the invention (e.g., the
number of IFN-encoding delivery vectors, viral or otherwise) or the
number of treatments using the compositions of the invention may be
increased or decreased based on the severity of, occurrence of, or
progression of, the disease or symptoms in the patient.
Additional Therapeutic Regimens
[0120] If desired, the subject may also receive additional
therapeutic regimens. For example, an additional therapeutic agent
may be admixed into a single formulation together with the
pharmaceutical compositions described herein at concentrations
known to be effective for such therapeutic agents. Additional
therapeutic agents may also be delivered separately. When agents
are present in different pharmaceutical compositions, different
routes of administration may be employed. Particularly useful
therapeutic agents include, e.g., antiviral agents,
immunostimulatory agents, and other immunization vaccines. When
treating cancer with the compositions of the invention,
particularly useful additional therapeutic agents include
chemotherapeutic agents, such as, e.g., camptothecin,
homocamptothecin, colchicine, thiocolchicine, combretastatin,
dolastatin, doxorubicin, methotrexate, podophyllotoxin, rhizoxin,
rhizoxin D, a taxol, paclitaxel, CC1065, and a maytansinoid.
[0121] In some instances, the pharmaceutical composition and
additional therapeutic agents are administered at least one hour,
two hours, four hours, six hours, 10 hours, 12 hours, 18 hours, 24
hours, three days, seven days, fourteen days, or one month apart.
The dosage and frequency of administration of each component can be
controlled independently. The additional therapeutic agents
described herein may be admixed with additional active or inert
ingredients, e.g., in conventional pharmaceutically acceptable
carriers. A pharmaceutical carrier can be any compatible, non-toxic
substance suitable for the administration of the compositions of
the invention to a subject. Pharmaceutically acceptable carriers
include, for example, water, saline, buffers and other compounds,
described, for example, in the Merck Index, Merck & Co.,
Rahway, N.J. A slow release formulation or a slow release apparatus
may be also be used for continuous administration. The additional
therapeutic regimen may involve other therapies, including
modification to the lifestyle of the subject being treated.
[0122] Antiviral Agents
[0123] Antiviral agents may be used as an additional therapeutic
agent, either in combination with the vaccine or in a separate
administration. Exemplary antiviral agents are abacavir, aciclovir,
acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir,
atripla, brivudine, cidofovir, combivir, darunavir, delavirdine,
didanosine, docosanol, edoxudine, efavirenz, emtricitabine,
enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed dose
combinations, fomivirsen, fosamprenavir, foscarnet, fosfonet,
fusion inhibitors, ganciclovir, gardasil, ibacitabine, immunovir,
idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors,
interferon type III, interferon type II, interferon type I,
interferon, lamivudine, lopinavir, loviride, MK-0518, maraviroc,
moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues,
oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin,
protease inhibitors, reverse transcriptase inhibitors, ribavirin,
rimantadine, ritonavir, saquinavir, stavudine, synergistic
enhancers, tenofovir, tenofovir disoproxil, tipranavir,
trifluridine, trizivir, tromantadine, truvada, valaciclovir,
valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,
zanamivir, and zidovudine. Exemplary antiviral agents are listed
in, e.g., U.S. Pat. Nos. 6,093,550 and 6,894,033, hereby
incorporated by reference.
[0124] Anti-Bacterial Agents
[0125] The compositions of the invention (e.g., Ad5-IFN.alpha.) can
be administered with an anti-bacterial agent, such as an
antibiotic, e.g., one or more penicillins, cephalosporins,
aminoglycosides, macrolides, sulfa compounds, fluoroquinolones, or
tetracyclines. Other examples of anti-bacterial agents include
penicillin G, penicillin V, methicillin, nafcillin, oxacillin,
cloxacillin, dicloxacillion, ampicillin, amoxicillin,
bacampicillin, cyclacillin, carbenicillin indanyl, ticarcillin,
mezlocillin, piperacillin, cephalothin, cefazolin, cephapirin,
cephradine, cephalexin, cefadroxil, cefamandole nafate, cefuroxime,
cefonicid, ceforanide, cefaclor, cefoxitin, cefotetan, cefmetazole,
cefataxime, ceftizoxime, ceftriaxone, ceftazidime, cefoperazone,
moxalactam, cefixime, erythromycin, stearate, ethylsuccinate,
estolate, lactobionate, gluceptate, azithromycin, clarithromycin
oxytetracycline, demeclocycline, doxycycline, minocycline, amikacin
sulfate, gentamicin sulfate, intrathecal, kanamycin sulfate,
netilmicin sulfate, streptomycin sulfate, tobramycin sulfate,
neomycin sulfate, sulfadiazine, sulfamethizole, sulfisoxazole,
sulfisoxazole acetyl, sulfamethoxazole, trisulfapyrimidines,
phenazopyridine, erythromycin ethylsuccinate, Trimethoprim,
Ciprofloxacin, Ciprofloxacin hydrochloride, enoxacin, Lomefloxacin
hydrochloride, Norfloxacin, Ofloxacin, vancomycin hydrochloride,
teicoplanin, rifampin, metronidazole, metronidazole hydrochloride,
polmyxins, bacitracin, methenamine, methenamine hippurate,
methenamine mandelate, nitrofurantoin, phenazopyridine
hydrochloride, silver nitrate, acetic acid, Domeboro solution,
m-cresyl acetate, Colymycin S otic, cortisporin, tridesilon,
ciclopiroxolamine, clioquinol, griseofulvin, fulvicin, grisactin,
grisactin ultra, grifulvin V, halaprogin, pyrithione zinc, selenium
sulfide, tolnaftate, undecylenic acid, naftfine, terbinafind,
imidazole, econazole, ketoconazole, miconaxole nitrate,
Monistat-Derm, oxiconazole nitrate, sulconazole nitrate,
bis-triazoles, intraconazole, amphotericin B, nystatin,
mycolstatin, nilstat, butoconazole, clotrimazole, tioconazold,
fluconazole, intraconazole, terconazole, nystatin, mycostatin, O-V
Statin, cantharidin, intralesional, podophyllin resin, podofilox,
salicylic acid, benzylbenzoate, crotamiton, lindane, malathion,
permethrin, phrethrins, piperonyl butoxide, sulfur, isoniazid,
pyrazinamide, ethambutol, capreomycin sulfate, cycloserine,
ethambutol hydrochloride, ethionamide, clofazimine, dapsone,
ethionamide, itraconazole, potassium iodide flucytosine,
chloroquine phosphate, hydroxychloroquine phosphate, chloroquine
hydrochloride, quinine sulfate, pyrimethamine/sulfadoxine,
mefloquine, quinidine gluconate, dilozanide furoate, eflornithine
hydrochloride, furazolidone, iodoquinol, melarsoprol,
metronidazole, nifurtimox, paramomycin sulfate, pentamidine
isethionate, primaquine phosphate, quinine sulfate, sodium
stibogluconate, meglumine antimoniate, trimetrexate glucuronate,
pyrimethamine, albendazole, diethycicarbamazine citrate,
ivermectin, mebendazole, metrifonate, niclosamide, oxamniquine,
pyrantel pamoate, suramin sodium, thiabendazole, cytarabine,
idoxuridine, trifluridine, vidarabine, acyclovir, Zidovudine,
ribavirin, bromovinyldeoxyuridine, fluoroiodoaracytosine,
amantadine, acemannan, amphotericin B methyl, Ampligen,
castanospermine, soluble CD.sub.4, dextran sulfate,
dideoxycytidine, dideoxyinosine, didihydrodideoxythymidine,
foscarnet sodium, fusidic acid, HPA-23, isoprinosine,
penicillamine, peptide T, ribavirin, rifabutin, didanosine,
zalcitabine, and the like.
[0126] Immunostimulatory Agents
[0127] Immunogenicity of the pharmaceutical compositions of the
invention may be significantly improved if the compositions of the
present invention (e.g., Ad5-IFN.alpha.) are co-administered with
an immunostimulatory agent or adjuvant. Exemplary immunostimulatory
agents include aluminum phosphate, aluminum hydroxide, QS21, Quil A
(and derivatives and components thereof), calcium phosphate,
calcium hydroxide, zinc hydroxide, glycolipid analogs, octodecyl
esters of an amino acid, muramyl dipeptides, polyphosphazene,
lipoproteins, ISCOM matrix, DC-Chol, DDA, cytokines, and other
adjuvants and derivatives thereof.
[0128] Immunization Vaccines
[0129] In some instances, it may be desirable to combine the
compositions of the present invention with compositions that induce
protective responses against other viruses. For example, the
compositions of the present invention (e.g., Ad5-IFN.alpha.) can be
administered simultaneously, separately, or sequentially with an
immunization vaccine, such as a vaccine for, e.g., influenza,
malaria, tuberculosis, smallpox, measles, rubella, mumps, or any
other vaccines known in the art.
[0130] For example, the vaccine can be, e.g., a bacterial, viral,
fungal, or parasite vaccine known in the art for treating a
bacterial, viral, fungal, or parasitic agent, respectively. The
vaccine may be directed against a bacterium selected from
Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli,
Klebsiella pneumoniae, Bruscella, Burkholderia mallei, Yersinia
pestis, and Bacillus anthracis; a virus selected from a member of
the Flaviviridae family (e.g., a member of the Flavivirus,
Pestivirus, and Hepacivirus genera), which includes the hepatitis C
virus, Yellow fever virus; Tick-borne viruses, such as the Gadgets
Gully virus, Kadam virus, Kyasanur Forest disease virus, Langat
virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm
virus, Karshi virus, tick-borne encephalitis virus, Neudoerfl
virus, Sofjin virus, Louping ill virus and the Negishi virus;
seabird tick-borne viruses, such as the Meaban virus, Saumarez Reef
virus, and the Tyuleniy virus; mosquito-borne viruses, such as the
Aroa virus, dengue virus, Kedougou virus, Cacipacore virus,
Koutango virus, Japanese encephalitis virus, Murray Valley
encephalitis virus, St. Louis encephalitis virus, Usutu virus, West
Nile virus, Yaounde virus, Kokobera virus, Bagaza virus, Ilheus
virus, Israel turkey meningoencephalo-myelitis virus, Ntaya virus,
Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edge Hill
virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus,
Wesselsbron virus, yellow fever virus; and viruses with no known
arthropod vector, such as the Entebbe bat virus, Yokose virus, Apoi
virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja
virus, San Perlita virus, Bukalasa bat virus, Carey Island virus,
Dakar bat virus, Montana myotis leukoencephalitis virus, Phnom Penh
bat virus, Rio Bravo virus, Tamana bat virus, and the Cell fusing
agent virus; a virus selected from a member of the Arenaviridae
family, which includes the Ippy virus, Lassa virus (e.g., the
Josiah, LP, or GA391 strain), lymphocytic choriomeningitis virus
(LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal virus,
Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliveros
virus, Parana virus, Pichinde virus, Pirital virus, Sabia virus,
Tacaribe virus, Tamiami virus, Whitewater Arroyo virus, Chapare
virus, and Lujo virus; a virus selected from a member of the
Bunyaviridae family (e.g., a member of the Hantavirus, Nairovirus,
Orthobunyavirus, and Phlebovirus genera), which includes the
Hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus,
Rift Valley fever virus, La Crosse virus, Punta Toro virus (PTV),
California encephalitis virus, and Crimean-Congo hemorrhagic fever
(CCHF) virus; a virus selected from a member of the Filoviridae
family, which includes the Ebola virus (e.g., the Zaire, Sudan,
Ivory Coast, Reston, and Uganda strains) and the Marburg virus
(e.g., the Angola, Ci67, Musoke, Popp, Ravn and Lake Victoria
strains); a member of the Togaviridae family (e.g., a member of the
Alphavirus genus), which includes the Venezuelan equine
encephalitis virus (VEE), Eastern equine encephalitis virus (EEE),
Western equine encephalitis virus (WEE), Sindbis virus, rubella
virus, Semliki Forest virus, Ross River virus, Barmah Forest virus,
O'nyong'nyong virus, and the chikungunya virus; a member of the
Poxyiridae family (e.g., a member of the Orthopoxvirus genus),
which includes the smallpox virus, monkeypox virus, and vaccinia
virus; a member of the Herpesviridae family, which includes the
herpes simplex virus (HSV; types 1, 2, and 6), human herpes virus
(e.g., types 7 and 8), cytomegalovirus (CMV), Epstein-Barr virus
(EBV), Varicella-Zoster virus, and Kaposi's sarcoma
associated-herpesvirus (KSHV); a member of the Orthomyxoviridae
family, which includes the influenza virus (A, B, and C), such as
the H5N1 avian influenza virus or H1N1 swine flu; a member of the
Coronaviridae family, which includes the severe acute respiratory
syndrome (SARS) virus; a member of the Rhabdoviridae family, which
includes the rabies virus and vesicular stomatitis virus (VSV); a
member of the Paramyxoviridae family, which includes the human
respiratory syncytial virus (RSV), Newcastle disease virus,
hendravirus, nipahvirus, measles virus, rinderpest virus, canine
distemper virus, Sendai virus, human parainfluenza virus (e.g., 1,
2, 3, and 4), rhinovirus, and mumps virus; a member of the
Picornaviridae family, which includes the poliovirus, human
enterovirus (A, B, C, and D), hepatitis A virus, and the
coxsackievirus; a member of the Hepadnaviridae family, which
includes the hepatitis B virus; a member of the Papillamoviridae
family, which includes the human papilloma virus; a member of the
Parvoviridae family, which includes the adeno-associated virus; a
member of the Astroviridae family, which includes the astrovirus; a
member of the Polyomaviridae family, which includes the JC virus,
BK virus, and SV40 virus; a member of the Calciviridae family,
which includes the Norwalk virus; a member of the Reoviridae
family, which includes the rotavirus; and a member of the
Retroviridae family, which includes the human immunodeficiency
virus (HIV; e.g., types 1 and 2), and human T-lymphotropic virus
Types I and II (HTLV-1 and HTLV-2, respectively); or a fungus
selected from Aspergillus, Blastomyces dermatitidis, Candida,
Coccidioides immitis, Cryptococcus neoformans, Histoplasma
capsulatum var. capsulatum, Paracoccidioides brasiliensis,
Sporothrix schenckii, Zygomycetes spp., Absidia corymbifera,
Rhizomucor pusillus, and Rhizopus arrhizus; or parasite selected
from Toxoplasma gondii, Plasmodium falciparum, P. vivax, P. ovale,
P. malariae, Trypanosoma spp., and Legionella spp.
[0131] Examples of vaccines known in the art that can be
administered in combination with the compositions of the present
invention (e.g., the Ad5-IFN.alpha. constructs described herein)
include AVA (BioThrax) for anthrax; VAR (Varivax) and MMRV
(ProQuad) for chickenpox; DTaP (Daptacel, Infanrix, Tripedia), Td
(Decavaca, generic), DT (-generic-), Tdap (Boostrix, Adacel),
DTaP-IPV (Kinrix), DTaP-HepB-IPV (Pediarix), DTaP-IPV/Hib
(Pentacel), and DTaP/Hib (TriHIBit) for Diphtheria; HepA (Havrix,
Vaqta) and HepA-HepB (Twinrix) for Hepatitis A; HepB (Engerix-B,
Recombivax HB), Hib-HepB (Comvax), DTaP-HepB-IPV (Pediarix), and
HepA-HepB (Twinrix) for Hepatitis B; Hib (ActHIB, PedvaxHlB,
Hiberix), Hib-HepB (Comvax), DTaP/Hib (TriHIBit), and DTaP-IPV/Hib
(Pentacel) for Haemophilus influenzae type b; HPV4 (Gardasil) and
HPV2 (Cervarix) for Human Papillomavirus (HPV); TIV (Afluria,
Agriflu, FluLaval, Fluarix, Fluvirin, Fluzone) and LAIV (FluMist)
for Influenza; JE (Ixiaro and JE-Vax) for Japanese encephalitis
(JE); MMR (M-M-R II) and MMRV (ProQuad) for Measles; MCV4
(Menactra), MPSV4 (Menomune), and MODC (Menveo) for Meningitis; MMR
(M-M-R II) and MMRV (ProQuad) for Mumps; DTaP (Daptacel, Infanrix,
Tripedia), Tdap (Adacel, Boostrix), DTaP-IPV (Kinrix),
DTaP-HepB-IPV (Pediarix), DTaP-IPV/Hib (Pentacel), and DTaP/Hib
(TriHIBit) for Pertussis; PCV7 (Prevnar), PCV13 (Prevnar13), and
PPSV23 (Pneumovax 23) for Bacterial Pneumonia; Polio (Ipol),
DTaP-IPV (Kinrix), DTaP-HepB-IPV (Pediarix), and DTaP-IPV/Hib
(Pentacel) for Polio; Rabies (Imovax Rabies and RabAvert); RV1
(Rotarix) and RV5 (RotaTeq) for Rotavirus; MMR (M-M-R II) and MMRV
(ProQuad) for Rubella; ZOS (Zostavax) for Shingles; Vaccinia
(ACAM2000, Dryvax) for Smallpox and Monkeypox; DTaP (Daptacel,
Infanrix, Tripedia), Td (Decavac, generic), DT (-generic-), TT
(-generic-), Tdap (Boostrix, Adacel), DTaP-IPV (Kinrix),
DTaP-HepB-IPV (Pediarix), DTaP-IPV/Hib (Pentacel), and DTaP/Hib
(TriHIBit) for Tetanus; BCG (TICE BCG, Mycobax) for Tuberculosis
(TB); Typhoid Oral (Vivotif) and Typhoid Polysaccharide (Typhim Vi)
for Typhoid; and YF (YF-Vax) for Yellow Fever.
[0132] Ebola Vaccine
[0133] Ad-CAGoptZGP is a vaccine that uses an Adenovirus 5 backbone
and encodes the surface proteins of the Ebola virus (see Richardson
et al. (PLoS 4:e5308, 2009)). Earlier versions of this vaccine have
been previously shown to protect mice, guinea pigs and nonhuman
primates from an otherwise lethal challenge of Zaire Ebola virus.
Ad-CAGoptZGP incorporates three improvements: codon optimization of
the gene insert, inclusion of a consensus Kozak sequence, and
reconfiguration of a CAG promoter. Transfection or transduction of
cells with Ad-CAGoptZGP results in high expression of the Ebola
glycoprotein from those cells, and allows for a functional dose
.about.100 times lower than with other adenovirus-based Ebola
vaccine constructs and with a faster time to immunity. Finally,
Ad-CAGoptZGP is capable of inducing full protection to mice
(partial protection to guinea pigs) when given 30 minutes
post-challenge, whereas previous vaccines were not functional
post-exposure. The strength of this vaccine is its lasting
immunity.
[0134] In an embodiment, a pharmaceutical composition of the
invention (e.g., the Ad5-IFN.alpha. constructs described herein)
can be administered simultaneously, separately, or sequentially
with the Ad-CAGoptZGP Ebola vaccine. Preferably, one or both of the
agents are formulated for intranasal or pulmonary administration.
Our experimental data shows significant synergy when, e.g.,
Ad5-IFN.alpha. and Ad-CAGoptZGP are combined (whether administered
in a single composition or in separate compositions; see, e.g.,
Example 14 herein). Specifically, complete treatment efficacy is
seen 30 min post-exposure with ZEBOV with no reduction in body
weight in both mouse and Guinea pig models. We expect to gain the
benefits of both rapid onset (3 hours) of Ad5-IFN.alpha. and long
lasting protection of Ad-CAGoptZGP in order to maximize the
protective benefit of both components, as is seen in Table 1. The
combination of an immune stimulator and Ebola vaccine contributes
to a highly effective, focused therapy, and a broad spectrum
antiviral makes this combination a superior treatment option.
TABLE-US-00001 TABLE 1 Summary of capabilities of Ad5-IFN.alpha.,
Ad-CAGoptZGP Ebola vaccine, and their combination as a prophylactic
for Ebola viruses Combination Fast acting AND long lasting immunity
Prophylactic Excellent efficacy pre-and post- exposure Needle-free
Cost effective manufacturing Ad-CAGoptZGP Long lasting immunity
Some efficacy post-exposure Needle-free Simple cost-effective
manufacturing Ad5-IFN.alpha. Rapid onset (3 hours) Broad spectrum
protection Needle-free Simple cost-effective manufacturing Efficacy
pre-and post-exposure Known and acceptable safety profiles of all
components
[0135] The combination of Ad5-IFN.alpha. and Ad-CAGoptZGP also
provides for rapid onset of therapeutic and prophylactic effects
and sustained protection against reinfection. The combination of
Ad5-IFN.alpha. and Ad-CAGoptZGP (either separately or in
combination) promotes direct stimulation of the innate immune
system within 1-10 hours (e.g., within 3 hours), which acts to
counter, e.g., viral hemorrhagic fever viruses present within the
recipient. Rapid onset to protection is one of the many benefits of
the combination therapy. The combination of Ad5-IFN.alpha. and
Ad-CAGoptZGP is also quickly fully functional with a single dose,
although multiple doses (e.g., 2, 3, 4, or 5 doses) of one or both
of the agents can be administered, as needed.
[0136] Expeditionary & Shelf Stable
[0137] To minimize logistical constraints, the combination of
Ad5-IFN.alpha. and Ad-CAGoptZGP can be formulated to be shelf
stable and expeditionarily rugged. Formulations described herein
allow for deployment of the agent(s) at >35.degree. C., if
necessary, for greater than, e.g., 30-90 days (e.g., at least 60
days) and for short periods of between 30 minutes and 5 hours
(e.g., at least 1 hour) at temperatures as high as 90.degree.
C.
Filovirus Efficacy Data
[0138] Ad5-IFN.alpha. and Ad-CAGoptZGP each have been tested
separately and in combination in well characterized animal models
of Filovirus infection (Zaire Ebola; ZEBOV). Mouse studies showed
that dosing with a range of 10.sup.4 to 10.sup.6 plaque forming
units (PFU) of Ad-CAGoptZGP was fully protective, and 10.sup.7 PFU
of Ad5-IFN.alpha. treated or pre-treated mice, resulting in
complete survival and negligible weight loss.
[0139] Similar results were obtained from a guinea pig model of
fatal ZEBOV infection in which intranasaldelivery of
2.times.10.sup.8 PFU mAd5-IFN.alpha. resulted in 100% survival and
slight weight loss for those treated compared to 100% fatal for
those untreated animals. 10.sup.10 PFU Ad-CAGoptZGP resulted in 33%
survival while the combination of Ad5-IFN.alpha. and Ad-CAGoptZGP
resulted in 100% survival with no weight loss. These results are
particularly impressive given the susceptibility of Guinea pigs to
ZEBOV. In this study the efficacy of daily injections of
recombinant IFN.alpha. protein was also assessed, and it was noted
that some survival benefit was observed (FIG. 10B).
Formulation and Administration of the Pharmaceutical Compositions
of the Invention
[0140] The compositions utilized in the methods described herein
can be formulated for administration by a route selected from,
e.g., parenteral, dermal, transdermal, ocular, inhalation, buccal,
sublingual, perilingual, nasal, rectal, topical administration, and
oral administration. Administration may be by, e.g., intranasal
release. Parenteral administration includes intravenous,
intraperitoneal, subcutaneous, and intramuscular administration.
Parenteral, intranasal or intraocular administration may be
provided by using, e.g., aqueous suspensions, isotonic saline
solutions, sterile and injectable solutions containing
pharmacologically compatible dispersants and/or solubilizers, for
example, propylene glycol or polyethylene glycol, lyophilized
powder formulations, and gel formulations. The preferred method of
administration can vary depending on various factors (e.g., the
components of the composition being administered and the severity
of the condition being treated). Formulations suitable for oral or
nasal administration may consist of liquid solutions, such as an
effective amount of the composition dissolved in a diluent (e.g.,
water, saline, or PEG-400), capsules, sachets, tablets, or gels,
each containing a predetermined amount of the IFN delivery vehicle
composition of the invention. The pharmaceutical composition may
also be an aerosol formulation for inhalation, e.g., to the
bronchial passageways. Aerosol formulations may be mixed with
pressurized, pharmaceutically acceptable propellants (e.g.,
dichlorodifluoromethane, propane, or nitrogen). In particular,
administration by inhalation can be accomplished by using, e.g., an
aerosol containing sorbitan trioleate or oleic acid, for example,
together with trichlorofluoromethane, dichlorofluoromethane,
dichlorotetrafluoroethane, or any other biologically compatible
propellant gas.
[0141] Immunogenicity of the composition of the invention may be
significantly improved if it is co-administered with an
immunostimulatory agent or adjuvant. Suitable adjuvants well-known
to those skilled in the art include, e.g., aluminum phosphate,
aluminum hydroxide, QS21, Quil A (and derivatives and components
thereof), calcium phosphate, calcium hydroxide, zinc hydroxide,
glycolipid analogs, octodecyl esters of an amino acid, muramyl
dipeptides, polyphosphazene, lipoproteins, ISCOM matrix, DC-Chol,
DDA, cytokines, and other adjuvants and derivatives thereof.
[0142] In some instances, it may be desirable to combine the
compositions of the invention with compositions that induce
protective responses against other viruses. For example, the
compositions of the present invention can be administered
simultaneously, separately, or sequentially with other immunization
vaccines, such as those for, e.g., influenza, malaria,
tuberculosis, or any other vaccines known in the art.
[0143] Pharmaceutical compositions according to the invention
described herein may be formulated to release the composition
immediately upon administration (e.g., targeted delivery) or at any
predetermined time period after administration using controlled or
extended release formulations. Administration of the pharmaceutical
composition in controlled or extended release formulations is
useful where the composition, either alone or in combination, has
(i) a narrow therapeutic index (e.g., the difference between the
plasma concentration leading to harmful side effects or toxic
reactions and the plasma concentration leading to a therapeutic
effect is small; generally, the therapeutic index, TI, is defined
as the ratio of median lethal dose (LD.sub.50) to median effective
dose (ED.sub.50)); (ii) a narrow absorption window at the site of
release (e.g., the gastro-intestinal tract); or (iii) a short
biological half-life, so that frequent dosing during a day is
required in order to sustain a therapeutic level.
[0144] Many strategies can be pursued to obtain controlled or
extended release in which the rate of release outweighs the rate of
metabolism of the pharmaceutical composition. For example,
controlled release can be obtained by the appropriate selection of
formulation parameters and ingredients, including, e.g.,
appropriate controlled release compositions and coatings. Suitable
formulations are known to those of skill in the art. Examples
include single or multiple unit tablet or capsule compositions, oil
solutions, suspensions, emulsions, microcapsules, microspheres,
nanoparticles, patches, and liposomes.
[0145] The compositions of the invention may be administered to
provide pre-exposure prophylaxis or after a subject has been
exposed to a pathogen, such as a virus. The composition may be
administered, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35,
40, 45, 50, 55, or 60 minutes, 2, 4, 6, 10, 15, or 24 hours, 2, 3,
5, or 7 days, 2, 4, 6 or 8 weeks, or even 3, 4, or 6 months
pre-exposure, or may be administered to the subject 15-30 minutes
or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 20, 24, 48, 72 hours, or
longer post-exposure to the pathogen (e.g., a viral pathogen).
[0146] When treating autoimmune disease or cancer, the compositions
of the invention may be administered to the subject either before
the occurrence of symptoms or a definitive diagnosis or after
diagnosis or symptoms become evident. For example, the composition
may be administered, e.g., immediately after diagnosis or the
clinical recognition of symptoms or 2, 4, 6, 10, 15, or 24 hours,
2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, or even 3, 4, or 6 months
after diagnosis or detection of symptoms.
[0147] The compositions may be sterilized by conventional
sterilization techniques, or may be sterile filtered. The resulting
aqueous solutions may be packaged for use as is, or lyophilized,
the lyophilized preparation may be administered in powder form or
combined with a sterile aqueous carrier prior to administration.
The pH of the preparations typically will be between 3 and 11, more
preferably between 5 and 9 or between 6 and 8, and most preferably
between 7 and 8, such as 7 to 7.5. The resulting compositions in
solid form may be packaged in multiple single dose units, each
containing a fixed amount of the IFN delivery vector (e.g., an Ad5
conIFN-.alpha. delivery vector) and, if desired, one or more
immunomodulatory agents, such as in a sealed package of tablets or
capsules, or in a suitable dry powder inhaler (DPI) capable of
administering one or more doses.
[0148] Nasal or Pulmonary Delivery
[0149] There are several benefits of intranasal or pulmonary
administration over, e.g., oral, intravascular, or intramuscular
administration. In particular, an intranasal or pulmonary
administration route is less harsh for an adenoviral vector system.
There are fewer proteolytic enzymes present in, e.g., the nasal
epithelium and the environment has a more neutral pH (i.e., it is
less acidic). Also, the uptake of particles of the viral delivery
vector would be more consistent in the nasal or pulmonary mucosa
than in the gut where there would be more variation in the content
of the intestinal lumen, and thus greater variability in the
ability of the vector to transduce/transfect cells in that
environment. Moreover, the nasal mucosa is well irrigated, and is
thus a permeable mucosal site.
[0150] Thus, in an embodiment, the IFN-.alpha. delivery vector of
the invention, and pharmaceutical compositions containing it, are
delivered via an intranasal or pulmonary route in, e.g.,
lyophilized powder form, in an aerosolized liquid form, or in a gel
form. These routes of administration avoid recognition of, e.g.,
the Ad5 vector by the host immune system, thereby bypassing any
pre-existing immunity the host may have. In addition, intranasal
and pulmonary delivery allow for easy administration in the event
of the need for mass distribution.
[0151] Pulmonary and/or intranasal administration of the
compositions of the invention includes, e.g., providing a mist
(aqueous or fine powder) to the lungs (upper and/or lower
respiratory tract) or nasal epithelium, respectively. This form of
administration has a number of benefits over conventional
needle-based injections. First, it does not involve the use of a
needle, which means better patient compliance because it is
"pain-free." Second, pulmonary and intranasal administration allows
for self-administration, which saves physicians' time, makes
instrumentation unnecessary, and eliminates apprehension for the
patient. Third, the use of sugar- or salt-based placebo powders or
solutions facilitates training for administration without pain.
Fourth, there is no risk of medical problems caused by, e.g.,
needle-borne contamination by bacteria/viruses or other problems
from an unclean injection site. Fifth, the distribution of the
aerosol or powder results in a thorough and more even application
of the vaccine. Sixth, the particle size of the vaccine can be
controlled so that effective deposition at, e.g., the upper and/or
lower respiratory tract, takes place based on the characteristics
of the administration device. Furthermore, needle-based
administrations typically require a trained medical professional to
insure that the injected medication is correctly delivered to the
right compartment of the body (i.e., intravenous versus
intramuscular). The preparation of aerosolized adenoviral vectors
is described in, e.g., U.S. Pat. No. 7,097,827, which is
incorporated by reference herein.
[0152] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise the vector (e.g., the
Ad5-conIFN-.alpha. vector) in an aqueous medium at a concentration
of, e.g., about 0.01 to 25 mg of vector per mL of solution,
preferably about 0.1 to 10 mg/mL. The formulation may also include
a buffer and a simple sugar (e.g., for protein stabilization and
regulation of osmotic pressure), and/or human serum albumin ranging
in concentration from 0.1 to 10 mg/ml. Examples of buffers that may
be used are sodium acetate, citrate and glycine. Preferably, the
buffer will have a composition and molarity suitable to adjust the
solution to a pH in the range of 3 to 9. Generally, buffer
molarities of from 1 mM to 50 mM are suitable for this purpose.
Examples of excipients, usually in amounts ranging from 1% to 90%
by weight (e.g., 1% to 50% by weight, more preferably 5% to 30% by
weight) of the formulation include, e.g., monosaccharides such as
fructose, maltose, galactose, glucose, D-mannose, sorbose, and the
like; disaccharides, such as lactose, sucrose, trehalose,
cellobiose, and the like; polysaccharides, such as raffinose,
melezitose, maltodextrins, dextrans, starches, and the like;
alditols, such as mannitol, xylitol, xylose, maltitol, lactitol,
xylitol sorbitol (glucitol), sorbitose, pyranosyl sorbitol,
myoinositol and the like; and glycine, CaCl.sub.2, hydroxyectoine,
ectoine, gelatin, di-myo-inositol phosphate (DIP), cyclic 2,3
diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate (DGP),
.beta.-mannosylglycerate (firoin), .beta.-mannosylglyceramide
(firoin A), proline betaine and/or derivatives as well as
combinations thereof.
[0153] The nebulizer formulation may also contain a surfactant to
reduce or prevent surface induced aggregation of the composition
components caused by atomization of the solution in forming the
aerosol. Various conventional surfactants can be employed, such as
polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene
sorbitan fatty acid esters. Amounts will generally range between
0.001% and 4% by weight of the formulation. An especially preferred
surfactant for purposes of this invention is polyoxyethylene
sorbitan monooleate.
[0154] Specific formulations and methods of generating suitable
dispersions of liquid particles of the invention are described in,
e.g., WO 94/20069, U.S. Pat. No. 5,915,378, U.S. Pat. No.
5,960,792, U.S. Pat. No. 5,957,124, U.S. Pat. No. 5,934,272, U.S.
Pat. No. 5,915,378, U.S. Pat. No. 5,855,564, U.S. Pat. No.
5,826,570, and U.S. Pat. No. 5,522,385, each of which is hereby
incorporated by reference.
[0155] The compositions of the invention (e.g., an adenoviral
vector that includes a nucleic acid molecule encoding an interferon
(e.g., Ad5-conIFN-.alpha.)) are preferentially administered
intranasally. The Ad5 virus is highly efficient in delivering genes
to the epithelial cells of the nasal membranes. Mucosal dosing is
efficient because it stimulates both the systemic and mucosal
immunity at the portal of entry (see, e.g., Gutierro et al.,
Vaccine 20:2181-2190, 2002; and Patel et al., J. Infect. Dis.
196:S413-420, 2007). In addition, utilizing live Ad5 virus to
deliver the IFN provides an additional route of immune stimulation,
thereby acting as an adjuvant in ensuring maximum effect is
achieved. Thus, delivering the compositions of the invention to a
site where the infectious agent (e.g., a virus) enters will likely
result in a lower required dose. Specific instrumentation has been
developed to effectively deliver aerosol droplets (diameter>2
um) to this compartment (see, e.g., the Mucosal Atomization Device
(MAD300), Wolfe Tory Medical). Droplet (or powdered particle) size
is important as aerosols<1 um penetrate further down the
respiratory tract and can cause adverse effects.
[0156] The compositions of the invention can also be delivered in
powder form using, e.g., a metered dose inhaler device. This powder
may be produced by lyophilization and may also contain a stabilizer
such as human serum albumin (HSA). Typically, more than 0.5% (w/w)
HSA is added. Additionally, one or more of the following may be
added as an excipient to the preparation, if necessary, to enhance
one or more features (e.g., to facilitate dispersal of the powder
from a device, to increase the shelf-life of the vaccine
composition, or to improve the stability of the vaccine composition
during lyophilization): monosaccharides such as fructose, maltose,
galactose, glucose, D-mannose, sorbose, and the like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and
the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; alditols, such as
mannitol, xylitol, xylose, maltitol, lactitol, xylitol sorbitol
(glucitol), sorbitose, pyranosyl sorbitol, myoinositol and the
like; and glycine, CaCl.sub.2, hydroxyectoine, ectoine, gelatin,
di-myo-inositol phosphate (DIP), cyclic 2,3 diphosphoglycerate
(cDPG), 1,1-di-glycerol phosphate (DGP), .beta.-mannosylglycerate
(firoin), .beta.-mannosylglyceramide (firoin A), proline betaine
and/or derivatives as well as combinations thereof. The amount
added to the formulation can range from about 0.01 to 200% (w/w),
preferably from approximately 1 to 50% (w/w), and more preferably
from about 5 to 30% (w/w) of the vector present. Such formulations
are then lyophilized and milled to the desired particle size. The
particles of the powder shall have aerodynamic properties in the
nasal cavities and lung corresponding to particles with a density
of about 1 g/cm.sup.2 having a median diameter less than 50 um,
preferably between 1.5 and 10 um, more preferably of between 1.8
and 7.0 um, and most preferably from about 2.0 to 4 um. The mean
particle diameter can be measured using conventional equipment,
such as a Cascade Impactor (Andersen, Ga.).
[0157] The dry powder formulations of the present invention may
conveniently be formulated by first suspending the vector (e.g., an
adenoviral vector that includes a nucleic acid molecule encoding an
interferon (e.g., Ad5-conIFN-.alpha.) or other nucleic acid
construct of the invention) in an aqueous solution. The relative
amounts of vector and any added excipient material will depend on
the desired final ratio of vector to excipient. Conveniently, the
ratio of vector to excipient will be in the range from about 2:1 to
1:100 (vector:excipient), preferably from 1:1 to 1:10, with a total
solids concentration in the aqueous suspension being usually less
than 5% by weight, more usually being less than 3% by weight.
[0158] The powder may be suspended in a propellant with the aid of
a surfactant. The propellant may be any conventional material
employed for this purpose, such as a chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,
including trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or
combinations thereof. Suitable surfactants include sorbitan
trioleate and soya lecithin. Oleic acid may also be useful as a
surfactant. This mixture is then loaded into the delivery
device.
[0159] In the case of compositions of the invention that include
viral vectors, it is usually desirable that the aqueous solution be
buffered in order to enhance the activity of the viral vectors
after drying. Buffers or pH-adjusting agents typically include a
salt prepared from, e.g., an organic acid or base. Representative
buffers include organic acid salts of citric acid, ascorbic acid,
gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic
acid, or phthalic acid, Tris, tromethamine hydrochloride, or
phosphate buffers.
[0160] Additional polymeric excipients/additives that can be
included in the formulations of the compositions of the invention
include, e.g., polyvinylpyrrolidones, derivatized celluloses such
as hydroxymethylcellulose, hydroxyethylcellulose, and
hydroxypropylmethylcellulose, Ficolls (a polymeric sugar),
hydroxyethylstarch, dextrates (e.g., cyclodextrins, such as
2-hydroxypropyl-.beta.-cyclodextrin and
sulfobutylether-.beta.-cyclodextrin), polyethylene glycols, and
pectin.
[0161] The powder compositions of the invention for use in these
devices may be generated and/or delivered by methods disclosed in
WO 96/32149, WO 97/41833, and WO 98/29096, and in U.S. Pat. Nos.
7,482,024; 7,481,212; 7,371,373; 6,303,582; 6,001,336; 5,997,848;
5,993,783; 5,985,248; 5,976,574; 5,922,354; 5,785,049; and U.S.
Pat. No. 5,654,007, each of which is incorporated by reference
herein. The powder form can also be administered using, e.g., a
prefilled administration device, such as the devices described in,
e.g., U.S. Pat. Nos. 5,437,267; 6,068,199; 6,715,485; 5,994,314;
7,235,391; and 6,398,774, each of which is incorporated by
reference herein. The powders will generally have moisture contents
below about 20% by weight, usually below about 10% by weight, and
preferably below about 6% by weight. Such low moisture-containing
solids tend to exhibit a greater stability upon packaging and
storage.
[0162] Mechanical devices designed for pulmonary and/or nasal
delivery of the compositions of the invention include but are not
limited to nebulizers, metered dose inhalers, and powder inhalers,
all of which are familiar to those of skill in the art. Specific
examples of commercially available devices suitable for the
practice of this invention are the Ultravent nebulizer,
manufactured by Mallinckrodt, Inc., St. Louis, Mo., USA; the
Mucosal Atomization Device (e.g., MAD300), Wolfe Tory Medical; the
Acorn II nebulizer, manufactured by Marquest Medical Products,
Englewood, Colo., USA; the Ventolin metered dose inhaler,
manufactured by Glaxo Inc., Research Triangle Park, N.C., USA; the
OptiNose device, manufactured by OptiNose, Oslo, Norway; the
Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford,
Mass., USA the "standing cloud" device of Nektar Therapeutics,
Inc., San Carlos, Calif., USA; the AIR inhaler manufactured by
Alkermes, Cambridge, Mass., USA; and the AERx pulmonary drug
delivery system manufactured by Aradigm Corporation, Hayward,
Calif., USA. See also the delivery devices described in, e.g., U.S.
Pat. Nos. 5,522,378; 5,775,320; 5,934,272; and 5,960,792; the
OptiNose devices in U.S. Pat. Nos. 6,715,485; 7,347,201; and
7,481,218; and U.S. Patent Application Publication Nos.
2004/0112378; 2005/0072430; 2004/0112379; 2004/0149289;
2005/0028812; 2008/0163874; 2008/0161771; 2008/0223363;
2005/0235992; 2006/0096589; 2006/0169278; 2007/0039614; and
2007/0186927); and the device in U.S. Pat. No. 7,669,597.
[0163] The compositions of the invention can also be formulated as
intranasal carriers in the form of nasal gels, creams, pastes or
ointments that provide a more sustained contact with the nasal
mucosal surfaces. These formulations can have a viscosity of, e.g.,
from about 10 to about 250,000 centipoise (cps), or from about 2500
to 100,000 cps, or from about 5,000 to 50,000 cps or greater. Such
carrier viscous formulations may be based upon, simply by way of
example, alkylcelluloses and/or other biocompatible carriers of
high viscosity well known to the art (see e.g., Remington, cited
supra. A preferred alkylcellulose is, e.g., methylcellulose in a
concentration ranging from about 5 to about 1000 or more mg per 100
ml of carrier. A more preferred concentration of methyl cellulose
is, simply by way of example, from about 25 to about mg per 100 ml
of carrier. The carrier containing the IFN delivery vehicle of the
invention can also be, e.g., soaked into a fabric material, such as
gauze, that can be applied to the nasal mucosal surfaces to allow
for penetration of the delivery vehicles therein.
[0164] Examples of gel formulations that can be used to prepare
compositions of the invention are also described in, e.g., U.S.
Pat. Nos. 7,538,122; 7,387,788; 7,166,575; 6,413,539; and
6,004,583; each of which is incorporated herein by reference. The
gel formulations of the invention may also further include a
permeation enhancer (penetration enhancer). Permeation enhancers
include, but are not limited to, sulfoxides such as
dimethylsulfoxide and decylmethylsulfoxide; surfactants such as
sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium
bromide, benzalkonium chloride, poloxamer (231, 182, 184), tween
(20, 40, 60, 80) and lecithin; the 1-substituted
azacycloheptan-2-ones, particularly
1-n-dodecylcyclazacycloheptan-2-one; fatty alcohols such as lauryl
alcohol, myristyl alcohol, oleyl alcohol and the like; fatty acids
such as lauric acid, oleic acid and valeric acid; fatty acid esters
such as isopropyl myristate, isopropyl palmitate, methylpropionate,
and ethyl oleate; polyols and esters thereof such as propylene
glycol, ethylene glycol, glycerol, butanediol, polyethylene glycol,
and polyethylene glycol monolaurate, amides and other nitrogenous
compounds such as urea, dimethylacetamide (DMA), dimethylformamide
(DMF), 2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine,
diethanolamine and triethanolamine, terpenes; alkanones, and
organic acids, particularly salicylic acid and salicylates, citric
acid and succinic acid. The permeation enhancer may be present from
about 0.1 to about 30% w/w. Preferred permeation enhancers are
fatty alcohols and fatty acids. The gel compositions may also
include a buffering agent, for example, carbonate buffers, citrate
buffers, phosphate buffers, acetate buffers, hydrochloric acid,
lactic acid, tartaric acid, inorganic and organic bases. The
buffering agent may be present in a concentration of about 1 to
about 10 weight percent, more preferred is a concentration of about
2 to about 5 weight percent, depending on the type of buffering
agent(s) used, as known by the one skilled in the art.
Concentrations of the buffering agent(s) may vary, however, and the
buffering agent may replace up to 100% of the water amount within
the composition.
Dosage
[0165] The pharmaceutical compositions of the invention can be
administered in a therapeutically effective amount that provides an
immunogenic and/or protective effect against infection by a
pathogen, such as a virus. For example, when the compositions
include a viral vector (e.g., an Ad5-based vector) that encodes an
IFN (e.g., IFN-.alpha., such as conIFN-.alpha.), at least about
1.times.10.sup.3 viral particles (vp)/dose or between
1.times.10.sup.1 and 1.times.10.sup.14 vp/dose, preferably between
1.times.10.sup.3 and 1.times.10.sup.12 vp/dose, and more preferably
between 1.times.10.sup.5 and 1.times.1011 vp/dose (e.g.,
1.5-3.0.times.10.sup.8 vp/ml, of the viral vector provides a
therapeutically effective amount of the IFN following expression in
host cells. A single viral particle includes one or more nucleic
acid molecules (either DNA or RNA) encoding viral and non-viral
proteins (e.g., viral structural and non-structural proteins and
including a non-endogenous IFN) and surrounded by a protective coat
(e.g., a lipid-based envelope or a protein-based capsid) that
includes protein subunits. Viral particle number can be measured
based on, e.g., lysis of vector particles, followed by measurement
of the absorbance at 260 nm (see, e.g., Steel, Curr. Opin. Biotech.
10:295-297, 1999).
[0166] When the composition is a non-viral vector that includes a
nucleic acid molecule that encodes an IFN (e.g., IFN-.alpha., such
as conIFN-.alpha.), the subject should be administered at least
about 1.times.10.sup.1 molecules/dose, e.g., between
1.times.10.sup.1 and 1.times.10.sup.15 molecules/dose, preferably
between 1.times.10.sup.3 and 1.times.10.sup.10 molecules/dose, and
more preferably between 1.times.10.sup.4 and 1.times.10.sup.8
molecules/dose, of the non-viral delivery vector. A single nucleic
acid molecule of a non-viral vector includes one or more nucleic
acid molecules (e.g., DNA or RNA) in the form of, e.g., a plasmid,
cosmid, yeast or bacterial artificial chromosome, and bacteriaphage
that is administered in a naked form or that has been surrounded by
or complexed with a protective substance (e.g., lipids or a lipid
based envelope, peptides, and polymers).
[0167] The dosage administered depends on the subject to be treated
(e.g., the age, body weight, capacity of the immune system, and
general health of the subject being treated), the form of
administration (e.g., as a solid or liquid), the manner of
administration (e.g., by injection, inhalation, dry powder
propellant), and the cells targeted (e.g., epithelial cells, such
as blood vessels epithelial cells, nasal epithelial cells, or
pulmonary epithelial cells). The composition is preferably
administered in an amount that provides a sufficient level of
expression of IFN that elicits an immune response without undue
adverse physiological effects in the host caused by the
treatment.
[0168] In addition, single or multiple administrations of the
compositions of the present invention may be given (pre- or
post-exposure) to a subject (e.g., one administration or
administration two or more times). For example, subjects who are
particularly susceptible to, e.g., viral infection may require
multiple treatments to establish and/or maintain protection against
the virus. Levels of induced immunity provided by the
pharmaceutical compositions described herein can be monitored by,
e.g., measuring amounts of neutralizing secretory and serum
antibodies. The dosages may then be adjusted or repeated as
necessary to maintain desired levels of protection against, e.g., a
viral infection.
[0169] Alternatively, the efficacy of treatment can be determined
by monitoring the level of IFN-.alpha. expressed in a subject
(e.g., a human) following administration of the compositions of the
invention (e.g., Ad5-IFN-.alpha. vectors). For example, the blood
or lymph of a subject can be tested for IFN-.alpha. levels using,
e.g., standard assays known in the art (see, e.g., Human
Interferon-Alpha Multi-Species ELISA kit (Product No. 41105) and
the Human Interferon-Alpha Serum Sample kit (Product No. 41110)
from Pestka Biomedical Laboratories (PBL), Piscataway, N.J.). The
efficacy of treatment can also be determined by monitoring the
level of expression or activation of IFN-.alpha. upregulated
factors, such as the double-stranded RNA (dsRNA)-dependent protein
kinase R (PKR), the 2'-5'-oligoadenylate synthetase (2'-5'-OAS),
IFN-inducible Mx proteins, a tryptophan-degrading enzyme (see,
e.g., Pfefferkorn, Proc. Natl. Acad. Sci. USA 81:908-912, 1984),
adenosine deaminase (ADAR1), IFN-stimulated gene 20 (ISG20), p 56,
ISG15, mGBP2, GBP-1, the APOBEC proteins, viperin, or other factors
(see, e.g., Zhang et al., J. Virol., 81:11246-11255, 2007, and U.S.
Pat. No. 7,442,527, which is incorporated by reference herein in
its entirety).
[0170] A single intranasal dose of the compositions of the
invention achieve protection, pre-exposure, from infectious agents
(e.g., viral agents). This is a dramatic improvement from the
several doses per week or even multiple daily doses that are
required with current IFN-.alpha. treatments. In addition, a single
dose administered directly post-exposure (e.g., within 24 hrs) to a
viral or other infectious agent can function as a treatment
according to the present invention. The effectiveness of a single
dose of the compositions of the invention eliminates the need to
track people to be treated and to retreat or revaccinate them,
which is a difficult problem in a pandemic or bioterrorist attack
where general panic typically ensues.
[0171] A single intranasal dose of the compositions of the
invention can also be used to achieve therapy in subjects being
treated for autoimmune disease or cancer. Multiple doses (e.g., 2,
3, 4, 5, or more doses) can also be administered, in necessary, to
these subjects.
Shelf Stability
[0172] Pharmaceutical formulations of the compositions of the
invention (e.g., a formulation that includes an Ad5-conIFN-.alpha.
delivery vector) demonstrate a significant shelf life, which
provides an advantage over other adenoviral, antiviral, or vaccine
products. In particular, the Ad5-based IFN-.alpha. delivery vector
of the invention, which can be manufactured and lyophilized
(freeze-dried), exhibits a shelf-life of at least about 1, 2, 3, or
4 weeks, preferably at least about 1, 2, 3, 4, 5, 6, 12, or 18
months, more preferably at least 20 months, still more preferably
at least about 22 months, and most preferably at least about 24
months when stored at room temperature. This is mission critical
for the military and in developing countries where public health
departments cannot guarantee refrigeration of medications. The
shelf life of the compositions of the invention can be extended by
storage at 4.degree. C.
[0173] The shelf life of the adenoviral vector-containing
compositions of the invention can be assessed by, e.g., determining
adenoviral vector titers (see, e.g., Croyle et al., Gene Therapy
8:1281-1290, 2001) or by assessing the biological activity (e.g.,
the ability to transfect a cell and express biologically active
IFN) of the IFN-containing delivery vehicle (e.g., viral or
non-viral delivery vehicle). In an embodiment, the compositions of
the invention exhibit a loss of less than 20% of the original titer
(or biological activity), more preferably less than 10%, and most
preferably less than 5%, after storage at room temperature for at
least 12 months. In other embodiments, the compositions of the
invention exhibit a loss of less than 40% of the original titer (or
biological activity), more preferably less than 30%, and most
preferably less than 20%, after storage at room temperature for at
least 24 months.
[0174] Pharmaceutical formulations of the compositions of the
invention also exhibit a shelf-life of at least about 1-15 days or
2-4 weeks or even at least about 2-6 months when stored at
temperatures in the range of about 30.degree. C. to about
55.degree. C. (e.g., .about.45.degree. C.). In an embodiment, the
composition is stored is a dry, unreconstituted powder form.
Preferably, a composition of the invention that is stored at a
temperature in the range of about 30.degree. C. to about 55.degree.
C. exhibits a loss of less than 40% (more preferably less than 30%,
20%, or 10%, and most preferably less than 5%) of the original
titer (or biological activity) when stored for a period of time in
the range of 1 week to 2 months.
[0175] In another embodiment, pharmaceutical formulations of the
compositions of the invention exhibit a shelf-life of at least
about 1, 2, 3, or 4 weeks, preferably at least about 1, 2, 3, 4, 5,
6, 12, or 18 months, more preferably at least 20 months, still more
preferably at least about 22 months, and most preferably at least
about 24 months when stored frozen (e.g., at a temperature in the
range of less than 4.degree. C. (e.g., 0.degree. C. to about
-1900.degree. C.)). In this embodiment, the composition can be
stored as a non-stabilized, frozen liquid. Preferably, a
composition of the invention that is stored at a temperature of
less than 4.degree. C. (e.g., 0.degree. C. to about -20.degree. C.)
exhibits a loss of less than 40% (more preferably less than 30%,
20%, or 10%, and most preferably less than 5%) of the original
titer (or biological activity) when stored for a period of time in
the range of 2 months to 2 years.
[0176] Benefits of the long-term stability and shelf-life of the
compositions of the invention include: a) ease of storage of the
compositions as no cold chain is required, which increases the
ability to disseminate and store the compositions in areas of the
world that lack consistent access to electricity (e.g., third world
economies and disaster or war zones) and improves military
operational tempo as less "stuff" must be carried or used in areas
without refrigeration; b) forward deployment is possible when the
drug can be thrown in a soldier's backpack or in the back of a WHO
disaster vehicle; c) less drug waste as losses due to thawing are
mitigated; and d) more cost effective use of Strategic National
Stockpile (SNS) storage space warehouse, which need not include
refrigeration for storage of the compositions.
[0177] Other benefits of the Ad5-based IFN-.alpha. delivery vector
of the invention are shown in FIG. 4.
Kits
[0178] The invention also provides kits including the IFN-.alpha.
delivery vector of the invention, in lyophilized powder form, and a
vial of hydration medium (e.g., sterile water or saline) that can
be used to reconstitute the powder. In another embodiment, the kit
includes a container of the IFN-.alpha. delivery vector of the
invention, in lyophilized powder form, and a separate delivery
device that can be combined with the container to allow release of
the contents of the container during administration. The kit may
also include a container of the IFN-.alpha. delivery vector of the
invention, in lyophilized powder form, a vial of hydration medium
(e.g., sterile water or saline) that can be used to reconstitute
the powder, if desired, and a delivery device that can be used to
release the IFN-.alpha. delivery vector as a powder or
reconstituted liquid in an aerosolized form (e.g., via pulmonary or
intranasal administration). Kits of the invention optionally
include instructions for practicing any method described herein,
including a therapeutic or prophylactic method, instructions for
using any composition identified herein, and/or instructions for
operating any apparatus, system, device, or component described
herein, as well as packaging materials.
EXAMPLES
[0179] The following examples are to illustrate the invention. They
are not meant to limit the invention in any way.
Example 1
Efficacy for Pre- and Post-Exposure Protection Against Western
Equine Encephalitis Virus and Venezuelan Equine Encephalitis
Virus
[0180] The use of an Ad5-IFN-.alpha. delivery vector has been shown
to provide both pre- and post-exposure protection against Western
Equine Encephalitis virus (WEEV; Wu et al., Virology 369:206-213,
2007), an arthropod (mosquito) borne alphavirus classified as a
Category B pathogen by the U.S. Centers for Disease Control (CDC).
In this study, mice were inoculated with 10.sup.7 PFU of
Ad5-mIFN.alpha. by intramuscular injection and challenged with
various WEEV strains at a range of timepoints. The Ad5-mIFN.alpha.
showed complete protection when administered 24 hr, 48 hr, and 1
week pre-exposure, and 38% protection when treated 13 weeks
pre-exposure. A single inoculation at 6 hr after the challenge
delayed the progress of WEEV infection and provided about 60%
protection.
[0181] A study using Venezuelan Equine Encephalitis Virus (VEEV)
yields similar results. VEEV is a more infections virus, and
intramuscular administration of Ad5-IFN-.alpha. resulted in
complete protection to 10LD50 when administered 24 hr pre-exposure
(other time points were not tested), and 75% survival to 100LD50.
In this case, Ad5-IFN did not protect when administered
post-exposure (O'Brien et al., J. Gen. Virol. 90:874-882,
2009).
Example 2
Uses for the Compositions of the Invention
[0182] Pre-exposure (post-event) prophylaxis: The compositions of
the invention can be used as a single administration broad-spectrum
antiviral prophylactic medical countermeasure against, e.g.,
viral-based bioweapon threats or risk from exposure to endemic
viral threats.
[0183] Military or Law Enforcement Operations
[0184] The compositions of the invention can be used as a
prophylaxis for military, law enforcement agents, or local
emergency coordinator (LEC) personnel who, during operations, are
exposed to viral-based biological weapons threats. The decision to
administer a composition of the invention (e.g., an Ad5 delivery
vector that contains a nucleic acid molecule encoding
conIFN-.alpha., and that is formulated as a lyophilized powder for
delivery to the nasal mucosa) to warfighters will be based on,
e.g., a) the presence of identifiable biowarfare agents as measured
by biosensors (as aerosols or surface contamination on equipment),
b) intelligence that such viral-based weapons have been deployed or
may be deployed by adversaries, or c) diseased sentinel animals, or
d) contact by the warfighter with victims expected to present
symptoms of viral disease.
[0185] Exposure During Research
[0186] A similar scenario is presented by researchers or
manufacturers who, by the very nature of their jobs, come in
regular contact with pathogenic viruses or other biological threats
and for which an additional precaution against equipment or
protocol failure. The compositions of the invention can be used as
a prophylaxis (pre- or post-exposure) for these individuals, as
well.
Example 3
Medical Chain
[0187] The compositions of the invention can be administered
prophylactically to medical chain personnel, e.g., physicians,
nurses, cleaning staff, and others who come into contact with
patients suffering from viral or bacterial infectious diseases or
who may have infectious diseases. The broad-spectrum nature of the
compositions of the invention allows for administration to the
subject before knowledge of the biological pathogen is available
and in cases where there is no time to positively identify the
viral pathogen. The compositions of the invention are also
beneficial in cases where a virus mutates during a pandemic leaving
the established vaccine ineffective or less protective.
Example 4
Public Health
[0188] Ring and Immediate Post Exposure Treatment
[0189] If a patient is known to have come in contact with a viral
threat in the preceding 24 hrs, a composition of the invention
(e.g., an Ad5 delivery vector that contains a nucleic acid molecule
encoding IFN-.alpha. (e.g., conIFN-.alpha.), and that is formulated
for nasal or pulmonary delivery, e.g., as an aerosolized powder or
liquid mist) can be administered as a post-exposure treatment. If
necessary, a composition of the invention can be administered,
e.g., as a "ring" treatment to all susceptible individuals in a
prescribed area around an outbreak of an infectious disease. Ring
treatment controls an outbreak by treating and monitoring a ring of
people around each infected individual.
[0190] Suspected Exposure Treatment
[0191] Even if exposure to a biological threat is not confirmed, a
composition of the invention can be administered to those people
thought to be exposed (the "worried well"), as the side effects of
IFN are minimal. For example, a cranberry grower in Massachusetts
is bitten by a mosquito and gets sick. For example, because there
is an endemic risk of Eastern equine encephalitis (EEE), the person
can be administered a composition of the invention, for example, by
nasal or pulmonary delivery (e.g., as an aerosolized powder or
liquid mist) and monitored for signs of improvement prior to
agricultural work near cranberry bogs.
[0192] Post-Exposure Prophylaxis
[0193] On a population level, if dissemination of a viral threat is
known or believed to have occurred, a composition of the invention
can be administered, for example, by nasal or pulmonary delivery
(e.g., as an aerosolized powder or liquid mist), stop the spread of
the viral threat. In this case, the invervention is administered
without knowing the infection status of the recipient, and thus the
function of prophylaxis and treatment would likely be applied.
Example 5
Veterinary Indications of Ad5-Vectored IFN
[0194] The broad spectrum anti-viral capabilities of interferon
polypeptide have been well recognized in veterinary medicine.
Indeed, the oral administration of IFN is an effective treatment
for shipping fever in thoroughbred race horses (Akai et al, J.
Equine Sci. 19:91, 2008) and cattle experiencing bovine respiratory
disease (BRDC; Cummins et al, J. Inf. & Cyto. Res. 19:907,
1999), and in the general treatment of respiratory illness in
horses (Moore et al, Can. Vet. J. 45:594, 2004). Intranasal or
pulmonary delivery of an Ad5-IFN could overcome the current
limitations of repeated dosing and high cost. An intranasal
delivery system for horses that could be used to administer
compositions of the present invention is described in, e.g., U.S.
Pat. No. 6,398,774, which is incorporated herein by reference. The
use of an Ad5-IFN production system has been shown to be safe and
effective in lab animals (see, e.g., Wu et al, Virology 369:206,
2007).
[0195] Other veterinary indications include the treatment or
prevention of pandemics by pathogens, such as Rift Valley Fever,
the treatment or prevention of endemic pathogens, and the treatment
or prevention of pathogens that are released intentionally. The
treatment or prevention in this context prevents or mitigates the
potential catastrophic loss of animals within the food chain.
Example 6
Ad5-VEE/WEE/EEE Equine Vaccine
[0196] To date, vaccination is the only means of combating highly
infectious, mosquito borne encephalitis alphaviruses. All horses in
North America are at risk and vaccination is recommended. Currently
marketed trivalent vaccines manufactured via traditional technology
require multiple yearly injections and boosters to provide
protection. A "live vaccine" approach using adenoviruses provides a
safe means of producing a rapid and persistent protection using
just a single intranasal administration.
Example 7
CoAdministration of Ad5-IFN with One or More Secondary Anti-Viral
Drugs
[0197] The Ad5-IFN delivery vehicle (e.g., encoding conIFN-.alpha.
or another IFN described herein) can be formulated with a
pharmaceutically acceptable excipient for intranasal dosing in
combination with an antihistamine and a neuraminidase inhibitor.
This composition can be administered to a subject either prior to
viral exposure or within 48 hours of exposure. The antihistamine
helps to reduce any nasal congestion, e.g., stuffed or blocked
nasal passages, caused by viral infection or rhinitis, thereby
maximizing the distribution of the Ad5-IFN and neuraminidase
inhibitor and their absorption by the epithelium of the upper
and/or lower respiratory tract. An example of such an antihistamine
would be H1 antagonists, such as fexofenadine or loratadine. A
neuraminidase inhibitor, such as Zanamivir (Relenza.RTM.,
GlaxoSmithKline), is a potent selective inhibitor of the viral
neuraminidase glycoprotein that is important for viral replication
of, e.g., influenza A and B and other viruses. The net effect of
this three drug combination is improved viral prophylaxis where the
IFN initiates a broad spectrum immune response, the neuraminidase
inhibitor blocks viral release from infected cells, and the
antihistamine ensures or improves delivery of the drugs to the
nasal epithelium.
[0198] Alternatively, the Ad5-IFN delivery vector can be
administered intransally as a separate composition and the
antihistamine and neuraminidase inhibitor (e.g., Oseltamivir
phosphate (Tamiflu.RTM., Roche Pharma)) can be administered orally
in separate compositions or in a single composition (see, e.g.,
U.S. Pat. No. 6,605,302, which is incorporated herein by
reference).
Example 8
Prophylaxis or Treatment of Punta Toro Virus (Family:
Bunyaviridiae)
[0199] Rift Valley fever virus (RVFV) is an arthropod-borne viral
fever that causes direct infection in humans and livestock. The
mode of transmission is via the bite of an infective Aedes or Culex
mosquito. Mechanical infection via aerosols or infected blood has
been reported in humans that work with, handle, or process
livestock or contaminated carcasses. Humans of both sexes and all
ages are susceptible and when infected with RVFV may develop
retinitis, encephalitis, or hepatitis associated with haemorrhages
that may be fatal (Heyman, Amercian Public Health Association,
Washington D.C., 2008). Recent outbreaks in Kenya resulted in 118
deaths and a case fatality rate of 29% (CDC, Morb. Motal. Wkly.
Rep. 56:73-76, 2007). There are no approved vaccines or effective
therapies for RVFV. Reflecting the concern of public health
officials, RVFV has been classified as a Category A pathogen by the
National Institute for Allergic and Infectious Diseases and has
received `Dual Agent` status by the Department of Health and Human
Services and the US Department of Agriculture.
[0200] Effective countermeasures that are highly stable, easily
administered, and elicit long lasting protective immunity are much
needed. Because direct work with RVFV is highly restricted and
requires enhanced BSL-3+ facilities, we have recently established
an intranasal (IN) respiratory route Punta Toro virus (PTV)
infection model in Syrian Hamsters. PTV is a BSL-2 surrogate for
RVFV, and produces disease in hamsters that models RVFV infection
and disease progression in humans (Gowen et al., Antiviral Res.
77:215-224, 2008).
[0201] The purpose of this experiment was to evaluate
Ad5-IFN.alpha. as a prophylactic agent to counter exposure to PTV.
The route of Ad5-IFN.alpha. exposure was by intranasal (IN) to
simulate respiratory mucosal surface delivery--a proposed route of
administration in humans. Doses of 10.sup.8, 10.sup.7, and 10.sup.6
PFU of Ad5-IFN.alpha. (n=15) were administered 24 hrs prior to
infectious challenge with PTV. The doses selected were based upon
previous studies demonstrating high-level protection and were
scaled to the hamster model based on typical dosing extrapolation
equations using body surface area. As is shown in FIG. 6,
administration of Ad5-IFN.alpha. at the indicated doses at least 24
hours prior to challenge with PTV resulted in 100% survival as
compared to the ribavarin treated, empty-vector treated, and
placebo controls.
[0202] In addition, we have demonstrated significant protection
against both respiratory and subcutaneous PTV challenge infections
in mice treated with Ad5-IFN.alpha.: a) prior to challenge as a
prophylactic (up to 21 days before challenge) and b) as a treatment
given up to +48 hr post-exposure.
Example 9
Prophylaxis or Treatment of Western Equine Encephalitis (Family:
Togaviridae)
[0203] Western Equine Encephalitis belongs to the Alphavirus genus
of the Togavirus family which represents a group of mosquito borne,
severely neuropathogenic, emerging pathogens in domestic animals
and humans. WEEV is endemic to the Western portion of North America
and is maintained in nature through a cycle involving wild birds as
reservoir hosts and Culex tarsalis mosquitoes as vectors (Wu et
al., Virology 369: 206-213, 2007) and have an overall case fatality
rate of 3%-8% depending on age.
[0204] As a weapon, WEEV can be easily transmitted through the
aerosol route with fatality rates as high as 40% in laboratory
accidents (Hanson et al., Science 158: 1283-1286, 1967). A closely
allied virus--Venezuelan Equine Encephalitis virus--was weaponized
by the U.S. and the former Soviet Union for aerosol dissemination
as an incapacitating agent on the battlefield. It was anticipated
that a biological weapons attack in a region populated by Equines
and mosquito vectors could initiate an epidemic (Eitzen et al.,
Medical Management of Biological Casualties 3.sup.rd Edition,
published for the Department of Defense by The US. Army Medical
Research Institute of Infectious Disease, Fort Detrick, Frederick
Md., 1998). The ongoing concern of these viruses as an existing
biological weapon and the lack of a safe and efficacious vaccine or
antiviral has prompted public health concern, and these viruses are
listed as a Category B Bioterrorist threat with the CDC (CDC,
Centers for Disease Control and Prevention; Public Health
Assessment of Potential Biological Terrorsm Agents Vol. 8,
2010).
[0205] One hundred forty (140) female Balb/c mice (10 per group)
were used in this study and divided into two studies; each used a
total of 70 mice. The first study tested the efficacy against WEEV
California strain and the second study against WEEV CBA87 strain.
The following treatment groups were used in both studies: [0206]
Groups 1-5: Single IN treatment with 10.sup.7 PFU Ad5-IFN.alpha. at
Day (-21, -14, -7, -1 or +4 hrs respectively) [0207] Group
6--IFN.alpha. B/D (recombinant mouse) 2.times.10.sup.7 IU/kg once
daily at Days 0 to 8, starting 4 hrs prior to challenge [0208]
Group 7--Control: untreated and challenged
[0209] All mice were challenged intranasally on Day 0 with lethal
dose of 2.5.times.10.sup.3 pfu of WEEV California strain in study 1
and 500 pfu of WEEV CBA87 strain in study 2 and followed for 14
days for clinical signs of disease and euthanized at
moribundity/morbidity. Administration of Ad5-IFN.alpha. (murine)
resulted in complete protection of all animals in the prophylactic
window, and 100% (California) & 70% (CBA87) survival in the +4
hrs treatment groups (FIGS. 7A and 7B).
Example 10
Prophylaxis or Treatment of Severe Acute Respiratory Syndrome
(Family: Coronaviridae)
[0210] SARS has recently emerged in the human population as a fatal
respiratory disease. Severely affected patients develop acute
respiratory distress syndrome, which corresponds with diffuse
alveolar damage at autopsy. A newly discovered Coronavirus, SCV,
has been identified as the primary cause of SARS. SARS patients
have been treated empirically with a combination of Ribavirin,
Oseltamivir, antibiotics and corticosteroids, with mixed results.
Treatment with recombinant human interferon (Alfacon.RTM.) has
shown clinical promise.
[0211] Groups of 10 mice were administered 50 .mu.l of
Ad5-IFN.alpha. (murine, 10.sup.6 PFU) IN once at 14, 7, 5, or 3
days pre-virus exposure (PVE). In addition, groups of 10 mice were
administered 50 .mu.l of Ad5-IFN.alpha. (murine) (10.sup.6 PFU or
10.sup.5 PFU) IN one time at 6, 12, 24 hours post virus exposure.
In both experiments Poly-ICLC was given at 1 mg/kg by the IN route
at 24 h before virus exposure and 8 h after exposure to virus and
served as a positive control for controlling the virus infection,
and 15 mice were treated with buffered saline at each timepoint
representing placebo controls. Animal deaths were recorded for up
to 21 days post virus exposure.
[0212] As shown in FIGS. 8A and 8B, treatment with Ad5-IFN.alpha.
(murine) resulted in complete protection of all animals in the
treatment groups.
Example 11
Prophylaxis or Treatment of Yellow Fever Virus (Family:
Flaviviridae)
[0213] Yellow Fever (YF) is an acute infectious viral disease with
a case fatality rate of 20-50% characterized by jaundice and
hemorrhagic symptoms. YF is transmitted by mosquitoes, typically
Aede spps in urban areas and Haemogogus spp or Sabethes spp in
forests with humans or primates serving as reservoirs. YF has an
endemic zone between 15.degree. N and 10.degree. S latitude which
encompasses 33 African and nine South African and Caribbean Island
with a combined population of >500 million people (Heymann,
Control of Communicable Disease Manual, Ammercian Public Health
Association, Washington, D.C., 2008). While an effective vaccine is
available, immunization coverage is variable, ranging from 30-95%
in Africa. No approved treatment exists.
[0214] Hamsters were injected (15-20/group) intraperitoneally (IP)
with 0.1 ml of the diluted virus (10 CClD.sub.50/animal).
Ad5-IFN.alpha. was administered by IN instillation at doses of
1.times.10.sup.8, 5.times.10.sup.7, 5.times.10.sup.6, or
5.times.10.sup.5 1.25.times.10.sup.6 PFU/animal one time at -4 h.
Mortality was observed daily for 21 days, and weight was recorded
on 0, 3, and 6 dpi. Liver tissue was taken at necropsy from 5
animals from each group for virus titration on 4 dpi. In a second
study, animals were administered 5.times.10.sup.7 PFU IN
Ad5-IFN.alpha. at -4 hr, or +1, +2 or +3 days post infection (dpi)
using the same controls as in the previous experiment.
[0215] Complete protection of hamsters was observed at the top two
doses of 1.times.10.sup.8 pfu and 5.times.10.sup.7 pfu of
Ad5-IFN.alpha. (FIG. 9A). A dose response was seen with increasing
mortality occurring at lower doses, although survival was
significantly improved in these groups over controls as well as a
delay in the mortality curve. Overall, all of the Ad5-IFN.alpha.
doses offered significant protection as compared with the empty
adenovirus vector control with efficacy similar to or greater than
that of the positive control. Using a dose of 5.times.10.sup.7 PFU
of Ad5-IFN.alpha. complete survival was seen with treatment at +1 d
and 90% survival at +2 dpi (FIG. 9B).
Example 12
Treatment of Ebola Virus (Family: Filoviridae)
[0216] Ebola hemorrhagic fever was first recognized in 1976 in two
simultaneous outbreaks in Sudan and Zaire which affected >600
people with case fatality rates of 55% and 90% respectively.
Person-to-person contact does occur through direct contact with
blood, secretions, organs, or semen from infected humans.
Nosocomial infections are frequent, and virtually all persons
infected from contaminated needles died. Despite extensive study,
the natural animal reservoir for Ebola remains unknown. There are
no approved vaccines or effective treatments for Filovirus
infections (Heymann, Control of Communicable Disease Manual,
Ammercian Public Health Association, Washington, D.C., 2008).
[0217] Ebola virus is considered a Category A bioterrorism agent by
the CDC(CDC, 2010, supra) and top priority public health biological
threat (PHEMCE, Public Health Emergency Medical Countermeasures
Enterprise, Health & Human Services, Washington D.C., 2007).
Such agents pose a risk to national security because they can be
easily disseminated or transmitted from person to person; result in
high mortality rates and have the potential for major public health
impact and require special action for public health
preparedness.
[0218] Here, Ad5-IFN.alpha. was tested in mouse and Guinea pig
models of the Ebola virus, Zaire strain (ZEBOV). Groups of 10 mice
were challenged by intraperitoneal (IP) injection with
1000.times.LD.sub.50 of the mouse-adapted Ebola virus. Thirty
minutes later they were dosed by either the IM (50 .mu.l per each
hind limb) or IN (50 .mu.l) route with a single dose of
1.times.10.sup.7 IFU (infectious units) mAd5-IFN.alpha. per mouse.
Control mice were injected IM with phosphate buffered saline (PBS)
(50 .mu.l per each hind limb). Complete survival benefit was seen
with administration of mouse mAd5-IFN.alpha. by either route, and
there was no significant weight loss in treated groups versus
control (FIG. 10A).
[0219] Following the success of the mouse study, Ad5-IFN.alpha. was
tested in a Guinea Pig (GP) model of Ebola virus, Zaire strain
(ZEBOV). The GP model more closely mimics the pathophysiology of
the disease in humans, and the animals are more susceptible to
challenge, thus making it a more difficult model to achieve
positive results. Eight Hartley guinea pigs were challenged by IP
injection with 100.times.LD.sub.50 of guinea pig-adapted ZEBOV. 30
minutes later two animals were dosed IN with 2.times.10.sup.8 PFU
Ad5-IFN.alpha. per guinea pig. In addition, recombinant IFN protein
was administered to three GPs daily for six days to assess the
therapeutic potential of the protein alone, while three animals
were untreated and served as a negative control group. All of the
animals treated with Ad5-IFN.alpha. survived, compared to 66% in
the interferon protein group, whereas all the control animals
perished (FIG. 10B).
Example 13
Prophylaxis for Pichinde Virus (Family: Arenaviridae)
[0220] Arenaviruses produce an acute viral illness which progresses
in 20% of patients to severe multisystem disease with hospitalized
case fatality rate up to 15%. The disease is severe in pregnancy
with fetal loss rates approaching 80% and associated frequent
maternal death. Arenaviruses are serologically divided into Old
World (e.g. Lassa fever) and New World (e.g. Machupo or Junin).
Lassa fever has had the greatest impact on public health by
hemorrhagic fever, with more than 100,000 endemic infections in
West Africa and 5,000 deaths annually (Fischer-Hoch et al., J.
Virol. 74:6777-6783, 2000). The mode of transmission is through
aerosol or direct contact with contaminated rodent excreta or via
person-to-person by pharyngeal secretions, semen or urine.
[0221] Arenaviruses are considered a Category A bioterrorism agent
by the CDC(CDC, 2010, supra) and a priority public health
biological weapons threat (PHEMCE, 2007, supra). Such agents pose a
risk to national security because they can be easily disseminated
or transmitted from person to person; result in high mortality
rates and have the potential for major public health impact and
require special action for public health preparedness. Pichinde
virus (PCV) is a New World Arenavirus that is highly pathogenic in
hamsters but is non-pathogenic in humans (Buchmeier et al., Infect.
Immun. 9:821-823, 1974). PCV infection in hamsters is a well
characterized animal model that produces a fulminating disease that
ends in terminal shock via vascular leakage syndrome with high
systemic viral titers. The distribution of viral antigens within
the infected host (Connolly et al., A. J. Trop. Med. Hyg. 4; 10-24,
1993) mimics the disease manifestations reported in human
Arenavirus cases (Walker et al., Am. J. Path. 107:349-356, 1982)
but can be utilized safely under BSL-2 conditions (Gowen and
Holbrook, Antiviral Res. 78:79-90, 2007).
[0222] Ad5-IFN.alpha. was tested in a hamster model of Pichinde
virus infection. One day prior to challenge, groups of 10 animals
were dosed via the IN route (200 .mu.l) with a single dose of
either: 10.sup.8, 10.sup.7, or 10.sup.6 PFU Ad5-IFN.alpha. per
hamster. Animals were challenged by intraperitoneal (IP) injection
with LD.sub.95 of the hamster-adapted PCV. Control mice were dosed
IN with phosphate buffered saline (PBS) (100 .mu.l per nostril).
Complete survival benefit was seen with administration of
Ad5-IFN.alpha. at the highest dose, with a dose dependent decline
in survival seen at lower levels (FIG. 11).
Example 14
Treatment with a Combination "Instant Acting Vaccine" for Ebola
(Family: Filovirus)
[0223] Ad5-IFN.alpha. Administered in Conjuction with a Vaccine
[0224] Vaccines have been a cornerstone for effective infectious
disease prevention since Jenner in 1796. Vaccines are
cost-effective, easily administered, generally safe and
longlasting. However, when facing bioweapons threats, broad
nation-wide vaccine campaigns have met with considerable
opposition. The bias against vaccination arises from the public's
balancing of the risk from a low-probability bioweapons threats vs
the certainty of adverse vaccine effects in a few patients. Indeed,
even the smallpox vaccination campaign which boasted the first and
only infectious disease ever irradicated, was discontinued some 30
years ago despite Presidential support for police and healthcare
worker vaccination. A second public health issue is the time delay.
Vaccines work slowly--often requiring 7 to 21 days--for a
vaccination and boosters to achieve protection. This time delay has
lethal consequences for most pathogenic viral bioweapon infections.
As such, current public health vaccination strategies and
stockpiles are directed toward disease mitigation and prevention of
secondary infection and disease spread. Infected individuals at
ground zero receive only supportive care. We propose the use of
Ad5-IFN.alpha. AND a vaccine to radically change this disease
management paradigm to include treatment AND prophylaxis. Further,
existing vaccine stockpiles can now be repurposed and utilized as
part of an "instant acting vaccine".
[0225] It is clear that Ad5-IFN.alpha. can act as a both a
prophylactic and a treatment. In this example, we combine
Ad5-IFN.alpha.-acting as a type of adjuvant--with a standard
vaccine to form an "Instant Acting Vaccine". The benefits of this
approach are significant. Ad5-IFN.alpha. functions as an immune
system stimulant, with the following benefits; a) administration of
Ad5-IFN.alpha. with a vaccine can protect the host against the
viral insult until the vaccine is functional and b) Ad5-IFN.alpha.
can stimulate the immune system to respond to the vaccine faster or
more vigorously and thus establish protective antibody levels
faster.
[0226] In the case of Ebola, we administered an Ad5-IFN.alpha. in
conjuction with an Ad5 vectored Ebola glycoprotein vaccine
(Ad-CAGoptZGP; vaccine described Richardson et al, 2009, supra;
Croyle et al, PLoS 3:1-9, 2008) to demonstrate the method and
benefit of the instant acting vaccine. Six Guinea pigs were
administered the vaccine (10.sup.9 or 10.sup.10 infectious units)
with Ad5-IFN.alpha. (2.times.10.sup.8 PFU) via IN administration 30
minutes after a 1000LD50 challenge with ZEBOV. These combined
treatments resulted in 100% survival of the animals (FIG. 12).
Ad5-IFN.alpha. alone was able to save 50% of the challenged animals
and the vaccine alone was only able to save 30% in a model with
1/10.sup.th the challenge. Thus, the two components work
synergistically to save animals that each component could not save
separately from challenge with Ebola.
[0227] Given this data, Ad5-IFN.alpha. has tremendous potential to
serve as a vaccine adjuvant for a wide range of vaccines, thereby
speeding the time to protection in either a prophylactic or
treatment model.
Example 15
Vaccine Stability
[0228] We have developed a rugged, shelf stable formulation of the
combination therapy, Ad5-IFN.alpha.+Ad-CAGoptZGP. Our preliminary
data illustrates Ad5 vector stability with no appreciable loss in
activity at 37.degree. C. for 84 days, and at 100.degree. C. for at
least an hour (ASM 2010).
Example 16
Safety Data
[0229] There is a wealth of clinical data showing that the Ad5
vector system and recombinant human IFN, separately, are safe
(including when administered using multiple repeat dosing). In
addition, Ad-CAGoptZGP alone has been used successfully to treat a
suspected Ebola infection of a lab worker in Germany. The patient
experienced a fever and headache commonly associated with antiviral
vaccines, but made a full recovery.
[0230] The doses of Ad5-IFN.alpha.+Ad-CAGoptZGP as evaluated in the
mouse and guinea pig ZEBOV models discussed above demonstrate
safety at even the highest expected doses. Our experience to date
indicates superior efficacy even at lower doses of Ad5-IFN.alpha.
(as low 1/1000th) used in animal models of other diseases (e.g.
Punta Toro, WEE, and SARS). This result, coupled with the
synergistic relationship of the 2 components
(Ad5-IFN.alpha.+Ad-CAGoptZGP) indicates that a lower dose should be
substantially effective against infection by a pathogen, such as,
e.g., an Ebola infection.
[0231] To date, more than 60 clinical trials have been conducted
with Ad5 as the gene delivery vector, thus providing a solid
toxicology framework for Ad5-IFN.alpha.-containing compositions of
the invention (including, e.g., the combination of Ad5-IFN.alpha.
and Ad-CAGoptZGP). For example, in humans a dosage in the range of
1.0.times.10.sup.6 to 1.0.times.10.sup.12 (e.g., 1.6.times.10.sup.9
PFU) for a 70 kg person for the combination of Ad5-IFN.alpha. and
Ad-CAGoptZGP is expected to provide therapeutic and prophylactic
benefit against challenge or exposure to a pathogen (e.g., a viral
agent). In our animal model studies, we have tested the combination
of Ad5-IFN.alpha. and Ad-CAGoptZGP at a viral particle (vp) to PFU
ratio of 10:1 with success. For example, with regard to a viral
particle (vp) to PFU ratio of 50:1, which is expected to be at the
higher end of the administration spectrum, the dose will be
8.times.10.sup.10 vp.
[0232] Safety of Replication Defective Ad5 Vectors
[0233] The safety of replication defective Ad5 vectors has been
confirmed during a dose escalation study involving 12 patients
where the Ad5 was delivered intranasally
(2.times.10.sup.7-2.times.10.sup.10 PFU/patient; see Knowles et
al., N.E. J. Med. 333:823-831, 1995). At the highest dose, adverse
effects were deemed moderate (ear ache and mucosal sensitivity) and
were resolved within three weeks. More recently, a pilot Phase I
safety study noted dose limiting toxicology at 2.times.10.sup.12
vp, with repeated doses of the Ad5 vector being well tolerated (see
Keedy et al., J. Clin. Oncol. 26:4166-4171, 2008). The NIH
Recombinant DNA Advisory Committee (NIH Report, Hum. Gene Ther.
13:3-13, 2002) reports the upper safe limit before toxicology of
replication defective Ad5 vectors as 7.times.10.sup.13 vp. Using
these studies as precedents, we expect the effective dose of a
combination therapy, such as Ad5-IFN.alpha. and Ad-CAGoptZGP, would
be at least 1-2 orders of magnitude lower than the low safe dose
threshold for Ad5 administration.
[0234] Safety of Interferons
[0235] Interferons are safely used clinically to treat Hepatitis C
and SARS, where the high dose side effects can be flu like symptoms
such as increased body temperature, headache, muscle pain,
convulsion, and dizziness. In some cases hair thinning and
depression has also been observed. In cases of high risk melanoma
the maximum tolerated dose was used (4.5.times.10.sup.5 U/kg) daily
for one month (see Jonasch et al., Cancer J. 6:1390145, 2000),
followed by a half dose three times a week for 48 weeks. The
resultant level of IFN in the bloodstream for 12 hours post
injection can be extrapolated as approximately 230 U/mL (see
Cantell et al., J. Gen. Virol. 22:453-455, 1974). The level of
serum IFN measured in our mouse model was 250 U/mL (see Wu et al.,
Virology 369:206-213, 2007). Again, this comparison illustrates
that our maximum expected dose produces a serum IFN level that is
consistent with those found in patients undergoing antiviral
therapy.
Other Embodiments
[0236] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure that come
within known or customary practice within the art to which the
invention pertains and may be applied to the essential features
hereinbefore set forth.
[0237] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each independent publication or patent application was
specifically and individually indicated as being incorporated by
reference in their entirety. Also incorporated by reference is
PCT/CA2010/000844, entitled "ADMINISTRATION OF INTERFERON FOR
PROPHYLAXIS AGAINST OR TREATMENT OF PATHOGENIC INFECTION," which
was filed on Jun. 9, 2010, naming Jeffrey D. Turner and Jane E.
Ennis as inventors.
TABLE-US-00002 APPENDIX Interferon Alpha 1b-IFNA1 Nucleotide: NCBI
Reference Sequence: NM_024013.1 Homo sapiens (SEQ ID NO: 1) 1
agaacctaga gcccaaggtt cagagtcacc catctcagca agcccagaag tatctgcaat
61 atctacgatg gcctcgccct ttgctttact gatggtcctg gtggtgctca
gctgcaagtc 121 aagctgctct ctgggctgtg atctccctga gacccacagc
ctggataaca ggaggacctt 181 gatgctcctg gcacaaatga gcagaatctc
tccttcctcc tgtctgatgg acagacatga 241 ctttggattt ccccaggagg
agtttgatgg caaccagttc cagaaggctc cagccatctc 301 tgtcctccat
gagctgatcc agcagatctt caacctcttt accacaaaag attcatctgc 361
tgcttgggat gaggacctcc tagacaaatt ctgcaccgaa ctctaccagc agctgaatga
421 cttggaagcc tgtgtgatgc aggaggagag ggtgggagaa actcccctga
tgaatgcgga 481 ctccatcttg gctgtgaaga aatacttccg aagaatcact
ctctatctga cagagaagaa 541 atacagccct tgtgcctggg aggttgtcag
agcagaaatc atgagatccc tctctttatc 601 aacaaacttg caagaaagat
taaggaggaa ggaataacat ctggtccaac atgaaaacaa 661 ttcttattga
ctcatacacc aggtcacgct ttcatgaatt ctgtcatttc aaagactctc 721
acccctgcta taactatgac catgctgata aactgattta tctatttaaa tatttattta
781 actattcata agatttaaat tatttttgtt catataacgt catgtgcacc
tttacactgt 841 ggttagtgta ataaaacatg ttccttatat ttactc Amino Acid:
NCBI Reference Sequence: NP_076918.1 Homo sapiens (SEQ ID NO: 2) 1
maspfallmv lvvlsckssc slgcdlpeth sldnrrtlml laqmsrisps sclmdrhdfg
61 fpqeefdgnq fqkapaisvl heliqqifnl fttkdssaaw dedlldkfct
elyqqlndle 121 acvmqeervg etplmnadsi lavkkyfrri tlyltekkys
pcawevvrae imrslslstn 181 lqerlrrke Interferon Alpha 2b-IFNA2
Nucleotide: NCBI Reference Sequence: NM_000605.3 Homo sapiens (SEQ
ID NO: 3) 1 gagaacctgg agcctaaggt ttaggctcac ccatttcaac cagtctagca
gcatctgcaa 61 catctacaat ggccttgacc tttgctttac tggtggccct
cctggtgctc agctgcaagt 121 caagctgctc tgtgggctgt gatctgcctc
aaacccacag cctgggtagc aggaggacct 181 tgatgctcct ggcacagatg
aggagaatct ctcttttctc ctgcttgaag gacagacatg 241 actttggatt
tccccaggag gagtttggca accagttcca aaaggctgaa accatccctg 301
tcctccatga gatgatccag cagatcttca atctcttcag cacaaaggac tcatctgctg
361 cttgggatga gaccctccta gacaaattct acactgaact ctaccagcag
ctgaatgacc 421 tggaagcctg tgtgatacag ggggtggggg tgacagagac
tcccctgatg aaggaggact 481 ccattctggc tgtgaggaaa tacttccaaa
gaatcactct ctatctgaaa gagaagaaat 541 acagcccttg tgcctgggag
gttgtcagag cagaaatcat gagatctttt tctttgtcaa 601 caaacttgca
agaaagttta agaagtaagg aatgaaaact ggttcaacat ggaaatgatt 661
ttcattgatt cgtatgccag ctcacctttt tatgatctgc catttcaaag actcatgttt
721 ctgctatgac catgacacga tttaaatctt ttcaaatgtt tttaggagta
ttaatcaaca 781 ttgtattcag ctcttaaggc actagtccct tacagaggac
catgctgact gatccattat 841 ctatttaaat atttttaaaa tattatttat
ttaactattt ataaaacaac ttatttttgt 901 tcatattatg tcatgtgcac
ctttgcacag tggttaatgt aataaaatat gttctttgta 961 tttggtaaat
ttattttgtg ttgttcattg aacttttgct atggaaactt ttgtacttgt 1021
ttattcttta aaatgaaatt ccaagcctaa ttgtgcaacc tgattacaga ataactggta
1081 cacttcattt atccatcaat attatattca agatataagt aaaaataaac
tttctgtaaa 1141 cca Amino Acid: NCBI Accession No. AAP20099 Homo
sapiens (SEQ ID NO: 4) 1 mcdlpqthsl gsrrtlmlla qmrrislfsc
lkdrhdfgfp 41 qeefgnqfqk aetipvlhem iqqifnlfst kdssaawdet 81
lldkfytely qqlndleacv iqgvgvtetp lmkedsilav 121 rkyfqritly
lkekkyspca wevvraeimr sfslstnlqe 161 slrske Interferon Beta
1a-IFNB1 Nucleotide: NCBI Reference Sequence: NM_002176.2 Homo
sapiens (SEQ ID NO: 5) 1 acattctaac tgcaaccttt cgaagccttt
gctctggcac aacaggtagt aggcgacact 61 gttcgtgttg tcaacatgac
caacaagtgt ctcctccaaa ttgctctcct gttgtgcttc 121 tccactacag
ctctttccat gagctacaac ttgcttggat tcctacaaag aagcagcaat 181
tttcagtgtc agaagctcct gtggcaattg aatgggaggc ttgaatactg cctcaaggac
241 aggatgaact ttgacatccc tgaggagatt aagcagctgc agcagttcca
gaaggaggac 301 gccgcattga ccatctatga gatgctccag aacatctttg
ctattttcag acaagattca 361 tctagcactg gctggaatga gactattgtt
gagaacctcc tggctaatgt ctatcatcag 421 ataaaccatc tgaagacagt
cctggaagaa aaactggaga aagaagattt caccagggga 481 aaactcatga
gcagtctgca cctgaaaaga tattatggga ggattctgca ttacctgaag 541
gccaaggagt acagtcactg tgcctggacc atagtcagag tggaaatcct aaggaacttt
601 tacttcatta acagacttac aggttacctc cgaaactgaa gatctcctag
cctgtgcctc 661 tgggactgga caattgcttc aagcattctt caaccagcag
atgctgttta agtgactgat 721 ggctaatgta ctgcatatga aaggacacta
gaagattttg aaatttttat taaattatga 781 gttattttta tttatttaaa
ttttattttg gaaaataaat tatttttggt gcaaaagtca Amino Acid: NCBI
Reference Sequence: NP_002167.1 Homo sapiens (SEQ ID NO: 6) 1
mtnkcllqia lllcfsttal smsynllgfl qrssnfqcqk llwqlngrle yclkdrmnfd
61 ipeelkqlqq fqkedaalti yemlqnifal frqdssstgw netivenlla
nvyhqinhlk 121 tvleekleke dftrgklmss lhlkryygri lhylkakeys
hcawtivrve ilrnfyfinr 181 ltgylrn Interferon Gamma-IFNG Nucleotide:
NCBI Reference Sequence: NM_000619.2 Homo sapiens (SEQ ID NO: 7) 1
cacattgttc tgatcatctg aagatcagct attagaagag aaagatcagt taagtccttt
61 ggacctgatc agcttgatac aagaactact gatttcaact tctttggctt
aattctctcg 121 gaaacgatga aatatacaag ttatatcttg gcttttcagc
tctgcatcgt tttgggttct 181 cttggctgtt actgccagga cccatatgta
aaagaagcag aaaaccttaa gaaatatttt 241 aatgcaggtc attcagatgt
agcggataat ggaactcttt tcttaggcat tttgaagaat 301 tggaaagagg
agagtgacag aaaaataatg cagagccaaa ttgtctcctt ttacttcaaa 361
ctttttaaaa actttaaaga tgaccagagc atccaaaaga gtgtggagac catcaaggaa
421 gacatgaatg tcaagttttt caatagcaac aaaaagaaac gagatgactt
cgaaaagctg 481 actaattatt cggtaactga cttgaatgtc caacgcaaag
caatacatga actcatccaa 541 gtgatggctg aactgtcgcc agcagctaaa
acagggaagc gaaaaaggag tcagatgctg 601 tttcgaggtc gaagagcatc
ccagtaatgg ttgtcctgcc tgcaatattt gaattttaaa 661 tctaaatcta
tttattaata tttaacatta tttatatggg gaatatattt ttagactcat 721
caatcaaata agtatttata atagcaactt ttgtgtaatg aaaatgaata tctattaata
781 tatgtattat ttataattcc tatatcctgt gactgtctca cttaatcctt
tgttttctga 841 ctaattaggc aaggctatgt gattacaagg ctttatctca
ggggccaact aggcagccaa 901 cctaagcaag atcccatggg ttgtgtgttt
atttcacttg atgatacaat gaacacttat 961 aagtgaagtg atactatcca
gttactgccg gtttgaaaat atgcctgcaa tctgagccag 1021 tgctttaatg
gcatgtcaga cagaacttga atgtgtcagg tgaccctgat gaaaacatag 1081
catctcagga gatttcatgc ctggtgcttc caaatattgt tgacaactgt gactgtaccc
1141 aaatggaaag taactcattt gttaaaatta tcaatatcta atatatatga
ataaagtgta 1201 agttcacaac aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa Amino
Acid: NCBI Reference Sequence: NP_000610.2 Homo sapiens (SEQ ID NO:
8) 1 mkytsyilaf qlcivlgslg cycqdpyvke aenlkkyfna ghsdvadngt
lflgilknwk 61 eesdrkimqs qivsfyfklf knfkddqsiq ksvetikedm
nvkffnsnkk krddfekltn 121 ysvtdlnvqr kalheliqvm aelspaaktg
krkrsqmlfr grrasq Interferon Tau-IFNT Nucleotide: NCBI Reference
Sequence: NM_001015511.2 Bos taurus (SEQ ID NO: 9) 1 gatccccgga
aactagaatt cacctgaagg ttcacccaga ccccatctca gccagcccag 61
cagcagccac atcttcccca tggccttcgt gctctctcta ctgatggccc tggtgctggt
121 cagctacggc cagggacgat ctctgggttg ttacctgtct gaggaccaca
tgctaggtgc 181 cagggagaac ctcaggctcc tggcccgaat gaacagactc
tctcctcatc cctgtctgca 241 ggacagaaaa gactttggtc ttcctcagga
gatggtggag ggcaaccagc tccagaagga 301 tcaggctatc tctgtgctcc
acgagatgct ccagcagtgc ctcaacctct tctacacaga 361 gcactcgtct
gctgcctgga acaccaccct cctggagcag ctctgcactg ggctccaaca 421
gcagctggag gacctggacg cctgcctggg cccagtgatg ggagagaaag actctgacat
481 gggaaggatg ggccccattc tgactgtgaa gaagtacttc cagggtatcc
atgtctacct 541 gaaagaaaaa gaatacagtg actgcgcctg ggaaatcatc
agagtggaga tgatgagagc 601 cctctcttca tcaaccacct tgcaaaaaag
gttaagaaag atgggtggag atctgaactc 661 actttgagat gactctcgct
gactaagatg ccacatcacc ttcgtacact cacctgtgtt 721 catttcagaa
gactctgatt tctgcttcag ccaccgaaat cattgaatta ctttaactga 781
tactttgtca gcagtaataa gcaagtagat ataaaagtac tcagctgtag gggcatgagt
841 ccttaagtga tgcctgccct gatgttatct gttgttgatt tatgtattcc
ttcttgcatc 901 taacatactt aaaatattag gaaatttgta aagttacatt
tcatttgtac atctattaaa 961 atttctaaaa catgtttacc attttgtgtt
attaaatttg tcctttgttc tatttattaa 1021 atcaaagaaa atc Amino Acid:
GenBank: AAK53058.1 Bos taurus (SEQ ID NO: 10) 1 mkytsyilaf
qlcivlgslg cycqdpyvke aenlkkyfna ghsdvadngt lflgilknwk 61
eesdrkimqs qivsfyfklf knfkddqsiq ksvetikedm nvkffnsnkk krddfekltn
121 ysvtdlnvqr kalheliqvm aelspaaktg krkrsqmlfr grrasq Consensus
Interferon (conIFN-.alpha.) Amino Acid: (SEQ ID NO: 11) 1
cdlpqthslg nrralillaq mrrispfscl kdrhdfgfpq eefdgnqfqk aqaisvlhem
61 iqqrfnlfst kdssaawdes llekfytely qqlndleacv iqevgveetp
lmnvdsilav 121 kkyfqritly ltekkyspca wevvraeimr sfslstnlqe rlrrke
Sequence CWU 1
1
111876DNAHomo sapiens 1agaacctaga gcccaaggtt cagagtcacc catctcagca
agcccagaag tatctgcaat 60atctacgatg gcctcgccct ttgctttact gatggtcctg
gtggtgctca gctgcaagtc 120aagctgctct ctgggctgtg atctccctga
gacccacagc ctggataaca ggaggacctt 180gatgctcctg gcacaaatga
gcagaatctc tccttcctcc tgtctgatgg acagacatga 240ctttggattt
ccccaggagg agtttgatgg caaccagttc cagaaggctc cagccatctc
300tgtcctccat gagctgatcc agcagatctt caacctcttt accacaaaag
attcatctgc 360tgcttgggat gaggacctcc tagacaaatt ctgcaccgaa
ctctaccagc agctgaatga 420cttggaagcc tgtgtgatgc aggaggagag
ggtgggagaa actcccctga tgaatgcgga 480ctccatcttg gctgtgaaga
aatacttccg aagaatcact ctctatctga cagagaagaa 540atacagccct
tgtgcctggg aggttgtcag agcagaaatc atgagatccc tctctttatc
600aacaaacttg caagaaagat taaggaggaa ggaataacat ctggtccaac
atgaaaacaa 660ttcttattga ctcatacacc aggtcacgct ttcatgaatt
ctgtcatttc aaagactctc 720acccctgcta taactatgac catgctgata
aactgattta tctatttaaa tatttattta 780actattcata agatttaaat
tatttttgtt catataacgt catgtgcacc tttacactgt 840ggttagtgta
ataaaacatg ttccttatat ttactc 8762189PRTHomo sapiens 2Met Ala Ser
Pro Phe Ala Leu Leu Met Val Leu Val Val Leu Ser Cys 1 5 10 15 Lys
Ser Ser Cys Ser Leu Gly Cys Asp Leu Pro Glu Thr His Ser Leu 20 25
30 Asp Asn Arg Arg Thr Leu Met Leu Leu Ala Gln Met Ser Arg Ile Ser
35 40 45 Pro Ser Ser Cys Leu Met Asp Arg His Asp Phe Gly Phe Pro
Gln Glu 50 55 60 Glu Phe Asp Gly Asn Gln Phe Gln Lys Ala Pro Ala
Ile Ser Val Leu 65 70 75 80 His Glu Leu Ile Gln Gln Ile Phe Asn Leu
Phe Thr Thr Lys Asp Ser 85 90 95 Ser Ala Ala Trp Asp Glu Asp Leu
Leu Asp Lys Phe Cys Thr Glu Leu 100 105 110 Tyr Gln Gln Leu Asn Asp
Leu Glu Ala Cys Val Met Gln Glu Glu Arg 115 120 125 Val Gly Glu Thr
Pro Leu Met Asn Ala Asp Ser Ile Leu Ala Val Lys 130 135 140 Lys Tyr
Phe Arg Arg Ile Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser 145 150 155
160 Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Leu Ser
165 170 175 Leu Ser Thr Asn Leu Gln Glu Arg Leu Arg Arg Lys Glu 180
185 31143DNAHomo sapiens 3gagaacctgg agcctaaggt ttaggctcac
ccatttcaac cagtctagca gcatctgcaa 60catctacaat ggccttgacc tttgctttac
tggtggccct cctggtgctc agctgcaagt 120caagctgctc tgtgggctgt
gatctgcctc aaacccacag cctgggtagc aggaggacct 180tgatgctcct
ggcacagatg aggagaatct ctcttttctc ctgcttgaag gacagacatg
240actttggatt tccccaggag gagtttggca accagttcca aaaggctgaa
accatccctg 300tcctccatga gatgatccag cagatcttca atctcttcag
cacaaaggac tcatctgctg 360cttgggatga gaccctccta gacaaattct
acactgaact ctaccagcag ctgaatgacc 420tggaagcctg tgtgatacag
ggggtggggg tgacagagac tcccctgatg aaggaggact 480ccattctggc
tgtgaggaaa tacttccaaa gaatcactct ctatctgaaa gagaagaaat
540acagcccttg tgcctgggag gttgtcagag cagaaatcat gagatctttt
tctttgtcaa 600caaacttgca agaaagttta agaagtaagg aatgaaaact
ggttcaacat ggaaatgatt 660ttcattgatt cgtatgccag ctcacctttt
tatgatctgc catttcaaag actcatgttt 720ctgctatgac catgacacga
tttaaatctt ttcaaatgtt tttaggagta ttaatcaaca 780ttgtattcag
ctcttaaggc actagtccct tacagaggac catgctgact gatccattat
840ctatttaaat atttttaaaa tattatttat ttaactattt ataaaacaac
ttatttttgt 900tcatattatg tcatgtgcac ctttgcacag tggttaatgt
aataaaatat gttctttgta 960tttggtaaat ttattttgtg ttgttcattg
aacttttgct atggaaactt ttgtacttgt 1020ttattcttta aaatgaaatt
ccaagcctaa ttgtgcaacc tgattacaga ataactggta 1080cacttcattt
atccatcaat attatattca agatataagt aaaaataaac tttctgtaaa 1140cca
11434166PRTHomo sapiens 4Met Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu 1 5 10 15 Met Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys 20 25 30 Asp Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe 35 40 45 Gln Lys Ala Glu
Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile 50 55 60 Phe Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80
Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu 85
90 95 Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
Met 100 105 110 Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr 115 120 125 Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu
165 5840DNAHomo sapiens 5acattctaac tgcaaccttt cgaagccttt
gctctggcac aacaggtagt aggcgacact 60gttcgtgttg tcaacatgac caacaagtgt
ctcctccaaa ttgctctcct gttgtgcttc 120tccactacag ctctttccat
gagctacaac ttgcttggat tcctacaaag aagcagcaat 180tttcagtgtc
agaagctcct gtggcaattg aatgggaggc ttgaatactg cctcaaggac
240aggatgaact ttgacatccc tgaggagatt aagcagctgc agcagttcca
gaaggaggac 300gccgcattga ccatctatga gatgctccag aacatctttg
ctattttcag acaagattca 360tctagcactg gctggaatga gactattgtt
gagaacctcc tggctaatgt ctatcatcag 420ataaaccatc tgaagacagt
cctggaagaa aaactggaga aagaagattt caccagggga 480aaactcatga
gcagtctgca cctgaaaaga tattatggga ggattctgca ttacctgaag
540gccaaggagt acagtcactg tgcctggacc atagtcagag tggaaatcct
aaggaacttt 600tacttcatta acagacttac aggttacctc cgaaactgaa
gatctcctag cctgtgcctc 660tgggactgga caattgcttc aagcattctt
caaccagcag atgctgttta agtgactgat 720ggctaatgta ctgcatatga
aaggacacta gaagattttg aaatttttat taaattatga 780gttattttta
tttatttaaa ttttattttg gaaaataaat tatttttggt gcaaaagtca
8406187PRTHomo sapiens 6Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu
Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Met Ser Tyr Asn
Leu Leu Gly Phe Leu Gln Arg 20 25 30 Ser Ser Asn Phe Gln Cys Gln
Lys Leu Leu Trp Gln Leu Asn Gly Arg 35 40 45 Leu Glu Tyr Cys Leu
Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu 50 55 60 Ile Lys Gln
Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile 65 70 75 80 Tyr
Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser 85 90
95 Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val
100 105 110 Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys
Leu Glu 115 120 125 Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser
Leu His Leu Lys 130 135 140 Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu
Lys Ala Lys Glu Tyr Ser 145 150 155 160 His Cys Ala Trp Thr Ile Val
Arg Val Glu Ile Leu Arg Asn Phe Tyr 165 170 175 Phe Ile Asn Arg Leu
Thr Gly Tyr Leu Arg Asn 180 185 71240DNAHomo sapiens 7cacattgttc
tgatcatctg aagatcagct attagaagag aaagatcagt taagtccttt 60ggacctgatc
agcttgatac aagaactact gatttcaact tctttggctt aattctctcg
120gaaacgatga aatatacaag ttatatcttg gcttttcagc tctgcatcgt
tttgggttct 180cttggctgtt actgccagga cccatatgta aaagaagcag
aaaaccttaa gaaatatttt 240aatgcaggtc attcagatgt agcggataat
ggaactcttt tcttaggcat tttgaagaat 300tggaaagagg agagtgacag
aaaaataatg cagagccaaa ttgtctcctt ttacttcaaa 360ctttttaaaa
actttaaaga tgaccagagc atccaaaaga gtgtggagac catcaaggaa
420gacatgaatg tcaagttttt caatagcaac aaaaagaaac gagatgactt
cgaaaagctg 480actaattatt cggtaactga cttgaatgtc caacgcaaag
caatacatga actcatccaa 540gtgatggctg aactgtcgcc agcagctaaa
acagggaagc gaaaaaggag tcagatgctg 600tttcgaggtc gaagagcatc
ccagtaatgg ttgtcctgcc tgcaatattt gaattttaaa 660tctaaatcta
tttattaata tttaacatta tttatatggg gaatatattt ttagactcat
720caatcaaata agtatttata atagcaactt ttgtgtaatg aaaatgaata
tctattaata 780tatgtattat ttataattcc tatatcctgt gactgtctca
cttaatcctt tgttttctga 840ctaattaggc aaggctatgt gattacaagg
ctttatctca ggggccaact aggcagccaa 900cctaagcaag atcccatggg
ttgtgtgttt atttcacttg atgatacaat gaacacttat 960aagtgaagtg
atactatcca gttactgccg gtttgaaaat atgcctgcaa tctgagccag
1020tgctttaatg gcatgtcaga cagaacttga atgtgtcagg tgaccctgat
gaaaacatag 1080catctcagga gatttcatgc ctggtgcttc caaatattgt
tgacaactgt gactgtaccc 1140aaatggaaag taactcattt gttaaaatta
tcaatatcta atatatatga ataaagtgta 1200agttcacaac aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 12408166PRTHomo sapiens 8Met Lys Tyr Thr Ser
Tyr Ile Leu Ala Phe Gln Leu Cys Ile Val Leu 1 5 10 15 Gly Ser Leu
Gly Cys Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu 20 25 30 Asn
Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn 35 40
45 Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp
50 55 60 Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys
Leu Phe 65 70 75 80 Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser
Val Glu Thr Ile 85 90 95 Lys Glu Asp Met Asn Val Lys Phe Phe Asn
Ser Asn Lys Lys Lys Arg 100 105 110 Asp Asp Phe Glu Lys Leu Thr Asn
Tyr Ser Val Thr Asp Leu Asn Val 115 120 125 Gln Arg Lys Ala Ile His
Glu Leu Ile Gln Val Met Ala Glu Leu Ser 130 135 140 Pro Ala Ala Lys
Thr Gly Lys Arg Lys Arg Ser Gln Met Leu Phe Arg 145 150 155 160 Gly
Arg Arg Ala Ser Gln 165 91033DNABos taurus 9gatccccgga aactagaatt
cacctgaagg ttcacccaga ccccatctca gccagcccag 60cagcagccac atcttcccca
tggccttcgt gctctctcta ctgatggccc tggtgctggt 120cagctacggc
cagggacgat ctctgggttg ttacctgtct gaggaccaca tgctaggtgc
180cagggagaac ctcaggctcc tggcccgaat gaacagactc tctcctcatc
cctgtctgca 240ggacagaaaa gactttggtc ttcctcagga gatggtggag
ggcaaccagc tccagaagga 300tcaggctatc tctgtgctcc acgagatgct
ccagcagtgc ctcaacctct tctacacaga 360gcactcgtct gctgcctgga
acaccaccct cctggagcag ctctgcactg ggctccaaca 420gcagctggag
gacctggacg cctgcctggg cccagtgatg ggagagaaag actctgacat
480gggaaggatg ggccccattc tgactgtgaa gaagtacttc cagggtatcc
atgtctacct 540gaaagaaaaa gaatacagtg actgcgcctg ggaaatcatc
agagtggaga tgatgagagc 600cctctcttca tcaaccacct tgcaaaaaag
gttaagaaag atgggtggag atctgaactc 660actttgagat gactctcgct
gactaagatg ccacatcacc ttcgtacact cacctgtgtt 720catttcagaa
gactctgatt tctgcttcag ccaccgaaat cattgaatta ctttaactga
780tactttgtca gcagtaataa gcaagtagat ataaaagtac tcagctgtag
gggcatgagt 840ccttaagtga tgcctgccct gatgttatct gttgttgatt
tatgtattcc ttcttgcatc 900taacatactt aaaatattag gaaatttgta
aagttacatt tcatttgtac atctattaaa 960atttctaaaa catgtttacc
attttgtgtt attaaatttg tcctttgttc tatttattaa 1020atcaaagaaa atc
103310166PRTBos taurus 10Met Lys Tyr Thr Ser Tyr Ile Leu Ala Phe
Gln Leu Cys Ile Val Leu 1 5 10 15 Gly Ser Leu Gly Cys Tyr Cys Gln
Asp Pro Tyr Val Lys Glu Ala Glu 20 25 30 Asn Leu Lys Lys Tyr Phe
Asn Ala Gly His Ser Asp Val Ala Asp Asn 35 40 45 Gly Thr Leu Phe
Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp 50 55 60 Arg Lys
Ile Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe 65 70 75 80
Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr Ile 85
90 95 Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys
Arg 100 105 110 Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp
Leu Asn Val 115 120 125 Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val
Met Ala Glu Leu Ser 130 135 140 Pro Ala Ala Lys Thr Gly Lys Arg Lys
Arg Ser Gln Met Leu Phe Arg 145 150 155 160 Gly Arg Arg Ala Ser Gln
165 11166PRTArtificial SequenceSynthetic Construct 11Cys Asp Leu
Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu
Leu Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln
Gln Arg 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Ser 65 70 75 80 Leu Leu Glu Lys Phe Tyr Thr Glu Leu Tyr
Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val
Gly Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu
Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155
160 Arg Leu Arg Arg Lys Glu 165
* * * * *