U.S. patent application number 12/843395 was filed with the patent office on 2011-01-20 for vaccine composition containing synthetic adjuvant.
This patent application is currently assigned to INFECTIOUS DISEASE RESEARCH INSTITUTE. Invention is credited to Darrick Carter, Steven G. Reed.
Application Number | 20110014274 12/843395 |
Document ID | / |
Family ID | 41057395 |
Filed Date | 2011-01-20 |
United States Patent
Application |
20110014274 |
Kind Code |
A1 |
Reed; Steven G. ; et
al. |
January 20, 2011 |
VACCINE COMPOSITION CONTAINING SYNTHETIC ADJUVANT
Abstract
Compositions and methods, including vaccines and pharmaceutical
compositions for inducing or enhancing an immune response are
disclosed based on the discovery of useful immunological adjuvant
properties in a synthetic, glucopyranosyl lipid adjuvant (GLA) that
is provided in substantially homogeneous form. Chemically defined,
synthetic GLA offers a consistent vaccine component from lot to lot
without the fluctuations in contaminants or activity that
compromise natural-product adjuvants. Also provided are vaccines
and pharmaceutical compositions that include GLA and one or more of
an antigen, a Toll-like receptor (TLR) agonist, a co-adjuvant and a
carrier such as a pharmaceutical carrier.
Inventors: |
Reed; Steven G.; (Bellevue,
WA) ; Carter; Darrick; (Seattle, WA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE, SUITE 5400
SEATTLE
WA
98104
US
|
Assignee: |
INFECTIOUS DISEASE RESEARCH
INSTITUTE
Seattle
WA
|
Family ID: |
41057395 |
Appl. No.: |
12/843395 |
Filed: |
July 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12351710 |
Jan 9, 2009 |
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12843395 |
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12134127 |
Jun 5, 2008 |
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12351710 |
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12154663 |
May 22, 2008 |
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12134127 |
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11862122 |
Sep 26, 2007 |
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12154663 |
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60847404 |
Sep 26, 2006 |
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Current U.S.
Class: |
424/450 ;
424/231.1 |
Current CPC
Class: |
A61K 39/39 20130101;
Y02A 50/30 20180101; A61P 39/06 20180101; A61K 39/0005 20130101;
A61P 37/00 20180101; A61K 45/06 20130101; C12N 2760/16034 20130101;
A61K 39/008 20130101; A61K 2039/55572 20130101; A61K 2039/57
20130101; A61P 31/22 20180101; A61K 2039/53 20130101; A61P 37/04
20180101; A61P 11/08 20180101; A61K 39/04 20130101; C12N 7/00
20130101; C12N 2760/16071 20130101; A61K 39/145 20130101; A61K
2039/55566 20130101 |
Class at
Publication: |
424/450 ;
424/231.1 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 39/245 20060101 A61K039/245; A61P 31/22 20060101
A61P031/22; A61P 37/04 20060101 A61P037/04; A61P 39/06 20060101
A61P039/06 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made in part with government support
under Grant No. AI-25038 awarded by the National Institutes of
Health. The government has certain rights in this invention.
Claims
1-34. (canceled)
35. A pharmaceutical composition for inducing or enhancing an
immune response against herpes simplex virus comprising: (a) at
least one herpes simplex virus antigen; and (b) a lipid adjuvant of
the formula: ##STR00005## wherein: R.sup.1, R.sup.3, R.sup.5 and
R.sup.6 are C.sub.11-C.sub.20 alkyl; and R.sup.2 and R.sup.4 are
C.sub.12-C.sub.20 alkyl; or a pharmaceutically acceptable salt
thereof.
36. The composition of claim 35, wherein the at least one herpes
simplex virus antigen comprises at least one HSV-1 or HSV-2
antigen.
37. The composition of claim 35, wherein the at least one herpes
simplex virus antigen comprises whole live or inactivated
virus.
38. The composition of claim 35, wherein the at least one herpes
simplex virus antigen comprises at least one purified or
recombinant herpes simplex virus antigen selected from the group
consisting of an HSV immediate early protein, an HSV ICP27 protein
and an HSV gD protein, or combinations thereof.
39. The composition of claim 35, wherein the alkyl is straight
chain saturated alkyl.
40. The composition of claim 35, wherein R.sup.1, R.sup.3, R.sup.5
and R.sup.6 are undecyl and R.sup.2 and R.sup.4 are tridecyl.
41. The composition of claim 35, wherein the lipid adjuvant is
synthetic.
42. The composition of claim 35, wherein the lipid adjuvant is at
least 80% pure based on the total weight of lipid adjuvant species
in the composition.
43. The composition of claim 35, wherein the lipid adjuvant is at
least 95% pure based on the total weight of lipid adjuvant species
in the composition.
44. The composition of claim 35, further comprising a
pharmaceutically acceptable carrier or excipient.
45. The composition of claim 35, which is in a form of an
oil-in-water emulsion, a water-in-oil emulsion, a liposome, or a
microparticle.
46. The composition of claim 35, which is in a form of an
oil-in-water emulsion and the oil is a metabolizable oil.
47. The composition of claim 46, wherein the oil is squalene.
48. The composition of claim 35, further comprising an
antioxidant.
49. The composition of claim 35, further comprising
alpha-tocopherol.
50. The composition of claim 35, further comprising oil and
alpha-tocopherol where the oil:alpha-tocopherol are present in a
ratio of equal to or less than 1.
51. The composition of claim 35, which has been sterile
filtered.
52. The composition of claim 35, which is in the form of a stable
aqueous suspension of less than 0.2 um and further comprises at
least one component selected from the group consisting of
phospholipids, fatty acids, surfactants, detergents, saponins, and
fluorodated lipids.
53. A method for inducing or enhancing an immune response against a
herpes simplex virus comprising the steps of administering to a
subject in need thereof a composition of any one of claims 35-52.
Description
CROSS-REFERENCE(S) TO RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/351,710, filed Jan. 9, 2009, now pending;
which is a continuation of U.S. patent application Ser. No.
12/134,127 filed Jun. 5, 2008, now abandoned; and a
continuation-in-part of U.S. application Ser. No. 12/154,663, filed
May 22, 2008, now abandoned; and a continuation-in-part of U.S.
application Ser. No. 11/862,122 filed Sep. 26, 2007, now pending,
which claims the benefit under 35 U.S.C. .sctn.119(e) of U.S.
Provisional Patent Application No. 60/847,404 filed Sep. 26, 2006;
all of these applications are incorporated herein by reference in
their entireties.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to the field of pharmaceutical
and vaccine compositions. More specifically, embodiments described
herein relate to pharmaceutical and vaccine compositions, as well
as related prophylactic and therapeutic methods, wherein the
compositions comprise a glucopyranosyl lipid adjuvant (GLA).
[0005] 2. Description of the Related Art
[0006] The immune system of higher organisms has been characterized
as distinguishing foreign agents (or "non-self") agents from
familiar or "self" components, such that foreign agents elicit
immune responses while "self" components are ignored or tolerated.
Immune responses have traditionally been characterized as either
humoral responses, in which antibodies specific for antigens are
produced by differentiated B lymphocytes known as plasma cells, or
cell mediated responses, in which various types of T lymphocytes
act to eliminate antigens by a number of mechanisms. For example,
CD4+ helper T cells that are capable of recognizing specific
antigens may respond by releasing soluble mediators such as
cytokines to recruit additional cells of the immune system to
participate in an immune response. Also, CD8+ cytotoxic T cells
that are also capable of specific antigen recognition may respond
by binding to and destroying or damaging an antigen-bearing cell or
particle. It is known in the immunological arts to provide certain
vaccines according to a variety of formulations, usually for the
purpose of inducing a desired immune response in a host.
[0007] Several strategies for eliciting specific immune responses
through the administration of a vaccine to a host include
immunization with heat-killed or with live, attenuated infectious
pathogens such as viruses, bacteria or certain eukaryotic
pathogens; immunization with a non-virulent infective agent capable
of directing the expression of genetic material encoding the
antigen(s) to which an immune response is desired; and immunization
with subunit vaccines that contain isolated immunogens (such as
proteins) from a particular pathogen in order to induce immunity
against the pathogen. (See, e.g., Liu, 1998 Nature Medicine 4(5
suppl.):515.) For certain antigens there may be one or more types
of desirable immunity for which none of these approaches has been
particularly effective, including the development of vaccines that
are effective in protecting a host immunologically against human
immunodeficiency viruses or other infectious pathogens, cancer,
autoimmune disease, or other clinical conditions.
[0008] It has long been known that enterobacterial
lipopolysaccharide (LPS) is a potent stimulator of the immune
system, although its use in adjuvants has been curtailed by its
toxic effects. A non-toxic derivative of LPS, monophosphoryl lipid
A (MPL), produced by removal of the core carbohydrate group and the
phosphate from the reducing-end glucosamine, has been described by
Ribi et al (1986, Immunology and Immunopharmacology of Bacterial
Endotoxins, Plenum Publ. Corp., NY, p 407-419).
[0009] A further detoxified version of MPL results from the removal
of the acyl chain from the 3-position of the disaccharide backbone,
and is called 3-O-deacylated monophosphoryl lipid A (3D-MPL). It
can be purified and prepared by the methods taught in GB 2122204B,
which reference also discloses the preparation of diphosphoryl
lipid A, and 3-O-deacylated variants thereof. For example, 3D-MPL
has been prepared in the form of an emulsion having a small
particle size less than 0.2 .mu.m in diameter, and its method of
manufacture is disclosed in WO 94/21292. Aqueous formulations
comprising monophosphoryl lipid A and a surfactant have been
described in WO9843670A2.
[0010] Bacterial lipopolysaccharide-derived adjuvants to be
formulated in adjuvant combinations may be purified and processed
from bacterial sources, or alternatively they may be synthetic. For
example, purified monophosphoryl lipid A is described in Ribi et at
1986 (supra), and 3-O-deacylated monophosphoryl or diphosphoryl
lipid A derived from Salmonella sp. is described in GB 2220211 and
U.S. Pat. No. 4,912,094. 3D-MPL and the .beta.(1-6) glucosamine
disaccharides as well as other purified and synthetic
lipopolysaccharides have been described (WO 98/01139; U.S. Pat. No.
6,005,099 and EP 0 729 473 B1, Hilgers et al., 1986 Int. Arch.
Allergy Immunol., 79(4):392-6; Hilgers et at., 1987, Immunology,
60(1); 141-6; and EP 0 549 074 B1). Combinations of 3D-MPL and
saponin adjuvants derived from the bark of Quillaja Saponaria
molina have been described in EP 0 761 231B. WO 95/17210 discloses
an adjuvant emulsion system based on squalene, .alpha.-tocopherol,
and polyoxyethylene sorbitan monooleate (TWEEN.TM.-80), formulated
with the immunostimulant QS21, and optionally including 3D-MPL.
Despite the accessibility of such combinations, the use of
adjuvants derived from natural products is accompanied by high
production costs, inconsistency from lot to lot, difficulties
associated with large-scale production, and uncertainty with
respect to the presence of impurities in the compositional make-up
of any given preparation.
[0011] Clearly there is a need for improved vaccines, and in
particular for vaccines that beneficially contain high-purity,
chemically defined adjuvant components that exhibit lot-to-lot
consistency and that can be manufactured efficiently on an
industrial scale without introducing unwanted or structurally
undefined contaminants. The present invention provides compositions
and methods for such vaccines, and offers other related
advantages.
BRIEF SUMMARY OF THE INVENTION
[0012] The present invention in its several embodiments is directed
to compositions and methods that advantageously employ the
synthetic glucopyranosyl lipid adjuvant (GLA) as an adjuvant and
vaccine component. According to one embodiment of the invention
described herein, there is provided a vaccine composition
comprising an antigen and a glucopyranosyl lipid adjuvant
(GLA).
[0013] In other embodiments there is provided a vaccine composition
comprising (a) an antigen; a glucopyranosyl lipid adjuvant (GLA);
and a toll-like receptor (TLR) agonist, wherein in certain further
embodiments the TLR agonist is selected from lipopolysaccharide,
peptidoglycan, polyl:C, CpG, 3M003, flagellin, Leishmania homolog
of eukaryotic ribosomal elongation and initiation factor 4a (LeIF)
and at least one hepatitis C antigen. In another embodiment there
is provided a vaccine composition comprising: an antigen; a
glucopyranosyl lipid adjuvant (GLA); and at least one co-adjuvant
that is selected from saponins and saponin mimetics. In another
embodiment there is provided a vaccine composition comprising an
antigen; a glucopyranosyl lipid adjuvant (GLA); and a carrier that
comprises at least one of an oil and ISCOMATRIX.TM.. In another
embodiment there is provided a vaccine composition comprising an
antigen; a glucopyranosyl lipid adjuvant (GLA); and one or more of:
(i) at least one co-adjuvant, (ii) at least one TLR agonist, (iii)
at least one imidazoquinoline immune response modifier, and (iv) at
least one double stem loop immune modifier (dSLIM). In certain
further embodiments (i) the co-adjuvant, when present, is selected
from alum, a plant alkaloid and a detergent, wherein the plant
alkaloid is selected from tomatine and the detergent is selected
from saponin, Polysorbate 80, Span 85 and Stearyl tyrosine, (ii)
the TLR agonist, when present, is selected from lipopolysaccharide,
peptidoglycan, polyl:C, CpG, 3M003, flagellin, Leishmania homolog
of eukaryotic ribosomal elongation and initiation factor 4a (LeIF)
and at least one hepatitis C antigen, and (iii) the
imidazoquinoline immune response modifier, when present, is
selected from resiquimod (R848), imiquimod and gardiquimod. In
another embodiment there is provided a vaccine composition
comprising: an antigen; a glucopyranosyl lipid adjuvant (GLA); and
at least one of a co-adjuvant and a pharmaceutically acceptable
carrier, wherein: the co-adjuvant is selected from a cytokine, a
detergent, and a block copolymer or biodegradable polymer, and the
pharmaceutically acceptable carrier comprises a carrier that is
selected from calcium phosphate, an oil-in-water emulsion, a
water-in-oil emulsion, a liposome, a novosome, a non-ionic
surfactant vesicle (e.g., niosome) and a microparticle. In a
particular embodiment, where a liposome or similar carrier is used,
the GLA is in the laminar structure of the liposome or is
encapsulated. In another particular embodiment, where a
microparticle is used, the microparticle is one that is based on or
comprises polymer fat lipids.
[0014] In certain further embodiments the cytokine is selected from
GM-CSF, IL-2, IL-7, IL-12, TNF-.alpha. and IFN-gamma, the block
copolymer or biodegradable polymer is selected from Pluronic L121,
CRL1005, PLGA, PLA, PLG, and polyl:C, and the detergent is selected
from the group consisting of saponin, Polysorbate 80, Span 85 and
Stearyl tyrosine.
[0015] In other embodiments there is provided a vaccine composition
comprising: at least one recombinant expression construct which
comprises a promoter operably linked to a nucleic acid sequence
encoding an antigen; and a glucopyranosyl lipid adjuvant (GLA). In
one embodiment the recombinant expression construct is present in a
viral vector, which in certain further embodiments is present in a
virus that is selected from an adenovirus, an adeno-associated
virus, a herpesvirus, a lentivirus, a poxvirus, and a
retrovirus.
[0016] According to certain of any of the above described
embodiments, the GLA is not 3'-de-O-acylated. According to certain
of any of the above described embodiments, the GLA comprises: (i) a
diglucosamine backbone having a reducing terminus glucosamine
linked to a non-reducing terminus glucosamine through an ether
linkage between hexosamine position 1 of the non-reducing terminus
glucosamine and hexosamine position 6 of the reducing terminus
glucosamine; (ii) an O-phosphoryl group attached to hexosamine
position 4 of the non-reducing terminus glucosamine; and (iii) up
to six fatty acyl chains; wherein one of the fatty acyl chains is
attached to 3-hydroxy of the reducing terminus glucosamine through
an ester linkage, wherein one of the fatty acyl chains is attached
to a 2-amino of the non-reducing terminus glucosamine through an
amide linkage and comprises a tetradecanoyl chain linked to an
alkanoyl chain of greater than 12 carbon atoms through an ester
linkage, and wherein one of the fatty acyl chains is attached to
3-hydroxy of the non-reducing terminus glucosamine through an ester
linkage and comprises a tetradecanoyl chain linked to an alkanoyl
chain of greater than 12 carbon atoms through an ester linkage.
[0017] According to certain of any of the above described
embodiments that include a TLR agonist, the TLR agonist is capable
of delivering a biological signal by interacting with at least one
TLR that is selected from TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7,
TLR-8 and TLR-9. In certain further embodiments the TLR agonist is
selected from lipopolysaccharide, peptidoglycan, polyl:C, CpG,
3M003, flagellin, Leishmania homolog of eukaryotic ribosomal
elongation and initiation factor 4a (LeIF) and at least one
hepatitis C antigen. In a particular embodiment, where a TLR-7
and/or TLR-8 agonist is used, the TLR-7 and/or TLR-8 agonist is
entrapped within a vesicle.
[0018] According to certain of any of the above described
embodiments, the GLA has the formula:
##STR00001##
where: R.sup.1, R.sup.3, R.sup.5 and R.sup.6 are C.sub.11-C.sub.20
alkyl; and R.sup.2 and R.sup.4 are C.sub.12-C.sub.20 alkyl.
[0019] According to certain of any of the above described
embodiments, the vaccine composition is capable of eliciting an
immune response in a host. In certain further embodiments the
immune response is specific for the antigen. According to certain
of any of the above described embodiments, the antigen is capable
of eliciting in a host an immune response that is selected from a
humoral response and a cell-mediated response. According to certain
of any of the above described embodiments, the vaccine composition
is capable of eliciting in a host at least one immune response that
is selected from a T.sub.H1-type T lymphocyte response, a
T.sub.H2-type T lymphocyte response, a cytotoxic T lymphocyte (CTL)
response, an antibody response, a cytokine response, a lymphokine
response, a chemokine response, and an inflammatory response.
According to certain of any of the above described embodiments, the
vaccine composition is capable of eliciting in a host at least one
immune response that is selected from (a) production of one or a
plurality of cytokines wherein the cytokine is selected from
interferon-gamma (IFN-.gamma.), tumor necrosis factor-alpha
(TNF-.alpha.), (b) production of one or a plurality of interleukins
wherein the interleukin is selected from IL-1, IL-2, IL-3, IL-4,
IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18 and IL-23, (c)
production one or a plurality of chemokines wherein the chemokine
is selected from MIP-1.alpha., MIP-1.beta., RANTES, CCL4 and CCL5,
and (d) a lymphocyte response that is selected from a memory T cell
response, a memory B cell response, an effector T cell response, a
cytotoxic T cell response and an effector B cell response.
[0020] According to certain of any of the above described
embodiments, the antigen is derived from at least one infectious
pathogen that is selected from a bacterium, a virus, and a
fungus.
[0021] In certain further embodiments the bacterium is an
Actinobacterium, and in certain still further embodiments the
Actinobacterium is a mycobacterium. In certain other related
embodiments the mycobacterium is selected from M. tuberculosis and
M. leprae. In certain other related embodiments the bacterium is
selected from Salmonella, Neisseria, Borrelia, Chlamydia and
Bordetella.
[0022] In certain other related embodiments the virus is selected
from a herpes simplex virus, a human immunodeficiency virus (HIV),
a feline immunodeficiency virus (FIV), cytomegalovirus, Varicella
Zoster Virus, hepatitis virus, Epstein Barr Virus (EBV),
respiratory syncytial virus, human papilloma virus (HPV) and a
cytomegalovirus. According to certain of any of the above described
embodiments, the antigen is derived from a human immunodeficiency
virus, which in certain further embodiments is selected from HIV-1
and HIV-2.
[0023] In certain other related embodiments the fungus is selected
from Aspergillus, Blastomyces, Coccidioides and Pneumocystis. In
certain other related embodiments the fungus is a yeast, which in
certain further embodiments is a Candida, wherein in certain still
further embodiments the Candida is selected from C. albicans, C.
glabrata, C. krusei, C. lusitaniae, C. tropicalis and C.
parapsilosis.
[0024] According to certain of any of the above described
embodiments, the antigen is derived from a parasite, which in
certain further embodiments is a protozoan, which in certain
further embodiments is a Plasmodium, which in certain still further
embodiments is selected from P. falciparum, P. vivax, P. malariae
and P. ovale. In certain other embodiments the parasite is selected
from Acanthamoeba, Entamoeba histolytica, Angiostrongylus,
Schistosoma mansonii, Schistosoma haematobium, Schistosoma
japonicum, Schistosoma mekongi, Cryptosporidium, Ancylostoma,
Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba
hartmanni, Entamoeba polecki, Wuchereria bancrofti, Giardia,
Leishmania, Enterobius vermicularis, Ascaris lumbricoides,
Trichuris trichuria, Necator americanus, Ancylostoma duodenale,
Brugia malayi, Onchocerca volvulus, Dracanculus medinensis,
Trichinella spiralis, Strongyloides stercoralis, Opisthorchis
sinensis, Paragonimus sp, Fasciola hepatica, Fasciola magna,
Fasciola gigantica), Taenia saginata and Taenia solium.
[0025] According to certain of any of the above described
embodiments, the antigen is derived from at least one cancer cell.
In certain further embodiments the cancer cell originates in a
primary solid tumor, and in certain other embodiments the cancer
cell originates in a cancer that is a metastatic or secondary solid
tumor, and in certain other embodiments the cancer cell originates
in a cancer that is a circulating tumor or an ascites tumor. In
certain related embodiments the cancer cell originates in a cancer
that is selected from cervical cancer, ovarian cancer, breast
cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, pseudomyxoma petitonei,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma and Wilms' tumor. In certain other related embodiments
the cancer cell originates in a cancer that is selected from
testicular tumor, lung carcinoma, small cell lung carcinoma,
bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oliodendroglioma, meningioma,
melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma,
multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain
disease.
[0026] According to certain of any of the above described
embodiments, the antigen is derived from, or is immunologically
cross-reactive with, at least one epitope, biomolecule, cell or
tissue that is associated with an autoimmune disease. In certain
further embodiments the epitope, biomolecule, cell or tissue that
is associated with an autoimmune disease is selected from snRNP
when the autoimmune disease is systemic lupus erythematosus, at
least one of thyroglobulin, thyrotropin receptor and a thyroid
epithelial cell when the autoimmune disease is Graves' disease, a
platelet when the autoimmune disease is thrombocytopenic purpura,
at least one of pemphigus antigen, desmoglein-3, desmoplakin,
envoplakin and bullous pemphigoid antigen 1 when the autoimmune
disease is pemphigus, myelin basic protein when the autoimmune
disease is multiple sclerosis, a pancreatic islet beta cell when
the autoimmune disease is type 1 diabetes, and an acetylcholine
receptor when the autoimmune disease is myasthenia gravis.
[0027] In another embodiment there is provided a pharmaceutical
composition for inducing or enhancing an immune response,
comprising a glucopyranosyl lipid adjuvant (GLA); and a
pharmaceutically acceptable carrier or excipient. In another
embodiment there is provided a pharmaceutical composition for
inducing or enhancing an immune response comprising an antigen; a
glucopyranosyl lipid adjuvant (GLA); and a pharmaceutically
acceptable carrier or excipient. In another embodiment there is
provided a pharmaceutical composition for inducing or enhancing an
immune response comprising an antigen; a glucopyranosyl lipid
adjuvant (GLA); a toll-like receptor (TLR) agonist; and a
pharmaceutically acceptable carrier or excipient. In a further
embodiment the TLR agonist is selected from lipopolysaccharide,
peptidoglycan, polyl:C, CpG, 3M003, flagellin, Leishmania homolog
of eukaryotic ribosomal elongation and initiation factor 4a (LeIF)
and at least one hepatitis C antigen. In another embodiment there
is provided a pharmaceutical composition for inducing or enhancing
an immune response comprising: an antigen; a glucopyranosyl lipid
adjuvant (GLA); at least one co-adjuvant that is selected from
saponins and saponin mimetics; and a pharmaceutically acceptable
carrier or excipient. In another embodiment there is provided a
pharmaceutical composition for inducing or enhancing an immune
response comprising antigen; a glucopyranosyl lipid adjuvant (GLA);
and a pharmaceutically acceptable carrier that comprises at least
one of an oil and ISCOMATRIX.TM.. In another embodiment there is
provided a pharmaceutical composition for inducing or enhancing an
immune response comprising: (a) an antigen; (b) a glucopyranosyl
lipid adjuvant (GLA); (c) one or more of: (i) at least one
co-adjuvant, (ii) at least one TLR agonist, (iii) at least one
imidazoquinoline immune response modifier, and (iv) at least one
double stem loop immune modifier (dSLIM); and (d) a
pharmaceutically acceptable carrier or excipient. In certain
further embodiments (i) the co-adjuvant, when present, is selected
from alum, a plant alkaloid and a detergent, wherein the plant
alkaloid is tomatine and the detergent is selected from saponin,
Polysorbate 80, Span 85 and Stearyl tyrosine, (ii) the TLR agonist,
when present, is selected from lipopolysaccharide, peptidoglycan,
polyl:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic
ribosomal elongation and initiation factor 4a (LeIF) and at least
one hepatitis C antigen, and (iii) the imidazoquinoline immune
response modifier, when present, is selected from resiquimod
(R848), imiquimod and gardiquimod.
[0028] In another embodiment there is provided a pharmaceutical
composition for inducing or enhancing an immune response,
comprising: an antigen; a glucopyranosyl lipid adjuvant (GLA); and
at least one co-adjuvant; and a pharmaceutically acceptable
carrier, wherein: the co-adjuvant is selected from a cytokine, a
block copolymer or biodegradable polymer, and a detergent, and the
pharmaceutically acceptable carrier comprises a carrier that is
selected from calcium phosphate, an oil-in-water emulsion, a
water-in-oil emulsion, a liposome, and a microparticle. In certain
further embodiments the cytokine is selected from GM-CSF, IL-2,
IL-7, IL-12, TNF and IFN-gamma, the block copolymer or
biodegradable polymer is selected from Pluronic.RTM. L121, CRL1005,
PLGA, PLA, PLG, and polyl:C, and the detergent is selected from the
group consisting of saponin, Polysorbate 80, Span 85 and Stearyl
tyrosine.
[0029] In another embodiment there is provided a pharmaceutical
composition comprising: at least one recombinant expression
construct which comprises a promoter operably linked to a nucleic
acid sequence encoding an antigen; a glucopyranosyl lipid adjuvant
(GLA); and a pharmaceutically acceptable carrier or excipient. In
certain further embodiments the recombinant expression construct is
present in a viral vector, which in certain further embodiments is
present in a virus that is selected from an adenovirus, an
adeno-associated virus, a herpesvirus, a lentivirus, a poxvirus,
and a retrovirus.
[0030] According to certain further embodiments of the
above-described pharmaceutical compositions, the antigen and the
GLA are in contact with one another, and according to certain other
further embodiments of the above-described pharmaceutical
compositions, the antigen and the GLA are not in contact with one
another. In certain further embodiments wherein the antigen and the
GLA are not in contact with one another, they are present in
separate containers. In other embodiments there is provided a
pharmaceutical composition for inducing or enhancing an immune
response comprising a first combination comprising an antigen and a
first pharmaceutically acceptable carrier or excipient; and a
second combination comprising a glucopyranosyl lipid adjuvant (GLA)
and a second pharmaceutically acceptable carrier or excipient,
wherein the antigen and the GLA are not in contact with one
another. In a further embodiment the antigen and the GLA are
present in separate containers. In certain related embodiments the
first pharmaceutically acceptable carrier or excipient is different
from the second pharmaceutically acceptable carrier or excipient.
In other related embodiments the first pharmaceutically acceptable
carrier or excipient is not different from the second
pharmaceutically acceptable carrier or excipient.
[0031] In another embodiment there is provided a method of treating
or preventing an infectious disease in a subject having or
suspected of being at risk for having the infectious disease, the
method comprising administering to the subject a vaccine
composition that comprises (a) an antigen; and (b) a glucopyranosyl
lipid adjuvant (GLA), wherein the antigen is derived from, or is
immunologically cross-reactive with, at least one infectious
pathogen that is associated with the infectious disease, and
thereby treating or preventing the infectious disease. In another
embodiment there is provided a method of treating or preventing an
infectious disease in a subject having or suspected of being at
risk for having the infectious disease, the method comprising
administering to the subject a vaccine composition that comprises
(a) an antigen; (b) a glucopyranosyl lipid adjuvant (GLA); and (c)
a toll-like receptor (TLR) agonist, wherein the antigen is derived
from, or is immunologically cross-reactive with, at least one
infectious pathogen that is associated with the infectious disease,
and thereby treating or preventing the infectious disease. In a
further embodiment the TLR agonist is selected from
lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003, flagellin,
Leishmania homolog of eukaryotic ribosomal elongation and
initiation factor 4a (LeIF) and at least one hepatitis C antigen.
In another embodiment there is provided a method of treating or
preventing an infectious disease in a subject having or suspected
of being at risk for having the infectious disease, the method
comprising administering to the subject a vaccine composition that
comprises (a) an antigen; (b) a glucopyranosyl lipid adjuvant
(GLA); and (c) at least one co-adjuvant that is selected from the
group consisting of saponins and saponin mimetics, wherein the
antigen is derived from, or is immunologically cross-reactive with,
at least one infectious pathogen that is associated with the
infectious disease, and thereby treating or preventing the
infectious disease. In another embodiment there is provided a
method of treating or preventing an infectious disease in a subject
having or suspected of being at risk for having the infectious
disease, the method comprising administering to the subject a
vaccine composition that comprises (a) an antigen; (b) a
glucopyranosyl lipid adjuvant (GLA); and (c) a carrier that
comprises at least one of an oil and ISCOMATRIX.TM., wherein the
antigen is derived from, or is immunologically cross-reactive with,
at least one infectious pathogen that is associated with the
infectious disease, and thereby treating or preventing the
infectious disease. In another embodiment there is provided a
method of treating or preventing an infectious disease in a subject
having or suspected of being at risk for having the infectious
disease, the method comprising administering to the subject a
vaccine composition that comprises (a) an antigen; (b) a
glucopyranosyl lipid adjuvant (GLA); and (c) one or more of: (i) at
least one co-adjuvant, (ii) at least one TLR agonist, (iii) at
least one imidazoquinoline immune response modifier, and (iv) at
least one double stem loop immune modifier (dSLIM), wherein the
antigen is derived from, or is immunologically cross-reactive with,
at least one infectious pathogen that is associated with the
infectious disease, and thereby treating or preventing the
infectious disease. In certain further embodiments, (i) the
co-adjuvant, when present, is selected from alum, a plant alkaloid
and a detergent, wherein the plant alkaloid is tomatine and the
detergent is selected from saponin, Polysorbate 80, Span 85 and
Stearyl tyrosine, (ii) the TLR agonist, when present, is selected
from the group consisting of lipopolysaccharide, peptidoglycan,
polyl:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic
ribosomal elongation and initiation factor 4a (LeIF) and at least
one hepatitis C antigen, and (iii) the imidazoquinoline immune
response modifier, when present, is selected from the group
consisting of resiquimod (R848), imiquimod and gardiquimod.
[0032] In another embodiment there is provided a method of treating
or preventing an infectious disease in a subject having or
suspected of being at risk for having the infectious disease, the
method comprising administering to the subject a vaccine
composition that comprises (a) an antigen; (b) a glucopyranosyl
lipid adjuvant (GLA); and (c) at least one of a co-adjuvant and a
pharmaceutically acceptable carrier, wherein: the co-adjuvant is
selected from a cytokine, a block copolymer or biodegradable
polymer, and a detergent, and the pharmaceutically acceptable
carrier comprises a carrier that is selected from the group
consisting of calcium phosphate, an oil-in-water emulsion, a
water-in-oil emulsion, a liposome, and a microparticle, wherein the
antigen is derived from, or is immunologically cross-reactive with,
at least one infectious pathogen that is associated with the
infectious disease, and thereby treating or preventing the
infectious disease. In certain further embodiments the cytokine is
selected from GM-CSF, IL-2, IL-7, IL-12, TNF-.alpha. and IFN-gamma,
the block copolymer or biodegradable polymer is selected from
Pluronic L121, CRL1005, PLGA, PLA, PLG, and polyl:C, and the
detergent is selected from the group consisting of saponin,
Polysorbate 80, Span 85 and Stearyl tyrosine.
[0033] In another embodiment there is provided a method of treating
or preventing an infectious disease in a subject having or
suspected of being at risk for having the infectious disease, the
method comprising administering to the subject a vaccine
composition that comprises (a) at least one recombinant expression
construct which comprises a promoter operably linked to a nucleic
acid sequence encoding an antigen; and (b) a glucopyranosyl lipid
adjuvant (GLA), wherein the antigen is derived from, or is
immunologically cross-reactive with, at least one infectious
pathogen that is associated with the infectious disease, and
thereby treating or preventing the infectious disease. In a further
embodiment the recombinant expression construct is present in a
viral vector, which in certain still further embodiments is present
in a virus that is selected from an adenovirus, an adeno-associated
virus, a herpesvirus, a lentivirus, a poxvirus, and a retrovirus.
According to certain embodiments relating to the above described
methods, the antigen is derived from at least one infectious
pathogen that is selected from a bacterium, a virus, and a
fungus.
[0034] In another embodiment there is provided a method of treating
or preventing autoimmune disease in a subject having or suspected
of being at risk for having an autoimmune disease, the method
comprising administering to the subject a vaccine composition that
comprises (a) an antigen; and (b) a glucopyranosyl lipid adjuvant
(GLA), wherein the antigen is derived from, or is immunologically
cross-reactive with, at least one epitope, biomolecule, cell or
tissue that is associated with the autoimmune disease, and thereby
treating or preventing the autoimmune disease. In another
embodiment there is provided a method of treating or preventing an
autoimmune disease in a subject having or suspected of being at
risk for having an autoimmune disease, the method comprising
administering to the subject a vaccine composition that comprises
(a) an antigen; (b) a glucopyranosyl lipid adjuvant (GLA); and (c)
a toll-like receptor (TLR) agonist, wherein the antigen is derived
from, or is immunologically cross-reactive with, at least one
epitope, biomolecule, cell or tissue that is associated with the
autoimmune disease, and thereby treating or preventing the
autoimmune disease. In certain further embodiments the TLR agonist
is selected from lipopolysaccharide, peptidoglycan, polyl:C, CpG,
3M003, flagellin, Leishmania homolog of eukaryotic ribosomal
elongation and initiation factor 4a (LeIF) and at least one
hepatitis C antigen. In another embodiment there is provided a
method of treating or preventing an autoimmune disease in a subject
having or suspected of being at risk for having an autoimmune
disease, the method comprising administering to the subject a
vaccine composition that comprises (a) an antigen; (b) a
glucopyranosyl lipid adjuvant (GLA); and (c) at least one
co-adjuvant that is selected from the group consisting of saponins
and saponin mimetics, wherein the antigen is derived from, or is
immunologically cross-reactive with, at least one epitope,
biomolecule, cell or tissue that is associated with the autoimmune
disease, and thereby treating or preventing the autoimmune disease.
In another embodiment there is provided a method of treating or
preventing an autoimmune disease in a subject having or suspected
of being at risk for having an autoimmune disease, the method
comprising administering to the subject a vaccine composition that
comprises (a) an antigen; (b) a glucopyranosyl lipid adjuvant
(GLA); and (c) a carrier that comprises at least one of an oil and
ISCOMATRIX.TM., wherein the antigen is derived from, or is
immunologically cross-reactive with, at least one epitope,
biomolecule, cell or tissue that is associated with the autoimmune
disease, and thereby treating or preventing the autoimmune disease.
In another embodiment there is provided a method of treating or
preventing an autoimmune disease in a subject having or suspected
of being at risk for having an autoimmune disease, the method
comprising administering to the subject a vaccine composition that
comprises (a) an antigen; (b) a glucopyranosyl lipid adjuvant
(GLA); and (c) one or more of: (i) at least one co-adjuvant, (ii)
at least one TLR agonist, (iii) at least one imidazoquinoline
immune response modifier, and (iv) at least one double stem loop
immune modifier (dSLIM), wherein the antigen is derived from, or is
immunologically cross-reactive with, at least one epitope,
biomolecule, cell or tissue that is associated with the autoimmune
disease, and thereby treating or preventing the autoimmune disease.
In certain further embodiments (i) the co-adjuvant, when present,
is selected from alum, a plant alkaloid and a detergent, wherein
the plant alkaloid is tomatine and the detergent is selected from
saponin, Polysorbate 80, Span 85 and Stearyl tyrosine, (ii) the TLR
agonist, when present, is selected from the group consisting of
lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003, flagellin,
Leishmania homolog of eukaryotic ribosomal elongation and
initiation factor 4a (LeIF) and at least one hepatitis C antigen,
and (iii) the imidazoquinoline immune response modifier, when
present, is selected from the group consisting of resiquimod
(R848), imiquimod and gardiquimod.
[0035] In another embodiment there is provided a method of treating
or preventing an autoimmune disease in a subject having or
suspected of being at risk for having an autoimmune disease, the
method comprising administering to the subject a vaccine
composition that comprises (a) an antigen; (b) a glucopyranosyl
lipid adjuvant (GLA); and (c) at least one of a co-adjuvant and a
pharmaceutically acceptable carrier, wherein: the co-adjuvant is
selected from a cytokine, a block copolymer or biodegradable
polymer, and a detergent, and the pharmaceutically acceptable
carrier comprises a carrier that is selected from the group
consisting of calcium phosphate, an oil-in-water emulsion, a
water-in-oil emulsion, a liposome, and a microparticle, wherein the
antigen is derived from, or is immunologically cross-reactive with,
at least one epitope, biomolecule, cell or tissue that is
associated with the autoimmune disease, and thereby treating or
preventing the autoimmune disease. In a further embodiment the
cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-.alpha.
and IFN-gamma, the block copolymer or biodegradable polymer is
selected from Pluronic L121, CRL1005, PLGA, PLA, PLG, and polyl:C,
and the detergent is selected from the group consisting of saponin,
Polysorbate 80, Span 85 and Stearyl tyrosine.
[0036] In another embodiment there is provided a method of treating
or preventing an autoimmune disease in a subject having or
suspected of being at risk for having an autoimmune disease, the
method comprising administering to the subject a vaccine
composition that comprises (a) at least one recombinant expression
construct which comprises a promoter operably linked to a nucleic
acid sequence encoding an antigen; and (b) a glucopyranosyl lipid
adjuvant (GLA), wherein the antigen is derived from, or is
immunologically cross-reactive with, at least one epitope,
biomolecule, cell or tissue that is associated with the autoimmune
disease, and thereby treating or preventing the autoimmune disease.
In a further embodiment the recombinant expression construct is
present in a viral vector, which in certain further embodiments is
present in a virus that is selected from an adenovirus, an
adeno-associated virus, a herpesvirus, a lentivirus, a poxvirus,
and a retrovirus.
[0037] In certain of the above described embodiments as relate to a
method of treating or preventing an autoimmune disease, the
autoimmune disease is selected from Type 1 diabetes, rheumatoid
arthritis, multiple sclerosis, systemic lupus erythematosus,
myasthenia gravis, Crohn's disease, Graves' disease,
thrombocytopenic purpura and pemphigus. In certain other of the
above described embodiments as relate to a method of treating or
preventing an autoimmune disease, the epitope, biomolecule, cell or
tissue that is associated with an autoimmune disease is selected
from snRNP when the autoimmune disease is systemic lupus
erythematosus, at least one of thyroglobulin, thyrotropin receptor
and a thyroid epithelial cell when the autoimmune disease is
Graves' disease, a platelet when the autoimmune disease is
thrombocytopenic purpura, at least one of pemphigus antigen,
desmoglein-3, desmoplakin, envoplakin and bullous pemphigoid
antigen 1 when the autoimmune disease is pemphigus, myelin basic
protein when the autoimmune disease is multiple sclerosis, a
pancreatic islet beta cell when the autoimmune disease is type 1
diabetes, and an acetylcholine receptor when the autoimmune disease
is myasthenia gravis.
[0038] According to other embodiments there is provided a method of
treating or preventing cancer in a subject having or suspected of
being at risk for having an cancer, the method comprising
administering to the subject a vaccine composition that comprises
(a) an antigen; and (b) a glucopyranosyl lipid adjuvant (GLA),
wherein the antigen is derived from, or is immunologically
cross-reactive with, at least one epitope, biomolecule, cell or
tissue that is associated with the cancer, and thereby treating or
preventing the cancer. According to other embodiments there is
provided a method of treating or preventing cancer in a subject
having or suspected of being at risk for having cancer, the method
comprising administering to the subject a vaccine composition that
comprises (a) an antigen; (b) a glucopyranosyl lipid adjuvant
(GLA); and (c) a toll-like receptor (TLR) agonist, wherein the
antigen is derived from, or is immunologically cross-reactive with,
at least one epitope, biomolecule, cell or tissue that is
associated with the cancer, and thereby treating or preventing the
cancer. In certain further embodiments the TLR agonist is selected
from lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003,
flagellin, Leishmania homolog of eukaryotic ribosomal elongation
and initiation factor 4a (LeIF) and at least one hepatitis C
antigen. According to other embodiments there is provided a method
of treating or preventing cancer in a subject having or suspected
of being at risk for having cancer, the method comprising
administering to the subject a vaccine composition that comprises
(a) an antigen; (b) a glucopyranosyl lipid adjuvant (GLA); and (c)
at least one co-adjuvant that is selected from the group consisting
of saponins and saponin mimetics, wherein the antigen is derived
from, or is immunologically cross-reactive with, at least one
epitope, biomolecule, cell or tissue that is associated with the
cancer, and thereby treating or preventing the cancer.
[0039] According to other embodiments there is provided a method of
treating or preventing cancer in a subject having or suspected of
being at risk for having cancer, the method comprising
administering to the subject a vaccine composition that comprises
(a) an antigen; (b) a glucopyranosyl lipid adjuvant (GLA); and (c)
a carrier that comprises at least one of an oil and ISCOMATRIX.TM.,
wherein the antigen is derived from, or is immunologically
cross-reactive with, at least one epitope, biomolecule, cell or
tissue that is associated with the cancer, and thereby treating or
preventing the cancer. According to other embodiments there is
provided a method of treating or preventing cancer in a subject
having or suspected of being at risk for having cancer, the method
comprising administering to the subject a vaccine composition that
comprises (a) an antigen; (b) a glucopyranosyl lipid adjuvant
(GLA); and (c) one or more of: (i) at least one co-adjuvant, (ii)
at least one TLR agonist, (iii) at least one imidazoquinoline
immune response modifier, and (iv) at least one double stem loop
immune modifier (dSLIM), wherein the antigen is derived from, or is
immunologically cross-reactive with, at least one epitope,
biomolecule, cell or tissue that is associated with the cancer, and
thereby treating or preventing the cancer. In certain further
embodiments (i) the co-adjuvant, when present, is selected from the
group consisting of alum, a plant alkaloid and a detergent, wherein
the plant alkaloid is tomatine and the detergent is selected from
saponin, Polysorbate 80, Span 85 and Stearyl tyrosine, (ii) the TLR
agonist, when present, is selected from the group consisting of
lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003, flagellin,
Leishmania homolog of eukaryotic ribosomal elongation and
initiation factor 4a (LeIF) and at least one hepatitis C antigen,
and (iii) the imidazoquinoline immune response modifier, when
present, is selected from the group consisting of resiquimod
(R848), imiquimod and gardiquimod. According to other embodiments
there is provided a method of treating or preventing cancer in a
subject having or suspected of being at risk for having cancer, the
method comprising administering to the subject a vaccine
composition that comprises (a) an antigen; (b) a glucopyranosyl
lipid adjuvant (GLA); and (c) at least one of a co-adjuvant and a
pharmaceutically acceptable carrier, wherein the co-adjuvant is
selected from the group consisting of a cytokine, a block copolymer
or biodegradable polymer, and a detergent, and the pharmaceutically
acceptable carrier comprises a carrier that is selected from the
group consisting of calcium phosphate, an oil-in-water emulsion, a
water-in-oil emulsion, a liposome, and a microparticle, wherein the
antigen is derived from, or is immunologically cross-reactive with,
at least one epitope, biomolecule, cell or tissue that is
associated with the cancer, and thereby treating or preventing the
cancer. In a further embodiment the cytokine is selected from
GM-CSF, IL-2, IL-7, IL-12, TNF-.alpha. and IFN-gamma, the block
copolymer or biodegradable polymer is selected from Pluronic L121,
CRL1005, PLGA, PLA, PLG, and polyl:C, and the detergent is selected
from saponin, Polysorbate 80, Span 85 and Stearyl tyrosine.
According to other embodiments there is provided a method of
treating or preventing cancer in a subject having or suspected of
being at risk for having cancer, the method comprising
administering to the subject a vaccine composition that comprises
(a) at least one recombinant expression construct which comprises a
promoter operably linked to a nucleic acid sequence encoding an
antigen; and (b) a glucopyranosyl lipid adjuvant (GLA), wherein the
antigen is derived from, or is immunologically cross-reactive with,
at least one epitope, biomolecule, cell or tissue that is
associated with the cancer, and thereby treating or preventing the
cancer. In a further embodiment the recombinant expression
construct is present in a viral vector, which in certain further
embodiments is present in a virus that is selected from an
adenovirus, an adeno-associated virus, a herpesvirus, a lentivirus,
a poxvirus, and a retrovirus.
[0040] In certain further embodiments of the above described
methods of treating or preventing cancer the antigen is derived
from at least one cancer cell, which in certain further embodiments
originates in a primary solid tumor, and in certain other further
embodiments originates in a cancer that is a metastatic or
secondary solid tumor, and in certain other further embodiments
originates in a cancer that is a circulating tumor or an ascites
tumor. In certain embodiments the cancer cell originates in a
cancer that is selected from cervical cancer, ovarian cancer,
breast cancer, prostate cancer, fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma, pseudomyxoma
petitonei, lymphangioendotheliosarcoma, synovioma, mesothelioma,
Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma and Wilms' tumor. In certain other embodiments the cancer
cell originates in a cancer that is selected from testicular tumor,
lung carcinoma, small cell lung carcinoma, bladder carcinoma,
epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma,
acoustic neuroma, oliodendroglioma, meningioma, melanoma,
neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple
myeloma, Waldenstrom's macroglobulinemia and heavy chain
disease.
[0041] According to certain further embodiments of any one of the
above-described methods of treating or preventing infectious
disease or autoimmune disease or cancer, the step of administering
is performed once, while in certain other further embodiments of
such methods the step of administering is performed at least two
times, and in certain other further embodiments the step of
administering is performed at least three times, and in certain
other further embodiments the step of administering is performed
four or more times. According to certain further embodiments of any
one of the above-described methods of treating or preventing
infectious disease or autoimmune disease or cancer, prior to the
step of administering, the subject is primed with a priming agent
that is selected from a bacterial extract, a live virus vaccine, at
least one recombinant expression construct which comprises a
promoter operably linked to a nucleic acid sequence encoding the
antigen, and a viral vector that comprises a promoter operably
linked to a nucleic acid sequence encoding the antigen. In a
further embodiment the bacterial extract is derived from Bacillus
Calmet-Guerin (BCG).
[0042] In another embodiment there is provided a method of
eliciting or enhancing a desired antigen-specific immune response
in a subject, comprising administering to the subject a vaccine
composition that comprises (a) an antigen, and (b) a glucopyranosyl
lipid adjuvant (GLA). In another embodiment there is provided a
method of eliciting or enhancing a desired antigen-specific immune
response in a subject, comprising administering to the subject a
vaccine composition that comprises (a) an antigen, (b) a
glucopyranosyl lipid adjuvant (GLA), and (c) a toll-like receptor
(TLR) agonist. In certain further embodiments the TLR agonist is
selected from lipopolysaccharide, peptidoglycan, polyl:C, CpG,
3M003, flagellin, Leishmania homolog of eukaryotic ribosomal
elongation and initiation factor 4a (LeIF) and at least one
hepatitis C antigen. In another embodiment there is provided a
method of eliciting or enhancing a desired antigen-specific immune
response in a subject, comprising administering to the subject a
vaccine composition that comprises (a) an antigen, (b) a
glucopyranosyl lipid adjuvant (GLA), and (c) at least one
co-adjuvant that is selected from the group consisting of saponins
and saponin mimetics. In another embodiment there is provided a
method of eliciting or enhancing a desired antigen-specific immune
response in a subject, comprising administering to the subject a
vaccine composition that comprises (a) an antigen, (b) a
glucopyranosyl lipid adjuvant (GLA), and (c) a carrier that
comprises at least one of an oil and ISCOMATRIX.TM.. In another
embodiment there is provided a method of eliciting or enhancing a
desired antigen-specific immune response in a subject, comprising
administering to the subject a vaccine composition that comprises
(a) an antigen; (b) a glucopyranosyl lipid adjuvant (GLA); and (c)
one or more of: (i) at least one co-adjuvant, (ii) at least one TLR
agonist, (iii) at least one imidazoquinoline immune response
modifier, and (iv) at least one double stem loop immune modifier
(dSLIM). In certain further embodiments, the co-adjuvant, when
present, is selected from alum, a plant alkaloid and a detergent,
wherein the plant alkaloid is selected from tomatine and the
detergent is selected from saponin, Polysorbate 80, Span 85 and
Stearyl tyrosine, (ii) the TLR agonist, when present, is selected
from the group consisting of lipopolysaccharide, peptidoglycan,
polyl:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic
ribosomal elongation and initiation factor 4a (LeIF) and at least
one hepatitis C antigen, and (iii) the imidazoquinoline immune
response modifier, when present, is selected from the group
consisting of resiquimod (R848), imiquimod and gardiquimod.
[0043] In another embodiment there is provided a method of
eliciting or enhancing a desired antigen-specific immune response
in a subject, comprising administering to the subject a vaccine
composition that comprises (a) an antigen; (b) a glucopyranosyl
lipid adjuvant (GLA); and (c) at least one of a co-adjuvant and a
pharmaceutically acceptable carrier, wherein: the co-adjuvant is
selected from a cytokine, a block copolymer, a biodegradable
polymer, and a detergent, and the pharmaceutically acceptable
carrier comprises a carrier that is selected from calcium
phosphate, an oil-in-water emulsion, a water-in-oil emulsion, a
liposome, and a microparticle. In certain further embodiments the
cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-.alpha.
and IFN-gamma, the block copolymer or biodegradable polymer is
selected from Pluronic L121, CRL1005, PLGA, PLA, PLG, and polyl:C,
and the detergent is selected from the group consisting of saponin,
Polysorbate 80, Span 85 and Stearyl tyrosine.
[0044] In another embodiment there is provided a method of
eliciting or enhancing a desired antigen-specific immune response
in a subject, comprising administering to the subject a vaccine
composition that comprises (a) at least one recombinant expression
construct which comprises a promoter operably linked to a nucleic
acid sequence encoding an antigen, and (b) a glucopyranosyl lipid
adjuvant (GLA). In certain further embodiments the recombinant
expression construct is present in a viral vector, which in certain
further embodiments is present in a virus that is selected from an
adenovirus, an adeno-associated virus, a herpesvirus, a lentivirus,
a poxvirus, and a retrovirus.
[0045] In certain further embodiments of the above described
methods of eliciting or enhancing a desired antigen-specific
response in a subject, the GLA is not 3'-de-O-acylated. In certain
other further embodiments of the above described methods of
eliciting or enhancing a desired antigen-specific response in a
subject, the GLA comprises:(i) a diglucosamine backbone having a
reducing terminus glucosamine linked to a non-reducing terminus
glucosamine through an ether linkage between hexosamine position 1
of the non-reducing terminus glucosamine and hexosamine position 6
of the reducing terminus glucosamine; (ii) an O-phosphoryl group
attached to hexosamine position 4 of the non-reducing terminus
glucosamine; and (iii) up to six fatty acyl chains; wherein one of
the fatty acyl chains is attached to 3-hydroxy of the reducing
terminus glucosamine through an ester linkage, wherein one of the
fatty acyl chains is attached to a 2-amino of the non-reducing
terminus glucosamine through an amide linkage and comprises a
tetradecanoyl chain linked to an alkanoyl chain of greater than 12
carbon atoms through an ester linkage, and wherein one of the fatty
acyl chains is attached to 3-hydroxy of the non-reducing terminus
glucosamine through an ester linkage and comprises a tetradecanoyl
chain linked to an alkanoyl chain of greater than 12 carbon atoms
through an ester linkage. In certain related further embodiments
the TLR agonist, when present, is capable of delivering a
biological signal by interacting with at least one TLR that is
selected from TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 and
TLR-9. In certain further embodiments the TLR agonist is selected
from lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003,
flagellin, Leishmania homolog of eukaryotic ribosomal elongation
and initiation factor 4a (LeIF) and at least one hepatitis C
antigen.
[0046] In certain further embodiments of the above described
methods of eliciting or enhancing a desired antigen-specific
response in a subject, the GLA has the formula:
##STR00002##
where: [0047] R.sup.1, R.sup.3, R.sup.5 and R.sup.6 are
C.sub.11-C.sub.20 alkyl; and [0048] R.sup.2 and R.sup.4 are
C.sub.12-C.sub.20 alkyl.
[0049] In certain further embodiments of the above described
methods of eliciting or enhancing a desired antigen-specific
response in a subject, the vaccine composition is capable of
eliciting an immune response in a host. In certain further
embodiments the immune response is specific for the antigen. In
certain further embodiments of the above described methods of
eliciting or enhancing a desired antigen-specific response in a
subject, the antigen is capable of eliciting in a host an immune
response that is selected from a humoral response and a
cell-mediated response. In certain further embodiments of the above
described methods of eliciting or enhancing a desired
antigen-specific response in a subject, the vaccine composition is
capable of eliciting in a host at least one immune response that is
selected from the group consisting of: a T.sub.H1-type T lymphocyte
response, a T.sub.H2-type T lymphocyte response, a cytotoxic T
lymphocyte (CTL) response, an antibody response, a cytokine
response, a lymphokine response, a chemokine response, and an
inflammatory response. In certain further embodiments of the above
described methods of eliciting or enhancing a desired
antigen-specific response in a subject, the vaccine composition is
capable of eliciting in a host at least one immune response that is
selected from the group consisting of: (a) production of one or a
plurality of cytokines wherein the cytokine is selected from the
group consisting of interferon-gamma (IFN-.gamma.) and tumor
necrosis factor-alpha (TNF-.alpha.), (b) production of one or a
plurality of interleukins wherein the interleukin is selected from
IL-1, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16,
IL-18 and IL-23, (c) production one or a plurality of chemokines
wherein the chemokine is selected from MIP-1.alpha., MIP-1.beta.,
RANTES, CCL4 and CCL5, and (d) a lymphocyte response that is
selected from a memory T cell response, a memory B cell response,
an effector T cell response, a cytotoxic T cell response and an
effector B cell response.
[0050] According to certain other embodiments, there is provided a
method of preparing a vaccine composition, comprising admixing (a)
an antigen and (b) a glucopyranosyl lipid adjuvant (GLA). According
to certain other embodiments, there is provided a method of
preparing a vaccine composition, comprising admixing (a) an
antigen, (b) a glucopyranosyl lipid adjuvant (GLA) and (c) a
toll-like receptor (TLR) agonist. In certain further embodiments
the TLR agonist is selected from lipopolysaccharide, peptidoglycan,
polyl:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic
ribosomal elongation and initiation factor 4a (LeIF) and at least
one hepatitis C antigen. According to certain other embodiments,
there is provided a method of preparing a vaccine composition,
comprising admixing (a) an antigen, (b) a glucopyranosyl lipid
adjuvant (GLA), and (c) at least one co-adjuvant that is selected
from the group consisting of saponins and saponin mimetics.
According to certain other embodiments, there is provided a method
of preparing a vaccine composition, comprising admixing (a) an
antigen, (b) a glucopyranosyl lipid adjuvant (GLA), and (c) a
carrier that comprises at least one of an oil and ISCOMATRIX.TM..
According to certain other embodiments, there is provided a method
of preparing a vaccine composition, comprising admixing (a) an
antigen; (b) a glucopyranosyl lipid adjuvant (GLA); and (c) one or
more of: (i) at least one co-adjuvant, (ii) at least one TLR
agonist, (iii) at least one imidazoquinoline immune response
modifier, and (iv) at least one double stem loop immune modifier
(dSLIM). In certain further embodiments, (i) the co-adjuvant, when
present, is selected from the group consisting of alum, a plant
alkaloid and a detergent, wherein the plant alkaloid is selected
from tomatine and the detergent is selected from saponin,
Polysorbate 80, Span 85 and Stearyl tyrosine, (ii) the TLR agonist,
when present, is selected from the group consisting of
lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003, flagellin,
Leishmania homolog of eukaryotic ribosomal elongation and
initiation factor 4a (LeIF) and at least one hepatitis C antigen,
and (iii) the imidazoquinoline immune response modifier, when
present, is selected from the group consisting of resiquimod
(R848), imiquimod and gardiquimod. According to certain other
embodiments, there is provided a method of preparing a vaccine
composition, comprising admixing (a) an antigen; (b) a
glucopyranosyl lipid adjuvant (GLA); and (c) at least one of a
co-adjuvant and a pharmaceutically acceptable carrier, wherein: the
co-adjuvant is selected from the group consisting of a cytokine, a
block copolymer or biodegradable polymer, and a detergent, and the
pharmaceutically acceptable carrier comprises a carrier that is
selected from the group consisting of calcium phosphate, an
oil-in-water emulsion, a water-in-oil emulsion, a liposome, and a
microparticle. In certain further embodiments the cytokine is
selected from GM-CSF, IL-2, IL-7, IL-12, TNF-.alpha. and IFN-gamma,
the block copolymer or biodegradable polymer is selected from
Pluronic L121, CRL1005, PLGA, PLA, PLG, and polyl:C, and the
detergent is selected from saponin, Polysorbate 80, Span 85 and
Stearyl tyrosine.
[0051] According to certain other embodiments, there is provided a
method of preparing a vaccine composition, comprising admixing (a)
at least one recombinant expression construct which comprises a
promoter operably linked to a nucleic acid sequence encoding an
antigen, and (b) a glucopyranosyl lipid adjuvant (GLA). In certain
further embodiments the recombinant expression construct is present
in a viral vector, which in certain further embodiments is present
in a virus that is selected from an adenovirus, an adeno-associated
virus, a herpesvirus, a lentivirus, a poxvirus, and a retrovirus.
In certain embodiments the GLA is not 3'-de-O-acylated. In certain
embodiments the GLA comprises: (i) a diglucosamine backbone having
a reducing terminus glucosamine linked to a non-reducing terminus
glucosamine through an ether linkage between hexosamine position 1
of the non-reducing terminus glucosamine and hexosamine position 6
of the reducing terminus glucosamine; (ii) an O-phosphoryl group
attached to hexosamine position 4 of the non-reducing terminus
glucosamine; and (iii) up to six fatty acyl chains; wherein one of
the fatty acyl chains is attached to 3-hydroxy of the reducing
terminus glucosamine through an ester linkage, wherein one of the
fatty acyl chains is attached to a 2-amino of the non-reducing
terminus glucosamine through an amide linkage and comprises a
tetradecanoyl chain linked to an alkanoyl chain of greater than 12
carbon atoms through an ester linkage, and wherein one of the fatty
acyl chains is attached to 3-hydroxy of the non-reducing terminus
glucosamine through an ester linkage and comprises a tetradecanoyl
chain linked to an alkanoyl chain of greater than 12 carbon atoms
through an ester linkage. In certain embodiments the TLR agonist is
capable of delivering a biological signal by interacting with at
least one TLR that is selected from TLR-2, TLR-3, TLR-4, TLR-5,
TLR-6, TLR-7, TLR-8 and TLR-9. In certain further embodiments the
TLR agonist is selected from lipopolysaccharide, peptidoglycan,
polyl:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic
ribosomal elongation and initiation factor 4a (LeIF) and at least
one hepatitis C antigen.
[0052] According to certain embodiments of the above-described
methods of preparing a vaccine composition, the GLA has the
formula:
##STR00003##
where: [0053] R.sup.1, R.sup.3, R.sup.5 and R.sup.6 are
C.sub.11-C.sub.20 alkyl; and [0054] R.sup.2 and R.sup.4 are
C.sub.12-C.sub.20 alkyl.
[0055] In certain further embodiments the step of admixing
comprises emulsifying, and in certain other further embodiments the
step of admixing comprises forming particles, which in certain
further embodiments comprise microparticles. In certain other
further embodiments the step of admixing comprises forming a
precipitate which comprises all or a portion of the antigen and all
or a portion of the GLA.
[0056] In certain other embodiments there is provided an
immunological adjuvant pharmaceutical composition comprising: a
glycopyranosyl lipid adjuvant (GLA); and a pharmaceutically
acceptable carrier or excipient. In certain other embodiments there
is provided an immunological adjuvant composition comprising a
glycopyranosyl lipid adjuvant (GLA); and a toll-like receptor (TLR)
agonist. In certain further embodiments the TLR agonist is selected
from lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003,
flagellin, Leishmania homolog of eukaryotic ribosomal elongation
and initiation factor 4a (LeIF) and at least one hepatitis C
antigen. In certain other embodiments there is provided an
immunological adjuvant composition comprising: a glycopyranosyl
lipid adjuvant (GLA); and at least one co-adjuvant that is selected
from saponins and saponin mimetics. In certain other embodiments
there is provided an immunological adjuvant pharmaceutical
composition comprising: a glycopyranosyl lipid adjuvant (GLA); and
a pharmaceutically acceptable carrier that comprises at least one
of an oil and ISCOMATRIX.TM.. In certain other embodiments there is
provided an immunological adjuvant composition comprising: (a) a
glycopyranosyl lipid adjuvant (GLA); and (b) one or more of: (i) at
least one co-adjuvant, (ii) at least one TLR agonist, (iii) at
least one imidazoquinoline immune response modifier, and (iv) at
least one double stem loop immune modifier (dSLIM).
[0057] In certain further embodiments, (i) the co-adjuvant, when
present, is selected from the group consisting of alum, a plant
alkaloid and a detergent, wherein the plant alkaloid is tomatine
and the detergent is selected from saponin, Polysorbate 80, Span 85
and Stearyl tyrosine, (ii) the TLR agonist, when present, is
selected from the group consisting of lipopolysaccharide,
peptidoglycan, polyl:C, CpG, 3M003, flagellin, Leishmania homolog
of eukaryotic ribosomal elongation and initiation factor 4a (LeIF)
and at least one hepatitis C antigen, and (iii) the
imidazoquinoline immune response modifier, when present, is
selected from the group consisting of resiquimod (R848), imiquimod
and gardiquimod.
[0058] In certain other embodiments there is provided an
immunological adjuvant composition comprising: a glycopyranosyl
lipid adjuvant (GLA); and at least one of a co-adjuvant and a
pharmaceutically acceptable carrier, wherein: the co-adjuvant is
selected from the group consisting of a cytokine, a block copolymer
or biodegradable polymer, and a detergent, and the pharmaceutically
acceptable carrier comprises a carrier that is selected from
calcium phosphate, an oil-in-water emulsion, a water-in-oil
emulsion, a liposome, and a microparticle. In certain further
embodiments the cytokine is selected from GM-CSF, IL-2, IL-7,
IL-12, TNF and IFN-gamma, the block copolymer or biodegradable
polymer is selected from Pluronic L121, CRL1005, PLGA, PLA, PLG,
and polyl:C, and the detergent is selected from the group
consisting of saponin, Polysorbate 80, Span 85 and Stearyl
tyrosine.
[0059] In certain other embodiments there is provided a method of
altering immunological responsiveness in a host, comprising:
administering to the host an immunological adjuvant pharmaceutical
composition that comprises a glycopyranosyl lipid adjuvant (GLA),
and a pharmaceutically acceptable carrier or excipient, and thereby
altering host immunological responsiveness. In certain other
embodiments there is provided a method of altering immunological
responsiveness in a host, comprising: administering to the host an
immunological adjuvant composition that comprises a glycopyranosyl
lipid adjuvant (GLA), and (b) a toll-like receptor (TLR) agonist,
and thereby altering host immunological responsiveness. In certain
further embodiments the TLR agonist is selected from
lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003, flagellin,
Leishmania homolog of eukaryotic ribosomal elongation and
initiation factor 4a (LeIF) and at least one hepatitis C antigen.
In certain other embodiments there is provided a method of altering
immunological responsiveness in a host, comprising: administering
to the host an immunological adjuvant composition that comprises a
glycopyranosyl lipid adjuvant (GLA), and at least one co-adjuvant
that is selected from the group consisting of saponins and saponin
mimetics, and thereby altering host immunological responsiveness.
In certain other embodiments there is provided a method of altering
immunological responsiveness in a host, comprising: administering
to the host an immunological adjuvant composition that comprises a
glycopyranosyl lipid adjuvant (GLA), and a pharmaceutically
acceptable carrier that comprises at least one of an oil and
ISCOMATRIX.TM., and thereby altering host immunological
responsiveness. In certain other embodiments there is provided a
method of altering immunological responsiveness in a host,
comprising: administering to the host an immunological adjuvant
composition that comprises a glycopyranosyl lipid adjuvant (GLA),
and one or more of: (i) at least one co-adjuvant, (ii) at least one
TLR agonist, (iii) at least one imidazoquinoline immune response
modifier, and (iv) at least one double stem loop immune modifier
(dSLIM), and thereby altering host immunological
responsiveness.
[0060] In certain further embodiments, the co-adjuvant, when
present, is selected from alum, a plant alkaloid and a detergent,
wherein the plant alkaloid is tomatine and the detergent is
selected from saponin, Polysorbate 80, Span 85 and Stearyl
tyrosine, the TLR agonist, when present, is selected from
lipopolysaccharide, peptidoglycan, polyl:C, CpG, 3M003, flagellin,
Leishmania homolog of eukaryotic ribosomal elongation and
initiation factor 4a (LeIF) and at least one hepatitis C antigen,
and the imidazoquinoline immune response modifier, when present, is
selected from the group consisting of resiquimod (R848), imiquimod
and gardiquimod.
[0061] In certain other embodiments there is provided a method of
altering immunological responsiveness in a host, comprising:
administering to the host an immunological adjuvant composition
that comprises a glycopyranosyl lipid adjuvant (GLA); and at least
one of a co-adjuvant and a pharmaceutically acceptable carrier,
wherein: the co-adjuvant is selected from the group consisting of a
cytokine, a block copolymer or biodegradable polymer, and a
detergent, and the pharmaceutically acceptable carrier comprises a
carrier that is selected from the group consisting of calcium
phosphate, an oil-in-water emulsion, a water-in-oil emulsion, a
liposome, and a microparticle, and thereby altering host
immunological responsiveness. In certain further embodiments the
cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-.alpha.
and IFN-gamma, the block copolymer or biodegradable polymer is
selected from Pluronic L121, CRL1005, PLGA, PLA, PLG, and polyl:C,
and the detergent is selected from the group consisting of saponin,
Polysorbate 80, Span 85 and Stearyl tyrosine.
[0062] In certain further embodiments of the above described
methods of altering immunological responsiveness in a host, the
step of administering is performed one, two, three, four or more
times. In certain other further embodiments of the above described
methods of altering immunological responsiveness in a host,
altering immunological responsiveness in the host comprises
inducing or enhancing an immune response. In certain other further
embodiments of the above described methods of altering
immunological responsiveness in a host, altering immunological
responsiveness in the host comprises down-regulating an immune
response. In certain further embodiments of the above described
methods of altering immunological responsiveness in a host, the
method further comprises administering simultaneously or
sequentially and in either order an antigen that is derived from,
or is immunologically cross-reactive with, at least one infectious
pathogen that is associated with an infectious disease against
which induced or enhanced immunological responsiveness is desired.
In certain further such embodiments the step of administering the
antigen is performed one, two, three, four or more times. In
certain other further embodiments of the above described methods of
altering immunological responsiveness in a host, the method
comprises administering simultaneously or sequentially and in
either order an antigen that is derived from, or is immunologically
cross-reactive with, at least one epitope, biomolecule, cell or
tissue that is associated with an autoimmune disease and against
which down-regulated immunological responsiveness is desired. In
certain further such embodiments the step of administering the
antigen is performed one, two, three, four or more times. In
certain other further embodiments of the above described methods of
altering immunological responsiveness in a host, the method
comprises administering simultaneously or sequentially and in
either order an antigen that is derived from, or is immunologically
cross-reactive with, at least one epitope, biomolecule, cell or
tissue that is associated with a cancer against which induced or
enhanced immunological responsiveness is desired. In certain
further such embodiments the step of administering the antigen is
performed one, two, three, four or more times.
[0063] In another embodiment there is provided a kit, comprising:
an immunological adjuvant composition as described above in a first
container; and an antigen in a second container, wherein the
immunological adjuvant composition is not in contact with the
antigen. In another embodiment there is provided a kit, comprising:
an immunological adjuvant composition as described above in a first
container; and at least one recombinant expression construct which
comprises a promoter operably linked to a nucleic acid sequence
encoding an antigen, in a second container, wherein the
immunological adjuvant composition is not in contact with the
recombinant expression construct. In certain further embodiments of
the just-described kit, the antigen is derived from at least one
infectious pathogen that is selected from a bacteria, a virus, a
yeast and a protozoan. In certain other further embodiments of the
just-described kit, the antigen is derived from at least one cancer
cell. In certain other further embodiments of the just-described
kit, the antigen is derived from, or is immunologically
cross-reactive with, at least one epitope, biomolecule, cell or
tissue that is associated with an autoimmune disease.
[0064] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings. In addition, various references are set forth
herein which describe in more detail certain aspects of this
invention, and are therefore incorporated by reference in their
entireties.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0065] FIG. 1 shows HPLC data demonstrating the number and amounts
of contaminating materials in MPL-AF and GLA-AF. These
chromatograms were collected using an Agilent 1100 system and an
ESA Corona CAD detector. The method was run using a methanol to
chloroform gradient on a Waters Atlantis C18 column. The injections
included 2.5 .mu.g of GLA and MPL respectively and 0.27 .mu.g of
synthetic phosphocholine (POPC) which is used as a solubilizing
agent.
[0066] FIG. 2 shows ELISA data demonstrating levels of cytokines
and chemokines expressed by human macrophages of the Mono Mac 6
cell line (panels a-e), and PBMC-derived DC (panels f-h) in
response to GLA stimulation. Cells were cultured at 1.times.105
cells/well with an aqueous formulation of GSK Biologicals MPL.RTM.
(MPL-AF), GLA (GLA-AF), or AF vehicle alone for 24 hrs. MIP-1b,
IP-10, IL-6, IL-23 and IL-1b levels in supernatants were measured
by sandwich ELISA.
[0067] FIG. 3 shows ELISA data demonstrating levels of anti-Fluzone
antibody production induced in mice one week after each
immunization (i.e., at day 7, panel A; and at day 28, panel B)
using two different doses of Fluzone vaccine formulated with
GLA-AF, or GLA-SE, compared to Fluzone alone. Panels A & B show
ELISA Ab titers of mice immunized twice at 3 weeks interval with 20
ml (1.8 .mu.g) or 2 ml (0.18 mg) of Fluzone (Flu) vaccine in a
formulation containing GLA-AF, GLA-SE or no adjuvant, one week
after the first (A) or second (B) injection. Panel C shows titers
of neutralizing antibody (HAI) in the sera of mice after the second
immunization.
[0068] FIG. 4 shows ELISA data demonstrating levels of anti-SMT
antibody production induced in mice one week after the third
immunization using SMT antigen alone, or formulated with GLA-SE.
C57BL/6 mice were immunized three times at three-week intervals
with SMT antigen (10 .mu.g per animal for each immunization)
formulated in a stable emulsion containing GLA (GLA-SE; 20 .mu.g
per animal for each immunization), or injected with SMT protein
alone. Sera were collected by bleeding one week after each
immunization, and serum levels of IgG1, and IgG2c antibodies
specific for SMT were examined by ELISA. Means and SEM of
reciprocal endpoint titers are shown.
[0069] FIG. 5 shows ELISA data demonstrating levels of
anti-Leish-110f antibody production induced in mice one week after
the first immunization using Leish-110f antigen formulated with
different amounts of GLA (40, 20, 5, or 1 .mu.g), compared to
saline controls. Balb/c mice were immunized three times at two-week
intervals with the Leish-110f antigen (10 .mu.g per animal for each
immunization) formulated in a stable emulsion containing 40, 20, 5,
or 1 mg of GLA (GLA-SE), or injected with saline. Sera were
collected by bleeding one week after each immunization, and serum
levels of IgG1 and IgG2a antibodies specific for Leish-110f were
examined by ELISA. Means and SEM of reciprocal endpoint titers are
shown for the sera collected 7 days after the 1st immunization.
[0070] FIG. 6 shows ELISA data demonstrating levels of
anti-Leish-110f IFN-.gamma. cytokine production induced in mice one
week after the third immunization using Leish-110f antigen
formulated with different amounts of GLA, compared to saline
controls. Splenocytes, from Balb/c mice immunized three times at
two-week intervals with Leish-110f antigen (10 .mu.g) formulated in
a stable emulsion containing 40, 5, or 1 .mu.g of MPL (MPL-SE) or
GLA (GLA-SE;), or from mice injected with a saline solution, were
cultured for 3 days in vitro in medium alone, or in medium
containing 10 mg/ml of Leish-110f, or 3 mg/ml of Concanavalin A
(ConA). IFN-g levels in supernatants were measured by ELISA. Means
and SEM are shown.
[0071] FIG. 7 shows ICS data demonstrating the frequencies of
ID83-specific IFN-.gamma., IL-2, and TNF cytokine producing CD4+
and CD8+ T cells induced in mice one week after the third
immunization using ID83 alone or adjuvanted with formulations
containing GLA (GLA-SE), GLA+CpG (GLA/CpG-SE), or GLA+GDQ
(GLA/GDQ-SE). Splenocytes from C57BL/6 mice, immunized three times
at three-week intervals with M. tuberculosis ID83 fusion protein (8
.mu.g) formulated with GLA-SE, GLA/CpG-SE, GLA/Gardiquimod
(GDQ)-SE, or injected with saline, were cultured in vitro for 12
hrs in medium containing 10 mg/ml of ID83. Cell levels of IL-2,
TNF, and IFN-g in CD3+CD4+ or CD3+CD8+ gated T cells were detected
by intracellular staining and measured by flow cytometry on a BD
LSRII FACS.
[0072] FIG. 8, panel A shows ICS data demonstrating the frequencies
of ML0276-specific IFN-.gamma. cytokine producing CD4+ T cells
induced in mice one week after the third immunization using ML0276
antigen formulated with aqueous formulations containing CpG, or
Imiquimod (IMQ), or a stable oil emulsion containing GLA (GLA-SE),
or the three mixed together, compared to saline and naive controls.
Splenocytes from C57BL/6 mice, immunized three times at three-week
intervals with M. leprea ML0276 antigen (10 .mu.g) formulated with
CpG, Imiquimod (IMQ), GLA-SE, a combination of the three, or
injected with saline, were cultured for 12 hrs in vitro in medium
containing 10 mg/ml of ML0276. Panel A shows cell levels of IFN-g
in CD3+CD4+ T cells were detected by intracellular staining and
measured by flow cytometry on a BD LSRII FACS. Panel B shows
draining lymph node cellularity as a correlate of protection.
[0073] FIG. 9 shows the surface expression of CD86 upon stimulation
with GLA. Donor N003 CD14+ monocytes-derived primary dendritic
cells were incubated for 44 hours with 10,000 ng/ml, 1000 ng/ml,
100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml, or 0.01 ng/ml GLA (panel
A) or MPL (panel B). A cytokine cocktail made of PGE.sub.2,
IL-1.beta., TNF.alpha., and IL-6 was run as a positive control.
Expression levels of the costimulatory molecule CD86 at the surface
of DC were used as an indicator of cell activation and measured by
flow cytometry on a LSRII instrument (BD Biosciences, San Jose,
Calif.) using CD86-specific fluorochrome--labeled antibody
(eBiosciences, San Diego, Calif.).
[0074] FIG. 10 shows cultured human dendritic cells (DC) from three
donors that show increased maturation with GLA stimulation. PBMC
from three normal donors was purified for human CD14+
monocytes-derived primary dendritic cells and stimulated with GLA
(panels A-C) or MPL (panels D-F). No stimulation was used as a
negative control and a standard cytokine maturation cocktail of
PGE2, IL-1.beta., TNF.alpha., and IL-6 was run as a positive
control. The percent maximum expression of CD86-specific
fluorochrome-labeled antibody (eBiosciences, San Diego, Calif.) was
used to monitor DC maturation by flow cytometry on a LSRII
instrument (BD Biosciences, San Jose, Calif.)
[0075] FIG. 11 shows the lesion development in mice vaccinated with
Leish-110f+GLA-SE upon challenge with L. major. Four Balb/c mice
per treatment group were immunized three times at two-week
intervals with either saline or the Leish-110f antigen (10 .mu.g
per animal for each immunization) formulated in a stable emulsion
containing 20 .mu.g of (i) GLA (Avanti Polar Lipids, Inc.,
Alabaster, Ala.; product number 699800; GLA-SE), or (ii) MPL.RTM.
in an emulsion as supplied by the manufacturer ("MPL-SE", GSK
Biologicals, Rixensart, Belgium). Three weeks after the last
injection, mice were challenged intradermally in the pinea of both
ears with 2.times.10.sup.3 purified Leishmania major clone V1
(MOHM/IL/80/Friedlin) metacyclic promastigotes. Development of
cutaneous lesions was monitored weekly for 6 weeks
post-infection.
[0076] FIG. 12 shows parasite burden in mice vaccinated with
Leish-110f
[0077] +GLA-SE upon challenge with L. major. Four Balb/c mice per
treatment group were immunized three times at two-week intervals
with either saline or the Leish-110f antigen (10 .mu.g per animal
for each immunization) formulated in a stable emulsion containing
20 .mu.g of (i) GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.;
product number 699800; GLA-SE), or (ii) MPL.RTM. in an emulsion as
supplied by the manufacturer ("MPL-SE", GSK Biologicals, Rixensart,
Belgium). Three weeks after the last injection, mice were
challenged intradermally in the pinea of both ears with
2.times.10.sup.3 purified Leishmania major clone V1
(MOHM/IL/80/Friedlin) metacyclic promastigotes. Parasite burden in
the ear and draining lymph nodes of infected mice were determined 6
weeks post-infection. Individual counts and mean are shown for each
group. Student's t test was performed and differences between
groups were considered statistically significant when
p<0.05.
[0078] FIG. 13 shows a flow cytometry analysis of OVA-specific
T-cells. Single-cell analysis of OVA-specific CD8+ and CD4+ T cells
producing single, double or triple Th1-type cytokines are shown.
IFN-.gamma., IL-2 and TNF-.alpha. production by CD8+ (panels A-C)
and CD4+ (panels D-F) T cells in response to in vitro stimulation
with medium, P/I or OVA were evaluated by flow cytometry.
Splenocytes were purified from mice that were injected with saline,
lentiviral vaccine or lentiviral vaccine plus GLA-SE, and were
incubated in the presence of anti-CD28 and anti-CD49d with the
addition of medium or OVA.
[0079] FIG. 14 shows anti-Fluzone IgG antibody levels seven days
following a boost immunization with Fluzone (two doses; 2 .mu.g and
0.2 .mu.g) given intradermally (i.d.) with and without adjuvant.
Panel A shows Fluzone-specific IgG antibody responses; panel B
shows Fluzone-specific IgG2a responses; panel C shows
Fluzone-specific IgG1 responses; and panel D shows IgG1:IgG2a
ratios (results <1.0 represent an IgG2a dominant response;
results >1.0 represent an IgG1 dominant response). Asterisks
represent statistical significance, p<0.05.
[0080] FIGS. 15A-D show cytokine levels for IFN-.gamma. (15A),
IL-10 (15B), IL-2 (15C), and IL-5 (15D) from ex vivo stimulated
splenocytes 3 weeks following an intramuscular (i.m.) boost
immunization with Fluzone (0.2 .mu.g) plus GLA-SE (5 .mu.g). The
average cytokine levels from three individual Balb/c mice per
treatment group.+-.s.d. are shown.
DETAILED DESCRIPTION OF THE INVENTION
[0081] The present invention in its several embodiments provides
vaccine compositions, adjuvant compositions, and related methods
that include the use of a synthetic glucopyranosyl lipid adjuvant
(GLA). GLA provides a synthetic immunological adjuvant which,
advantageously relative to adjuvants of the prior art, and in
particular, relative to natural product adjuvants, can be prepared
in substantially homogeneous form. Moreover, GLA can be prepared
efficiently and economically through large-scale synthetic chemical
manufacturing, unlike natural product-derived adjuvants. As a
synthetic adjuvant that is chemically synthesized from defined
starting materials to obtain a chemically defined product that
exhibits qualitative and quantitative batch-to-batch consistency,
GLA thus offers unprecedented benefits including improved product
quality control. Surprisingly, although 3-acylated
monophosphorylated lipid A has been associated with certain
toxicities, it has been found that when the 2 amine position
contains a single acyl chain, the molecules retain acceptable
safety profiles. Further, the synthesis of such compounds is
simplified because specific deacylation at the 3 position presents
technical challenges. Thus, the invention offers further advantages
in terms of safety and ease of synthesis.
[0082] As described herein, GLA-containing compositions and methods
for their use include in some embodiments the use of GLA by itself
with a pharmaceutically acceptable carrier or excipient for
immunological adjuvant activity, including "adjuvanting" in which
GLA administration to a subject may be wholly independent of,
and/or separated temporally and/or spatially from, administration
to the subject of one or more antigens against which elicitation or
enhancement of an immune response (e.g., an antigen-specific
response) in the subject is desired. Other embodiments include the
use of GLA in a vaccine composition that also includes one or a
plurality of antigens to which an immune response elicited or
enhanced by such a vaccine is desired. As described herein, these
vaccine compositions may in certain related embodiments also
include one or more toll-like receptor (TLR) agonist and/or one or
a plurality of one or more of a co-adjuvant, an imidazoquinoline
immune response modifier, and a double stem loop immune modifier
(dSLIM). In other related embodiments, a vaccine composition as
provided herein may comprise GLA and one or more recombinant
expression constructs each comprising a promoter operably linked to
a nucleic acid sequence encoding the antigen against which
elicitation or enhancement of an immune response (e.g., an
antigen-specific response) in the subject is desired.
GLA
[0083] As also noted above, as a chemically synthesized adjuvant
GLA can be prepared in substantially homogeneous form, which refers
to a GLA preparation that is at least 80%, preferably at least 85%,
more preferably at least 90%, more preferably at least 95% and
still more preferably at least 96%, 97%, 98% or 99% pure with
respect to the GLA molecule.
[0084] In certain embodiments, GLA comprises (i) a diglucosamine
backbone having a reducing terminus glucosamine linked to a
non-reducing terminus glucosamine through an ether linkage between
hexosamine position 1 of the non-reducing terminus glucosamine and
hexosamine position 6 of the reducing terminus glucosamine; (ii) an
O-phosphoryl group attached to hexosamine position 4 of the
non-reducing terminus glucosamine; and (iii) up to six fatty acyl
chains; wherein one of the fatty acyl chains is attached to
3-hydroxy of the reducing terminus glucosamine through an ester
linkage, wherein one of the fatty acyl chains is attached to a
2-amino of the non-reducing terminus glucosamine through an amide
linkage and comprises a tetradecanoyl chain linked to an alkanoyl
chain of greater than 12 carbon atoms through an ester linkage, and
wherein one of the fatty acyl chains is attached to 3-hydroxy of
the non-reducing terminus glucosamine through an ester linkage and
comprises a tetradecanoyl chain linked to an alkanoyl chain of
greater than 12 carbon atoms through an ester linkage.
Determination of the degree of purity of a given GLA preparation
can be readily made by those familiar with the appropriate
analytical chemistry methodologies, such as by gas chromatography,
liquid chromatography, mass spectroscopy and/or nuclear magnetic
resonance analysis.
[0085] In certain embodiments, a GLA as used herein may have the
following general structural formula:
##STR00004##
where R.sup.1, R.sup.3, R.sup.5 and R.sup.6 are C.sub.11-C.sub.20
alkyl; and R.sup.2 and R.sup.4 are C.sub.12-C.sub.20 alkyl.
[0086] In a more specific embodiment, the GLA has the formula set
forth above wherein R.sup.1, R.sup.3, R.sup.5 and R.sup.6 are
C.sub.11-14 alkyl; and R.sup.2 and R.sup.4 are C.sub.12-15
alkyl.
[0087] In a more specific embodiment, the GLA has the formula set
forth above wherein R.sup.1, R.sup.3, R.sup.5 and R.sup.6 are
C.sub.11 alkyl; and R.sup.2 and R.sup.4 are C.sub.13 alkyl.
[0088] As demonstrated herein, it has unexpectedly been found that
GLA has surprisingly superior immunostimulatory activity when
compared to MPL, while maintaining similar or reduced toxicity. For
example, in certain embodiments, the GLA is effective for inducing
an immune response that is at least 2-fold, at least 3-fold, at
least 5-fold or at least 10-fold more potent than is induced using
MPL at substantially the same or similar concentration. In other
specific embodiments, the GLA has substantially the same or similar
activity as MPL at concentrations at least 5-fold, at least
10-fold, at least 25-fold or at least 100-fold lower than MPL.
[0089] Immune responses may be measured using any of a variety of
known immunological assays or parameters known in the art and/or
described herein. For example, immune responses may be detected by
any of a variety of well known parameters, including but not
limited to in vivo or in vitro determination of: soluble
immunoglobulins or antibodies; soluble mediators such as cytokines,
lymphokines, chemokines, hormones, growth factors and the like as
well as other soluble small peptide, carbohydrate, nucleotide
and/or lipid mediators; cellular activation state changes as
determined by altered functional or structural properties of cells
of the immune system, for example cell proliferation, altered
motility, induction of specialized activities such as specific gene
expression or cytolytic behavior; cellular differentiation by cells
of the immune system, including altered surface antigen expression
profiles or the onset of apoptosis (programmed cell death); or any
other criterion by which the presence of an immune response may be
detected. In a specific embodiment, an immune response is detected
by measuring the induction of soluble mediators such as cytokines
and/or chemokines (e.g., IFN-.gamma., IL-2, TNF, IL-1.beta., etc.).
In another particular embodiment, an immune response may be
detected by measuring in vivo protection from disease in an
appropriate animal model.
[0090] GLA can be obtained commercially, for example, from Avanti
Polar Lipids, Inc. (Alabaster, Ala.; product number 699800, wherein
where R.sup.1, R.sup.3, R.sup.5 and R.sup.6 are undecyl and R.sup.2
and R.sup.4 are dodecyl).
[0091] "Alkyl" means a straight chain or branched, noncyclic or
cyclic, unsaturated or saturated aliphatic hydrocarbon containing
from 1 to 20 carbon atoms, and in certain preferred embodiments
containing from 11 to 20 carbon atoms. Representative saturated
straight chain alkyls include methyl, ethyl, n-propyl, n-butyl,
n-pentyl, n-hexyl, and the like, including undecyl, dodecyl,
tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl,
etc.; while saturated branched alkyls include isopropyl, sec-butyl,
isobutyl, tert-butyl, isopentyl, and the like. Representative
saturated cyclic alkyls include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic
alkyls include cyclopentenyl and cyclohexenyl, and the like. Cyclic
alkyls are also referred to herein as "homocycles" or "homocyclic
rings." Unsaturated alkyls contain at least one double or triple
bond between adjacent carbon atoms (referred to as an "alkenyl" or
"alkynyl", respectively). Representative straight chain and
branched alkenyls include ethylenyl, propylenyl, 1-butenyl,
2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,
3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and
the like; while representative straight chain and branched alkynyls
include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl,
2-pentynyl, 3-methyl-1-butynyl, and the like.
[0092] Accordingly, in certain embodiments contemplated herein GLA
may have any of the above described structures, and in certain
embodiments it is expressly contemplated that GLA may, and in
certain other embodiments it is expressly contemplated that GLA may
not, have any structure of a lipid adjuvant that is disclosed in
one or more of U.S. Pat. No. 6,544,518, EP 1531158, WO 2001/036433,
WO 97/11708, WO 95/14026, U.S. Pat. No. 4,987,237, JP 63010728, JP
07055906, WO 2000/013029, U.S. Pat. No. 5,530,113, U.S. Pat. No.
5,612,476, U.S. Pat. No. 5,756,718, U.S. Pat. No. 5,843,918, WO
96/09310, U.S. Pub. 2004/161776, U.S. Pub. No. 2003/170249, U.S.
Pub. No. 2002/176867, WO 2002/032450, WO 2002/028424, WO
2002/016560, WO 2000/042994, WO 2000/025815, WO 2000?018929, JP
10131046, WO 93/12778, EP 324455, DE 3833319, U.S. Pat. No.
4,844,894, U.S. Pat. No. 4,629,722. According to certain
embodiments GLA is not 3'-de-O-acylated.
Antigen
[0093] An antigen, for use in certain embodiments of the herein
described vaccine compositions and methods employing GLA, may be
any target epitope, molecule (including a biomolecule), molecular
complex (including molecular complexes that contain biomolecules),
subcellular assembly, cell or tissue against which elicitation or
enhancement of immunreactivity in a subject is desired. Frequently,
the term antigen will refer to a polypeptide antigen of interest.
However, antigen, as used herein, may also refer to a recombinant
construct which encodes a polypeptide antigen of interest (e.g, an
expression construct). In certain preferred embodiments the antigen
may be, or may be derived from, or may be immunologically
cross-reactive with, an infectious pathogen and/or an epitope,
biomolecule, cell or tissue that is associated with infection,
cancer, autoimmune disease, allergy, asthma, or any other condition
where stimulation of an antigen-specific immune response would be
desirable or beneficial.
[0094] Preferably and in certain embodiments the vaccine
formulations of the present invention contain an antigen or
antigenic composition capable of eliciting an immune response
against a human or other mammalian pathogen, which antigen or
antigenic composition may include a composition derived from a
virus such as from HIV-1, (such as tat, nef, gp120 or gp160), human
herpes viruses, such as gD or derivatives thereof or Immediate
Early protein such as ICP27 from HSV1 or HSV2, cytomegalovirus
((esp. Human)(such as gB or derivatives thereof), Rotavirus
(including live-attenuated viruses), Epstein Barr virus (such as
gp350 or derivatives thereof), Varicella Zoster Virus (such as gpI,
II and IE63), or from a hepatitis virus such as hepatitis B virus
(for example Hepatitis B Surface antigen or a derivative thereof),
hepatitis A virus, hepatitis C virus and hepatitis E virus, or from
other viral pathogens, such as paramyxoviruses: Respiratory
Syncytial virus (such as F and G proteins or derivatives thereof),
parainfluenza virus, measles virus, mumps virus, human papilloma
viruses (for example HPV6, 11, 16, 18, etc.), flaviviruses (e.g.,
Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus,
Japanese Encephalitis Virus) or Influenza virus (whole live or
inactivated virus, split influenza virus, grown in eggs or MDCK
cells, or whole flu virosomes (as described by Gluck, Vaccine,
1992, 10, 915-920) or purified or recombinant proteins thereof,
such as HA, NP, NA, or M proteins, or combinations thereof).
[0095] In certain other preferred embodiments the vaccine
formulations of the present invention contain an antigen or
antigenic composition capable of eliciting an immune response
against a human or other mammlian pathogen, which antigen or
antigenic composition may include a composition derived from one or
more bacterial pathogens such as Neisseria spp, including N.
gonorrhea and N. meningitidis (for example capsular polysaccharides
and conjugates thereof, transferrin-binding proteins, lactoferrin
binding proteins, PiIC, adhesins); S. pyogenes (for example M
proteins or fragments thereof, C5A protease, lipoteichoic acids),
S. agalactiae, S. mutans: H. ducreyi; Moraxella spp, including M.
catarrhalis, also known as Branhamella catarrhalis (for example
high and low molecular weight adhesins and invasins); Bordetella
spp, including B. pertussis (for example pertactin, pertussis toxin
or derivatives thereof, filamenteous hemagglutinin, adenylate
cyclase, fimbriae), B. parapertussis and B. bronchiseptica;
Mycobacterium spp., including M. tuberculosis (for example ESAT6,
Antigen 85A, --B or --C), M. bovis, M. leprae, M. avium, M.
paratuberculosis, M. smegmatis; Legionella spp, including L.
pneumophila; Escherichia spp, including enterotoxic E. coli (for
example colonization factors, heat-labile toxin or derivatives
thereof, heat-stable toxin or derivatives thereof),
enterohemorragic E. coli, enteropathogenic E. coli (for example
shiga toxin-like toxin or derivatives thereof); Vibrio spp,
including V. cholera (for example cholera toxin or derivatives
thereof); Shigella spp, including S. sonnei, S. dysenteriae, S.
flexnerii; Yersinia spp, including Y. enterocolitica (for example a
Yop protein), Y. pestis, Y. pseudotuberculosis; Campylobacter spp,
including C. jejuni (for example toxins, adhesins and invasins) and
C. coli; Salmonella spp, including S. typhi, S. paratyphi, S.
choleraesuis, S. enteritidis; Listeria spp., including L.
monocytogenes; Helicobacter spp, including H. pylori (for example
urease, catalase, vacuolating toxin); Pseudomonas spp, including P.
aeruginosa; Staphylococcus spp., including S. aureus, S.
epidermidis; Enterococcus spp., including E. faecalis, E. faecium;
Clostridium spp., including C. tetani (for example tetanus toxin
and derivative thereof), C. botulinum (for example botulinum toxin
and derivative thereof), C. difficile (for example clostridium
toxins A or B and derivatives thereof); Bacillus spp., including B.
anthracis (for example botulinum toxin and derivatives thereof);
Corynebacterium spp., including C. diphtheriae (for example
diphtheria toxin and derivatives thereof); Borrelia spp., including
B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii
(for example OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA,
OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC, DbpA,
DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agent
of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including
R. rickettsii; Chlamydia spp. including C. trachomatis (for example
MOMP, heparin-binding proteins), C. pneumoniae (for example MOMP,
heparin-binding proteins), C. psittaci; Leptospira spp., including
L. interrogans; Treponema spp., including T. pallidum (for example
the rare outer membrane proteins), T. denticola, T. hyodysenteriae;
or other bacterial pathogens.
[0096] In certain other preferred embodiments the vaccine
formulations of the present invention contain an antigen or
antigenic composition capable of eliciting an immune response
against a human or other mammalian pathogen, which antigen or
antigenic composition may include a compostion derived from one or
more parasites (See, e.g., John, D. T. and Petri, W. A., Markell
and Voge's Medical Parasitology-9.sup.th Ed., 2006, WB Saunders,
Philadelphia; Bowman, D. D., Georgis' Parasitology for
Veterinarians-8.sup.th Ed., 2002, WB Saunders, Philadelphia) such
as Plasmodium spp., including P. falciparum; Toxoplasma spp.,
including T. gondii (for example SAG2, SAG3, Tg34); Entamoeba spp.,
including E. histolytica; Babesia spp., including B. microti;
Trypanosoma spp., including T. cruzi; Giardia spp., including G.
lamblia; Leshmania spp., including L. major; Pneumocystis spp.,
including P. carinii; Trichomonas spp., including T. vaginalis; or
from a helminth capable of infecting a mammal, such as: (i)
nematode infections (including, but not limited to, Enterobius
vermicularis, Ascaris lumbricoides, Trichuris trichuria, Necator
americanus, Ancylostoma duodenale, Wuchereria bancrofti, Brugia
malayi, Onchocerca volvulus, Dracanculus medinensis, Trichinella
spiralis, and Strongyloides stercoralis); (ii) trematode infections
(including, but not limited to, Schistosoma mansoni, Schistosoma
haematobium, Schistosoma japonicum, Schistosoma mekongi,
Opisthorchis sinensis, Paragonimus sp, Fasciola hepatica, Fasciola
magna, Fasciola gigantica); and (iii) cestode infections
(including, but not limited to, Taenia saginata and Taenia solium).
Certain embodiments may therefore contemplate vaccine compositions
that include an antigen derived from Schisostoma spp., Schistosoma
mansonii, Schistosoma haematobium, and/or Schistosoma japonicum, or
derived from yeast such as Candida spp., including C. albicans;
Cryptococcus spp., including C. neoformans.
[0097] Other preferred specific antigens for M. tuberculosis are
for example Th Ra12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL,
mTTC2 and hTCC1 (WO 99/51748). Proteins for M. tuberculosis also
include fusion proteins and variants thereof where at least two,
preferably three polypeptides of M. tuberculosis are fused into a
larger protein. Preferred fusions include Ra12-TbH9-Ra35,
Erd14-DPV-MTI, DPV-MTI-MSL, Erd14DPV-MTI-MSL-mTCC2,
Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, TbH9-DPV-MTI (WO
99151748).
[0098] Most preferred antigens for Chlamydia include for example
the High Molecular Weight Protein (HWMP) (WO 99/17741), ORF3 (EP
366 412), and putative membrane proteins (Pmps). Other Chlamydia
antigens of the vaccine formulation can be selected from the group
described in WO 99128475. Preferred bacterial vaccines comprise
antigens derived from Streptococcus spp, including S. pneumoniae
(for example capsular polysaccharides and conjugates thereof, PsaA,
PspA, streptolysin, choline-binding proteins) and the protein
antigen Pneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins
et al., Microbial Pathogenesis, 25, 337-342), and mutant detoxified
derivatives thereof (WO 90/06951; WO 99/03884). Other preferred
bacterial vaccines comprise antigens derived from Haemophilus spp.,
including H. influenzae type B (for example PRP and conjugates
thereof), non typeable H. influenzae, for example OMP26, high
molecular weight adhesins, P5, P6, protein D and lipoprotein D, and
fimbrin and fimbrin derived peptides (U.S. Pat. No. 5,843,464) or
multiple copy varients or fusion proteins thereof.
[0099] Derivatives of Hepatitis B Surface antigen are well known in
the art and include, inter alia, those PreS1, Pars2 S antigens set
forth described in European Patent applications EP-A414 374;
EP-A-0304 578, and EP 198474. In one preferred aspect the vaccine
formulation of the invention comprises the HIV-1 antigen, gp120,
especially when expressed in CHO cells. In a further embodiment,
the vaccine formulation of the invention comprises gD2t as
hereinabove defined.
[0100] In a preferred embodiment of the present invention vaccines
containing the claimed adjuvant comprise antigen derived from the
Human Papilloma Virus (HPV) considered to be responsible for
genital warts (HPV 6 or HPV 11 and others), and the HPV viruses
responsible for cervical cancer (HPV16, HPV18 and others).
Particularly preferred forms of genital wart prophylactic, or
therapeutic, vaccine comprise L1 particles or capsomers, and fusion
proteins comprising one or more antigens selected from the HPV 6
and HPV 11 proteins E6, E7, L1, and L2. Certain preferred forms of
fusion protein include L2E7 as disclosed in WO 96/26277, and
proteinD(1/3)-E7 disclosed in GB 9717953.5 (PCT/EP98/05285). A
preferred HPV cervical infection or cancer, prophylaxis or
therapeutic vaccine, composition may comprise HPV 16 or 18
antigens. For example, L1 or L2 antigen monomers, or L1 or L2
antigens presented together as a virus like particle (VLP) or the
L1 alone protein presented alone in a VLP or caposmer structure.
Such antigens, virus like particles and capsomer are per se known.
See for example WO94/00152, WO94/20137, WO94/05792, and
WO93/02184.
[0101] Additional early proteins may be included alone or as fusion
proteins such as E7, E2 or preferably F5 for example; particularly
preferred embodiments include a VLP comprising L1 E7 fusion
proteins (WO 96/11272). Particularly preferred HPV 16 antigens
comprise the early proteins E6 or F7 in fusion with a protein D
carrier to form Protein D-E6 or E7 fusions from HPV 16, or
combinations thereof; or combinations of E6 or E7 with L2 (WO
96/26277). Alternatively the HPV 16 or 18 early proteins E6 and E7,
may be presented in a single molecule, preferably a Protein D-E6/E7
fusion. Such vaccine may optionally contain either or both E6 and
E7 proteins front HPV 18, preferably in the form of a Protein D-E6
or Protein D-E7 fusion protein or Protein D E6/E7 fusion protein.
The vaccine of the present invention may additionally comprise
antigens from other HPV strains, preferably from strains HPV 31 or
33.
[0102] Vaccines of the present invention further comprise antigens
derived from parasites that cause Malaria. For example, preferred
antigens from Plasmodia falciparum include RTS,S and TRAP. RTS is a
hybrid protein comprising substantially all the C-terminal portion
of the circumsporozoite (CS) protein of P. falciparum linked via
four amino acids of the preS2 portion of Hepatitis B surface
antigen to the surface (S) antigen of hepatitis B virus. Its full
structure is disclosed in the International Patent Application No.
PCT/EP92/02591, published as WO 93/10152 claiming priority from UK
patent application No. 9124390.7. When expressed in yeast RTS is
produced as a lipoprotein particle, and when it is co-expressed
with the S antigen from HBV it produces a mixed particle known as
RTS,S.
[0103] TRAP antigens are described in the International Patent
Application No. PCT/GB89/00895 published as WO 90/01496. A
preferred embodiment of the present invention is a Malaria vaccine
wherein the antigenic preparation comprises a combination of the
RTS,S and TRAP antigens. Other plasmodia antigens that are likely
candidates to be components of a multistage Malaria vaccine are P.
faciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin,
PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25, Pfs28,
PFS27125, Pfs16, Pfs48/45, Pfs230 and their analogues in Plasmodium
spp.
[0104] Accordingly, certain herein disclosed embodiment contemplate
an antigen that is derived from at least one infectious pathogen
such as a bacterium, a virus or a fungus, including an
Actinobacterium such as M. tuberculosis or M. leprae or another
mycobacterium; a bacterium such as a member of the genus
Salmonella, Neisseria, Borrelia, Chlamydia or Bordetella; a virus
such as a herpes simplex virus, a human immunodeficiency virus
(HIV), a feline immunodeficiency virus (FIV), cytomegalovirus,
Varicella Zoster Virus, hepatitis virus, Epstein Barr Virus (EBV),
respiratory syncytial virus, human papilloma virus (HPV) and a
cytomegalovirus; HIV such as HIV-1 or HIV-2; a fungus such as
Aspergillus, Blastomyces, Coccidioides and Pneumocysti or a yeast,
including Candida species such as C. albicans, C. glabrata, C.
krusei, C. lusitaniae, C. tropicalis and C. parapsilosis; a
parasite such as a protozoan, for example, a Plasmodium species
including P. falciparum, P. vivax, P. malariae and P. ovale; or
another parasite such as one or more of Acanthamoeba, Entamoeba
histolytica, Angiostrongylus, Schistosoma mansonii, Schistosoma
haematobium, Schistosoma japonicum, Cryptosporidium, Ancylostoma,
Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba
hartmanni, Entamoeba polecki, Wuchereria bancrofti, Giardia, and
Leishmania.
[0105] For example, in GLA-containing vaccine embodiments
containing antigens derived from Borrelia sp., the antigens may
include nucleic acid, pathogen derived antigen or antigenic
preparations, recombinantly produced protein or peptides, and
chimeric fusion proteins. One such antigen is OspA. The OspA may be
a full mature protein in a lipidated form by virtue of its
biosynthesis in a host cell (Lipo-OspA) or may alternatively be a
non-lipidated derivative. Such non-lipidated derivatives include
the non-lipidated NS1-OspA fusion protein which has the first 81
N-terminal amino acids of the non-structural protein (NS1) of the
influenza virus, and the complete OspA protein, and another,
MDP-OspA is a non-lipidated form of OspA carrying 3 additional
N-terminal amino acids.
[0106] Compositions and methods are known in the art for
identifying subjects having, or suspected of being at risk for
having, an infection with an infectious pathogen as described
herein.
[0107] For example, the bacterium Mycobacterium tuberculosis cases
tuberculosis (TB). The bacteria usually attack the lungs but can
also attack the kidney, spine, and brain. If not treated properly,
TB disease can be fatal. The disease is spread from one person to
another in the air when an infected person sneezes or coughs. In
2003, more than 14,000 cases of TB were reported in the United
States.
[0108] Although tuberculosis can generally be controlled using
extended antibiotic therapy, such treatment is not sufficient to
prevent the spread of the disease and concerns exist regarding the
potential selection for antibiotic-resistant strains. Infected
individuals may be asymptomatic, but contagious, for some time. In
addition, although compliance with the treatment regimen is
critical, patient behavior is difficult to monitor. Some patients
do not complete the course of treatment, which can lead to
ineffective treatment and the development of drug resistance.
(e.g., U.S. Pat. No. 7,087,713)
[0109] Currently, vaccination with live bacteria is the most
efficient method for inducing protective immunity against
tuberculosis. The most common Mycobacterium employed for this
purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of
Mycobacterium bovis. However, the safety and efficacy of BCG is a
source of controversy and some countries, such as the United
States, do not vaccinate the general public. Diagnosis is commonly
achieved using a skin test, which involves intradermal exposure to
tuberculin PPD (protein-purified derivative). Antigen-specific T
cell responses result in measurable induration at the injection
site by 48 72 hours after injection, which indicates exposure to
Mycobacterial antigens. Sensitivity and specificity have, however,
been a problem with this test, and individuals vaccinated with BCG
cannot be distinguished from infected individuals. (e.g., U.S. Pat.
No. 7,087,713)
[0110] While macrophages have been shown to act as the principal
effectors of M. tuberculosis immunity, T cells are the predominant
inducers of such immunity. The essential role of T cells in
protection against M. tuberculosis infection is illustrated by the
frequent occurrence of M. tuberculosis in AIDS patients, due to the
depletion of CD4 T cells associated with human immunodeficiency
virus (HIV) infection. Mycobacterium-reactive CD4 T cells have been
shown to be potent producers of gamma-interferon (IFN-gamma),
which, in turn, has been shown to trigger the anti-mycobacterial
effects of macrophages in mice. While the role of IFN-gamma in
humans is less clear, studies have shown that
1,25-dihydroxy-vitamin D3, either alone or in combination with
IFN-gamma or tumor necrosis factor-alpha, activates human
macrophages to inhibit M. tuberculosis infection. Furthermore, it
is known that IFN-gamma stimulates human macrophages to make
1,25-dihydroxy-vitamin D3. Similarly, IL-12 has been shown to play
a role in stimulating resistance to M. tuberculosis infection. For
a review of the immunology of M. tuberculosis infection, see Chan
and Kaufmann, in Tuberculosis: Pathogenesis, Protection and
Control, Bloom (ed.), ASM Press. Washington, D.C. (1994).
[0111] Existing compounds and methods for diagnosing tuberculosis
or for inducing protective immunity against tuberculosis include
the use of polypeptides that contain at least one immunogenic
portion of one or more Mycobacterium proteins and DNA molecules
encoding such polypeptides. Diagnostic kits containing such
polypeptides or DNA sequences and a suitable detection reagent may
be used for the detection of Mycobacterium infection in patients
and biological samples. Antibodies directed against such
polypeptides are also provided. In addition, such compounds may be
formulated into vaccines and/or pharmaceutical compositions for
immunization against Mycobacterium infection. (U.S. Pat. Nos.
6,949,246 and 6,555,653).
[0112] Malaria was eliminated in many parts of the world in the
1960s, but the disease still persists and new strains of the
disease are emerging that are resistant to existing drugs. Malaria
is a major public health problem in more than 90 countries. Nine
out of ten cases of malaria occur in sub-Saharan Africa. More than
one third of the world's population is at risk, and between 350 and
500 million people are infected with malaria each year. Forty-five
million pregnant women are at risk of contracting malaria this
year. Of those individuals already infected, more than 1 million of
those infected die each year from what is a preventable disease.
The majority of those deaths are children in Africa.
[0113] Malaria is usually transmitted when a person is bitten by an
infected female Anopheles mosquito. To transmit the mosquito must
have been infected by having drawn blood from a person already
infected with malaria. Malaria is caused by a parasite and the
clinical symptoms of the disease include fever and flu-like
illness, such as chills, headache, muscle aches, and tiredness.
These symptoms may be accompanied by nausea, vomiting, and
diarrhea. Malaria can also cause anemia and jaundice because of the
loss of red blood cells. Infection with one type of malaria,
Plasmodium falciparum, if not promptly treated, may cause kidney
failure, seizures, mental confusion, coma, and death.
[0114] An in vitro diagnostic method for malaria in an individual
is known, comprising placing a tissue or a biological fluid taken
from an individual in contact with a molecule or polypeptide
composition, wherein said molecule or polypeptide composition
comprises one or more peptide sequences bearing all or part of one
or more T epitopes of the proteins resulting from the infectious
activity of P. falciparum, under conditions allowing an in vitro
immunological reaction to occur between said composition and the
antibodies that may be present in the tissue or biological fluid,
and in vitro detection of the antigen-antibody complexes formed
(see, e.g., U.S. Pat. No. 7,087,231). Expression and purification
of a recombinant Plasmodium falciparum (3D7) AMA-1 ectodomain have
been described. Previous methods have produced a highly purified
protein which retains folding and disulfide bridging of the native
molecule. The recombinant AMA-1 is useful as a diagnostic reagentas
well as in antibody production, and as a protein for use alone, or
as part of, a vaccine to prevent malaria. (U.S. Pat. No.
7,029,685)
[0115] Polynucleotides have been described in the art that encode
species-specific P. vivax malarial peptide antigens which are
proteins or fragments of proteins secreted into the plasma of a
susceptible mammalian host after infection, as have monoclonal or
polyclonal antibodies directed against these antigens. The peptide
antigens, monoclonal antibodies, and/or polyclonal antibodies are
utilized in assays used to diagnose malaria, as well as to
determine whether Plasmodium vivax is the species responsible for
the infection. (U.S. Pat. No. 6,706,872) Species-specific P. vivax
malarial peptide antigens have also been reported which are
proteins or fragments of proteins secreted into the plasma of a
susceptible mammalian host after infection, as have monoclonal or
polyclonal antibodies directed against these antigens. The peptide
antigens, monoclonal antibodies, and/or polyclonal antibodies are
utilized in assays used to diagnose malaria, as well as to
determine whether Plasmodium vivax is the species responsible for
the infection (see, e.g., U.S. Pat. No. 6,231,861).
[0116] A recombinant Plasmodium falciparum (3D7) AMA-1 ectodomain
has also been expressed by a method that produces a highly purified
protein which retains folding and disulfide bridging of the native
molecule. The recombinant AMA-1 is useful as a diagnostic reagent,
for use in antibody production, and as a vaccine. (U.S. Pat. No.
7,060,276) Similarly known are the expression and purification of a
recombinant Plasmodium falciparum (3D7) MSP-1.sub.42, which retains
folding and disulfide bridging of the native molecule. The
recombinant MSP-1.sub.42 is useful as a diagnostic reagent, for use
in antibody production, and as a vaccine. (U.S. Pat. No.
6,855,322)
[0117] Diagnostic methods for the detection of human malaria
infections to identify a subject having or suspected of being at
risk for having an infection with a malaria infectious pathogen are
thus known according to these and related disclosures.
Specifically, for example, blood samples are combined with a
reagent containing 3-acetyl pyridine adenine dinucleotide (APAD), a
substrate (e.g. a lactate salt or lactic acid), and a buffer. The
reagent is designed to detect the presence of a unique glycolytic
enzyme produced by the malaria parasite. This enzyme is known as
parasite lactic acid dehydrogenase (PLDH). PLDH is readily
distinguishable from host LDH using the above-described reagent.
Combination of the reagent with a parasitized blood sample results
in the reduction of APAD. However, APAD is not reduced by host LDH.
The reduced APAD may then be detected by various techniques,
including spectral, fluorimetric, electrophoretic, or colorimetric
analysis. Detection of the reduced APAD in the foregoing manner
provides a positive indication of malaria infection (e.g., U.S.
Pat. No. 5,124,141). In another methodology for diagnosing malaria,
a polypeptide comprising a characteristic amino acid sequence
derived from the Plasmodium falciparum antigen GLURP, is recognized
in a test sample by a specific antibody raised against or reactive
with the polypeptide. (U.S. Pat. No. 5,231,168)
[0118] Leishmaniasis is a widespread parasitic disease with
frequent epidemics in the Indian subcontinent, Africa, and Latin
America and is a World Health Organization priority for vaccine
development. A complex of different diseases, Leishmania parasites
cause fatal infections of internal organs, as well as serious skin
disease. One of the most devastating forms of leishmaniasis is a
disfiguring infection of the nose and mouth. The number of cases of
leishmaniasis are increasing, and it is now out of control in many
areas. Leishmaniasis is also on the rise in some developed
countries, specifically southern Europe as a result of HIV
infection. Available drugs are toxic, expensive, and require
long-term daily injections.
[0119] Leishmania are protozoan parasites that inhabit macrophages
or the white blood cells of the immune system. The parasites are
transmitted by the bite of small blood sucking insects (sand
flies), which are difficult to control, as they inhabit vast areas
of the planet.
[0120] Visceral leishmaniasis is the most dangerous of the three
manifestations of the disease. It is estimated that about 500,000
new cases of the visceral form (kala-azar or "the killing disease")
occur each year. More than 200 million people are currently at risk
for contracting visceral leishmaniasis. Over 90 percent of visceral
leishmaniasis cases occur in India, Bangladesh, Sudan, Brazil, and
Nepal. Most of the deaths occur in children. Those with the
cutaneous forms are often left permanently disfigured.
[0121] Leishmania infections are difficult to diagnose and
typically involve histopathologic analysis of tissue biopsy
specimens. Several serological and immunological diagnostic assays
have, however, been developed. (U.S. Pat. No. 7,008,774; Senaldi et
al., (1996)J. Immunol. Methods 193:9 5; Zijlstra, et al., (1997)
Trans. R. Soc. Trop. Med. Hyg. 91:671 673; Badaro, et al., (1996)
J. Inf. Dis. 173:758 761; Choudhary, S., et al., (1992) J. Comm.
Dis. 24:32 36; Badaro, R., et al., (1986) Am. J. Trop. Med. Hyg.
35:72 78; Choudhary, A., et al., (1990) Trans. R. Soc. Trop. Med.
Hyg. 84:363 366; and Reed, S. G., et al., (1990) Am. J. Trop. Med.
Hyg. 43:632 639). The promastigotes release metabolic products into
the culture medium to produce conditioned medium. These metabolic
products are immunogenic to the host. See Schnur, L. F., et al.,
(1972) Isrl. J. Med. Sci. 8:932 942; Sergeiev, V. P., et al.,
(1969) Med. Parasitol. 38:208 212; E1-On, J., et al., (1979) Exper.
Parasitol. 47:254 269; and Bray, R. S., et al., (1966) Trans. R.
Soc. Trop. Med. Hyg. 60:605 609; U.S. Pat. No. 6,846,648, U.S. Pat.
No. 5,912,166; U.S. Pat. No. 5,719,263; U.S. Pat. No.
5,411,865).
[0122] About 40 million people around the world are infected with
HIV, the virus that causes AIDS. Around 3 million people die of the
disease each year, 95 percent of them in the developing world. Each
year, close to 5 million people become infected with HIV.
Currently, sub-Saharan African carries the highest burden of
disease, but it is quickly spreading to other countries such as
India, China, and Russia. The epidemic is growing most rapidly
among minority populations. In the United States there have been
more than 950,000 cases of AIDS reported since 1981. AIDS hits
people during their most productive years. Women, for both
biological and social reasons, have an increased risk for
HIV/AIDS.
[0123] AIDS is caused by human immunodeficiency virus (HIV), which
kills and damages cells of the body's immune system and
progressively destroys the body's ability to fight infections and
certain cancers. HIV is spread most commonly by having unprotected
sex with an infected partner. The most robust solution to the
problem is preventing the virus from spreading. Making a safe,
effective, and affordable HIV vaccine is one way to reach this
goal. Across the world, fewer than one in five people at high risk
for HIV infection have access to effective prevention.
[0124] Methods for diagnosing HIV infections are known, including
by virus culture, PCR of definitive nucleic acid sequences from
patient specimens, and antibody tests for the presence of anti-HIV
antibodies in patient sera, (see e.g., U.S. Pat. Nos. 6,979,535,
6,544,728, 6,316,183, 6,261,762, 4,743,540.)
[0125] According to certain other embodiments as disclosed herein,
the vaccine compositions and related formulations and methods of
use may include an antigen that is derived from a cancer cell, as
may be useful for the immunotherapeutic treatment of cancers. For
example, the adjuvant formulation may finds utility with tumor
rejection antigens such as those for prostate, breast, colorectal,
lung, pancreatic, renal or melanoma cancers. Exemplary cancer or
cancer cell-derived antigens include MAGE 1, 3 and MAGE 4 or other
MAGE antigens such as those disclosed in WO99/40188, PRAME, BAGE,
Lage (also known as NY Eos 1) SAGE and HAGE (WO 99/53061) or GAGE
(Robbins and Kawakami, 1996 Current Opinions in Immunology 8, pps
628-636; Van den Eynde et al., International Journal of Clinical
& Laboratory Research (1997 & 1998); Correale et al.
(1997), Journal of the National Cancer Institute 89, p. 293. These
non-limiting examples of cancer antigens are expressed in a wide
range of tumor types such as melanoma, lung carcinoma, sarcoma and
bladder carcinoma. See, e.g., U.S. Pat. No. 6,544,518.
[0126] Other tumor-specific antigens are suitable for use with GLA
according to certain presently disclosed embodiments include, but
are not restricted to, tumor-specific or tumor-associated
gangliosides such as GM.sub.2, and GM.sub.3 or conjugates thereof
to carrier proteins; or an antigen for use in a GLA vaccine
composition for eliciting or enhancing an anti-cancer immune
response may be a self peptide hormone such as whole length
Gonadotrophin hormone releasing hormone (GnRH, WO 95/20600), a
short 10 amino acid long peptide, useful in the treatment of many
cancers. In another embodiment prostate antigens are used, such as
Prostate specific antigen (PSA), PAP, PSCA (e.g., Proc. Nat. Acad.
Sci. USA 95(4) 1735-1740 1998), PSMA or, in a preferred embodiment
an antigen known as Prostase. (e.g., Nelson, et al., Proc. Natl.
Acad. Sci. USA(1999) 96: 3114-3119; Ferguson, et al. Proc. Natl.
Acad. Sci. USA 1999. 96, 3114-3119; WO 98/12302; U.S. Pat. No.
5,955,306; WO 98/20117; U.S. Pat. Nos. 5,840,871 and 5,786,148; WO
00/04149. Other prostate specific antigens are known from WO
98/137418, and WO/004149. Another is STEAP (PNAS 96 14523 14528
7-12 1999).
[0127] Other tumor associated antigens useful in the context of the
present invention include: Plu-1 (J. Biol. Chem. 274 (22)
15633-15645, 1999), HASH-1, HasH-2, Cripto (Salomon et al Bioessays
199, 21:61-70, U.S. Pat. No. 5,654,140) and Criptin (U.S. Pat. No.
5,981,215). Additionally, antigens particularly relevant for
vaccines in the therapy of cancer also comprise tyrosinase and
survivin.
[0128] The herein disclosed embodiments pertaining to
GLA-containing vaccine compositions comprising a cancer antigen
will be useful against any cancer characterised by tumor associated
antigen expression, such as HER-2/neu expression or other
cancer-specific or cancer-associated antigens.
[0129] Diagnosis of cancer in a subject having or suspected of
being at risk for having cancer may be accomplished by any of a
wide range of art-accepted methodologies, which may vary depending
on a variety of factors including clinical presentation, degree of
progression of the cancer, the type of cancer, and other factors.
Examples of cancer diagnostics include histopathological,
histocytochemical, immunohistocytochemical and
immunohistopathological examination of patient samples (e.g.,
blood, skin biopsy, other tissue biopsy, surgical specimens, etc.),
PCR tests for defined genetic (e.g., nucleic acid) markers,
serological tests for circulating cancer-associated antigens or
cells bearing such antigens, or for antibodies of defined
specificity, or other methodologies with which those skilled in the
art will be familiar. See, e.g., U.S. Pat. Nos. 6,734,172;
6,770,445; 6,893,820; 6,979,730; 7,060,802; 7,030,232; 6,933,123;
6,682,901; 6,587,792; 6,512,102; 7,078,180; 7,070,931; JP5-328975;
Waslylyk et al., 1993 Eur. J. Bloch. 211(7):18.
[0130] Vaccine compositions and methods according to certain
embodiments of the present invention may also be used for the
prophylaxis or therapy of autoimmune diseases, which include
diseases, conditions or disorders wherein a host's or subject's
immune system detrimentally mediates an immune response that is
directed against "self" tissues, cells, biomolecules (e.g.,
peptides, polypeptides, proteins, glycoproteins, lipoproteins,
proteolipids, lipids, glycolipids, nucleic acids such as RNA and
DNA, oligosaccharides, polysaccharides, proteoglycans,
glycosaminoglycans, or the like, and other molecular components of
the subjects cells and tissues) or epitopes (e.g., specific
immunologically defined recognition structures such as those
recognized by an antibody variable region complementarity
determining region (CDR) or by a T cell receptor CDR.
[0131] Autoimmune diseases are thus characterized by an abnormal
immune response involving either cells or antibodies, that are in
either case directed against normal autologous tissues. Autoimmune
diseases in mammals can generally be classified in one of two
different categories: cell-mediated disease (i.e., T-cell) or
antibody-mediated disorders. Non-limiting examples of cell-mediated
autoimmune diseases include multiple sclerosis, rheumatoid
arthritis, Hashimoto thyroiditis, type I diabetes mellitus
(Juvenile onset diabetes) and autoimmune uvoretinitis.
Antibody-mediated autoimmune disorders include, but are not limited
to, myasthenia gravis, systemic lupus erythematosus (or SLE),
Graves' disease, autoimmune hemolytic anemia, autoimmune
thrombocytopenia, autoimmune asthma, cryoglobulinemia, thrombic
thrombocytopenic purpura, primary biliary sclerosis and pernicious
anemia. The antigen(s) associated with: systemic lupus
erythematosus is small nuclear ribonucleic acid proteins (snRNP);
Graves' disease is the thyrotropin receptor, thyroglobulin and
other components of thyroid epithelial cells (Akamizu et al., 1996;
Kellerman et al., 1995; Raju et al., 1997; and Texier et al.,
1992); pemphigus is cadherin-like pemphigus antigens such as
desmoglein 3 and other adhesion molecules (Memar et al., 1996:
Stanley, 1995; Plott et al., 1994; and Hashimoto, 1993); and
thrombic thrombocytopenic purpura is antigens of platelets. (See,
e.g., U.S. Pat. No. 6,929,796; Gorski et al. (Eds.), Autoimmunity,
2001, Kluwer Academic Publishers, Norwell, M A; Radbruch and
Lipsky, P. E. (Eds.) Current Concepts in Autoimmunity and Chronic
Inflammation (Curr. Top. Microbiol. and Immunol.) 2001, Springer,
N.Y.)
[0132] Autoimmunity plays a role in more than 80 different
diseases, including type 1 diabetes, multiple sclerosis, lupus,
rheumatoid arthritis, scleroderma, and thyroid diseases. Vigorous
quantitative estimates of morbidity for most autoimmune diseases
are lacking. Most recent studies done in the late 1990s reveal that
autoimmune diseases are the third most common major illness in the
United States; and the most common autoimmune diseases affect more
than 8.5 million Americans. Current estimates of the prevalence of
the disease range from 5 to 8 percent of the United States
population. Most autoimmune diseases disproportionately affect
women. Women are 2.7 times more likely than men to acquire an
autoimmune disease. Women are more susceptible to autoimmune
diseases; men appear to have higher levels of natural killer cell
activity than do women. (Jacobsen et al, Clinical Immunology and
Immunopathology, 84:223-243, 1997.)
[0133] Autoimmune diseases occur when the immune system mistakes
self tissues for nonself and mounts an inappropriate attack. The
body can be affected in different ways from autoimmune diseases,
including, for example, the gut (Crohn's disease) and the brain
(multiple sclerosis). It is known that an autoantibody attacks
self-cells or self-tissues to injure their function and as a result
causes autoimmune diseases, and that the autoantibody may be
detected in the patient's serum prior to the actual occurrence of
an autoimmune disease (e.g., appearance of clinical signs and
symptoms). Detection of an autoantibody thus permits early
discovery or recognition of presence or risk for developing an
autoimmune disease. Based on these findings, a variety of
autoantibodies against autoantigens have been discovered and the
autoantibodies against autoantigens have been measured in clinical
tests (e.g., U.S. Pat. Nos. 6,919,210, 6,596,501, 7,012,134,
6,919,078) while other autoimmune diagnostics may involve detection
of a relevant metabolite (e.g., U.S. Pat. No. 4,659,659) or
immunological reactivity (e.g., U.S. Pat. Nos. 4,614,722 and
5,147,785, 4,420,558, 5,298,396, 5,162,990, 4,420,461, 4,595,654,
5,846,758, 6,660,487).
[0134] In certain embodiments, the compositions of the invention
will be particularly applicable in treatment of the elderly and/or
the immunosuppressed, including subjects on kidney dialysis,
subjects on chemo-therapy and/or radiation therapy, transplant
recipients, and the like. Such individuals generally exhibit
diminished immune responses to vaccines and therefore use of the
compositions of the invention can enhance the immune responses
achieved in these subjects.
[0135] In other embodiments, the antigen or antigens used in the
compositions of the invention include antigens associated with
respiratory diseases, such as those caused or exacerbated by
bacterial infection (e.g. pneumococcal), for the prophylaxis and
therapy of conditions such as chronic obstructive pulmonary disease
(COPD). COPD is defined physiologically by the presence of
irreversible or partially reversible airway obstruction in patients
with chronic bronchitis and/or emphysema (Am J Respir Crit. Care
Med. 1995 November; 152(5 Pt 2):S77-121). Exacerbations of COPD are
often caused by bacterial (e.g. pneumococcal) infection (Clin
Microbiol Rev. 2001 April; 14(2):336-63). In a particular
embodiment, a composition of the invention comprises a GLA
adjuvant, as described herein, in combination with the Pneumococcal
vaccine Prevnar.RTM. (Wyeth).
[0136] In still other embodiments, the compositions of the
invention, comprising GLA as described herein, are used in the
treatment of allergic conditions. For example, in a particular
embodiment, the compositions are used in allergy desensitization
therapy. Such therapy involves the stimulation of the immune system
with gradually increasing doses of the substances to which a person
is allergic, wherein the substances are formulated in compositions
comprising GLA. In specific embodiments, the compositions are used
in the treatment of allergies to food products, pollen, mites, cats
or stinging insects (e.g., bees, hornets, yellow jackets, wasps,
velvet ants, fire ants).
TLR
[0137] As described herein, certain embodiments of the present
invention contemplate vaccine compositions and immunological
adjuvant compositions, including pharmaceutical compositions, that
include one or more toll-like receptor agonist (TLR agonist).
Toll-like receptors (TLR) include cell surface transmembrane
receptors of the innate immune system that confer early-phase
recognition capability to host cells for a variety of conserved
microbial molecular structures such as may be present in or on a
large number of infectious pathogens. (e.g., Armant et al., 2002
Genome Biol. 3(8):reviews 3011.1-3011.6; Fearon et al., 1996
Science 272:50; Medzhitov et al., 1997 Curr. Opin. Immunol. 9:4;
Luster 2002 Curr. Opin. Immunol. 14:129; Lien et al. 2003 Nat.
Immunol. 4:1162; Medzhitov, 2001 Nat. Rev. Immunol. 1:135; Takeda
et al., 2003 Ann Rev Immunol. 21:335; Takeda et al. 2005 Int.
Immunol. 17:1; Kaisho et al., 2004 Microbes Infect. 6:1388; Datta
et al., 2003 J. Immunol. 170:4102).
[0138] Induction of TLR-mediated signal transduction to potentiate
the initiation of immune responses via the innate immune system may
be effected by TLR agonists, which engage cell surface TLR. For
example, lipopolysaccharide (LPS) may be a TLR agonist through TLR2
or TLR4 (Tsan et al., 2004 J. Leuk. Biol. 76:514; Tsan et al., 2004
Am. J. Physiol. Cell Phsiol. 286:C739; Lin et al., 2005 Shock
24:206); poly(inosine-cytidine) (polyl:C) may be a TLR agonist
through TLR3 (Salem et al., 2006 Vaccine 24:5119); CpG sequences
(oligodeoxynucleotides containing unmethylated cytosine-guanosine
or "CpG" dinucleotide motifs, e.g., CpG 7909, Cooper et al., 2005
AIDS 19:1473; CpG 10101 Bayes et al. Methods Find Exp Clin
Pharmacol 27:193; Vollmer et al. Expert Opinion on Biological
Therapy 5:673; Vollmer et al., 2004 Antimicrob. Agents Chemother.
48:2314; Deng et al., 2004 J. Immunol. 173:5148) may be TLR
agonists through TLR9 (Andaloussi et al., 2006 Glia 54:526; Chen et
al., 2006 J. Immunol. 177:2373); peptidoglycans may be TLR2 and/or
TLR6 agonists (Soboll et al., 2006 Biol. Reprod. 75:131; Nakao et
al., 2005 J. Immunol. 174:1566); 3M003
(4-amino-2-(ethoxymethyl)-.alpha.,.alpha.-dimethyl-6,7,8,9-tetrahydro-1H--
imidazo[4,5-c]quinoline-1-ethanol hydrate, Mol. Wt. 318 Da from 3M
Pharmaceuticals, St. Paul, Minn., which is also a source of the
related compounds 3M001 and 3M002; Gorden et al., 2005 J. Immunol.
174:1259) may be a TLR7 agonist (Johansen 2005 Clin. Exp. Allerg.
35:1591) and/or a TLR8 agonist (Johansen 2005); flagellin may be a
TLR5 agonist (Feuillet et al., 2006 Proc. Nat. Acad. Sci. USA
103:12487); and hepatitis C antigens may act as TLR agonists
through TLR7 and/or TLR9 (Lee et al., 2006 Proc. Nat. Acad. Sci.
USA 103:1828; Horsmans et al., 2005 Hepatol. 42:724). Other TLR
agonists are known (e.g., Schirmbeck et al., 2003 J. Immunol.
171:5198) and may be used according to certain of the presently
described embodiments.
[0139] For example, and by way of background (see, e.g., U.S. Pat.
No. 6,544,518) immunostimulatory oligonucleotides containing
ummethylated CpG dinucleotides ("CpG") are known as being adjuvants
when administered by both systemic and mucosal routes (WO 96/02555,
EP 468520, Davis et al., J. Immunol, 1998. 160(2):870-876;
McCluskie and Davis, J. Immunol., 1998, 161(9):4463-6). CpG is an
abbreviation for cytosine-guanosine dinucleotide motifs present in
DNA. The central role of the CG motif in immunostimulation was
elucidated by Krieg, Nature 374, p 546 1995. Detailed analysis has
shown that the CG motif has to be in a certain sequence context,
and that such sequences are common in bacterial DNA but are rare in
vertebrate DNA. The immunostimulatory sequence is often: Purine,
Purine, C, G, pyrimidine, pyrimidine; wherein the dinucleotide CG
motif is not methylated, but other unmethylated CpG sequences are
known to be immunostimulatory and may be used in certain
embodiments of the present invention. CpG when formulated into
vaccines, may be administered in free solution together with free
antigen (WO 96/02555; McCluskie and Davis, supra) or covalently
conjugated to an antigen (PCT Publication No. WO 98/16247), or
formulated with a carrier such as aluminium hydroxide (e.g., Davis
et al. supra, Brazolot-Millan et al., Proc. Natl. Acad. Sci., USA,
1998, 95(26), 15553-8).
[0140] The preferred oligonucleotides for use in adjuvants or
vaccines of the present invention preferably contain two or more
dinucleotide CpG motifs separated by at least three, more
preferably at least six or more nucleotides. The oligonucleotides
of the present invention are typically deoxynucleotides. In a
preferred embodiment the internucleotide in the oligonucleotide is
phosphorodithioate, or more preferably a phosphorothioate bond,
although phosphodiester and other internucleotide bonds are within
the scope of the invention including oligonucleotides with mixed
internucleotide linkages. Methods for producing phosphorothioate
oligonucleotides or phosphorodithioate are described in U.S. Pat.
Nos. 5,666,153, 5,278,302 and WO95/26204.
[0141] Examples of preferred oligonucleotides have sequences that
are disclosed in the following publications; for certain herein
disclosed embodiments the sequences preferably contain
phosphorothioate modified internucleotide linkages:
[0142] CPG 7909: Cooper et al., "CPG 7909 adjuvant improves
hepatitis B virus vaccine seroprotection in antiretroviral-treated
HIV-infected adults." AIDS, 2005 Sep. 23; 19(14):1473-9.
[0143] CpG 10101: Bayes et al., "Gateways to clinical trials."
Methods Find. Exp. Clin. Pharmacol. 2005 April; 27(3):193-219.
[0144] Vollmer J., "Progress in drug development of
immunostimula-tory CpG oligodeoxynucleotide ligands for TLR9."
Expert Opinion on Biological Therapy. 2005 May; 5(5): 673-682
[0145] Alternative CpG oligonucleotides may comprise variants of
the preferred sequences described in the above-cited publications
that differ in that they have inconsequential nucleotide sequence
substitutions, insertions, deletions and/or additions thereto. The
CpG oligonucleotides utilized in certain embodiments of the present
invention may be synthesized by any method known in the art (e.g.,
EP 468520). Conveniently, such oligonucleotides may be synthesized
utilising an automated synthesizer. The oligonucleotides are
typically deoxynucleotides. In a preferred embodiment the
internucleotide bond in the oligonucleotide is phosphorodithioate,
or more preferably phosphorothioate bond, although phosphodiesters
are also within the scope of the presently contemplated
embodiments. Oligonucleotides comprising different internucleotide
linkages are also contemplated, e.g., mixed phosphorothioate
phophodiesters. Other internucleotide bonds which stabilize the
oligonucleotide may also be used.
Co-Adjuvant
[0146] Certain embodiments as provided herein include vaccine
compositions and immunological adjuvant compositions, including
pharmaceutical compositions, that contain, in addition to GLA, at
least one co-adjuvant, which refers to a component of such
compositions that has adjuvant activity but that is other than GLA.
A co-adjuvant having such adjuvant activity includes a composition
that, when administered to a subject such as a human (e.g., a human
patient), a non-human primate, a mammal or another higher
eukaryotic organism having a recognized immune system, is capable
of altering (i.e., increasing or decreasing in a statistically
significant manner, and in certain preferred embodiments, enhancing
or increasing) the potency and/or longevity of an immune response.
(See, e.g., Powell and Newman, "Vaccine design--The Subunit and
Adjuvant Approach", 1995, Plenum Press, New York) In certain
embodiments disclosed herein GLA and a desired antigen, and
optionally one or more co-adjuvants, may so alter, e.g., elicit or
enhance, an immune response that is directed against the desired
antigen which may be administered at the same time as GLA or may be
separated in time and/or space (e.g., at a different anatomic site)
in its administration, but certain invention embodiments are not
intended to be so limited and thus also contemplate administration
of GLA in a composition that does not include a specified antigen
but which may also include one or more of a TLR agonist, a
co-adjuvant, an imidazoquinline immune response modifier, and a
double stem loop immune modifier (dSLIM).
[0147] Accordingly and as noted above, co-adjuvants include
compositions other than GLA that have adjuvant effects, such as
saponins and saponin mimetics, including QS21 and QS21 mimetics
(see, e.g., U.S. Pat. No. 5,057,540; EP 0 362 279 B1; WO 95/17210),
alum, plant alkaloids such as tomatine, detergents such as (but not
limited to) saponin, polysorbate 80, Span 85 and stearyl tyrosine,
one or more cytokines (e.g., GM-CSF, IL-2, IL-7, IL-12, TNF-alpha,
IFN-gamma), an imidazoquinoline immune response modifier, and a
double stem loop immune modifier (dSLIM, e.g., Weeratna et al.,
2005 Vaccine 23:5263).
[0148] Detergents including saponins are taught in, e.g., U.S. Pat.
No. 6,544,518; Lacaille-Dubois, M and Wagner H. (1996 Phytomedicine
2:363-386), U.S. Pat. No. 5,057,540, Kensil, Crit. Rev Ther Drug
Carrier Syst, 1996, 12 (1-2):1-55, and EP 0 362 279 B1. Particulate
structures, termed Immune Stimulating Complexes (ISCOMS),
comprising fractions of Quil A (saponin) are haemolytic and have
been used in the manufacture of vaccines (Morein, B., EP 0 109 942
B1). These structures have been reported to have adjuvant activity
(EP 0 109 942 B1; WO 96/11711). The haemolytic saponins QS21 and
QS17 (HPLC purified fractions of Quil A) have been described as
potent systemic adjuvants, and the method of their production is
disclosed in U.S. Pat. No. 5,057,540 and EP 0 362 279 B1. Also
described in these references is the use of QS7 (a non-haemolytic
fraction of Quil-A) which acts as a potent adjuvant for systemic
vaccines. Use of QS21 is further described in Kensil et al. (1991.
J. Immunology 146:431-437). Combinations of QS21 and polysorbate or
cyclodextrin are also known (WO 99/10008). Particulate adjuvant
systems comprising fractions of QuilA, such as QS21 and QS7 are
described in WO 96/33739 and WO 96/11711. Other saponins which have
been used in systemic vaccination studies include those derived
from other plant species such as Gypsophila and Saponaria (Bomford
et al., Vaccine, 10(9):572-577, 1992).
[0149] Escin is another detergent related to the saponins for use
in the adjuvant compositions of the embodiments herein disclosed.
Escin is described in the Merck index (12.sup.th Ed.: entry 3737)
as a mixture of saponin occurring in the seed of the horse chestnut
tree, Aesculus hippocastanum. Its isolation is described by
chromatography and purification (Fiedler, Arzneimittel-Forsch. 4,
213 (1953)), and by ion-exchange resins (Erbring et al., U.S. Pat.
No. 3,238,190). Fractions of escin (also known as aescin) have been
purified and shown to be biologically active (Yoshikawa M, et al.
(Chem Pharm Bull (Tokyo) 1996 August; 44(8): 1454-1464)). Digitonin
is another detergent, also being described in the Merck index (12th
Ed., entry 3204) as a saponin, being derived from the seeds of
Digitalis purpurea and purified according to the procedure
described by Gisvold et al., J. Am. Pharm. Assoc., 1934, 23, 664;
and Rubenstroth-Bauer, Physiol. Chem., 1955, 301, 621.
[0150] Other co-adjuvants for use according to certain herein
disclosed embodiments include a block co-polymer or biodegradable
polymer, which refers to a class of polymeric compounds with which
those in the relevant art will be familiar. Examples of a block
co-polymer or biodegradable polymer that may be included in a GLA
vaccine composition or a GLA immunological adjuvant include
Pluronic.RTM. L121 (BASF Corp., Mount Olive, N.J.; see, e.g., Yeh
et al., 1996 Pharm. Res. 13:1693; U.S. Pat. No. 5,565,209), CRL1005
(e.g., Triozzi et al., 1997 Clin Canc. Res. 3:2355),
poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA),
poly-(D,L-lactide-co-glycolide) (PLG), and polyl:C. (See, e.g.,
Powell and Newman, "Vaccine design--The Subunit and Adjuvant
Approach", 1995, Plenum Press, New York)
[0151] Certain embodiments contemplate GLA vaccines and GLA
immunological adjuvants that include an oil, which in some such
embodiments may contribute co-adjuvant activity and in other such
embodiments may additionally or alternatively provide a
pharmaceutically acceptable carrier or excipient. Any number of
suitable oils are known and may be selected for inclusion in
vaccine compositions and immunological adjuvant compositions based
on the present disclosure. Examples of such oils, by way of
illustration and not limitation, include squalene, squalane,
mineral oil, olive oil, cholesterol, and a mannide monooleate.
[0152] Immune response modifiers such as imidazoquinoline immune
response modifiers are also known in the art and may also be
included as co-adjuvants in certain presently disclosed
embodiments. Certain preferred imidazoquinoline immune response
modifiers include, by way of non-limiting example, resiquimod
(R848), imiquimod and gardiquimod (Hemmi et al., 2002 Nat. Immunol.
3:196; Gibson et al., 2002 Cell. Immunol. 218:74; Gorden et al.,
2005 J. Immunol. 174:1259); these and other imidazoquinoline immune
response modifiers may, under appropriate conditions, also have TLR
agonist activity as described herein. Other immune response
modifiers are the nucleic acid-based double stem loop immune
modifiers (dSLIM). Specific examples of dSLIM that are contemplated
for use in certain of the presently disclosed embodiments can be
found in Schmidt et al., 2006 Allergy 61:56; Weihrauch et al. 2005
Clin Cancer Res. 11(16):5993-6001; Modern Biopharmaceuticals, J.
Knablein (Editor). John Wiley & Sons, Dec. 6, 2005. (dSLIM
discussed on pages 183 to .about.200), and from Mologen AG (Berlin,
FRG: [retrieved online on Aug. 18, 2006 at
http://www.mologen.com/English/04.20-dSLIM.shtml].
[0153] As also noted above, one type of co-adjuvant for use with
GLA as described herein may be the aluminum co-adjuvants, which are
generally referred to as "alum." Alum co-adjuvants are based on the
following: aluminum oxy-hydroxide; aluminum hydroxyphosphoate; or
various proprietary salts. Vaccines that use alum co-adjuvants may
include vaccines for tetanus strains, HPV, hepatitis A, inactivated
polio virus, and other antigens as described herein. Alum
co-adjuvants are advantageous because they have a good safety
record, augment antibody responses, stabilize antigens, and are
relatively simple for large-scale production. (Edelman 2002 Mol.
Biotechnol. 21:129-148; Edelman, R. 1980 Rev. Infect. Dis.
2:370-383.)
[0154] Other co-adjuvants that may be combined with GLA for
effective immune stimulation include saponins and saponin mimetics,
including QS21 and structurally related compounds conferring
similar effects and referred to herein as QS21 mimetics. QS21 has
been recognized as a preferred co-adjuvant. QS21 may comprise an
HPLC purified non-toxic fraction derived from the bark of Quillaja
Saponaria Molina. The production of QS21 is disclosed in U.S. Pat.
No. 5,057,540. (See also U.S. Pat. Nos. 6,936,255, 7,029,678 and
6,932,972.)
[0155] GLA may also in certain embodiments be combined with
"immunostimulatory complexes" known as ISCOMS (e.g., U.S. Pat. Nos.
6,869,607, 6,846,489, 6,027,732, 4,981,684), including
saponin-derived ISCOMATRIX.RTM., which is commercially available,
for example, from Iscotec (Stockholm, Sweden) and CSL Ltd.
(Parkville, Victoria, Australia).
[0156] In still other embodiments, adjuvant/delivery systems may be
used in combination with GLA which are lipid assemblies (liposomes
and other formulations) comprising, for example, positively charged
polycationic lipids. Illustratively, such systems may include a
synthetic biocompatible polycationic sphingolipid (e.g.,
D-Erythro-N-palmitoyl sphingosyl-1-0 carbamoyl-spermine; also
referred to as CCS; available from NasVax Ltd.) or may include
cholesterol (C) in addition to the CCS lipid (referred to as CCS/C
or Vaxisome.RTM.; available from NasVax Ltd.). The vaccine
liposomal formulations may be formed, for example, by mixing a dry
powder of CCS or CCS/C with vaccine proteins or polynucleotides
(antigens), such as in aqueous suspension. Antigen-loaded liposomes
can then be sprayed into the nose (i.n.) or injected
intramuscularly (i.m.) or subcutaneously (s.c.), for example.
[0157] In further embodiments, a vesicular adjuvant/delivery system
consisting of unilamellar or multilamellar vesicles, called
niosomes, can be used in conjunction with GLA. In this case, an
aqueous solution is generally enclosed in a highly ordered bilayer
made up of non-ionic surfactant, with or without cholesterol and
dicetyl phosphate, and exhibit a behaviour similar to liposomes in
vivo. The bilayered vesicular structure is an assembly of
hydrophobic tails of surfactant monomer, shielded away from the
aqueous space located in the center and hydrophilic head group, in
contact with the same. Addition of cholesterol results in an
ordered liquid phase formation which gives the rigidity to the
bilayer, and results in less leaky niosomes. Dicetyl phosphate is
known to increase the size of vesicles, provide charge to the
vesicles, and thus shows increase entrapment efficiency. Other
charge-inducers are stearylamine and diacylglycerol, that also help
in electrostatic stabilization of the vesicles.
[0158] Niosomes have unique advantages over liposomes. Nisomes are
quite stable structures, even in the emulsified form. They require
no special conditions such as low temperature or inert atmosphere
for protection or storage, and are chemically stable. Relatively
low cost of materials makes it suitable for industrial manufacture.
A number of non-ionic surfactants have been used to prepare
vesicles, e.g., polyglycerol alkyl ether, glucosyl dialkyl ethers,
crown ethers, ester linked surfactants, polyoxyethylene alkyl
ether, Brij, and various spans and tweens.
[0159] Similar to liposomes, there are 3 major types of
niosomes--multilamellar vesicles (MLV, size >0.05 .mu.m), small
unilamellar vesicles (SUV, size -0.025-0.05 .mu.m), and large
unilamellar vesicles (LUV, size >0.10 .mu.m). MLVs vesicles
exhibit increased-trapped volume and equilibrium solute
distribution, and generally require hand-shaking method. They show
variations in lipid compositions. SUVs are commonly produced by
sonication, and French Press procedures. Ultrasonic
electrocapillary emulsification or solvent dilution techniques can
also be used to prepare SUVs. The injections of lipids solubilized
in an organic solvent into an aqueous buffer can result in
spontaneous formation of LUV. Another method of preparation of LUV
is reverse phase evaporation, or by detergent solubilization
method.
Recombinant Expression Construct
[0160] According to certain herein disclosed embodiments, the GLA
vaccine composition may contain at least one recombinant expression
construct which comprises a promoter operably linked to a nucleic
acid sequence encoding an antigen. In certain further embodiments
the recombinant expression construct is present in a viral vector,
such as an adenovirus, adeno-associated virus, herpesvirus,
lentivirus, poxvirus or retrovirus vector. Compositions and methods
for making and using such expression constructs and vectors are
known in the art, for the expression of polypeptide antigens as
provided herein, for example, according to Ausubel et al. (Eds.),
Current Protocols in Molecular Biology, 2006 John Wiley & Sons,
NY. Non-limiting examples of recombinant expression constructs
generally can be found, for instance, in U.S. Pat. Nos. 6,844,192;
7,037,712; 7,052,904; 7,001,770; 6,106,824; 5,693,531; 6,613,892;
6,875,610; 7,067,310; 6,218,186; 6,783,981; 7,052,904; 6,783,981;
6,734,172; 6,713,068; 5,795,577 and 6,770,445 and elsewhere, with
teachings that can be adapted to the expression of polypeptide
antigens as provided herein, for use in certain presently disclosed
embodiments.
Immune Response
[0161] The invention thus provides compositions for altering (i.e.,
increasing or decreasing in a statistically significant manner, for
example, relative to an appropriate control as will be familiar to
persons skilled in the art) immune responses in a host capable of
mounting an immune response. As will be known to persons having
ordinary skill in the art, an immune response may be any active
alteration of the immune status of a host, which may include any
alteration in the structure or function of one or more tissues,
organs, cells or molecules that participate in maintenance and/or
regulation of host immune status. Typically, immune responses may
be detected by any of a variety of well known parameters, including
but not limited to in vivo or in vitro determination of: soluble
immunoglobulins or antibodies; soluble mediators such as cytokines,
lymphokines, chemokines, hormones, growth factors and the like as
well as other soluble small peptide, carbohydrate, nucleotide
and/or lipid mediators; cellular activation state changes as
determined by altered functional or structural properties of cells
of the immune system, for example cell proliferation, altered
motility, induction of specialized activities such as specific gene
expression or cytolytic behavior; cellular differentiation by cells
of the immune system, including altered surface antigen expression
profiles or the onset of apoptosis (programmed cell death); or any
other criterion by which the presence of an immune response may be
detected.
[0162] Immune responses may often be regarded, for instance, as
discrimination between self and non-self structures by the cells
and tissues of a host's immune system at the molecular and cellular
levels, but the invention should not be so limited. For example,
immune responses may also include immune system state changes that
result from immune recognition of self molecules, cells or tissues,
as may accompany any number of normal conditions such as typical
regulation of immune system components, or as may be present in
pathological conditions such as the inappropriate autoimmune
responses observed in autoimmune and degenerative diseases. As
another example, in addition to induction by up-regulation of
particular immune system activities (such as antibody and/or
cytokine production, or activation of cell mediated immunity)
immune responses may also include suppression, attenuation or any
other down-regulation of detectable immunity, which may be the
consequence of the antigen selected, the route of antigen
administration, specific tolerance induction or other factors.
[0163] Determination of the induction of an immune response by the
vaccines of the present invention may be established by any of a
number of well known immunological assays with which those having
ordinary skill in the art will be readily familiar. Such assays
include, but need not be limited to, to in vivo or in vitro
determination of: soluble antibodies; soluble mediators such as
cytokines, lymphokines, chemokines, hormones, growth factors and
the like as well as other soluble small peptide, carbohydrate,
nucleotide and/or lipid mediators; cellular activation state
changes as determined by altered functional or structural
properties of cells of the immune system, for example cell
proliferation, altered motility, induction of specialized
activities such as specific gene expression or cytolytic behavior;
cellular differentiation by cells of the immune system, including
altered surface antigen expression profiles or the onset of
apoptosis (programmed cell death). Procedures for performing these
and similar assays are widely known and may be found, for example
in Lefkovits (Immunology Methods Manual: The Comprehensive
Sourcebook of Techniques, 1998; see also Current Protocols in
Immunology; see also, e.g., Weir, Handbook of Experimental
Immunology, 1986 Blackwell Scientific, Boston, Mass.; Mishell and
Shigii (eds.) Selected Methods in Cellular Immunology, 1979 Freeman
Publishing, San Francisco, Calif.; Green and Reed, 1998 Science
281:1309 and references cited therein.).
[0164] Detection of the proliferation of antigen-reactive T cells
may be accomplished by a variety of known techniques. For example,
T cell proliferation can be detected by measuring the rate of DNA
synthesis, and antigen specificity can be determined by controlling
the stimuli (such as, for example, a specific desired antigen- or a
control antigen-pulsed antigen presenting cells) to which candidate
antigen-reactive T cells are exposed. T cells which have been
stimulated to proliferate exhibit an increased rate of DNA
synthesis. A typical way to measure the rate of DNA synthesis is,
for example, by pulse-labeling cultures of T cells with tritiated
thymidine, a nucleoside precursor which is incorporated into newly
synthesized DNA. The amount of tritiated thymidine incorporated can
be determined using a liquid scintillation spectrophotometer. Other
ways to detect T cell proliferation include measuring increases in
interleukin-2 (IL-2) production, Ca.sup.2+ flux, or dye uptake,
such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium.
Alternatively, synthesis of lymphokines (such as interferon-gamma)
can be measured or the relative number of T cells that can respond
to a particular antigen may be quantified.
[0165] Detection of antigen-specific antibody production may be
achieved, for example, by assaying a sample (e.g., an
immunoglobulin containing sample such as serum, plasma or blood)
from a host treated with a vaccine according to the present
invention using in vitro methodologies such as radioimmunoassay
(RIA), enzyme linked immunosorbent assays (ELISA), equilibrium
dialysis or solid phase immunoblotting including Western blotting.
In preferred embodiments ELISA assays may further include
antigen-capture immobilization of the target antigen with a solid
phase monoclonal antibody specific for the antigen, for example, to
enhance the sensitivity of the assay. Elaboration of soluble
mediators (e.g., cytokines, chemokines, lymphokines,
prostaglandins, etc.) may also be readily determined by
enzyme-linked immunosorbent assay (ELISA), for example, using
methods, apparatus and reagents that are readily available from
commercial sources (e.g., Sigma, St. Louis, Mo.; see also R & D
Systems 2006 Catalog, R & D Systems, Minneapolis, Minn.).
[0166] Any number of other immunological parameters may be
monitored using routine assays that are well known in the art.
These may include, for example, antibody dependent cell-mediated
cytotoxicity (ADCC) assays, secondary in vitro antibody responses,
flow immunocytofluorimetric analysis of various peripheral blood or
lymphoid mononuclear cell subpopulations using well established
marker antigen systems, immunohistochemistry or other relevant
assays. These and other assays may be found, for example, in Rose
et al. (Eds.), Manual of Clinical Laboratory Immunology, 5.sup.th
Ed., 1997 American Society of Microbiology, Washington, D.C.
[0167] Accordingly it is contemplated that the vaccine and adjuvant
compositions provided herein will be capable of eliciting or
enhancing in a host at least one immune response that is selected
from a T.sub.H1-type T lymphocyte response, a T.sub.H2-type T
lymphocyte response, a cytotoxic T lymphocyte (CTL) response, an
antibody response, a cytokine response, a lymphokine response, a
chemokine response, and an inflammatory response. In certain
embodiments the immune response may comprise at least one of
production of one or a plurality of cytokines wherein the cytokine
is selected from interferon-gamma (IFN-.gamma.), tumor necrosis
factor-alpha (TNF-.alpha.), production of one or a plurality of
interleukins wherein the interleukin is selected from IL-1, IL-2,
IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18 and
IL-23, production one or a plurality of chemokines wherein the
chemokine is selected from MIP-1.alpha., MIP-1.beta., RANTES, CCL4
and CCL5, and a lymphocyte response that is selected from a memory
T cell response, a memory B cell response, an effector T cell
response, a cytotoxic T cell response and an effector B cell
response. See, e.g., WO 94/00153; WO 95/17209; WO 96/02555; U.S.
Pat. No. 6,692,752; U.S. Pat. No. 7,084,256; U.S. Pat. No.
6,977,073; U.S. Pat. No. 6,749,856; U.S. Pat. No. 6,733,763; U.S.
Pat. No. 6,797,276; U.S. Pat. No. 6,752,995; U.S. Pat. No.
6,057,427; U.S. Pat. No. 6,472,515; U.S. Pat. No. 6,309,847; U.S.
Pat. No. 6,969,704; U.S. Pat. No. 6,120,769; U.S. Pat. No.
5,993,800; U.S. Pat. No. 5,595,888; Smith et al., 1987 J Biol Chem.
262:6951; Kriegler et al., 1988 Cell 53:45 53;Beutler et al., 1986
Nature 320:584; U.S. Pat. No. 6,991,791; U.S. Pat. No. 6,654,462;
U.S. Pat. No. 6,375,944.
Pharmaceutical Compositions
[0168] Pharmaceutical compositions generally comprise GLA
(available from Avanti Polar Lipids, Inc., Alabaster, Ala.; product
number 699800) and may further comprise one or more components as
provided herein that are selected from antigen, TLR agonist,
co-adjuvant (including optionally a cytokine, an imidazoquinoline
immune response modifier and/or a dSLIM), and/or a recombinant
expression construct, in combination with a pharmaceutically
acceptable carrier, excipient or diluent.
[0169] Therefore, in certain aspects, the present invention is
drawn to GLA "monotherapy" wherein GLA, as described herein, is
formulated in a composition that is substantially devoid of other
antigens, and is administered to a subject in order to stimulate an
immune response, e.g., a non-specific immune response, for the
purpose of treating or preventing a disease or other condition,
such as for treating an infection by an organism, for treating
seasonal rhinitis, or the like. In one embodiment, for example, the
compositions and methods of the invention employ a
monophosphorylated disaccharide for stimulating an immune response
in a subject. In another particular embodiment, the compositions
and methods employ a 2-monoacyl form of Lipid A for stimulating an
immune response in a subject. In another particular embodiment, the
GLA is in the form of a spray, optionally provided in a kit.
[0170] The GLA may be preferably formulated in a stable emulsion.
In one particular embodiment, for example, a composition is
provided comprising a lipid A derivative in a stable emulsion
substantially devoid of other antigens. In another particular
embodiment, a composition is provided comprising a derivative of
3-acylated monophosphorylated lipid A, suitable for use in mammals,
wherein the 2 amine position has a single acyl chain, and that is
substantially devoid of other antigens.
[0171] In certain other embodiments, the pharmaceutical composition
is a vaccine composition that comprises both GLA and an antigen and
may further comprise one or more components, as provided herein,
that are selected from TLR agonist, co-adjuvant (including, e.g., a
cytokine, an imidazoquinoline immune response modifier and/or a
dSLIM) and the like and/or a recombinant expression construct, in
combination with a pharmaceutically acceptable carrier, excipient
or diluent.
[0172] Illustrative carriers will be nontoxic to recipients at the
dosages and concentrations employed. For GLA-plus-nucleic
acid-based vaccines, or for vaccines comprising GLA plus an
antigen, about 0.01 .mu.g/kg to about 100 mg/kg body weight will be
administered, typically by the intradermal, subcutaneous,
intramuscular or intravenous route, or by other routes.
[0173] A preferred dosage is about 1 .mu.g/kg to about 1 mg/kg,
with about 5 .mu.g/kg to about 200 .mu.g/kg particularly preferred.
It will be evident to those skilled in the art that the number and
frequency of administration will be dependent upon the response of
the host. "Pharmaceutically acceptable carriers" for therapeutic
use are well known in the pharmaceutical art, and are described,
for example, in Remingtons Pharmaceutical Sciences, Mack Publishing
Co. (A. R. Gennaro edit. 1985). For example, sterile saline and
phosphate-buffered saline at physiological pH may be used.
Preservatives, stabilizers, dyes and even flavoring agents may be
provided in the pharmaceutical composition. For example, sodium
benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be
added as preservatives. Id. at 1449. In addition, antioxidants and
suspending agents may be used. Id.
[0174] "Pharmaceutically acceptable salt" refers to salts of the
compounds of the present invention derived from the combination of
such compounds and an organic or inorganic acid (acid addition
salts) or an organic or inorganic base (base addition salts). The
compositions of the present invention may be used in either the
free base or salt forms, with both forms being considered as being
within the scope of the present invention.
[0175] The pharmaceutical compositions may be in any form which
allows for the composition to be administered to a patient. For
example, the composition may be in the form of a solid, liquid or
gas (aerosol). Typical routes of administration include, without
limitation, oral, topical, parenteral (e.g., sublingually or
buccally), sublingual, rectal, vaginal, and intranasal (e.g., as a
spray). The term parenteral as used herein includes iontophoretic
(e.g., U.S. Pat. Nos. 7,033,598; 7,018,345; 6,970,739),
sonophoretic (e.g., U.S. Pat. Nos. 4,780,212; 4,767,402; 4,948,587;
5,618,275; 5,656,016; 5,722,397; 6,322,532; 6,018,678), thermal
(e.g., U.S. Pat. Nos. 5,885,211; 6,685,699), passive transdermal
(e.g., U.S. Pat. Nos. 3,598,122; 3,598,123; 4,286,592; 4,314,557;
4,379,454; 4,568,343; 5,464,387; UK Pat. Spec. No. 2232892; U.S.
Pat. Nos. 6,871,477; 6,974,588; 6,676,961), microneedle (e.g., U.S.
Pat. Nos. 6,908,453; 5,457,041; 5,591,139; 6,033,928)
administration and also subcutaneous injections, intravenous,
intramuscular, intrasternal, intracavernous, intrathecal,
intrameatal, intraurethral injection or infusion techniques. In a
particular embodiment, a composition as described herein (including
vaccine and pharmaceutical compositions) is administered
intradermally by a technique selected from iontophoresis,
microcavitation, sonophoresis or microneedles.
[0176] The pharmaceutical composition is formulated so as to allow
the active ingredients contained therein to be bioavailable upon
administration of the composition to a patient. Compositions that
will be administered to a patient take the form of one or more
dosage units, where for example, a tablet may be a single dosage
unit, and a container of one or more compounds of the invention in
aerosol form may hold a plurality of dosage units.
[0177] For oral administration, an excipient and/or binder may be
present. Examples are sucrose, kaolin, glycerin, starch dextrins,
sodium alginate, carboxymethylcellulose and ethyl cellulose.
Coloring and/or flavoring agents may be present. A coating shell
may be employed.
[0178] The composition may be in the form of a liquid, e.g., an
elixir, syrup, solution, emulsion or suspension. The liquid may be
for oral administration or for delivery by injection, as two
examples. When intended for oral administration, preferred
compositions contain one or more of a sweetening agent,
preservatives, dye/colorant and flavor enhancer. In a composition
intended to be administered by injection, one or more of a
surfactant, preservative, wetting agent, dispersing agent,
suspending agent, buffer, stabilizer and isotonic agent may be
included.
[0179] A liquid pharmaceutical composition as used herein, whether
in the form of a solution, suspension or other like form, may
include one or more of the following carriers or excipients:
sterile diluents such as water for injection, saline solution,
preferably physiological saline, Ringer's solution, isotonic sodium
chloride, fixed oils such as squalene, squalane, mineral oil, a
mannide monooleate, cholesterol, and/or synthetic mono or
digylcerides which may serve as the solvent or suspending medium,
polyethylene glycols, glycerin, propylene glycol or other solvents;
antibacterial agents such as benzyl alcohol or methyl paraben;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. The parenteral
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic. An injectable
pharmaceutical composition is preferably sterile.
[0180] In a particular embodiment, a pharmaceutical or vaccine
composition of the invention comprises a stable aqueous suspension
of less than 0.2 um and further comprises at least one component
selected from the group consisting of phospholipids, fatty acids,
surfactants, detergents, saponins, fluorodated lipids, and the
like.
[0181] In another embodiment, a composition of the invention is
formulated in a manner which can be aerosolized.
[0182] It may also be desirable to include other components in a
vaccine or pharmaceutical composition, such as delivery vehicles
including but not limited to aluminum salts, water-in-oil
emulsions, biodegradable oil vehicles, oil-in-water emulsions,
biodegradable microcapsules, and liposomes. Examples of additional
immunostimulatory substances (co-adjuvants) for use in such
vehicles are also described above and may include
N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucan, IL-12,
GM-CSF, gamma interferon and IL-12.
[0183] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention, the type of carrier will vary depending on the mode
of administration and whether a sustained release is desired. For
parenteral administration, such as subcutaneous injection, the
carrier preferably comprises water, saline, alcohol, a fat, a wax
or a buffer. For oral administration, any of the above carriers or
a solid carrier, such as mannitol, lactose, starch, magnesium
stearate, sodium saccharine, talcum, cellulose, glucose, sucrose,
and magnesium carbonate, may be employed. Biodegradable
microspheres (e.g., polylactic galactide) may also be employed as
carriers for the pharmaceutical compositions of this invention.
Suitable biodegradable microspheres are disclosed, for example, in
U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, it is
preferable that the microsphere be larger than approximately 25
microns.
[0184] Pharmaceutical compositions (including GLA vaccines and GLA
immunological adjuvants) may also contain diluents such as buffers,
antioxidants such as ascorbic acid, low molecular weight (less than
about 10 residues) polypeptides, proteins, amino acids,
carbohydrates including glucose, sucrose or dextrins, chelating
agents such as EDTA, glutathione and other stabilizers and
excipients. Neutral buffered saline or saline mixed with
nonspecific serum albumin are exemplary appropriate diluents.
Preferably, product may be formulated as a lyophilizate using
appropriate excipient solutions (e.g., sucrose) as diluents.
[0185] As described above, in certain embodiments the subject
invention includes compositions capable of delivering nucleic acid
molecules encoding desired antigens. Such compositions include
recombinant viral vectors (e.g., retroviruses (see WO 90/07936, WO
91/02805, WO 93/25234, WO 93/25698, and
[0186] WO 94/03622), adenovirus (see Berkner, Biotechniques
6:616-627, 1988; Li et al., Hum. Gene Ther. 4:403-409, 1993;
Vincent et al., Nat. Genet. 5:130-134, 1993; and Kolls et al.,
Proc. Natl. Acad. Sci. USA 91:215-219, 1994), pox virus (see U.S.
Pat. No. 4,769,330; U.S. Pat. No. 5,017,487; and WO 89/01973)),
recombinant expression construct nucleic acid molecules complexed
to a polycationic molecule (see WO 93/03709), and nucleic acids
associated with liposomes (see Wang et al., Proc. Natl. Acad. Sci.
USA 84:7851, 1987). In certain embodiments, the DNA may be linked
to killed or inactivated adenovirus (see Curiel et al., Hum. Gene
Ther. 3:147-154, 1992; Cotton et al., Proc. Natl. Acad. Sci. USA
89:6094, 1992). Other suitable compositions include DNA-ligand (see
Wu et al., J. Biol. Chem. 264:16985-16987, 1989) and lipid-DNA
combinations (see Feigner et al., Proc. Natl. Acad. Sci. USA
84:7413-7417, 1989).
[0187] In addition to direct in vivo procedures, ex vivo procedures
may be used in which cells are removed from a host, modified, and
placed into the same or another host animal. It will be evident
that one can utilize any of the compositions noted above for
introduction of antigen-encoding nucleic acid molecules into tissue
cells in an ex vivo context. Protocols for viral, physical and
chemical methods of uptake are well known in the art.
[0188] Accordingly, the present invention is useful for enhancing
or eliciting, in a host, a patient or in cell culture, an immune
response. As used herein, the term "patient" refers to any
warm-blooded animal, preferably a human. A patient may be afflicted
with an infectious disease, cancer, such as breast cancer, or an
autoimmune disease, or may be normal (i.e., free of detectable
disease and/or infection). A "cell culture" is any preparation
containing immunocompetent cells or isolated cells of the immune
system (including, but not limited to, T cells, macrophages,
monocytes, B cells and dendritic cells). Such cells may be isolated
by any of a variety of techniques well known to those of ordinary
skill in the art (e.g., Ficoll-hypaque density centrifugation). The
cells may (but need not) have been isolated from a patient
afflicted with cancer, and may be reintroduced into a patient after
treatment.
[0189] In certain embodiments a liquid composition intended for
either parenteral or oral administration should contain an amount
of GLA vaccine composition such that a suitable dosage will be
obtained. Typically, this amount is at least 0.01 wt % of an
antigen in the composition. When intended for oral administration,
this amount may be varied to be between 0.1 and about 70% of the
weight of the composition. Preferred oral compositions contain
between about 4% and about 50% of the antigen. Preferred
compositions and preparations are prepared so that a parenteral
dosage unit contains between 0.01 to 1% by weight of active
composition.
[0190] The pharmaceutical composition may be intended for topical
administration, in which case the carrier may suitably comprise a
solution, emulsion, ointment or gel base. The base, for example,
may comprise one or more of the following: petrolatum, lanolin,
polyethylene glycols, beeswax, mineral oil, diluents such as water
and alcohol, and emulsifiers and stabilizers. Thickening agents may
be present in a pharmaceutical composition for topical
administration. If intended for transdermal administration, the
composition may include a transdermal patch or iontophoresis
device. Topical formulations may contain a concentration of the
antigen (e.g., GLA-antigen vaccine composition) or GLA (e.g.,
immunological adjuvant composition; GLA is available from Avanti
Polar Lipids, Inc., Alabaster, Ala.; e.g., product number 699800)
of from about 0.1 to about 10% w/v (weight per unit volume).
[0191] The composition may be intended for rectal administration,
in the form, e.g., of a suppository which will melt in the rectum
and release the drug. The composition for rectal administration may
contain an oleaginous base as a suitable nonirritating excipient.
Such bases include, without limitation, lanolin, cocoa butter and
polyethylene glycol. In the methods of the invention, the vaccine
compositions/adjuvants may be administered through use of
insert(s), bead(s), timed-release formulation(s), patch(es) or
fast-release formulation(s).
[0192] Also contemplated in certain embodiments are kits comprising
the herein described GLA vaccine compositions and/or GLA
immunological adjuvant compositions, which may be provided in one
or more containers. In one embodiment all components of the GLA
vaccine compositions and/or GLA immunological adjuvant compositions
are present together in a single container, but the invention
embodiments are not intended to be so limited and also contemplate
two or more containers in which, for example, a GLA immunological
adjuvant composition is separate from, and not in contact with, the
antigen component. By way of non-limiting theory, it is believed
that in some cases administration only of the GLA immunological
adjuvant composition may be performed beneficially, whilst in other
cases such administration may beneficially be separated temporally
and/or spatially (e.g., at a different anatomical site) from
administration of the antigen, whilst in still other cases
administration to the subject is beneficially conducted of a GLA
vaccine composition as described herein and containing both antigen
and GLA, and optionally other herein described components as
well.
[0193] A container according to such kit embodiments may be any
suitable container, vessel, vial, ampule, tube, cup, box, bottle,
flask, jar, dish, well of a single-well or multi-well apparatus,
reservoir, tank, or the like, or other device in which the herein
disclosed compositions may be placed, stored and/or transported,
and accessed to remove the contents. Typically such a container may
be made of a material that is compatible with the intended use and
from which recovery of the contained contents can be readily
achieved. Preferred examples of such containers include glass
and/or plastic sealed or re-sealable tubes and ampules, including
those having a rubber septum or other sealing means that is
compatible with withdrawal of the contents using a needle and
syringe. Such containers may, for instance, by made of glass or a
chemically compatible plastic or resin, which may be made of, or
may be coated with, a material that permits efficient recovery of
material from the container and/or protects the material from,
e.g., degradative conditions such as ultraviolet light or
temperature extremes, or from the introduction of unwanted
contaminants including microbial contaminants. The containers are
preferably sterile or sterilizable, and made of materials that will
be compatible with any carrier, excipient, solvent, vehicle or the
like, such as may be used to suspend or dissolve the herein
described vaccine compositions and/or immunological adjuvant
compositions and/or antigens and/or recombinant expression
constructs, etc.
[0194] Emulsion systems may also be used in formulating
compositions of the present invention. For example, many single or
multiphase emulsion systems have been described. Oil in water
emulsion adjuvants per se have been suggested to be useful as
adjuvant composition (EP 0 399 843B), also combinations of oil in
water emulsions and other active agents have been described as
adjuvants for vaccines (WO 95/17210; WO 98/56414; WO 99/12565; WO
99/11241). Other oil emulsion adjuvants have been described, such
as water in oil emulsions (U.S. Pat. No. 5,422,109; EP 0 480 982
B2) and water in oil in water emulsions (U.S. Pat. No. 5,424,067;
EP 0 480 981 B). The oil emulsion adjuvants for use in the present
invention may be natural or synthetic, and may be mineral or
organic. Examples of mineral and organic oils will be readily
apparent to the man skilled in the art.
[0195] In a particular embodiment, a composition of the invention
comprises an emulsion of oil in water wherein the GLA is
incorporated in the oil phase. In another embodiment, a composition
of the invention comprises an emulsion of oil in water wherein the
GLA is incorporated in the oil phase and wherein an additional
component is present, such as a co-adjuvant, TLR agonist, or the
like, as described herein.
[0196] In order for any oil in water composition to be suitable for
human administration, the oil phase of the emulsion system
preferably comprises a metabolizable oil. The meaning of the term
metabolizable oil is well known in the art. Metabolizable can be
defined as "being capable of being transformed by metabolism"
(Dorland's illustrated Medical Dictionary, W. B. Saunders Company,
25th edition (1974)). The oil may be any vegetable oil, fish oil,
animal oil or synthetic oil, which is not toxic to the recipient
and is capable of being transformed by metabolism. Nuts (such as
peanut oil), seeds, and grains are common sources of vegetable
oils. Synthetic oils are also part of this invention and can
include commercially available oils such as NEOBEE.RTM. and
others.
[0197] Squalene
(2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene), for
example, is an unsaturated oil which is found in large quantities
in shark-liver oil, and in lower quantities in olive oil, wheat
germ nil, rice bran oil, and yeast, and is a particularly preferred
oil for use in this invention. Squalene is a metabolizable oil
virtue of the fact that it is an intermediate in the biosynthesis
of cholesterol (Merck index, 10th Edition, entry no. 8619).
Particularly preferred oil emulsions are oil in water emulsions,
and in particular squalene in water emulsions. In addition, the
most preferred oil emulsion adjuvants of the present invention
comprise an antioxidant, which is preferably the oil
.alpha.-tocopherol (vitamin E, EP 0 382 271 B1). WO 95/17210 and WO
99/11241 disclose emulsion adjuvants based on squalene,
alpha-tocopherol, and TWEEN.RTM. 80, optionally formulated with the
immunostimulants QS21 and/or 3D-MPL (which are discussed above). WO
99/12565 discloses an improvement to these squalene emulsions with
the addition of a sterol into the oil phase. Additionally, a
triglyceride, such as tricaprylin (C.sub.27H.sub.50O.sub.6), may be
added to the oil phase in order to stabilize the emulsion (WO
98/56414).
[0198] The size of the oil droplets found within the stable oil in
water emulsion are preferably less than 1 micron, may be in the
range of substantially 30-600 nm, preferably substantially around
30-500 nm in diameter, and most preferably substantially 150-500 nm
in diameter, and in particular about 150 nm in diameter as measured
by photon correlation spectroscopy. In this regard, 80% of the oil
droplets by number should be within the preferred ranges, more
preferably more than 90% and most preferably more than 95% of the
oil droplets by number are within the defined size ranges The
amounts of the components present in the oil emulsions of the
present invention are conventionally in the range of from 2 to 10%
oil, such as squalene; and when present, from 2 to 10% alpha
tocopherol; and from 0.3 to 3% surfactant, such as polyoxyethylene
sorbitan monooleate. Preferably the ratio of oil:alpha tocopherol
is equal or less than 1 as this provides a more stable emulsion.
Span 85 may also be present at a level of about 1%. In some cases
it may be advantageous that the vaccines of the present invention
will further contain a stabiliser.
[0199] The method of producing oil in water emulsions is well known
to the person skilled in the art. Commonly, the method comprises
the mixing the oil phase with a surfactant such as a
PBS/TWEEN80.RTM. solution, followed by homogenization using a
homogenizer. For instance, a method that comprises passing the
mixture once, twice or more times through a syringe needle would be
suitable for homogenizing small volumes of liquid. Equally, the
emulsification process in a microfluidiser (M110S microfluidics
machine, maximum of 50 passes, for a period of 2 minutes at maximum
pressure input of 6 bar (output pressure of about 850 bar)) could
be adapted to produce smaller or larger volumes of emulsion. This
adaptation could be achieved by routine experimentation comprising
the measurement of the resultant emulsion until a preparation was
achieved with oil droplets of the required diameter.
[0200] The following Examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
GLA Aqueous Formulation
[0201] This example describes the preparation of a GLA-containing
adjuvant aqueous formulation. The aqueous formulation of GLA
(GLA-AF) contains Water For Injection (WFI), GLA (Avanti Polar
Lipids, Inc., Alabaster, Ala.; product number 699800), and
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The
formulation was prepared by adding a solution of ethanol and POPC
to a pre-weighed amount of GLA. This wetted GLA was sonicated for
10 minutes to disperse the GLA as much as possible. The GLA was
then dried under nitrogen gas. The dried GLA and POPC were
reconstituted with WFI to the correct volume. This solution was
sonicated at 60.degree. C. for 15-30 minutes until all the GLA and
POPC were in solution. For long term storage, GLA-AF formulations
must be lyophilized. The lyophilization process consisted of adding
glycerol to the solution until it was 2% of the total volume. Then
the solution was placed in vials in 1-10 mL amounts. The vials were
then run through the lyophilization process which consisted of
freezing the solution and then putting it under vacuum to draw off
the frozen water by sublimation.
Example 2
GLA HPLC Analysis
[0202] This example describes HPLC analysis of a GLA-containing
adjuvant aqueous formulation. After the formulation was
manufactured (see Example 1 above), certain release and stability
tests were conducted to ensure product quality and reproducibility.
All formulations were tested for release and long-term stability
using High Performance Liquid Chromatography (HPLC), Dynamic Light
Scattering (DLS) and a visual examination. HPLC chromatograms were
collected using an Agilent 1100 system and an ESA Corona CAD
detector. The method was run using a methanol to chloroform
gradient on a Waters Atlantis C18 column. The injections included
2.5 .mu.g of GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.;
product number 699800, GLA-AF) or MPL.RTM. (GSK Biologicals,
Rixensart, Belgium, MPL-AF) respectively, and 0.27 .mu.g of
synthetic phosphocholine (POPC) which was used as a solubilizing
agent.
[0203] FIG. 1 shows HPLC data demonstrating the number and amounts
of contaminating materials in MPL-AF and GLA-AF.
[0204] The HPLC profiles showed that GLA-AF was substantially purer
than MPL-AF. That is, there were fewer contaminant peaks in the
GLA-AF than in the MPL-AF adjuvant formulation. A purer starting
product is of tremendous benefit to researchers as the biological
response obtained is from the single major component used in the
formulations of the GLA.
Example 3
GLA Oil Formulation
[0205] This example describes preparation of one milliliter of a
GLA-containing adjuvant oil formulation. GLA (100 micrograms;
Avanti Polar Lipids, Inc., Alabaster, Ala.; product number 699800)
was emulsified in squalene (34.3 mg) with glycerol (22.7 mg),
phosphotidylcholine or lecithin (7.64 mg), Pluronic.RTM. F-68 (BASF
Corp., Mount Olive, N.J.) or similar block co-polymer (0.364 mg) in
25 millimolar ammonium phosphate buffer (pH=5.1) using 0.5 mg
D,L-alpha-tocopherol as an antioxidant. The mixture was processed
under high pressure until an emulsion formed that did not separate
and that had an average particle size of less than 180 nm. The
emulsion was then sterile-filtered into glass unidose vials and
capped for longer term storage. This preparation may be used for at
least three years when stored at 2-8.degree. C.
Example 4
GLA Stimulation of Murine Macrophages and Dendritic Cells
[0206] This example describes an in vitro model demonstrating an
adjuvant effect of GLA. Standard tissue culture methodologies and
reagents were employed. Cells of the murine J774 and RAW267.4
macrophage cell line (American Type Culture Collection, Manassas,
Va.) were maintained according to the supplier's recommendations
and cultured as adherent cell monolayers in multiwell dishes.
Dendritic cells were derived from bone marrow progenitor cells
following a protocol by Xiong et al. (J. Biol. Chem. 2004, 279, pp
10776-83). Various adjuvant concentrations of synthetic GLA (Avanti
Polar Lipids, Inc., Alabaster, Ala.; product number 699800) were
achieved by diluting an aqueous adjuvant preparation in cell
culture medium (DMEM containing 10% fetal bovine serum), and cells
were maintained for 24 hours at 37.degree. C. in a humidified
atmosphere containing 5% CO.sub.2, prior to collection of cell-free
culture supernatants. Supernatant fluids were assayed for soluble
murine cytokines such as IL-12, IL-6, and TNF, and chemokines such
as RANTES, using specific sandwich ELISA assay kits (eBiosciences,
San Diego, Calif. for cytokines, and R&D Systems, Minneapolis,
Minn. for chemokines) according to the manufacturer's
instructions.
[0207] GLA-AF induced dose-dependent immune responses in mouse
macrophage cell lines and primary murine DC, characterized by the
secretion of cytokines such as IL-12p40, IL-6, and TNF, and
chemokines like RANTES.
Example 5
GLA Stimulation of Human Macrophages and Dendritic Cells
[0208] This example describes an in vitro model demonstrating the
adjuvant effects of GLA. Standard tissue culture methodologies and
reagents were employed.
[0209] Cells of the human Mono Mac 6 macrophage cell line (American
Type Culture Collection, Manassas, Va.) were maintained according
to the supplier's recommendations and cultured as adherent cell
monolayers in multiwell plates. Dendritic cells were derived from
peripheral blood mononuclear cells (PBMC) following a standard
protocol. Various adjuvant concentrations of either synthetic GLA
(Avanti Polar Lipids, Inc., Alabaster, Ala.; product number 699800)
or the natural product MPL.RTM. (GSK Biologicals, Rixensart,
Belgium) were achieved by diluting an aqueous adjuvant preparation
in cell culture medium (DMEM containing 10% fetal bovine serum, for
MonoMac 6, or 10% human serum, for DC), and cells were maintained
for 24 hours at 37.degree. C. in a humidified atmosphere containing
5% CO.sub.2, prior to collection of cell-free culture supernatants.
Supernatant fluids were assayed for soluble human cytokines such as
IL-1.beta., IL-23, and IL-6, and chemokines such as IP-10, RANTES
and MIP-1 .beta. using specific sandwich ELISA assay kits
(eBiosciences, San Diego, Calif. for cytokines, and Invitrogen,
Carlsbad, Calif., for chemokines) according to the manufacturer's
instructions.
[0210] FIG. 2 shows ELISA data demonstrating levels of cytokines
and chemokines expressed by human macrophages of the Mono Mac 6
cell line (panels a-e), and monocyte-derived DC (panels f-h) in
response to GLA stimulation.
[0211] GLA-AF induced a dose-dependent immune response in the human
macrophage cell line Mono Mac 6 (FIG. 2, panels a-e), and primary
DC (FIG. 2, panels f-h), characterized by the secretion of
cytokines such as IL-1 .beta., IL-6, IL-23, and chemokines such as
RANTES, IP-10, MIP-1.beta.. GLA-AF was active at concentrations
5-500 lower compared to MPL-AF for all the cytokines and chemokines
that were tested.
Example 6
GLA Stimulation of Human Blood Cells
[0212] This example describes an in vitro model demonstrating
adjuvant effects of GLA. Standard tissue culture methodologies and
reagents were employed.
[0213] Human whole blood cells were cultured with various adjuvant
concentrations of either synthetic GLA (Avanti Polar Lipids, Inc.,
Alabaster, Ala.; product number 699800) or the natural product
MPL.RTM. (GSK Biologicals, Rixensart, Belgium), achieved by
diluting an aqueous adjuvant preparation in cell culture medium
(DMEM containing 10% fetal bovine serum). Blood cells were
maintained for 16 hours at 37.degree. C. in a humidified atmosphere
containing 5% CO.sub.2, prior to collection of cell-free culture
supernatants. Supernatant fluids were assayed for soluble human
cytokine IL-1.beta. using specific sandwich ELISA assay kit
(eBiosciences, San Diego, Calif.) according to the manufacturer's
instructions.
[0214] GLA-AF induced a dose-dependent immune response in human
whole blood cells, characterized by the secretion of IL-1.beta.
cytokine. In this assay, 92 nM of GLA was equivalent in potency to
57,000 nM of MPL-AF.
Example 7
Use of GLA-Containing Vaccine In Vivo
[0215] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine against Influenza. Standard
immunological methodologies and reagents were employed (Current
Protocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley
& Sons, NY).
[0216] Mice (three Balb/c animals per group) were immunized twice
at three-week intervals with the Fluzone vaccine (Sanofi-Aventis,
Swiftwater, Pa., at 1/25 (20 .mu.l) and 1/250 (2 .mu.l) of the
human dosage, alone, or formulated in (i) an aqueous emulsion
containing GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; product
number 699800; 20 .mu.g per animal for each immunization) according
to the procedure used in Example 1 above ("GLA-AF"), or (ii) a
stable emulsion containing GLA (Avanti Polar Lipids, Inc.,
Alabaster, Ala.; product number 699800; 20 .mu.g per animal for
each immunization) according to the procedure used in Example 3
above ("GLA-SE"). Sera were collected by bleeding animals one week
after each immunization, and serum levels of total IgG antibodies
specific for Fluzone were examined by ELISA according to published
methods (Id.). Serum levels of virus neutralizing antibodies were
also examined by Hemagglutination Inhibition Assay (HAI) according
to published methods.
[0217] FIG. 3 shows ELISA data demonstrating levels of anti-Fluzone
antibody production induced in mice one week after each
immunization (i.e., at day 7, panel A; and at day 28, panel B)
using two different doses of Fluzone vaccine formulated with
GLA-AF, or GLA-SE, compared to Fluzone alone. Means and SEM of
reciprocal endpoint titers in each group/time point are shown. FIG.
3, panel C shows HAI data demonstrating levels of virus
neutralizing antibody production induced in mice one week after the
second immunization using two different doses of Fluzone vaccine
formulated with GLA-AF, or GLA-SE, compared to Fluzone alone. Means
and SEM of reciprocal endpoint titers in each group/time point are
shown.
[0218] Total IgG and neutralizing antibody titers in response to
Fluzone vaccination were enhanced by adding GLA, either in an
aqueous or stable oil formulation. The adjuvanting effect of GLA
was more pronounced with the 2 .mu.l dose of Fluzone vaccine, and
induced antigen-specific humoral responses similar to (GLA-AF) or
greater than (GLA-SE) 20 .mu.l of Fluzone vaccine alone. These
results suggest that it is possible to reduce the dose of Fluzone
vaccine by adjuvanting it with GLA-containing formulations, and
still induce high levels of IgG and neutralizing antibody titers.
This is of particular importance in the context of a world pandemic
infection such as Bird Flu.
Example 8
Use of GLA-Containing Vaccine In Vivo
[0219] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine containing a specific
Leishmania antigen. Standard immunological methodologies and
reagents were employed (Current Protocols in Immunology, Coligan et
al. (Eds.) 2006 John Wiley & Sons, NY).
[0220] Mice (three C57BL/6 animals per group) were immunized three
times at three-week intervals with the SMT antigen (10 .mu.g per
animal for each immunization) used alone or formulated in a stable
emulsion containing GLA (Avanti Polar Lipids, Inc., Alabaster,
Ala.; product number 699800; 20 .mu.g per animal for each
immunization) according to the procedure used in Example 3 above,
GLA-SE). Sera were collected by bleeding animals one week after the
third immunization, and serum levels of IgG1 and IgG2c antibodies
specific for SMT antigen were examined by ELISA according to
published methods.
[0221] FIG. 4 shows ELISA data demonstrating levels of anti-SMT
antibody production induced in mice one week after the third
immunization using SMT antigen alone, or formulated with GLA-SE.
Means and SEM of reciprocal endpoint titers in each group are
shown.
[0222] Predominance of either IgG1 or IgG2c antibody isotype is
associated with TH2 or TH1 responses respectively. It has been
demonstrated that a TH1 response is necessary for protection
against Leishmania infection. SMT alone vaccination induced
predominantly SMT-specific IgG1 antibody. SMT+GLA-SE vaccination
induced higher antibody titers, and reverted the phenotype to a
predominantly IgG2c antigen-specific antibody response, associated
with protection against the disease.
Example 9
Use of GLA-Containing Vaccine In Vivo
[0223] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine containing a specific
Leishmania antigen. Standard immunological methodologies and
reagents were employed (Current Protocols in Immunology, Coligan et
al. (Eds.) 2006 John Wiley & Sons, NY).
[0224] Mice (three Balb/c animals per group) were immunized three
times at two-week intervals with the Leish-110f antigen (10 .mu.g
per animal for each immunization) formulated in a stable emulsion
containing different amounts of GLA (Avanti Polar Lipids, Inc.,
Alabaster, Ala.; product number 699800; 40, 20, 5, or 1 .mu.g per
animal for each immunization according to the procedure used in
Example 3 above, GLA-SE). Sera were collected by bleeding animals
one week after the first immunization, and serum levels of IgG1 and
IgG2a antibodies specific for Leish-110f were examined by ELISA
according to published methods (Id.).
[0225] FIG. 5 shows ELISA data demonstrating levels of
anti-Leish-110f antibody production induced in mice one week after
the first immunization using Leish-110f antigen formulated with
different amounts of GLA (40, 20, 5, or 1 .mu.g), compared to
saline controls. Means and SEM of reciprocal endpoint titers in
each group are shown.
[0226] Leish-110f-specific IgG1 and IgG2a antibody titers were GLA
dose-dependent. Predominance of TH1 associated IgG2a antibody was
observed at all concentrations of GLA tested.
Example 10
Use of GLA-Containing Vaccine In Vivo
[0227] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine containing a specific
Leishmania antigen. Standard immunological methodologies and
reagents were employed (Current Protocols in Immunology, Coligan et
al. (Eds.) 2006 John Wiley & Sons, NY).
[0228] Mice (three Balb/c animals per group) were immunized three
times at three-week intervals with saline or the Leish-111f antigen
(10 .mu.g per animal for each immunization) formulated in a stable
emulsion containing GLA (Avanti Polar Lipids, Inc., Alabaster,
Ala.; product number 699800; 20 .mu.g per animal for each
immunization, according to the procedure used in Example 3 above,
GLA-SE). Two weeks after the last injection, mice were sacrificed
and spleen collected to analyze T cell-dependent IFN-.gamma. and
IL-4 cytokine responses to in vitro antigen stimulation by ELISA
according to published methods.
[0229] Predominance of either IL-4 or IFN-.gamma. cytokine is
associated with TH2 or TH1 responses respectively. We and others
have demonstrated that a TH1 response is necessary for protection
against Leishmania infection. All animals responded well to ConA, a
potent mitogen. Leish-111f+GLA-SE vaccination induced Leish-111f
antigen-specific cytokine responses while no such responses were
observed in the saline control group. When compared to ConA,
Leish-111f+GLA-SE vaccination induced much more IFN-.gamma. than
IL-4, a TH1:TH2 ratio or phenotype associated with protection
against the disease.
Example 11
Use of GLA-Containing Vaccine In Vivo
[0230] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine containing a specific
Leishmania antigen. Standard immunological methodologies and
reagents were employed (Current Protocols in Immunology, Coligan et
al. (Eds.) 2006 John Wiley & Sons, NY).
[0231] Mice (three Balb/c animals per group) were immunized three
times at two-week intervals with saline or the Leish-110f antigen
(10 .mu.g per animal for each immunization) formulated in a stable
emulsion containing different amounts of (i) GLA (Avanti Polar
Lipids, Inc., Alabaster, Ala.; product number 699800; 40, 5, or 1
.mu.g per animal for each immunization) according to the procedure
used in Example 3 above (GLA-SE), or (ii) MPL.RTM. (40, 5, or 1
.mu.g per animal for each immunization) in an emulsion as supplied
by the manufacturer ("MPL-SE", GSK Biologicals, Rixensart,
Belgium). One week after the last injection, mice were sacrificed
and spleen collected to analyze T cell-dependent IFN-.gamma.
cytokine responses to in vitro antigen stimulation by ELISA
according to published methods (Id.). IFN-.gamma. cytokine
responses have been associated with a TH1 protective phenotype
against Leishmania infection.
[0232] FIG. 6 shows ELISA data demonstrating levels of
anti-Leish-110f IFN-.gamma. cytokine production induced in mice one
week after the third immunization using Leish-110f antigen
formulated with different amounts of GLA, compared to saline
controls. Means and SEM in each group are shown.
[0233] All animals responded well to ConA, a potent cell activator
and mitogen. Leish-110f+GLA-SE vaccination induced Leish-110f
antigen-specific cytokine responses, in a dose-dependent manner,
while no such responses were observed in the saline control group.
At all concentration tested, GLA-SE was more potent than MPL-SE, in
inducing higher levels of IFN-.gamma. secreted by antigen-specific
T cells
[0234] In conclusion, the addition of GLA in a stable oil
formulation to Leishmania vaccine antigen candidate Leish-110f
induced predominantly antigen-specific immune responses of the
cellular type (T cell) associated with the protective TH1
phenotype. In addition, GLA-SE was more potent than MPL-SE in
inducing protection-associated cytokines like IFN-.gamma..
Example 12
Use of GLA-Containing Vaccine In Vivo
[0235] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine containing a specific
Leishmania antigen. Standard immunological methodologies and
reagents were employed (Current Protocols in Immunology, Coligan et
al. (Eds.) 2006 John Wiley & Sons, NY).
[0236] Mice (three Balb/c animals per group) were immunized three
times at two-week intervals with saline or the Leish-110f antigen
(10 .mu.g per animal for each immunization) formulated in a stable
emulsion containing different amounts of (i) GLA (Avanti Polar
Lipids, Inc., Alabaster, Ala.; product number 699800; 20 .mu.g or 5
.mu.g per animal for each immunization) according to the procedure
used in Example 3 above (GLA-SE), or (ii) MPL.RTM. (20 .mu.g or 5
.mu.g per animal for each immunization) in an emulsion as supplied
by the manufacturer ("MPL-SE", GSK Biologicals, Rixensart,
Belgium). One week after the last injection, mice were sacrificed
and spleen collected to analyze T cell-dependent IFN-.gamma., IL-2,
and TNF cytokine responses to in vitro antigen stimulation by
intracellular cell staining (ICS) and Flow cytometry according to
published methods (Id.). These three cytokines have been associated
with a TH1 protective phenotype against Leishmania infection.
[0237] When analyzed at the single cell level, the frequency of
CD4+ T cells expressing all three cytokines IFN-.gamma., IL-2, and
TNF or a combination of IFN-.gamma. and IL-2 was higher in the
Leish-110f+GLA-SE group compared to the Leish-110f+MPL-SE group,
and this was observed at both 20 and 5 .mu.g doses. It has been
reported (Seder et al.) that high frequencies of CD4+ T cells
expressing all three cytokines IFN-.gamma., IL-2, and TNF
correlates with protection against Leishmania infection.
[0238] In conclusion, the addition of GLA in a stable oil
formulation to Leishmania vaccine antigen candidate Leish-110f
induced predominantly antigen-specific immune responses of the
cellular type (T cell) associated with the protective TH1
phenotype. In addition, GLA-SE was more potent than MPL-SE in
inducing protection-associated cytokines like IFN-.gamma., IL-2,
and TNF.
Example 13
Use of GLA-Containing Vaccine In Vivo
[0239] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine containing a specific
Mycobacterium tuberculosis antigen. Standard immunological
methodologies and reagents were employed (Current Protocols in
Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,
NY).
[0240] Mice (three C57BL/6 animals per group) were immunized three
times at three-week intervals with the ID83 antigen (8 .mu.g per
animal for each immunization) used alone or formulated in a stable
emulsion containing GLA (Avanti Polar Lipids, Inc., Alabaster,
Ala.; product number 699800; 20 .mu.g per animal for each
immunization, according to the procedure used in Example 3 above,
GLA-SE). Sera were collected by bleeding animals one week after the
third immunization, and serum levels of IgG1 and IgG2c antibodies
specific for ID83 were examined by ELISA according to published
methods (Id.) Predominance of either IgG1 or IgG2c antibody isotype
is associated with TH2 or TH1 responses, respectively. It has been
demonstrated that a TH1 response is necessary for protection
against Mycobacterium tuberculosis infection.
[0241] Vaccination with ID83 alone induced predominantly
antigen-specific IgG1 antibody. In contrast, ID83+ GLA-SE
vaccination induced higher antibody titers, and reverted the
phenotype to a predominantly IgG2c antigen-specific antibody
response, associated with protection against the disease.
Example 14
Use of GLA-Containing Vaccine In Vivo
[0242] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine containing a specific
Mycobacterium tuberculosis antigen. Standard immunological
methodologies and reagents were employed (Current Protocols in
Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,
NY).
[0243] Mice (three C57BL/6 animals per group) were immunized three
times at three-week intervals with the ID83 antigen (8 .mu.g per
animal for each immunization) used alone or formulated in a stable
emulsion containing GLA (GLA-SE), GLA+CpG (CpG.sub.1826, Coley
Pharmaceuticals, 25 .mu.g) (GLA/CpG-SE), or GLA+Gardiquimod (GDQ)
(Invivogen, 20 .mu.g) (GLA/GDQ-SE). Three weeks after the last
injection, mice were sacrificed and spleens collected to analyze
CD4+ and CD8+ T cell-dependent IFN-.gamma., IL-2, and TNF cytokine
responses to in vitro ID83 antigen stimulation by ICS and Flow
cytometry according to published methods. Expression of
IFN-.gamma., IL-2, and TNF cytokines have been associated with
protective TH1 responses against M. tuberculosis infection.
[0244] FIG. 7 shows ICS data demonstrating the frequencies of
ID83-specific IFN-.gamma., IL-2, and TNF cytokine producing CD4+
and CD8+ T cells induced in mice one week after the third
immunization using ID83 alone or adjuvanted with formulations
containing GLA (GLA-SE), GLA+CpG (GLA/CpG-SE), or GLA+GDQ
(GLA/GDQ-SE).
[0245] Frequencies of ID83 specific cytokine producing CD4+ or CD8+
T cells were at background levels for the saline and ID83 alone
vaccine groups. ID83 antigen specific cytokine producing T cells,
both CD4+ and CD8+, were induced by ID83+GLA-SE vaccination, and
their frequency further increased by the addition of a second TLR
ligand like GDQ (TLR7/8) or CpG (TLR9). T cells expressing
IFN-.gamma.+TNF or IFN-.gamma.+IL-2 were the predominant
populations.
[0246] In conclusion, adjuvanting an antigen against M.
tuberculosis with GLA-SE greatly enhanced the antigen specific
cellular response (T cells) as measured by the frequencies of T
cells expressing IFN-.gamma., IL-2, and/or TNF cytokines. Combining
GLA-SE with another TLR ligand further increased the frequency of
antigen specific cytokine producing cells, a phenotype associated
with protection against this disease.
Example 15
Use of GLA-Containing Vaccine In Vivo
[0247] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine containing a specific
Mycobacterium leprae antigen. Standard immunological methodologies
and reagents were employed (Current Protocols in Immunology,
Coligan et al. (Eds.) 2006 John Wiley & Sons, NY).
[0248] Mice (three C57BL/6 animals per group) were immunized three
times at three-week intervals with the ML0276 antigen (10 .mu.g per
animal for each immunization) adjuvanted with aqueous formulations
containing CpG (CpG.sub.1826, Coley Pharmaceutical, 25 .mu.g per
animal for each immunization), or Imiquimod (IMQ) (3M Pharma, 25
.mu.g per animal for each immunization), or GLA (Avanti Polar
Lipids, Inc., Alabaster, Ala.; product number 699800; 25 .mu.g per
animal for each immunization according to the procedure used in
Example 3 above, GLA-SE), a mix of the three, or saline as negative
control. Sera were collected by bleeding animals three weeks after
the second immunization, and serum levels of IgG antibodies
specific for ML0276 were examined by ELISA according to published
methods (Id.).
[0249] Animals from the saline control group did not show ML0276
specific IgG, and those from the ML0276+CpG and ML0276+IMQ groups
showed a very low level of antigen specific antibody. In contrast,
ML0276+GLA-SE induced a significant level of ML0276 specific IgG,
that was further increased when the three adjuvants were used
together.
[0250] In conclusion, the data support the adjuvanting effect of
GLA-SE and/or a combination of GLA-SE with additional TLR ligands
when used with antigen ML0276 for the induction of antigen specific
antibodies.
Example 16
Use of GLA-Containing Vaccine In Vivo
[0251] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine containing a specific
Mycobacterium leprae antigen. Standard immunological methodologies
and reagents were employed (Current Protocols in Immunology,
Coligan et al. (Eds.) 2006 John Wiley & Sons, NY).
[0252] Mice (three C57BL/6 animals per group) were immunized three
times at three-week intervals with the ML0276 antigen (10 .mu.g per
animal for each immunization) adjuvanted with aqueous formulations
containing CpG (CpG.sub.1826, Coley Pharmaceutical, 25 .mu.g per
animal for each immunization), or Imiquimod (IMQ) (3M Pharma, 25
.mu.g per animal for each immunization), or GLA (Avanti Polar
Lipids, Inc., Alabaster, Ala.; product number 699800; 25 .mu.g per
animal for each immunization, according to the procedure used in
Example 3 above, GLA-SE), a mix of the three, or saline as negative
control. Three weeks after the last injection, mice were sacrificed
and spleen collected to analyze CD4+T cell-dependent IFN-.gamma.
cytokine responses to in vitro ML0276 antigen stimulation by ICS
and Flow cytometry according to published methods. Expression of
IFN-.gamma. cytokine has been associated with protective TH1
responses against M. leprae infection.
[0253] FIG. 8, panel A shows ICS data demonstrating the frequencies
of ML0276-specific IFN-.gamma. cytokine producing CD4+ T cells
induced in mice one week after the third immunization using ML0276
antigen formulated with aqueous formulations containing CpG, or
Imiquimod (IMQ), or a stable oil emulsion containing GLA (GLA-SE),
or the three mixed together, compared to saline and naive controls.
Means in each group are shown. FIG. 8, panel B shows data
demonstrating the cellularity of lymph nodes draining the site of
M. leprae infection in mice immunized with ML0276 antigen
formulated with aqueous formulations containing CpG, or Imiquimod
(IMQ), or a stable oil emulsion containing GLA (GLA-SE), or a
mixture of the three, compared to saline and naive controls. Means
and SEM in each group are shown.
[0254] Animals from the saline control group did not show ML0276
specific IFN-.gamma. responses with a background frequency of 0.04%
positive cells. Those from the CpG and IMQ groups showed a slightly
increased frequency of antigen specific cytokine producing cells
with 0.17% and 0.11% respectively. In contrast, a significantly
higher number of ML0276 specific IFN-.gamma.+ CD4+ T cells (0.66%)
were observed when GLA-SE was used as an adjuvant, a frequency that
was further increased when the three adjuvants were mixed together
(2.14%).
[0255] A subset of mice was subsequently challenged with M. leprae
and found to be protected by ML0276+GLA-SE as measured by the
reduction in the number of cells in the lymph nodes draining the
site of challenge as compared to infected saline controls.
Vaccination with ML0276+CpG and ML0276+IMQ induced only a modest
decrease in cell numbers compared to saline.
[0256] In conclusion, the data support the adjuvanting effect of
GLA-SE and/or a combination of GLA-SE with additional TLR ligands
when used with antigen ML0276 for the induction of antigen specific
cellular responses.
Example 17
GLA Stimulation of Human Dendritic Cells
[0257] This example describes an in vitro model demonstrating an
adjuvant effect of GLA. Standard tissue culture methodologies and
reagents were employed.
[0258] Dendritic cells were derived from purified blood CD14+
monocytes following a published protocol. Various adjuvant
concentrations of either synthetic GLA (Avanti Polar Lipids, Inc.,
Alabaster, Ala.; product number 699800) or the natural product
MPL.RTM. (GSK Biologicals, Rixensart, Belgium) were achieved by
diluting an aqueous adjuvant preparation in cell culture medium
(RPMI containing 5% human serum). Prior to collection and analysis
of the expression of activation markers, cells were maintained for
44 hours at 37.degree. C. in a humidified atmosphere containing 5%
CO.sub.2. Expression levels of the costimulatory molecule CD86 at
the surface of DC were used as an indicator of cell activation and
measured by flow cytometry on a LSRII instrument (BD Biosciences,
San Jose, Calif.) using CD86-specific fluorochrome-labeled antibody
(eBiosciences, San Diego, Calif.) according to the manufacturer's
instructions.
[0259] FIG. 9 shows data obtained by flow cytometry demonstrating
levels of CD86 molecule expressed at the surface of human DC in
response to 44 h of stimulation with 10,000 ng/ml to 0.010 ng/ml
GLA (panel A) or MPL (panel B). A positive stimulation control
consisting of PGE2, IL-1.beta., TNF, and IL-6 was also
included.
[0260] GLA-AF induced a dose-dependent immune response in the human
primary DC (FIG. 9, panel A), characterized by the increased
expression of CD86. Maximal CD86 expression was seen with GLA at
10,000 ng/ml, 1000 ng/ml, and 100 ng/ml, while MPL had only maximal
expression at 10,000 ng/ml and 1000 ng/mL with DCs generated from
Donor N003. An additional three donors were used to generate DC
cultures and evaluated in a similar manner with GLA and MPL
stimulation at 1000 ng/ml to 1 ng/mL, see FIG. 10. The dendritic
cells from these donors showed different levels of sensitivity, but
in every case lower doses of GLA achieved higher CD86 expression
than MPL. This demonstrated that GLA was active at concentrations
at least 10-fold lower compared to MPL for human dendritic cell
maturation.
Example 18
Use of GLA-Containing Vaccine In Vivo
[0261] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine containing a specific
Leishmania antigen. Standard immunological methodologies and
reagents were employed (Current Protocols in Immunology, Coligan et
al. (Eds.) 2006 John Wiley & Sons, NY).
[0262] Four Balb/c mice per treatment group were immunized three
times at two-week intervals either with saline or the Leish-110f
antigen (10 .mu.g per animal for each immunization) formulated in a
stable emulsion containing 20 .mu.g of (i) GLA (Avanti Polar
Lipids, Inc., Alabaster, Ala.; product number 699800; per animal
for each immunization) according to the procedure used in Example 3
above (GLA-SE), or (ii) MPL.RTM. in an emulsion as supplied by the
manufacturer ("MPL-SE", GSK Biologicals, Rixensart, Belgium, per
animal for each immunization). Three weeks after the last
injection, mice were challenged intradermally in the pinea of both
ears with 2.times.10.sup.3 purified Leishmania major clone V1
(MOHM/IL/80/Friedlin) metacyclic promastigotes according to
published methods (Id.). Development of cutaneous lesions was
monitored weekly for 6 weeks post-infection.
[0263] FIG. 11 shows diameter of lesions in mice immunized using
Leish-110f antigen formulated with GLA-SE or MPL-SE, compared to
saline controls. Means in each group are shown.
[0264] All animals in the saline control groups developed
progressive non-healing lesions. Ear lesions that started to
develop during the first 2 weeks, were controlled at week 3, and
eventually resolved by week 4 in mice immunized with
Leish-110f+GLA-SE. Lesions of mice immunized with Leish-110f+MPL-SE
were reduced compared to the saline group but did not resolve.
GLA-SE was more potent than MPL-SE in preventing the development of
lesions.
[0265] In conclusion, the addition of GLA in a stable oil
formulation to Leishmania vaccine antigen candidate Leish-110f
prevented the development of cutaneous lesions upon challenge with
Leishmania major parasites. In addition, GLA-SE was more potent
than MPL-SE in controlling lesion development.
Example 19
Use of GLA-Containing Vaccine In Vivo
[0266] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a vaccine containing a specific
Leishmania antigen. Standard immunological methodologies and
reagents were employed (Current Protocols in Immunology, Coligan et
al. (Eds.) 2006 John Wiley & Sons, NY).
[0267] Four Balb/c mice per treatment group were immunized three
times at two-week intervals with saline or the Leish-110f antigen
(10 .mu.g per animal for each immunization) formulated in a stable
emulsion containing 20 .mu.g of (i) GLA (Avanti Polar Lipids, Inc.,
Alabaster, Ala.; product number 699800; per animal for each
immunization) according to the procedure used in Example 3 above
(GLA-SE), or (ii) MPL.RTM. in an emulsion as supplied by the
manufacturer ("MPL-SE", GSK Biologicals, Rixensart, Belgium, per
animal for each immunization). Three weeks after the last
injection, mice were challenged intradermally in the pinea of both
ears with 2.times.10.sup.3 purified Leishmania major clone V1
(MOHM/IL/80/Friedlin) metacyclic promastigotes according to
published methods (Id.). Parasite burden in the ear and draining
lymph nodes of infected mice were determined 6 weeks
post-infection.
[0268] FIG. 12 shows the number of parasites recovered in the ear
and draining lymph nodes of mice immunized using Leish-110f antigen
formulated with GLA-SE or MPL-SE, compared to saline controls.
Number of parasites in individual organs and means in each group
are shown. Differences in parasite numbers between groups were
evaluated for statistical significance using the Student's t test.
A difference was considered significant when the p value was
<0.05.
[0269] Animals in the saline control groups showed an average
parasite load of 10.sup.5 and 5.times.10.sup.4 in ears and draining
lymph nodes respectively. In mice immunized with Leish-110f+GLA-SE,
no parasites could be detected in 8/8 ears and in 6/8 draining
lymph nodes. In contrast, in mice immunized with Leish-110f+MPL-SE,
no parasites could be detected in 4/8 ears and 2/8 draining lymph
nodes. GLA-SE was more potent than MPL-SE in controlling infection
with Leishmania major.
[0270] In conclusion, the addition of GLA in a stable oil
formulation to Leishmania vaccine antigen candidate Leish-110f
controlled parasite burden in both ear and draining lymph nodes
upon challenge with Leishmania major parasites. In addition, GLA-SE
was more potent than MPL-SE in controlling the number of parasites
recovered from the infected tissues.
Example 20
Use of GLA-Containing Vaccine In Vivo
[0271] This example describes an in vivo model demonstrating an
adjuvant effect of GLA in a lentiviral vaccine containing OVA
antigen. Standard immunological methodologies and reagents were
employed (Current Protocols in Immunology, Coligan et al. (Eds.)
2006 John Wiley & Sons, NY).
[0272] Five C57BL/6 mice per treatment group were immunized one
time with saline, the lentiviral vaccine or the lentiviral vaccine
administered at the same time as a stable emulsion containing 20
.mu.g of GLA (Avanti Polar Lipids, Inc., Alabaster, Ala.; product
number 699800; per animal for each immunization) according to the
procedure used in Example 3 above (GLA-SE). Two weeks after the
injection, splenocytes were removed and T cell immunogenicity
(phenotype and effector functions) was evaluated by multiparameter
flow cytometry.
[0273] Splenocytes were plated in 24-well plates and stimulated ex
vivo overnight with 2 .mu.g/ml anti-CD28 (eBioscience, San Diego,
Calif.), anti-CD49d (eBioscience), and 20 .mu.g/ml OVA as antigen
(or with PMA/ionomycin, or without antigen as a negative control)
in complete media at 37.degree. C. After a 2 hour incubation at
37.degree. C., brefeldin A (GolgiPlug: BD Biosciences, San Jose,
Calif.) was added to the wells and the incubation resumed for an
additional 12 hrs at 37.degree. C. Cells were blocked with
anti-CD16/32 (eBioscience) 1:50 in 50 .mu.l and then stained with
AlexaFluor 700-anti-CD3 (eBioscience), PerCP-anti-CD4 (BD
Biosciences), and PE-anti-CD8 (BD Biosciences). The cells were
fixed using the Cytofix/Cytoperm kit (BD Biosciences) and
intracellular staining was performed according to the BD
Biosciences protocol. Cells were blocked with anti-CD16/32 and
intracellularly stained with FITC-anti-TNF-.alpha. (eBioscience),
Pacific Blue-anti-IL-2 (eBioscience) and PE-Cy7-anti-IFN-.gamma.
(BD Biosciences). Cells were analyzed with a LSRII FACS machine (BD
Biosciences) and the DIVA software to quantify CD4+T cells
producing IFN-.gamma., IL-2 and TNF-.alpha..
[0274] FIG. 13 shows CD8 (panels A-C) and CD4 (panels D-F)
responses in mice immunized with the lentivirus vaccine expressing
OVA antigen in the presence and absence of GLA-SE, compared to
saline controls. Means in each group are shown.
[0275] Splenocytes from animals in the lentivector vaccine plus
GLA-SE had higher percentages of CD8+CD44+ as well as CD4+CD44+
cytokine secreting cells than splenocytes from animals receiving
the lentivaccine alone. The addition of GLA-SE to the lentivaccine
immunization resulted in an increased frequency of antigen-specific
triple cytokine (IFN-.gamma., TNF-.alpha., IL-2) producing T
cells.
[0276] In conclusion, the addition of GLA in a stable oil
formulation to the lentiviral vector vaccine improved both the
quantity and quality of effector T cells which may have an
essential role in protection against a variety of infections.
Example 21
Use of GLA-Containing Vaccine In Vivo
[0277] This example describes an in vivo model demonstrating an
adjuvant effect of GLA-SE in a vaccine against Influenza. Standard
immunological methodologies and reagents were employed (e.g.,
Current Protocols in Immunology, Coligan et al. (Eds.) 2006 John
Wiley & Sons, NY).
[0278] Three Balb/c mice treatment group were immunized i.d. with
microneedles (NanoPass Technologies Ltd., Nes Ziona, Israel) 2
times at 3 week intervals (total volume was 100 .mu.l; 50 .mu.l per
hind leg) with i) a stable emulsion containing GLA, "GLA-SE",
(Avanti Polar Lipids, Inc., Alabaster, Ala.; product number 699800;
5 .mu.g per animal for each immunization) and Fluzone.RTM. (Sanofi
Pasteur, Stillwater Pa.; 2 .mu.g and 0.2 .mu.g treatment groups);
ii) stable emulsion (SE) plus Fluzone; or iii) Fluzone only. Mice
were immunized with the 2006-2007 commercial Fluzone formula which
includes components from A/New Calcdonia/20/99 (H1N1),
A/Wisconsin/67/2005 (H3N2), and B/Malaysia/2506/2004. Mice were
bled one week after the second immunization and serum was analyzed
for antibody responses to Fluzone.
[0279] IgG2a responses were dominant in mice immunized using
Fluzone combined with GLA-SE. One week following a boost
immunization, significantly greater levels of anti-Fluzone
antibodies (total IgG and IgG2a) were induced in this treatment
group (see FIG. 14, panels A-B). IgG1 antibody levels were similar
in all treatment groups (see FIG. 14, panel C). A significant
increase in endpoint antibody titers were seen in mice that
received a boost immunization of Fluzone plus GLA-SE as compared to
mice that received a boost immunization of Fluzone vaccine alone
(e.g., FIG. 14, compare "GLA-SE" treatment group (Fluzone plus
GLA-SE) with "(-)" treatment group (Fluzone)). No differences in
antibody titers were observed in mice that received a boost
immunization of Fluzone combined with a stable emulsion (SE) as
compared to mice that received a boost immunization of Fluzone
vaccine alone (e.g., FIG. 14, compare "SE" treatment group (Fluzone
plus SE) with "(-)" treatment group (Fluzone)).
[0280] Total IgG and IgG2a antibody titers were enhanced in
response to Fluzone vaccination combined with GLA-SE. The
adjuvanting effect of GLA-SE was similar for both the 2 .mu.g and
0.2 .mu.g doses of Fluzone vaccine in combination with GLA-SE.
These results suggest that it is possible to reduce the dose of
Fluzone vaccine by adjuvanting it with GLA-SE, and still induce
high levels of IgG and IgG2a antibody titers.
Example 22
Use of GLA-Containing Vaccine In Vivo
[0281] This example describes an in vivo model demonstrating an
adjuvant effect of GLA-SE in a vaccine against Influenza. Standard
immunological methodologies and reagents were employed (Current
Protocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley
& Sons, NY).
[0282] Three Balb/c mice treatment group were immunized i.m. 2
times at 3 week intervals (total volume was 100 .mu.l; 50 .mu.l per
hind leg) with i) a stable emulsion containing GLA, "GLA-SE",
(Avanti Polar Lipids, Inc., Alabaster, Ala.; product number 699800;
5 .mu.g per animal for each immunization) and Fluzone.RTM. (Sanofi
Pasteur, Stillwater Pa.; 0.2 .mu.g); ii) stable emulsion (SE) plus
Fluzone; iii) Fluzone only; or iv) saline. Mice were immunized with
the 2006-2007 commercial Fluzone formula which includes components
from A/New Calcdonia/20/99 (H1N1), A/Wisconsin/67/2005 (H3N2), and
B/Malaysia/2506/2004. Spleens were harvested three weeks following
an i.m. boost with saline, Fluzone alone, Fluzone plus SE, or
Fluzone plus GLA-SE.
[0283] Splenocytes were plated in 48-well plates and were
stimulated ex vivo with H1N1 antigen (Influenza virus A H1N1, New
Calcdonia, Fitzgerald Industries, Concord Mass.) or media (e.g.,
negative control) and supernatants were collected 72 hours later.
Supernatants were assayed for soluble murine cytokines including
IFN-gamma, IL-2, IL-10 and IL-5, using specific sandwich ELISA
assay kits (eBiosciences, San Diego, Calif.) according to the
manufacturer's instructions.
[0284] Mice immunized using Fluzone (0.2 .mu.g) plus GLA-SE (5
.mu.g) had significantly higher levels of IFN-.gamma. and IL-2
cytokines following H1N1 antigen stimulation of splenocytes as
compared to unstimulated controls (FIG. 15). Furthermore, treatment
groups ii-iv failed to show significant increases in IFN-.gamma.
and IL-2 after H1N1 antigen stimulation. In contrast, mice
immunized with Fluzone (0.2 .mu.g) and SE displayed a different
cytokine profile e.g., significantly higher levels of IL-5 and
IL-10 as compared to other treatment groups.
[0285] These results indicate that mice immunized with Fluzone plus
GLA-SE induced a significant Th.sub.1 type T-cell cytokine response
compared to other treatment groups. Th.sub.1 cytokine responses are
believed have an essential role in protection against a variety of
infections.
Example 23
USP Rabbit Pyrogen Test
[0286] GLA and MPL were evaluated for pyrogenicity/toxicity using
the United States Pharmacopeial (USP) rabbit pyrogen test method.
GLA was tested at the same doses and conditions commonly used for
MPL. Both GLA and MPL passed the USP<151> requirements for
the absence of pyrogens at the doses evaluated. These data indicate
that the unexpected high potency of GLA compared to MPL is not
associated with increased toxicity. Accordingly, lower doses of GLA
can be used to achieve the same or improved potency relative to MPL
while also having correspondingly reduced pyrogenicity due to the
lower doses needed.
[0287] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
[0288] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
* * * * *
References