U.S. patent application number 16/220557 was filed with the patent office on 2019-04-25 for methods of treating allergies and autoimmune diseases with homogenate of axenic c. elegans.
This patent application is currently assigned to The Henry M. Jackson Foundation for the Advcement of Military Medicine, Inc.. The applicant listed for this patent is The Henry M. Jackson Foundation for the Advancemen of Military Medicine, Inc.. Invention is credited to Belinda Jackson, Edward E. Mitre, Marina Torrero.
Application Number | 20190117747 16/220557 |
Document ID | / |
Family ID | 53180087 |
Filed Date | 2019-04-25 |
United States Patent
Application |
20190117747 |
Kind Code |
A1 |
Mitre; Edward E. ; et
al. |
April 25, 2019 |
METHODS OF TREATING ALLERGIES AND AUTOIMMUNE DISEASES WITH
HOMOGENATE OF AXENIC C. ELEGANS
Abstract
Methods of using axenic C. elegans homogenate for treating
allergies or an autoimmune disease are disclosed. Also disclosed is
a composition comprising a homogenate of C. elegans, wherein the
homogenate is obtained from C. elegans cultured in axenic media,
for use in treating an allergy or autoimmune disease.
Inventors: |
Mitre; Edward E.;
(Rockville, MD) ; Torrero; Marina; (Fairfax,
VA) ; Jackson; Belinda; (Burtonsville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Henry M. Jackson Foundation for the Advancemen of Military
Medicine, Inc. |
Bethesda |
MD |
US |
|
|
Assignee: |
The Henry M. Jackson Foundation for
the Advcement of Military Medicine, Inc.
Bethesda
MD
|
Family ID: |
53180087 |
Appl. No.: |
16/220557 |
Filed: |
December 14, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15036982 |
May 16, 2016 |
10213495 |
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PCT/US2014/066320 |
Nov 19, 2014 |
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16220557 |
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61906739 |
Nov 20, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/55516
20130101; A61K 39/39 20130101; A61P 37/06 20180101; A61K 35/62
20130101; A61P 11/02 20180101; A61K 39/0008 20130101; A61K 39/0003
20130101; A61P 37/00 20180101; A61K 39/35 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 35/62 20060101 A61K035/62 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under grant
number DK083131 awarded by the National Institutes of Health. The
government has certain rights in this invention.
Claims
1. A method of treating an allergy or an autoimmune disease in a
subject, the method comprising administering to the subject an
effective amount of a composition comprising a homogenate of C.
elegans, wherein the homogenate is obtained from C. elegans
cultured in axenic media that is free of E. coli.
2. The method of claim 2, wherein the autoimmune disease is a
Th1-mediated autoimmune disease.
3. The method of claim 3, wherein the Th1-mediated autoimmune
disease is selected from type 1 insulin-dependent diabetes
mellitus, scleroderma, multiple sclerosis, posterior uveitis,
Crohn's disease, inflammatory bowel disease, and rheumatoid
arthritis.
4. The method of claim 3, wherein the Th1-mediated autoimmune
disease is type 1 insulin-dependent diabetes mellitus.
5. The method of claim 1, wherein the allergy is an environmental
allergy or asthma.
6. The method of claim 1, wherein the subject is a mammal.
7. The method of claim 1, wherein the subject is a human.
8. The method of claim 1, wherein the composition is administered
chronically to the subject.
9. The method of claim 1, wherein the composition is administered
orally, parentally, or subcutaneously.
10. The method of claim 1, wherein the composition is free of E.
coli endotoxins.
11. The method of claim 10, wherein the composition is free of
lipopolysaccharides.
12. (canceled)
13. (canceled)
14. A homogenate of C. elegans and a pharmaceutically acceptable
excipient, wherein the homogenate is obtained from C. elegans
cultured in axenic media that is free of E. coli.
15. A method of increasing levels of IL-10 in a subject having a
Th1-mediated autoimmune disease, the method comprising
administering to the subject an effective amount of a composition
comprising a homogenate of C. elegans, wherein the homogenate is
obtained from C. elegans cultured in axenic media that is free of
E. coli.
16. The method of claim 15, wherein the Th1-mediated autoimmune
disease is type 1 insulin dependent diabetes mellitus.
17. The method of claim 15, wherein the subject is a mammal.
18. The method of claim 15, wherein the subject is a human.
19. The method of claim 15, wherein the composition is administered
chronically to the subject.
20. The method of claim 15, wherein the composition is administered
orally, parentally, or subcutaneously.
21. The method of claim 15, wherein the composition is free of E.
coli endotoxins.
22. The method of claim 15, wherein the composition is free of
lipopolysaccharides.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/036,982 (Allowed), filed 16 May 2016, which is a U.S.
National Stage application of PCT/US2014/066320, filed 19 Nov.
2014, which claims the benefit of, and relies on the filing date
of, U.S. provisional patent application No. 61/906,739, filed 20
Nov. 2013, the entire disclosure of which is incorporated herein by
reference.
BACKGROUND
[0003] The disease burden of the more than 80 distinct autoimmune
diseases in the United States is enormous, collectively affecting
14 to 22 million people, an estimated 5-8% of the U.S. population.
In addition, an estimated 50 million people in the U.S. suffer
allergies. Allergy is the fifth leading chronic disease in the U.S.
among all ages, and the third most common chronic disease among
children under 18 years old.
[0004] Several types of medications are available to treat
allergies symptoms, including antihistamines, decongestants,
corticosteroids, and others. It is estimated that the annual cost
for prescription and over-the-counter medications in the U.S. to
treat allergies is $11 billion.
[0005] For most autoimmune diseases, immunosuppression is the
therapy of choice. Conventional agents that induce non-specific
immunosuppression, such as non-steroidal anti-inflammatory drugs,
glucocorticoids, and methotrexate have traditionally been the
mainstay of therapy for many autoimmune diseases. While helpful,
these medications are not always fully efficacious and are
associated with significant toxicity when used chronically.
Additionally, by non-specifically suppressing the immune system,
these medications substantially increase patient susceptibility to
infections.
[0006] Over the past few years, a number of new medications have
become available which are able to specifically target certain arms
of the immune response. While such approaches clearly represent a
step forward in focusing immunosuppressive therapy, none does so in
an antigen-specific manner. Consequently, these new medications
still increase patient susceptibility to infections, albeit to a
smaller range of organisms than non-specific immunosuppressive
agents. This phenomenon is exemplified by the recent findings that
tumor necrosis factor inhibitors, despite blocking the activity of
only one cytokine, increase the risk of pneumonia, severe skin
infections, and reactivation of prior tuberculosis.
[0007] Given the increased risk to infection that occurs when even
specific facets of the immune system are inhibited, an alternative
therapy for autoimmune diseases would be one that suppresses only
self-reactive immune responses. Such a therapy would, ideally, be
efficacious without compromising the body's ability to fight off
infections. Several autoimmune diseases appear to be caused in
large part by Th1-driven inflammation. Examples include type 1
diabetes, multiple sclerosis, Crohn's disease, inflammatory bowel
disease, rheumatoid arthritis, and posterior uveitis. While the
Th1/Th2 paradigm has evolved somewhat since its first description
in 1986, it is still generally accepted that Th1 and Th2 responses
have the ability to counter regulate each other. In particular,
IL-4 suppresses differentiation of naive T-cells into Th1 cells,
resulting in decreased Th1 cytokine production and decreased Th1
cell proliferation in response to Th1-inducing antigens. In
addition to Th2 responses, immunoregulatory networks can also
suppress Th1 responses. These mechanisms include downregulatory
cytokines such as IL-10 and TGFb, and regulatory cells such as
T-regulatory cells and B-regulatory cells. Thus, there is a need
for new treatment modalities that selectively down regulate Th1
responses.
[0008] Other therapies have also become available which use
exposure to parasitic worms to treat or prevent hyperinflammatory
diseases. To date, two types of parasitic worm-mediated therapy
have been used in human clinical trials--Trichuris suis (pig
whipworm) to treat ulcerative colitis and Crohn's disease, and live
hookworm to treat allergies. While the exact mechanism(s) by which
parasitic worms protect against inflammatory diseases is not
completely understood, evidence suggests that parasitic worms
induce strong immunoregultory signals. Patients exposed to
parasitic worms demonstrated increased IgE antibody levels,
increased numbers of circulating basophils and eosinophils, and
increased IL-10 production which can suppress excessive
inflammatory responses.
[0009] However, a number of factors have limited the widespread use
of parasitic worm infections in commercial therapeutic
applications. Not only might patients potentially express concern
about being infected with parasitic worms, but if so infected, they
incur the risk of suffering from pathology induced by live worm
infection. Additionally, the use of animal hosts to cultivate
parasitic worms can compromise batch purity and homogeneity during
production. Due to the complex life cycle of parasitic worms, it is
also often difficult to obtain sufficiently large quantities of
antigen necessary for commercial therapeutic applications. Thus,
there is also a need for new treatment modalities that can suppress
excessive inflammatory responses without the risks associated with
parasitic infections.
SUMMARY
[0010] The present disclosure provides methods of treating an
allergy or autoimmune disease by administering to a subject a
composition comprising Caenorhabditis elegans (C. elegans) or a
homogenate thereof containing C. elegans antigens, where the C.
elegans has been cultured in axenic media. C. elegans is a
non-parasitic, free living nematode that is non-pathogenic to
humans. Administering axenic C. elegans antigen activates basophils
and eosinophils, increases IgE antibody levels, and increases
production of IL-10. Without intending to be bound by any theory,
activated basophils and eosinophils may protect against
Th1-mediated autoimmune disease, for example, through the release
of histamine or the synthesis of IL-4 and/or IL-10, all of which
have been shown to counteract or suppress Th1-driven immune
responses, or through the induction of negative feedback pathways
that down regulate immune responses.
[0011] One embodiment is directed to a method of treating an
allergy or an autoimmune disease in a subject, the method
comprising administering to the subject an effective amount of a
composition comprising a homogenate of C. elegans, wherein the
homogenate is obtained from C. elegans cultured in axenic
media.
[0012] Another embodiment is directed to a method of increasing
levels of IgE antibody in a subject, the method comprising
administering to the subject an effective amount of a composition
comprising a homogenate of C. elegans, wherein the homogenate is
obtained from C. elegans cultured in axenic media.
[0013] A further embodiment is directed to a method of increasing
levels of IL-10 in a subject, the method comprising administering
to the subject an effective amount of a composition comprising a
homogenate of C. elegans, wherein the homogenate is obtained from
C. elegans cultured in axenic media.
[0014] In yet another embodiment, the autoimmune disease can be a
Th-1 mediated autoimmune disease. In a further embodiment, the
Th1-mediated autoimmune disease is selected from type 1
insulin-dependent diabetes mellitus, scleroderma, multiple
sclerosis, posterior uveitis, Crohn's disease, inflammatory bowel
disease, and rheumatoid arthritis.
[0015] In a further embodiment, the allergy can be environmental
allergies and/or asthma.
[0016] In an embodiment, the subject is a mammal, preferably a
human.
[0017] In an embodiment, the composition is administered orally,
parenterally, or subcutaneously.
[0018] In yet another embodiment, the composition is free of E.
coli endotoxins. In a further embodiment, the composition is free
of lipopolysaccharides.
[0019] In another embodiment, the composition comprising axenic C.
elegans homogenate is administered chronically to the subject. In
one embodiment, the chronic administration comprises administering
the composition with axenic C. elegans homogenate to the subject
every week for at least 20 weeks. In another embodiment, the
chronic administration comprises administering the composition with
axenic C. elegans homogenate to the subject one or more times a day
for at least 5, 7, or 10 days.
[0020] Another aspect is directed to a composition comprising
axenic C. elegans homogenate for use in therapy. In one embodiment
the composition comprises axenic C. elegans homogenate for use in
treating an autoimmune disease, including a Th1-mediated autoimmune
disease, such as type 1 insulin-dependent diabetes mellitus,
scleroderma, multiple sclerosis, posterior uveitis, Crohn's
disease, inflammatory bowel disease, and rheumatoid arthritis. In
another embodiment, the composition comprises axenic C. elegans
homogenate for use in treating allergies, including environmental
allergies and asthma. The composition optionally comprises a
pharmaceutically acceptable excipient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate certain
embodiments, and together with the written description, serve to
explain certain principles of the antibodies and methods disclosed
herein.
[0022] FIGS. 1A-B: Effects of axenic C. elegans antigen injections
in vivo. FIG. 1 shows percentages of circulating white blood cells
that are eosinophils (A) and basophils (B) in mice after receiving
24 intraperitoneal injections of 100 .mu.g of axenic C. elegans
antigen or phosphate buffered saline as control over a period of 12
weeks. Statistical significance between groups was assessed by the
Mann-Whitney test (*<0.05).
[0023] FIGS. 2A-C: Treatment with axenic C. elegans antigen delays
the onset of diabetes. (A) Mean blood glucose levels and (B)
percentages of NOD mice with diabetes during treatment with twice
weekly injections of 100 .mu.g axenic C. elegans antigen (n=10) or
PBS control (n=10). (C) Mean total numbers of pancreatic islets
from axenic C. elegans antigen or control treated mice at 20 weeks
of age (9-10 animals per group). Pancreatic islets were classified
as non-infiltrated, periinsulitis, and intrainsulitis with less
than or more than 50% infiltrated lymphocytes. Statistical
significance between groups was assessed by the Mann-Whitney test
(*<0.05).
[0024] FIGS. 3A-D: Cytokine and antibody response after 10 weeks of
axenic C. elegans antigen/control treatment at 20 weeks of age. (A)
IFN.gamma. and (B) IL-10 cytokine production from spleen cells
after in vitro stimulation with 20 .mu.g/ml axenic C. elegans
antigen. (C) Plasma levels of polyclonal IgE and (D) axenic C.
elegans antigen-specific IgE. Statistical significance between
groups was assessed by the Mann-Whitney test.
[0025] FIG. 4: TLR4 activation in response to LPS, C. elegans
antigen, and axenic C. elegans antigen. TLR4 activation by 10 ng/ml
of purified Salmonella lipopolysaccharide (positive control), 20
.mu.g/ml C. elegans antigen prepared from C. elegans grown with
OP50 Escherichiae coli, and 20 .mu.g/ml axenic C. elegans antigen.
TLR4 activation was determined using an HEK293 cell line (obtained
from Invivogen) expressing TLR4 in which a secreted alkaline
phosphatase gene under the control of an NF-kB promoter is
expressed when TLR4 is activated.
DETAILED DESCRIPTION
[0026] Reference will now be made in detail to various exemplary
embodiments, examples of which are illustrated in the accompanying
drawings. It is to be understood that the following detailed
description is provided to give the reader a fuller understanding
of certain embodiments, features, and details of aspects of the
invention, and should not be interpreted as a limitation of the
scope of the invention.
[0027] 1. Definitions
[0028] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0029] The term "axenic media" as used in this disclosure refers to
culture media for growing Caenorhabditis elegans that does not
contain Escherichia coli. Thus, an axenic homogenate of C. elegans
is a homogenate obtained from C. elegans cultured in axenic
media.
[0030] The term "effective amount" refers to a dosage or amount
that is sufficient for treating an indicated disease or
condition.
[0031] The terms "treatment" or "treating" and the like refer to
any treatment of any disease or condition in a mammal, e.g.
particularly a human or a mouse, and includes inhibiting a disease,
condition, or symptom of a disease or condition, e.g., arresting
its development and/or delaying its onset or manifestation in the
patient or relieving a disease, condition, or symptom of a disease
or condition, e.g., causing regression of the condition or disease
and/or its symptoms.
[0032] The terms "subject," "host," "patient," and "individual" are
used interchangeably herein to refer to any mammalian subject for
whom diagnosis or therapy is desired, particularly humans.
[0033] The term "pharmaceutically acceptable carrier" or
"pharmaceutically acceptable excipient" means solvents, dispersion
media, coatings, antibacterial agents and antifungal agents,
isotonic agents, and absorption delaying agents, and the like, that
are compatible with pharmaceutical administration. The use of such
media and agents for pharmaceutically active substances is well
known in the art.
[0034] As understood in the art, the term "Th1-mediated autoimmune
disease" means an autoimmune disease that is associated with a type
1 helper T cell (Th1) response. Such diseases include, but are not
limited to, type 1 insulin-dependent diabetes mellitus,
scleroderma, multiple sclerosis, posterior uveitis, Crohn's
disease, inflammatory bowel disease, and rheumatoid arthritis.
[0035] The term "chronically administered" or the like means that
the composition is administered to the subject at least as
frequently as once a week for at least 10 weeks or at least as
frequently as one or more times a day for at least 5 days.
[0036] 2. Th1/Th2 Immune Responses
[0037] CD4 T helper cell responses to antigens can be classified
based on the cytokines they produce. Type 1 helper T cells (Th1)
produce inflammatory cytokines, such as IFN-.gamma., IL-2,
TNF-.alpha., and TNF-.beta.. Th1 cells activate macrophages and are
associated with cell-mediated immune responses. Type 2 helper cells
(Th2), on the other hand, typically produce cytokines, such as
IL-4, IL-5, IL-10, and IL-13. Th2 cells activate B cells and are
associated with antibody-mediated immune responses.
[0038] 3. Autoimmune Diseases and Allergies
[0039] Autoimmune diseases are characterized by overactive immune
responses to self antigens expressed on cells. In essence, the
immune system mistakes a self antigen in the body as a pathogen and
mounts an attack against the cell or tissue expressing the self
antigen. Autoimmune diseases usually involve chronic autoimmune
responses, leading to long-term tissue damage. Several autoimmune
diseases appear to be caused in large part by Th1-driven
inflammation based, in part, on cytokine profiles, including
predominantly high levels of IFN-.gamma.. Examples of Th1-mediated
autoimmune diseases, include, but are not limited to, type-1
insulin-dependent diabetes mellitus, multiple sclerosis, Crohn's
disease, inflammatory bowel disease, rheumatoid arthritis, and
posterior uveitis.
[0040] Allergic reactions occur when a body's immune system reacts
to normally harmless substances in the environment. Allergies
usually involve excessive activation of mast cells and basophils by
IgE. This results in an inflammatory response which can range from
uncomfortable to life-threatening anaphylaxis.
[0041] 4. Axenic C. elegans Homogenate Therapy for Autoimmune
Diseases and Allergies
[0042] The prevalence of allergies, type 1 diabetes, and other
autoimmune diseases has increased sharply over the past few
decades. While genetic factors play a role in susceptibility to
type 1 diabetes, the dramatic worldwide increase in the prevalence
of type 1 diabetes is probably due to changes in environmental
factors.
[0043] One environmental change that may play a part in the recent
increase in autoimmune diseases is the loss of chronic parasitic
worm infections in developed countries. Multiple studies have found
that individuals infected with chronic parasitic worm infections
have lower rates of autoimmune diseases than others living in the
same environment. Experimentally, parasitic worms have been shown
to protect against type 1 diabetes and other autoimmune diseases in
several animal models. In humans, oral administration of porcine
whipworm eggs has been shown to protect against inflammatory bowel
disease.
[0044] Until recently, most people had lifelong infections with
parasitic worms. As helminths have been identified in neolithic and
pre-Columbian mummies, it is likely that the human immune system
evolved in the setting of chronic infection with these parasites.
Consequently, it has been posited that the loss of parasitic worm
infections is partially responsible for the increased prevalence of
autoimmune and allergic diseases in developed countries--the notion
being that now, in the absence of the immunomodulatory responses
triggered by helminths, our immune systems have become
hyperresponsive.
[0045] Unlike most bacterial or viral pathogens, helminth
infections induce the production of specific IgE. This IgE binds to
basophils and mast cells through the Fc epsilon receptor I
(Fc.epsilon.RI), the high affinity IgE receptor. Helminth specific
antigens can then activate basophils and mast cells by
cross-linking IgE molecules and aggregating Fc.epsilon.RIs. As
helminths are large organisms that release substantial amounts of
antigen, and as these infections last for years, helminth
infections likely induce a state of chronic basophil and mast cell
activation. Indeed, recent time course studies in our lab
demonstrate that both chronic basophil activation and chronic mast
cell activation occur during infection of mice with the filarial
nematode Litomosoides sigmodontis.
[0046] Helminth infections also induce production of the cytokine
IL-10. IL-10 is an anti-inflammatory cytokine that was initially
described as a Th2-type or Treg-type cytokine but it is now known
that IL-10 is more broadly expressed. IL-10 acts as a crucial
feedback regulator of diverse immune responses, including
Th1-mediated and Th2-mediated immune responses.
[0047] Without intending to be bound by any theory, there are at
least two likely mechanistic rationales for postulating that
chronic activation of basophils and mast cells may protect against
Th1-driven autoimmune disease. First, factors released by basophils
and mast cells may have direct immunomodulatory properties that are
protective against Th1-mediated autoimmune diseases. Basophils, for
example, release large quantities of IL-4 when activated and have
been shown to do so in response to parasite antigen in
filaria-infected patients as well as in animal models of helminth
infection. As destruction of .beta.-islet cells in type 1 diabetes
is driven by IFN-.gamma. release from Th1 cells, and as IL-4
counter regulates Th1 responses and has been shown to improve
Th1-driven autoimmune diseases, chronic basophil activation may
protect against type 1 diabetes by release of IL-4. Similarly,
histamine, which is released from both basophils and mast cells,
has been shown in vitro to suppress Th1 responses by signaling
through the H2 receptor on lymphocytes. Alternatively, chronic
activation of basophils and mast cells could induce negative
feedback pathways that tamp down ongoing autoimmune responses.
Interestingly, there is substantial evidence that chronic
immunotherapy, in which patients with IgE-mediated allergies are
given weekly injections of allergen, augments immune regulatory
networks such as the suppressive cytokine IL-10 and natural
T-regulatory cells.
[0048] To determine whether recapitulation of the IgE-mediated
immune responses induced by helminths can afford protection against
autoimmunity in the absence of actual infection, non-obese diabetic
(NOD) mice were repeatedly administered intraperitoneal injections
of axenic C. elegans homogenate. NOD mice spontaneously develop
type 1 diabetes (also known as insulin dependent diabetes
mellitus), a form of diabetes that develops from the autoimmune
destruction of the insulin-producing beta islet cells of the
pancreas. NOD mice are an art recognized animal model for type 1
diabetes.
[0049] To mimic chronic helminth in vivo without using live
parasitic worms, NOD mice were treated with twice weekly injections
of axenic C. elegans homogenate. Treated mice exhibited increases
in circulating basophils, eosinophils, total IgE, and C.
elegans-specific IgE. These immunologic changes are consistent with
those observed in chronic helminth infections. Additionally,
splenocytes of C. elegans-treated mice released substantial
quantities of the downregulatory cytokine IL-10, whereas those from
control-treated mice did not. Consistent with our hypothesis, mice
given axenic C. elegans antigen injections were significantly
protected from developing autoimmune diabetes (10% disease rate vs
80% in controls) and exhibited less inflammation in the pancreatic
islets.
[0050] These results demonstrate that axenic C. elegans homogenate
therapy can protect against the onset of type I diabetes in NOD
mice and suggests that repeated administration of axenic C. elegans
homogenate represents a new avenue of therapy for Th1-associated
autoimmune diseases.
[0051] 6. Axenic Media
[0052] This disclosure provides a composition comprising a
homogenate of C. elegans, wherein the homogenate is obtained from
C. elegans cultured in axenic media, for use in treating allergies
and autoimmune diseases, such as type 1 diabetes. C. elegans is
commonly cultured on Nematode Growth Medium (NGM) agar using E.
coli as a food source. However, this method of culturing C. elegans
introduces undesirable E. coli endotoxins into the end homogenate.
E. coli endotoxin, including lipopolysaccharides (LPS), is
extremely difficult to eliminate through purification methods. Yet
if endotoxin is not eliminated in the homogenate that is
administered to a subject, the endotoxin will trigger an
undesirable immune response, such as increased IFN.gamma.
production.
[0053] Axenic media for culturing C.elegans can include various
salt solutions, essential amino acids, non-essential amino acids,
vitamins and growth factors, nucleic acid substitutes, and energy
sources, as described in "Chemically defined medium and
Caenorhabditis elegans," Szewczyk et al., BMC Biotechnology 2003,
3:19, which is hereby incorporated by reference in its entirety.
The axenic media can further include hemin chloride, preferably 20
uM hemin chloride; ultra-pasteurized (UHT) skim milk, preferably
10-20% UHT skim milk; cholesterol, preferably 5 ug/mL cholesterol;
and antibiotics, preferably tetracycline, streptomycin, and
nalidixic acid, more preferably 100 to 250 ug of tetracycline,
streptomycin, and nalidixic acid. Addition of heme and cholesterol
enhances worm growth, as C. elegans worms need to obtain heme and
cholesterol from the environment ("Lack of heme synthesis in a
free-living eukaryote", Rao et al. PNAS 2005, 102:12, "The
requirement of sterol and various sterol precursors in free-living
nematodes", N. C. Lu, et al., Nematologica 1977, 23:57-61).
[0054] 7. Methods of Making Axenic C. elegans Homogenate
[0055] C. elegans can be grown in axenic media as described above.
Whole C. elegans worms can be pulverized in solution by methods
known in the art. The pulverized worms can then be centrifuged and
the supernatant decanted and saved using methods known in the art.
Optionally, the pellet can be resuspended and centrifuged.
Centrifugation and supernatant decanting/saving can be repeated as
many times as necessary or desired. Saved supernatants can be
combined to form the final homogenate composition comprising axenic
C. elegans antigen.
[0056] 8. Methods of Use
[0057] The axenic C. elegans homogenate described herein can be
used in a variety of research and medical applications. In one
aspect, the disclosure provides a method of treating an allergy or
autoimmune disease in a subject, comprising administering to said
subject an effective amount of a composition comprising a
homogenate of C. elegans, wherein the homogenate is obtained from
C. elegans cultured in axenic media. The composition can further
include a pharmaceutically acceptable carrier and other excipients.
In certain embodiments, the composition includes at least one
non-naturally occurring pharmaceutically acceptable carrier or
other excipient. Preferably, the composition is one that does not
occur in nature. Preferably, the autoimmune disease is a
Th1-mediated autoimmune disease, including, but not limited to
type-1 insulin-dependent diabetes mellitus, multiple sclerosis,
Crohn's disease, inflammatory bowel disease, rheumatoid arthritis,
and posterior uveitis.
[0058] 9. Formulations and Administration
[0059] The disclosure provides compositions comprising a homogenate
of C. elegans, wherein the homogenate is obtained from C. elegans
cultured in axenic media. In certain embodiments, the compositions
are suitable for pharmaceutical use and administration to patients.
These compositions comprise an axenic homogenate of C. elegans and
a pharmaceutically acceptable excipient. The compositions may also
contain other active compounds providing supplemental, additional,
or enhanced therapeutic functions. Preferably, the composition is
one that does not occur in nature. The pharmaceutical compositions
may also be included in a container, pack, or dispenser together
with instructions for administration. In one embodiment, the
composition comprises an axenic homogenate of C. elegans for use in
treating an autoimmune disease, including a Th1-mediated autoimmune
disease, such as type-1 insulin-dependent diabetes mellitus,
multiple sclerosis, Crohn's disease, inflammatory bowel disease,
rheumatoid arthritis, and posterior uveitis.
[0060] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration. Methods
to accomplish the administration are known to those of ordinary
skill in the art. This includes, for example, injections, by
parenteral routes such as intravenous, intravascular,
intraarterial, subcutaneous, intramuscular, intratumor,
intraperitoneal, intraventricular, intraepidural, or others as well
as oral, nasal, ophthalmic, rectal, or topical. Sustained release
administration is also specifically contemplated, by such means as
depot injections or erodible implants. Localized delivery is
particularly contemplated, by such means as delivery via a catheter
to one or more arteries, such as the renal artery or a vessel
supplying a localized tumor. The composition can also be admixed
with food as a food additive.
[0061] In one embodiment a composition including axenic C. elegans
homogenate is administered to a patient by injection (e.g.,
intraperitoneally, intravenously, subcutaneously, intramuscularly,
etc.). The composition may be administered, for example, by bolus
injunction or by slow infusion. Slow infusion over a period of 30
minutes to 2 hours may be used. Generally, an initial candidate
dosage can be about 5-100 .mu.g/kg (0.005-0.1 mg/kg). A typical
daily dosage might range from about any of 0.001 mg/kg to about 100
mg/kg; about 0.01 mg/kg to about 10 mg/kg; or about 0.1 mg/kg to
about 5 mg/kg. The appropriate dosage of the axenic C. elegans
homogenate will depend on various factors, including the type of
axenic C. elegans homogenate used (or compositions thereof), route
of administration, frequency of administration, patient's health,
age, or size, the type and severity of the disease to be treated,
whether the agent is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the antibody, and the discretion of the attending
physician. Typically, the clinician will administer the axenic C.
elegans homogenate until a dosage is reached that achieves the
desired result.
[0062] For repeated administrations over several days or longer,
depending on the condition, the treatment is sustained until a
desired suppression of symptoms occurs or until sufficient
therapeutic end points are achieved. An exemplary dosage regimen
comprises administering a daily dose of about 5-100 .mu.g/kg for
about 5-14 days (or longer), with or without weekly maintenance
doses of about 100-250 .mu.g/kg. Alternatively, an exemplary dosing
regimen comprises administering an initial dose of about 5-100
.mu.g/kg, followed by a weekly maintenance dose of about 5-250
.mu.g/kg of the axenic C. elegans homogenate, or followed by a
maintenance dose of about 5-250 .mu.g/kg every other week. However,
other dosage regimens may be useful, depending on the
pharmacokinetic parameters that the practitioner wishes to achieve.
For example, dosing from one to four times a week is contemplated.
In some embodiments, dosing frequency is once every day, every
other day, every third day, every fourth day, every fifth day,
every sixth day; once every week, every 2 weeks, every 4 weeks,
every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9
weeks, or every 10 weeks; or once every month, every 2 months, or
every 3 months, or longer. The progress of this therapy is easily
monitored by conventional techniques and assays.
[0063] The dosing regimen can also vary over time. One risk of
axenic C. elegans homogenate therapy is the induction of
anaphylaxis, resulting from chronic mast cell and basophil
activation. To reduce this risk, it is possible to initiate
treatment with smaller doses of axenic C. elegans homogenate
followed by a gradual increase in treatment dosage. Such an
approach would be similar to allergen immunotherapy, where the
allergen dose is gradually increased over time. Allergen
immunotherapy induces repeated basophil and mast cell activation
and is routinely conducted in the outpatient arena for diseases as
benign as allergic rhinitis. Thus, in one embodiment, the axenic C.
elegans homogenate therapy can be administered at a smaller initial
dose followed by gradually increasing doses. The particular dosage
regimen, i.e., dose, timing and repetition, will depend on the
particular individual and that individual's medical history, as
well as the properties of the axenic C. elegans homogenate.
[0064] Toxicity and therapeutic efficacy of the composition can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., determining the LD50 (the dose
lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Axenic C.
elegans homogenates that exhibit large therapeutic indices may be
less toxic and/or more therapeutically effective.
[0065] To test whether repeated administration of axenic C. elegans
antigen (aCeAg) can protect against a Th1-driven autoimmune
disease, the effects of repeated aCeAg injections on the
development of type 1 diabetes in the NOD mouse model were studied.
The studies demonstrate that aCeAg delays type 1 diabetes onset and
decreases autoimmune inflammation against insulin-producing cells.
Immunological tests demonstrate that aCeAg injections induce
immunologic changes similar to those observed in chronic helminth
infections, with increases in IgE, eosinophils, basophils, and
IL-10 production. Further, in vitro testing demonstrates that
aCeAg, in contrast to soluble antigen prepared from C. elegans
worms grown on E. coli plates, does not induce TLR4 activation.
[0066] Material & Methods
[0067] Mice: Female NOD and IL-4-deficient
NOD.129P2(B6)Il4.sup.tm1Cgn/DvsJ mice (The Jackson Laboratory, Bar
Harbor, Me.) were maintained at the Uniformed Services University
(USU) animal facility with free access to food and water. All
experiments were performed under protocols approved by the USU
Institutional Animal Care and Use Committee.
[0068] Assessment of diabetes: Glucose levels of mice were
determined from blood taken by orbital bleeds every other week
using a standard blood glucose meter (Accu-Check.RTM. Advantage,
Roche Diagnostics GmbH). Mice with glucose levels greater than 230
mg/dl in two consecutive measurements were considered diabetic.
[0069] Preparation of axenic C. elegans antigen: C. elegans worms
were grown in chemically defined axenic media. The axenic media
included all the ingredients listed by Szewczyk and colleagues [1],
plus 20 mM of hemin chloride, 15% ultra-pasteurized skim milk, 5
mg/ml cholesterol, 100 mg/ml tetracycline, 250 mg/ml streptomycin,
and 250 mg/ml nalidixic acid. Whole C. elegans worms are then
mechanically homogenized by placing into "D" tubes from
BioPulverizer System 1 (MP Biomedicals) and then run on a
FastPrep-24 system (MP Biomedicals) at 4M/s.sup.2 for 20 s. Samples
were then centrifuged at 16, 100 g for 1 min, supernatant saved,
and the pellet resuspended in another 1 ml PBS. The fast-prep run,
centrifugation, and removal of supernatant were then repeated on
the resuspended pellet. Saved supernatants were combined,
centrifuged at 15,000 g for 5 min, and the final supernatant used
as aCeAg. Final protein concentration was determined using the
Pierce BCA Protein Assay Kit.
[0070] Treatment with axenic C. elegans antigen: Beginning at 7
weeks of age, mice were given twice weekly i.p. injections of 100
.mu.g aCeAg or PBS as control until 17 weeks of age, at which point
they received one injection per week until study endpoint at 20
weeks. Animals were euthanized at 20 weeks of age to assess
pancreas inflammation, splenocyte cytokine production, and
circulating IgE, basophil, and eosinophil levels.
[0071] Assessment of pancreas inflammation: Pancreases were
isolated and fixed in 10% formalin. Haematoxylin-eosin stained
slices were assessed for inflammation by a pathologist blinded to
the intervention group. Total numbers of islets of three
longitudinal sections 400 .mu.m apart of each pancreas were
assessed. The severity of insulitis was scored as non-infiltrated,
periinsulitis (lymphocytes at the periphery of islets), or
intrainsulitis (lymphocyte infiltration into the interior of the
islets lesser or greater than 50%).
[0072] Spleen cell culture: At study endpoint animals were
euthanized and spleens were isolated. Spleen cells were prepared
and cultured. In brief, single cell suspensions were obtained, red
blood lysis performed for spleen cells (ACK Lysing Buffer, Quality
Biological, Inc., Gaithersburg, Md.), and cells were plated at a
concentration of 2.times.10.sup.6 cells/ml in enriched media
(Iscove's Dulbecco modified medium (Mediatech, Manassas, Va.)
including 10% fetal calf serum (Valley Biomedical, Winchester,
Va.), 1% L-glutamine (Mediatech, Gaithersburg, Md.), 1%
insulin-transferrin-selenium medium (Invitrogen Inc., Carlsbad,
Calif.) and 80 .mu.g/ml gentamicin (Quality Biological, Inc.,
Gaithersburg, Md.)), stimulated with PBS as control or 20 .mu.g/ml
aCeAg, and cultured at 37.degree. C., 5% CO.sub.2.
[0073] Measurement of cytokines by ELISA: Cytokine enzyme-linked
immunosorbent assays (ELISAs) were performed from spleen cell
cultures. Culture supernatants from cells that were cultured as
described above were collected after 72 h of incubation.
IFN-.gamma., IL-4, IL-5, and IL-10 were quantified according to the
manufacturer's instructions (BD Biosciences, Franklin Lake,
N.J.).
[0074] Measurement of total and aCeAg-specific IgE levels by ELISA:
Blood was collected from aCeAg or PBS treated NOD mice at study
endpoint immediately after euthanasia by cardiac puncture and
analyzed for total and aCeAg-specific IgE by colorimetric sandwich
ELISA. Flat-bottom Immulon 4 plates (Thomas Scientific, Swedesboro,
N.J.) were coated overnight at 4.degree. C. with 10 mg/ml
anti-mouse IgE (clone R35-72) for total IgE or 20 mg/ml aCeAg for
aCeAg-specific IgE. Blocking was performed by 1 h incubation of
plates with 5% bovine serum albumin in PBS. Prior to testing, IgG
was adsorbed from serum samples by incubation of serum with
GammaBind G Sepharose (Amersham Biosciences, Uppsala, Sweden)
overnight at 4.degree. C. Serum samples were then diluted 1:5 and
1:50 for total IgE measurements. Plates were washed and incubated
with biotinylated rat anti-mouse IgE (clone R35-118) in PBS.
Following washing, 1/1000 dilution of alkaline
phosphatase-conjugated streptavidin (BD Pharmingen) was added, and
plates were incubated for 1 h at 37.degree. C. Nitrophenyl
phosphate disodium (Sigma-Aldrich, St. Louis, Mo.) was used as
substrate. Purified mouse IgE was used as standard for total IgE
(BD Biosciences). For aCeAg-IgE measurements, samples from control
and experimental groups were analyzed as duplicates on the same
plate to allow for accurate comparison between groups by OD.
Absorbance was detected at 405 nm using a PerkinElmer Victor3 V
microplate reader (PerkinElmer, Waltham, Mass.). All samples were
analyzed as duplicates at the same time on the same plate to allow
accurate comparison between groups by OD.
[0075] Flow cytometric detection of basophils and eosinophils:
Whole blood (100 ml) obtained by cardiac puncture after animal
euthanasia at 20 weeks was aliquotted in 5 ml polypropylene
round-bottom tubes (BD Falcon). Red blood cells were lysed and
leukocytes fixed with whole blood lysing reagent (Beckman Coulter,
Galway, Ireland). Cells were washed twice with 2 ml of PBS and
centrifuged at 500.times.g for 5 min. Supernatants were aspirated
and cells resuspended in 100 ml of 1% bovine serum albumin/PBS
followed by incubation at 4.degree. C. for 1 h. Cells were stained
with anti-IgE FITC (R35-71), anti-CD4 PerCP (RM4-5) and anti-B220
PerCP (RA3-6B2) to identify basophils; or SiglecF PE(E50-2440),
CD45 FITC (30-F11) and CD11c APC (HL3) to identify eosinophils. All
the antibodies were purchased from BD Pharmingen. Cells were washed
and resuspended in 200 ml of PBS for analysis using a BD LSR II
Optical Bench flow cytometer.
[0076] Statistics: Statistical analyses were performed with
GraphPad Prism software (GraphPad Software). Differences between
two unpaired groups were tested for significance with the
Mann-Whitney-U-test. P-values<0.05 were considered significant.
All experiments were performed at least twice.
EXAMPLE 1
Axenic C. elegans Antigen Protects Against Type 1 Diabetes in NOD
Mice
[0077] To test the hypothesis that aCeAg can protect against
autoimmune disease, development of type I diabetes was monitored in
NOD mice that were given repeated aCeAg injections. Repeated
administration of aCeAg significantly reduced blood glucose
concentrations in NOD mice at 20 weeks of age compared to control
animals (FIG. 3A) and delayed the onset of diabetes (80% mice
diabetic at 20 wks of age in control group vs 10% in aCeAg group,
FIG. 1B). Histological analysis of the pancreas revealed that mice
treated with aCeAg mice had greater total numbers of .beta.-islet
cells and less lymphocyte infiltration of the pancreatic islets
compared to PBS-treated mice at study endpoint (FIG. 2).
EXAMPLE 2
Axenic C. elegans Antigen Induces Immunological Changes Similar to
Those Observed in Response to Helminth Infection
[0078] Helminth infections characteristically induce increased
levels of eosinophils, basophils, and IgE antibodies. To determine
whether aCeAg injections induce these changes, at study endpoint
levels of circulating eosinophils and basophils were quantified by
flow cytometry and plasma total (polyclonal) IgE and aCeAg-specific
IgE levels measured by ELISA. Both eosinophil (4.5% in aCeAg
treated mice vs 3.1% in NOD mice) and basophil (0.60% vs 0.37%)
were increased in mice given aCeAg injections, though the
difference was only statistically significant for basophils (FIG.
3A, 3B). Similarly, total and aCeAg-specific IgE levels were also
markedly greater in aCeAg-treated mice than controls (FIG. 3C,
3D).
EXAMPLE 3
Axenic C. elegans Antigen Induces Production of IL-10 From
Splenocytes
[0079] The effect repeated aCeAg injections have on cellular
cytokine production was evaluated from spleen cells of 20 week old
NOD mice after stimulation with aCeAg in vitro. While IL-4 and
IL-13 production levels were below the level of detection (data not
shown), IL-10 levels were significantly greater from splenocytes of
aCeAg-treated NOD mice (FIG. 4A). As IL-10 is a an
immunosuppressive cytokine, it is possible that some of the
protective effect conferred by aCeAg injections is due to induction
of host IL-10 production. IFN.gamma. levels were similar between
control and aCeAg-treated animals (FIG. 4B).
EXAMPLE 4
Axenic C. elegans Antigen Does Not Activate TLR4
[0080] C. elegans nematode worms are typically grown on a culture
plate using E. coli bacteria (strain OP-50) as a food source. The
limitation to that approach for the purpose of making soluble worm
antigen is that the final product will be a mixture of bacterial
and worm products. Of particular concern is the E. coli product
lipopolysaccharide (LPS), which interacts with TLR4 receptors on
human cells to induce immediate inflammatory responses. To evaluate
whether soluble antigen produced from axenically grown C. elegans
worms is free of pro-inflammatory TLR-4 activating substances, we
tested the ability of aCeAg to activate TLR4 using a reporter cell
line for TLR4 activation. In contrast to purified Salmonella
lipopolysaccharide (positive control) and soluble antigen derived
from C. elegans worms grown with OP-50 E. coli, aCeAg does not
induce TLR4 activation.
[0081] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. In case of conflict, the present
specification, including definitions, will control. It will be
readily apparent to one of ordinary skill in the relevant arts that
other suitable modifications and adaptations to the methods and
applications described herein are obvious and may be made without
departing from the scope of the invention or any embodiment
thereof. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting. All patents,
patent applications, and published references cited herein are
hereby incorporated by reference in their entirety. [0082] 1.
Szewczyk, N. J., E. Kozak, and C. A. Conley, Chemically defined
medium and Caenorhabditis elegans. BMC Biotechnol, 2003. 3: p.
19.
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