U.S. patent application number 17/231544 was filed with the patent office on 2022-03-17 for mixed allergen compositions and methods for using the same.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Kari C. Nadeau.
Application Number | 20220080041 17/231544 |
Document ID | / |
Family ID | 1000005990371 |
Filed Date | 2022-03-17 |
United States Patent
Application |
20220080041 |
Kind Code |
A1 |
Nadeau; Kari C. |
March 17, 2022 |
MIXED ALLERGEN COMPOSITIONS AND METHODS FOR USING THE SAME
Abstract
Mixed allergen compositions of two or more different allergens
are provided. In some instances, the mixed allergen compositions
include: a nut allergen; an animal allergen; and at least one of: a
non-nut plant allergen; a biotic agent; and a vitamin. Also
provided are methods of administering the mixed allergen
compositions to a subject. The mixed allergen compositions find use
in a variety of applications, including health maintenance, immune
balance, gut balance, immune support, health improvement and
therapeutic applications.
Inventors: |
Nadeau; Kari C.; (Los Altos
Hills, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Stanford |
CA |
US |
|
|
Family ID: |
1000005990371 |
Appl. No.: |
17/231544 |
Filed: |
April 15, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16228953 |
Dec 21, 2018 |
11007264 |
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17231544 |
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15224004 |
Jul 29, 2016 |
10166286 |
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16228953 |
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15048609 |
Feb 19, 2016 |
10064936 |
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15224004 |
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62119014 |
Feb 20, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/55 20130101;
A61K 2035/115 20130101; A61K 45/06 20130101; A61K 2039/70 20130101;
A23L 33/155 20160801; A23L 33/10 20160801; A61K 2039/54 20130101;
A61K 2039/58 20130101; A61K 2039/542 20130101; A61K 39/35 20130101;
A61K 35/60 20130101; A23V 2002/00 20130101; A61K 31/375 20130101;
A61K 2039/57 20130101; A61K 31/593 20130101; A23L 5/00 20160801;
A61K 2039/545 20130101; A61K 2039/575 20130101; A61K 35/747
20130101; A61K 2039/572 20130101 |
International
Class: |
A61K 39/35 20060101
A61K039/35; A61K 31/593 20060101 A61K031/593; A61K 45/06 20060101
A61K045/06; A61K 31/375 20060101 A61K031/375; A61K 35/60 20060101
A61K035/60; A61K 35/747 20060101 A61K035/747; A23L 5/00 20060101
A23L005/00; A23L 33/10 20060101 A23L033/10; A23L 33/155 20060101
A23L033/155 |
Claims
1. A method of treating an autoimmune disease or inflammatory
disease in a human subject in need thereof comprising administering
to the subject a composition comprising: 1 to 20 different protein
allergens, and optionally a vitamin selected from vitamin D and
vitamin C; wherein the composition is administered to the subject
at least weekly or at least every other week.
2. The method of claim 1, further comprising allowing sufficient
time for the administration of the composition to inhibit the
symptoms of the autoimmune disease or inflammatory disease in the
human subject.
3. The method of claim 1, wherein the method further comprises
inhibiting the symptoms of one or more food allergies.
4. The method of claim 1, wherein the one or more food allergies is
a single food allergy.
5. The method of claim 1, wherein the one or more food allergies is
a multiple food allergy.
6. The method of claim 1, wherein the treating is prophylactically
treating.
7. The method of claim 1, wherein after administration the subject
has an improved immune protection against autoimmune inflammation
as measured by the subject's Th1 proliferation to the allergen, as
compared to levels before administration.
8. A method of reducing the incidence of, or for prophylactic
treating, an inflammatory disease or an autoimmune disease in a
pediatric or pregnant subject in need thereof, comprising
administering a composition comprising: 1 to 20 different protein
allergens, and optionally a vitamin selected from vitamin D and
vitamin C; wherein the composition is administered to the subject
at least weekly or at least every other week.
9. The method of claim 8, wherein administering the composition is
daily for a period of time, followed by weekly, twice weekly and/or
three times a week for a period of time.
10. The method of claim 1, wherein administering comprises
administering about 1 to about 10 mg (by weight protein allergen)
of the composition, or of each allergen, daily for about 1 week to
about 30 weeks, and then about 1 to about 300 mg or more (by weight
protein allergen) weekly for about 1 to about 30 or more weeks.
11. The method of claim 1, wherein administering comprises two,
three or more step-wise, increasing dose levels of the composition,
wherein each dose level is about 5 to about 100% more than the
preceding dose level by total weight protein level, wherein each
dose level is independently administered daily or weekly for about
1 to about 30 weeks.
12. The method of claim 1, wherein the 1 to 20 different protein
allergens are each selected from the group consisting of shellfish,
peanuts, tree nuts, fish, milk, egg, legume, grain, sesame, and
fruit allergens.
13. The method of claim 12, wherein the protein allergens are each
selected from the group consisting of cashew, pistachio, walnut,
almond, pecan, chicken egg, cow milk, peanut, shrimp, salmon,
wheat, cod, crab, soy, and sesame protein allergens.
14. The method of claim 13, wherein the composition is a food
product.
15. The method of claim 14, wherein the food product is pureed food
product or infant formula.
16. (canceled)
17. The method of claim 1, wherein the human subject is a pediatric
subject.
18. The method of claim 17, wherein the pediatric subject is 1
month to 7 years old.
19. The method of claim 17, wherein the pediatric subject is 1
month to 6 months, or 6 months to 7 years old.
20. The method of claim 1, wherein the composition comprises 6 to
20 different allergens.
21. The method of claim 1, wherein the subject has an increased
risk to develop the autoimmune disease or inflammatory disease
relative to the general population based on diagnostic criteria.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/224,004, filed Jul. 29, 2016, which is a
continuation-in-part of U.S. patent application Ser. No.
15/048,609, filed Feb. 19, 2016 (U.S. Pat. No. 10,064,936, issued
Sep. 4, 2018), which claims priority to U.S. Patent Application No.
62/119,014, filed Feb. 20, 2015; each of which is hereby
incorporated by reference in their entirety.
BACKGROUND
[0002] Allergy is a disorder of the immune system and is
characterized by the occurrence of allergic reactions to normally
harmless environmental substances. Allergies are caused by
allergens, which may be present in a wide variety of sources,
including but not limited to pollens or other plant components,
dust, molds or fungi, foods, additives, latex, transfusion
reactions, animal or bird danders, insect venoms, radiocontrast
medium, medications or chemicals. Common allergic reactions include
eczema, hives, hay fever, asthma, and reactions to venoms. Mild
allergies like hay fever are highly prevalent in the human
population and cause symptoms such as allergic conjunctivitis,
itchiness, and runny nose. In some people, severe allergies to
environmental or dietary allergens or to medication may result in
life-threatening anaphylactic reactions and potentially death, if
left untreated. Allergic reactions can occur in three distinct
patterns: a) an early phase reaction or acute response, b) late
phase reactions and c) potentially chronic allergic inflammation.
The early phase of the allergic reaction typically occurs within
minutes, or even seconds, following a first allergen exposure,
where this early phase is also commonly referred to as the
immediate allergic reaction. In the early stages of allergy, a
hypersensitivity reaction against an allergen, encountered for the
first time, causes a response in Th2 cells, which are a subset of T
cells that produce the cytokine interleukin-4 (IL-4). The Th2 cells
interact with B cells (lymphocytes that produce antibodies against
antigens) and, coupled with the effects of IL-4, stimulate the B
cells to begin production and secretion of Immunoglobulin E
(IgE).
[0003] IgE plays an important role in allergies and allergic
reactions. Upon introduction of an allergen, B cells of the
respective subject produce large amounts of IgE. The IgE elicits an
immune response by binding onto receptors found on basophils and
mast cells. When activated, these cells release chemical mediators
such as histamine and cytokines that cause the characteristic
symptoms of allergy.
[0004] A food allergy is an adverse immune response to a food
allergen, e.g., a food protein. Common food allergens are found in
shellfish, peanuts, tree nuts, fish, milk, eggs, soy and fresh
fruits such as strawberries, mango, banana, and apple.
Immunoglobulin E (IgE)-mediated food allergies are classified as
type-I immediate hypersensitivity reactions. These allergic
reactions have an acute onset (from seconds to one hour) and the
accompanying symptoms may include angioedema (soft tissue swelling
of the eyelids, face, lips, tongue, larynx and trachea); hives;
itching of the mouth, throat, eyes, or skin; gastrointestinal
symptoms such as nausea, vomiting, diarrhea, stomach cramps, or
abdominal pain; rhinorrhea or nasal congestion; wheezing, shortness
of breath, or difficulty swallowing; and even anaphylaxis, a
severe, whole-body allergic reaction that can result in death.
Gupta, et al. (PEDIATRICS Vol. 128 No. 1 Jul. 1, 2011 pp. e9-e17)
demonstrated that 1 out of 12 children under the age of 21 years of
age have a doctor's diagnosis of food allergies. This epidemic has
been reported to be doubling every 10 years for certain nuts (CDC
2009). Moreover, there are still deaths that occur every year due
fatal food allergies. Importantly, over $24 billion is spent per
year on health care/care costs for food allergic reactions (Gupta,
et al. JAMA PEDIATRICS November 2013, Vol. 167, No. 11). This cost
is largely due to about 90,000 visits to the ER per year in the
U.S. due to food induced anaphylactic symptoms.
SUMMARY
[0005] Mixed allergen compositions of two or more different
allergens are provided. In some instances, the mixed allergen
compositions include: a nut allergen; an animal allergen; and at
least one of: a non-nut plant allergen; a biotic agent; and a
vitamin. Also provided are methods of administering the mixed
allergen compositions to a subject. The mixed allergen compositions
find use in a variety of applications, including health
maintenance, immune balance, gut balance, immune support, health
improvement and therapeutic applications.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 depicts Th2 cell proliferation in ex vivo blood
samples from subjects fed different food allergen mixtures (units
are cells/mL). (It is noted that with respect to each of FIGS. 1 to
6 and 9 to 10, for each group of bars on the graphs the order of
test compositions from left to right corresponds to the order of
test compositions listed in the column at the right of the figure
from top to bottom. As such, in FIG. 1, 4-6 months group, the bars
from left to right are the data for each of the compositions tested
from top to bottom in the column at the right hand side of the
figure that lists the test compositions)
[0007] FIG. 2 depicts specific IgG4 levels in plasma from subjects
fed different food allergen mixtures (units are mg IgG4/L).
[0008] FIG. 3 depicts specific IgE levels in plasma from subjects
fed different food allergen mixtures (units are mg IgE/L).
[0009] FIG. 4 depicts Th2 cell proliferation in ex vivo blood
samples from subjects fed different food allergen mixtures (units
are cells/mL).
[0010] FIG. 5 depicts specific IgG4 levels in plasma from subjects
fed different food allergen mixtures (units are mg IgG4/L).
[0011] FIG. 6 depicts specific IgE levels in plasma from subjects
fed the food allergen mixtures (units are mg IgE/L).
[0012] FIG. 7 depicts T cell proliferation in response to gluten,
insulin, tetanus and bacterial flagellin in ex vivo blood samples
from subjects fed different food allergen mixtures.
[0013] FIG. 8 depicts Th2 cell proliferation in ex vivo blood
samples from subjects fed different food allergen mixtures (units
are cells/mL).
[0014] FIG. 9 depicts specific IgG4 levels in plasma from subjects
fed different food allergen/mixtures (units are mg IgG4/L).
[0015] FIG. 10 depicts specific IgE levels in plasma from subjects
fed different food allergen/mixtures (units are mg IgE/L).
DETAILED DESCRIPTION
[0016] Mixed allergen compositions of two or more different
allergens are provided. Mixed allergen compositions described
herein comprise, e.g., 2 to 20 or more (e.g., 2 to 100 or more)
different allergens, and optionally a vitamin such as a vitamin
selected from vitamin D, E, B6, C, A or combinations thereof. In
some instances, the mixed allergen compositions include: a nut
allergen; an animal allergen; and at least one of: a non-nut plant
allergen; a biotic agent; and a vitamin. In certain embodiments, a
composition may comprise allergens selected from the group
consisting of shellfish, peanuts, tree nuts, fish, milk, egg,
legume, grain, sesame, and fruit allergen. In further embodiments,
a composition may comprise allergens or allergens mixes selected
from the group consisting of cashew, pistachio, walnut, almond,
pecan, chicken egg, cow milk, peanut, shrimp, salmon, wheat, cod,
crab, soy, sesame protein allergens, and an allergen mix comprising
two or more thereof. The mixed allergen compositions find use in a
variety of applications, including health maintenance, immune
balance, gut balance, immune support, health improvement and
therapeutic applications.
[0017] Also provided are methods of administering the mixed
allergen compositions to a subject. Described herein are methods,
e.g., of treating an autoimmune disease or inflammatory disease in
a human subject in need thereof, which, e.g., may comprise
administering a composition comprising 1 to 20 or more (e.g., 1 to
100 more) different protein allergens, and optionally a vitamin,
such as a vitamin selected from vitamin D and vitamin C. Further
described are methods, e.g., for inhibiting symptoms of one or more
food allergies in a human subject, which, e.g., may comprise
administering a composition comprising 2 to 20 or more (e.g., 2 to
100 more) different protein allergens, and optionally a vitamin,
such as a vitamin selected from vitamin D and vitamin C. Such
methods are surprisingly efficacious in treating, e.g. symptoms of
allergies, e.g., even as compared to a composition having a single
protein allergen. Also provided are methods of reducing the
probability of developing one or more allergies in an unborn child
of a pregnant woman or a child of a nursing mother, and methods of
reducing the probability of developing one or more allergies in a
child having a developing immune system.
[0018] Before the present methods and compositions are described,
it is to be understood that this invention is not limited to a
particular method or composition described, as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
invention will be limited only by the appended claims.
[0019] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0020] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supersedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0021] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0022] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the peptide" includes reference to one or more
peptides and equivalents thereof, e.g., polypeptides, known to
those skilled in the art, and so forth.
[0023] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Mixed Allergen Compositions
[0024] As summarized above, aspects of the invention include mixed
allergen compositions. Such compositions may have uses in a variety
of applications including, for example, immune balance and immune
support. By mixed allergen composition is meant a composition that
includes two or more different allergens, where any two given
allergens are different if they are distinct from each other, e.g.,
they are compounds described by different chemical formula or
compositions described by different components (e.g., constituent
compounds) and/or amounts thereof. The number of different
allergens in a composition may vary, as desired. In certain
embodiments, the mixed allergen compositions include 2 more
different allergens, such as 3 or more different allergens, 4 or
more different allergens, 5 or more different allergens, 6 or more
different allergens, 7 or more different allergens, 8 or more
different allergens, 9 or more different allergens, 10 or more
different allergens, 15 or more different allergens, 20 or more
different allergens, 25 or more different allergens, 30 or more
different allergens, 40 or more different allergens, 50 or more
different allergens, 75 or more different allergens, 100 or more
different allergens, where in some instances the number of
different allergens in a given composition is 100 or less, such as
75 or less, including 50 or less, e.g., 25 or less, 15 or less,
including 10 or less. In certain embodiments, a composition may
comprise 2 to 20 different protein allergens, or e.g., 2 to 100
different allergens, or e.g., 2 to 1000 different allergens. In
further embodiments, a composition may comprise 6 to 20 different
protein allergens/protein allergen compositions. In certain
embodiments, a composition may consist essentially of 2 to 20
different protein allergens, or e.g., 2 to 100 different allergens,
or e.g., 2 to 1000 different allergens.
[0025] Allergens present in the composition may vary, where in some
instances the allergen present in the composition is one that
induces an allergy in a susceptible subject. Allergens include any
antigen, or active derivative thereof, that elicits a specific IgE
response. Antigens include any substance that can stimulate the
production of antibodies and can combine specifically with them.
Such antigens include those capable of eliciting an autoimmune
response. In the context of autoimmunity, the terms antigens,
protein allergens and allergens are used interchangeably to include
any substance that elicits an autoimmune response. Antigenic
determinants or epitopes are antigenic sites on a molecule.
Allergens may have little or no intrinsic toxicity by themselves,
but cause a pathological condition due to their ability to elicit
an IgE-associated immune response, and, upon subsequent exposure,
due to their ability to elicit IgE- and/or T cell-dependent
hypersensitivity reactions. As such, an "allergen" includes any
substance which is capable of stimulating a typical
hypersensitivity reaction in atopic subjects. Allergens that may be
present in a given mixed allergen composition include any substance
found in a variety of different sources, such as but not limited
to: foods, drugs, perfume, plants, the environment or biological
systems (e.g., prokaryotic or eukaryotic cells or viruses), as well
as chemical allergens. It is appreciated that reference to allergen
or an allergen composition (e.g., such as part of a provided food
product or composition) may each include a plurality of different
proteins as found in the naturally occurring allergen (either raw
or cooked). For example, a provided food product may include a
peanut allergen composition (which would include substantially all
peanut proteins present in e.g., defatted peanuts, ground peanuts,
etc.)
[0026] Allergens of interest include nut allergens. Nut allergens
are allergens that include one or more compounds found in nuts,
e.g., dry fruits that include an edible kernel or meat enclosed in
a woody or leathery shell. Nut allergens of interest include, but
are not limited to: peanut allergens, e.g., rAra h 1, rAra h 2,
rAra h 3, rAra h 8 PR-10, rAra h 9 LTP, peanut complete allergen
(the phrase "complete allergen" as used herein refers to all
possible antigenic components of a given food protein), etc.;
brazil nut allergens, e.g., rBer e 1, brazil nut complete allergen,
etc.; hazelnut or filbert allergens, e.g., rCor a 1 PR-10, rCor a 8
LTP, hazel nut complete allergen, nCor a 9, rCor a 14, etc.; walnut
allergens, e.g., rJug r 1, rJug r 3 LTP, walnut complete allergen,
etc.; cashew allergens; pistachio allergens, e.g., pistachio
component allergens, pistachio complete allergen, etc.; pecan
allergens, e.g., pecan component allergens, pecan complete
allergen, etc.; tree nut component package allergens, such as one
or more allergens from cashew nut, walnut, hazelnut, brazil nut;
etc.
[0027] Allergens of interest include animal allergens. Animal
allergens are allergens that include one or more compounds found in
animals, including both vertebrates and invertebrates. Vertebrate
animal allergens that may be present in mixed allergen compositions
include avian allergens, such as egg allergens, e.g., nGal d 1
Ovomucoid, n Gal d 2 Ovalbumin, nGal d 3 Conalbumin, egg white
complete allergen, etc.; mammalian allergens, such as milk
allergens, e.g., nBos d 4 alpha-lactalbumin, nBos d 5
beta-lactoglobulin, nBos d 8 Casein, nBos d Lactoferrin, milk
complete allergen, etc.; fish allergens, e.g., rCyp c 1, rGad c 1,
cod complete allergen, white fish allergens, pink fish allergens,
etc. Invertebrate animal allergens that may be present in mixed
allergen compositions include: crustacean allergens, such as shrimp
allergens, e.g., rPen a 1 tropomyosin, shrimp complete allergen,
etc.; insect allergens, e.g., bee sting venom allergen, wasp sting
venom allergen, mosquito bite allergen, etc.; and the like.
[0028] Allergens of interest include non-nut plant allergens, i.e.,
plant allergens that are not nut allergens. Plant allergens are
allergens that include one or more compounds found in plants. Plant
allergens of interest include: wheat allergens, e.g., rTri a 19
Omega-5 Gliadin, wheat complete allergen, gliadin wheat, rTri a 14
LTP, etc.; fruit allergens, e.g., kiwi allergens, e.g., rAct d 8
PR-10, kiwi complete allergen, etc.; vegetable allergens, e.g.,
carrot allergens, celery allergens etc., e.g., rApi g 1.01 PR-10,
rPhl p 12, celery complete allergen, CCD MUXF3 from Bromelain,
etc.; legume allergens, e.g., soy allergens, chickpea allergens,
etc., e.g., rGly m 4 PR-10, soy complete allergen, nGly m 5
Beta-conglycinin, nGly m 6 Glycinin, etc.; stone fruit allergens,
e.g., f419, f420, f421, f95, f242, o214 rPru p 1 PR-10, rPru p 3
LTP, stone fruit primary complete allergen, CCD MUXF3 from
Bromelain, etc.; oat allergens, e.g., oat component allergens, oat
complete allergen, etc.; seed allergens, e.g., sesame allergens,
e.g., sesame seed component allergens, sesame see complete
allergen, etc.
[0029] Additional types of allergens that may be present in mixed
allergen compositions include, but are not limited to: non-food
animal allergens, e.g., cats or dog fur and dander, cockroach
calyx, dust mite excretion, etc.; drug allergens, e.g., penicillin,
sulfonamides, salicylates, local anesthetics, etc.; mold spore
allergens; latex allergens; metal allergens; plant pollen
allergens, e.g., grass--ryegrass, timothy-grass, weeds--ragweed,
plantago, nettle, Artemisia, vulgaris, Chenopodium album, sorrel,
trees--birch alder, hazel, hornbeam, aesculus, willow, poplar,
platanus, tilia, olea, etc.; etc.
[0030] In certain embodiments, a composition may comprise one, two,
or more allergens each independently selected from the allergens
disclosed in Examples 1 or 2 herein. For example, in certain
embodiments, a composition may comprise one, two, or more allergens
selected from a group consisting of cashew (e.g., finely ground
cashew from "Wellbee's", Spring Valley, N.Y. 10977), pistachio
(e.g., pistachio flour from "nuts.com", Cranford, N.J. 07016),
walnut (e.g. roasted walnut from "Holmquist Hazelnut Orchards",
Lynden Wash. 98264), pecan (e.g., ground pecan from "King Arthur
Flour", Norwich, Vt., 05055), white fish (e.g., pacific cod from
"Seattle Seafoods", Bellevue, Wash. 98008), pink fish (e.g.,
sockeye salmon from "Seattle Seafoods", Bellevue, Wash. 98008),
shrimp (e.g., white shrimp from "Seattle Seafoods", Bellevue, Wash.
98008), peanut (e.g., defatted peanuts from "Byrd Mill Company",
Ashland, Va. 23005), soy (e.g., stone ground soy from "Bob's Red
Mill", Milwaukie, Oreg. 97222), hazelnut (e.g., natural hazelnuts
from "Holmquist Hazelnut Orchards", Lynden Wash. 98264), almond
(e.g., blanched almond flour from "Honeyville" Rancho Cucamonga,
Calif. 91730), milk (e.g., organic non-fat dry milk powder from
"Now Foods" Bloomingdale, Ill. 60108), egg (e.g., powdered egg
whites with sodium lauryl sulphate as an anti-caking agent from
"Honeyville Food Products", Honeyville, Utah 84314), crab (e.g.,
crab from "Seattle Seafoods", Bellevue, Wash. 98008), wheat (e.g.,
wheat flour from "Arrowhead Mills", USA. Boulder, Colo. 80301), and
sesame (Sesame flour from "Dipasa", Brownsville, Tex. 78526), and
optionally, vitamin D.
[0031] The amount of a given allergen in a mixed allergen
composition may vary, as desired. In some instances, the amount of
a given allergen ranges from about 1 to about 15,000 mg, about 5 to
about 15,000 mg, about 10 to about 10,000 mg, about 15 to about
5,000 mg, about 10 to about 100 mg, or about 15 to about 100 mg.
The weight percentage of a given allergen in a mixed allergen
composition may vary, ranging in some instances from about 0.1 to
about 99.9 wt. %, about 0.1 to about 15 wt. %, about 0.1 to about
99.9 wt. %, about 15 to about 99.9 wt. %, or about 25 to about 65
wt. %. The amount of a given allergen in a mixed allergen
composition may be recited by total mass, or by protein mass, which
may vary for a given allergen depending upon the weight percentage
of protein in that allergen.
[0032] A mixed allergen composition may be a unit dosage
composition, by which is meant that it is present in a composition
that is configured to be administered to a subject as a single
dose, which single dose may or may not be part of a dosing schedule
made up of two or more unit dosages that are administered to a
subject over a given a period of time. While the mass of a given
unit dosage may vary, in some instances unit dosages have a mass
ranging from 300 mg to 20 grams, such as 300 mg, 400 mg, 500 mg,
600 mg, 700 mg, 800 mg, 900 mg, 1000 mg (1 g), 1.5 g, 2 g, 3 g, 4
g, 5 g, 10 g, 15 g, 20 g, and anywhere in between. In certain
embodiments, any two of the mixed allergens are present in equal
parts, e.g., in a 1:1 ratio, such that each allergen is present in
the composition in equal weight. In such embodiments where the
mixed allergen composition includes three different allergens, the
three different allergens are present in a 1:1:1 ratio. For
example, a mixed allergen composition may include the following
allergens in equal parts (e.g., 1:1:1 etc. ratio): walnut,
hazelnut, shrimp, salmon, hen's egg, cow's milk, peanut, cashew,
almond, and wheat (e.g., about 30 mg of protein each; about 90 mg
of each protein; or about 300 mg of each protein). In certain
embodiments, the mixed allergen composition comprises almond,
cashew, walnut, hazelnut, peanut, soy, wheat, hen's egg, cow's
milk, white fish, pink fish, shrimp, and sesame allergens in equal
parts with a total estimated protein of 400 mg and 400 IU of
vitamin D3.
[0033] In some embodiments, the mixed allergen compositions include
a nut allergen and an animal allergen; and at least one of: a
non-nut plant allergen; a biotic agent; and a vitamin. As such, in
some embodiments, the mixed allergen compositions include a nut
allergen, an animal allergen and a non-nut plant allergen. In such
embodiments, the mixed allergen compositions may further include a
biotic agent or vitamin or both a biotic agent and a vitamin. In
some embodiments, the mixed allergen compositions include a nut
allergen, an animal allergen, and further include a biotic agent.
In such embodiments, the mixed allergen compositions may further
include a vitamin. In some embodiments, the mixed allergen
compositions include a nut allergen, an animal allergen, and
further include a vitamin. In such embodiments, the mixed allergen
compositions may further include a biotic agent.
[0034] Biotic agents may vary, and include both probiotics and
prebiotics. A probiotic is generally a live eukaryotic or a
prokaryotic organism which has a beneficial property when given to
a subject. In one aspect, the probiotic complements the existing
microflora in the subject. Hence, the probiotic agent is a live
microorganism which can confer a health benefit to a host subject.
The probiotic agent may be a culture of microorganisms or provided
in a dietary supplement or may be freeze dried and reconstituted
prior to use. A prebiotic is an agent that facilitates or confers
growth, maintenance and/or beneficial properties of or on the
subject's microflora. A prebiotic may include an oligosaccharide
and soluble or insoluble fiber material. Examples of probiotic
agents include, but are not limited to, species of Lactobacillus
spp., Escherichia spp., Bacillus spp., Bifidobacterium spp.,
Saccharomyces spp. and Streptococcus spp. Specific probiotic agents
that may be present in the mixed allergen compositions include:
Lactobacillus spp., such as Lactobacillus acidophilus,
Lactobacillus casei, Lactobacillus casei Shirota, Lactobacillus
casei immunitass, Lactobacillus johnsonii, Lactococcus lactis,
Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus
rhamnosus (lactobacillus GG), Lactobacillus salivarius and
Lactobacillus helvetirus. The probiotic microorganisms may be
naturally occurring, attenuated or genetically modified to
introduce new or to alter existing traits. In one embodiment, the
probiotic has been genetically modified to introduce an allergen
gene or part or fragment or portion thereof which is expressed to
produce recombinant microorganisms which release or expose the
subject's immune system to the allergen or an antigenic fragment
thereof. Hence, the probiotic and allergen may be combined into a
single component of the mixed allergen composition. When present,
the amount of the biotic agent in the mixed allergen composition
may vary. In some instances, the biotic agent is present in an
amount ranging from 1.5 to 99.9 wt. %, such as 10 to 25 wt. %.
[0035] The mixed allergen compositions may include one or more
vitamins, as desired. Vitamins that may be present in the
compositions include, but are not limited to: vitamin A, e.g., in
an amount ranging from 1 to 35,000 IU; vitamin C, e.g., in an
amount ranging from 1 to about 1,000 mg; vitamin D, e.g., in an
amount ranging from 1 to 4,000 IU; vitamin E, e.g., in an amount
ranging from 1 to 450 IU; vitamin K, e.g., in an amount ranging
from 1 to 250 mcg; vitamin B-1 (thiamin), e.g., in amount ranging
from 1 to 15 mg; vitamin B-2 (riboflavin), e.g., in an amount
ranging from 1 to 17 mg; vitamin B-3 (niacin), e.g., in an amount
ranging from 1 to 200 mg; vitamin B-5 (pantothenic acid), e.g., in
an amount ranging from 1 to 100 mg; vitamin B-6 (pyridoxine), e.g.,
in an amount ranging from 1 to 30 mg; vitamin B-9 (folic acid),
e.g., in an amount ranging from 1 to 4,000 mcg; vitamin B-12
(cobalamin), e.g., in an amount ranging from 1 to 250 mcg; vitamin
H (biotin), e.g., in an amount ranging from 1 to 1,000 mcg of
vitamin H (biotin); etc.; and combinations thereof.
[0036] The mixed allergen compositions of the invention may be
present in different configurations. In some instances the mixed
allergen composition is present in a solid configuration, e.g., as
a powder. When present as a powder, the dimensions of the particles
making up the powder may vary, ranging in some instances from 0.1
to 1000 microns, such as 1 to 500 microns.
[0037] Also provided are physiological acceptable compositions that
include the mixed allergen compositions and a physiologically
acceptable delivery vehicle. The mixed allergen compositions can be
incorporated into a variety of formulations for administration to a
subject. More particularly, the mixed allergen compositions can be
formulated into physiological acceptable compositions by
combination with appropriate, physiologically acceptable carriers
or diluents, and may be formulated into preparations in solid,
semi-solid, liquid or gaseous forms, such as tablets, capsules,
powders, granules, ointments, solutions, suppositories, injections,
inhalants and aerosols and topical compositions. The formulations
may be designed for administration via a number of different
routes, including oral, buccal, sublingual, rectal, parenteral,
intraperitoneal, intradermal, transdermal, intracheal, etc.,
administration.
[0038] The physiological compositions may be in a form suitable for
oral use, for example, as foods, tablets, troches, lozenges,
aqueous or oily suspensions, dispersible powders or granules,
emulsions, hard or soft capsules, or syrups or elixirs, gums, etc.
Compositions intended for oral use may be prepared according to any
convenient protocol for the manufacture of pharmaceutical
compositions and such compositions may contain one or more agents
selected from the group consisting of sweetening agents, flavoring
agents, coloring agents and preserving agents in order to provide
palatable preparations.
[0039] In certain embodiments the composition is a food product.
Food products of interest include a disclosed mixed allergen
composition in combination with a food delivery vehicle. By food
delivery vehicle is meant a delivery vehicle that is a nourishing
substance that is eaten, drunk, or otherwise taken into the body to
sustain life, provide energy, promote growth, etc. Examples of food
delivery vehicles or food products of interest include, but are not
limited to: baby or infant formula, baby food (e.g., pureed food
suitable for infant or toddler consumption), chips, cookies,
breads, spreads, creams, yogurts, liquid drinks, chocolate
containing products, candies, ice creams, cereals, coffees, pureed
food products, etc.
[0040] Also of interest as oral formulations are food supplements.
Where the oral formulation is provided as a food supplement, the
food supplement may further include one or more of a sweetener, a
stabilizer, a flavoring or a colorant, etc. An oral formulation
according to the present disclosure may be provided in the form of
sugar-coated tablets or lozenges, pills, gelatin capsules, or
syrups. Oral formulations may be provided as a bulk sample, e.g., a
container having multiple doses in powder form that can be measured
out by a subject, or in unit dose form, e.g., a pill, pouch, single
use container, and the like.
[0041] Tablets may contain the active ingredient in admixture with
non-toxic physiologically acceptable excipients which are suitable
for the manufacture of tablets. These excipients may be for
example, inert diluents, such as calcium carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example, magnesium stearate,
stearic acid or talc. The tablets may be uncoated or they may be
coated by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monostearate or glyceryl distearate may be
employed. They may also be coated by the technique described in the
U.S. Pat. Nos. 4,256,108; 4,166,452; and U.S. Pat. No. 4,265,874 to
form osmotic therapeutic tablets for control release.
[0042] Formulations for oral use may also be presented as hard
gelatin capsules wherein the mixed allergen component is mixed with
an inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the mixed
allergen component is mixed with water or an oil medium, for
example peanut oil, liquid paraffin, or olive oil.
[0043] Aqueous suspensions contain the mixed allergen component in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients may include suspending agents, for
example sodium carboxymethyl-cellulose, methylcellulose,
hydroxy-propylmethylcellulose, sodium alginate,
polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethylene-oxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more preservatives, for example ethyl, or n-propyl,
p-hydroxybenzoate, one or more coloring agents, one or more
flavoring agents, and one or more sweetening agents, such as
sucrose, saccharin or aspartame.
[0044] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavoring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0045] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, may also be present.
[0046] The physiologically acceptable compositions of the invention
may also be in the form of oil-in-water emulsions. The oily phase
may be a vegetable oil, for example olive oil or arachis oil, or a
mineral oil, for example liquid paraffin or mixtures of these.
Suitable emulsifying agents may be naturally-occurring
phosphatides, for example soy bean, lecithin, and esters or partial
esters derived from fatty acids and hexitol anhydrides, for example
sorbitan monooleate, and condensation products of the said partial
esters with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening and flavoring
agents.
[0047] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, preservative and
flavoring and coloring agents. The pharmaceutical compositions may
be in the form of a sterile injectable aqueous or oleagenous
suspension. This suspension may be formulated according to the
known art using those suitable dispersing or wetting agents and
suspending agents which have been mentioned above. The sterile
injectable preparation may also be a sterile injectable solution or
suspension in a non-toxic parenterally-acceptable diluent or
solvent, for example as a solution in 1,3-butane diol. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid find use in the
preparation of injectables.
[0048] The mixed allergen components can be formulated into
preparations for injection by dissolving, suspending or emulsifying
them in an aqueous or nonaqueous solvent, such as vegetable or
other similar oils, synthetic aliphatic acid glycerides, esters of
higher aliphatic acids or propylene glycol; and if desired, with
conventional additives such as solubilizers, isotonic agents,
suspending agents, emulsifying agents, stabilizers and
preservatives.
[0049] The mixed allergen components can be utilized in aerosol
formulation to be administered via inhalation. The compounds of the
present invention can be formulated into pressurized acceptable
propellants such as dichlorodifluoromethane, propane, nitrogen and
the like.
[0050] Furthermore, the mixed allergen compositions can be made
into suppositories by mixing with a variety of bases such as
emulsifying bases or water-soluble bases. The compounds of the
present invention can be administered rectally via a suppository.
The suppository can include vehicles such as cocoa butter,
carbowaxes and polyethylene glycols, which melt at body
temperature, yet are solidified at room temperature.
[0051] Also of interest are topical compositions, e.g., where the
mixed allergen composition is combined with a topical delivery
vehicle component. The topical delivery vehicle component of the
delivery compositions of the invention may vary, as desired, where
the particular ingredients of a given delivery vehicle component
will depend, at least in part, on the nature of the particular
composition. Delivery compositions of interest include liquid
formulations, such as lotions (liquids containing insoluble
material in the form of a suspension or emulsion, intended for
external application, including spray lotions) and aqueous
solutions, semi-solid formulations, such as gels (colloids in which
the disperse phase has combined with the dispersion medium to
produce a semisolid material, such as a jelly), creams (soft solids
or thick liquids) and ointments (soft, unctuous preparations), and
solid formulations, such as topical patches. As such, delivery
vehicle components of interest include, but are not limited to:
emulsions of the oil-in-water (O/W) and the water in-oil (W/O)
type, milk preparations, lotions, creams, ointments, gels, serum,
powders, masks, packs, sprays, aerosols or sticks.
[0052] The amount of mixed allergen composition that may be
combined with the carrier materials to produce a single dosage form
will vary depending upon the host treated and the particular mode
of administration. For example, a unit formulation intended for the
oral administration of humans may contain from 0.5 mg to 5 g of
mixed allergen composition compounded with an appropriate and
convenient amount of carrier material which may vary from about 0.1
to about 95 percent of the total composition, or e.g. about 0.1 to
about 10 percent, about 0.1 to about 4 weight percent, or about 5
to about 95 percent of the total composition. It will be
understood, however, that the specific dose level for any
particular patient will depend upon a variety of factors including
the age, body weight, general health, sex, diet, time of
administration, route of administration, rate of excretion, drug
combination and the severity of the particular disease undergoing
therapy. As such, unit dosage forms for oral or rectal
administration such as syrups, elixirs, and suspensions may be
provided wherein each dosage unit, for example, teaspoonful,
tablespoonful, tablet or suppository, contains a predetermined
amount of the composition containing one or more inhibitors.
Similarly, unit dosage forms for injection or intravenous
administration may include the inhibitor(s) in a composition as a
solution in sterile water, normal saline or another
pharmaceutically acceptable carrier. As described above "unit
dosage forms," include physically discrete units suitable as
unitary dosages for human and animal subjects, each unit containing
a predetermined quantity of mixed allergen composition calculated
in an amount sufficient to produce the desired effect in
association with a pharmaceutically acceptable diluent, carrier or
vehicle.
Methods
[0053] Aspects of the invention also include methods of
administering a mixed allergen composition, e.g., as described
above, to a subject. The administration route employed in a given
method may vary, e.g., depending on the nature of the mixed
allergen composition. As reviewed above, physiologically acceptable
compositions that include a mixed allergen composition may be
formulated for delivery to a subject using a variety of different
administration routes, such as but not limited to: oral, buccal,
sublingual, rectal, parenteral, intraperitoneal, intradermal,
transdermal, intracheal, etc., administration. As such, aspects of
the methods may include orally, buccally, sublingually, rectally,
parenterally, intraperitonealy, intradermally, transdermally,
intracecally, etc., administering a mixed allergen composition or
physiologically acceptable composition that includes the same,
e.g., as described above, to a subject.
[0054] The methods described herein may be employed with a variety
of different types of subjects, i.e., animals, where the animals
are typically "mammals" or "mammalian," where these terms are used
broadly to describe organisms which are within the class mammalia,
including the orders carnivore (e.g., dogs and cats), rodentia
(e.g., mice, guinea pigs, and rats), lagomorpha (e.g., rabbits) and
primates (e.g., humans, chimpanzees, and monkeys). In embodiments
of the invention, the subjects are humans. The subject may be
infant, juveniles or adults, where in some instances the subject
may be pregnant adults, e.g., as described in greater detail below.
As such, in certain embodiments, the subject is a pediatric subject
younger than 1 year of age, whereas in other embodiments the
subject is older, e.g., 1 year old or more, 5 years old or more,
etc., and including adults. In certain embodiments the pediatric
subject is 1 month to 7 years old, e.g., 1 month to 1 year old, 6
months to 7 years old, 6 months to 1 year old, 2 years to 4 years
old, or 3 to 7 years old. In certain embodiments, the pediatric
subject is an unborn child of a pregnant woman or a child of a
nursing mother.
[0055] The dosing schedule may vary as desired, and may depend on a
number of different factors, e.g., purpose of the administration,
age of the subject, condition of the subject, nature of the
physiologically acceptable composition, etc. In certain
embodiments, a mixed allergen composition or physiologically
acceptable composition that includes the same is administered to a
subject on an hourly basis, on an every few hours basis (e.g.,
every 2, 3, 4, 6 hours), on a daily basis, on an every few days
basis (e.g., every 2, 3, 4, 5, or 6 days, or, e.g., 2, 3, 4, 5, or
6 times a week), on a weekly basis, on a bi-weekly basis, on a
monthly basis, one a bimonthly basis, on a quarter annual basis, on
a semi-annual basis, on an annual basis, etc., for a treatment
period of time, which treatment period of time may also vary, where
in some instances the treatment period of time is 1 day or longer,
1 week or longer, 2 weeks or longer, 1 month or longer, 3 months or
longer, 6 months or longer, 1 year or longer, 2 years or longer, 3
years or longer, 5 years or longer, 10 years or longer, etc., up to
the life of the subject. In certain embodiments, the composition is
administered daily for a period of time, followed by weekly, twice
weekly and/or three times a week for a period of time. In certain
embodiments, the dose of the composition is varied over time, e.g.,
about 1 to about 10 mg (by weight protein allergen) of the
composition, or of each allergen, can be administered daily for
about 1 week to about 30 weeks, and then about 1 to about 300 mg or
more (by weight protein allergen) of the composition, or of each
allergen, can be administered weekly for about 1 week to about 30
weeks. In further embodiments, the dose and frequency of the
administration can be varied two, three or more times, e.g., the
administration may comprise two, three or more step-wise,
increasing dose levels, wherein each dose level is about 5 to about
100% more than the preceding dose level by total weight protein
level, wherein each dose level is independently administered daily
or weekly for about 1 to about 30 weeks. In certain embodiments,
the composition is administered to a subject for a time or
frequency to allow sufficient time for the administration of the
composition to inhibit the symptoms of an autoimmune disorder or
inflammatory disorder in a subject.
[0056] In some embodiments, the methods are methods of increasing
immune health of the subject. As the methods of these embodiments
are methods of increasing the immune health of a subject,
embodiments of such methods result in the subject being better able
to have a healthy immune response to a given challenge. Immune
health or immune balance may be characterized as a state in the
body where there is not an imbalance of inflammation. For example,
when IgE is decreased and/or IgG4 increased in plasma and/or Th2
decreased (e.g., as described in greater detail below in the
Examples section and accompanying figures). The magnitude of immune
health enhancement may vary, where in some instances the magnitude
is 2-fold or greater, e.g., 5-fold or greater, including 10-fold or
greater, e.g., as compared to a suitable control.
[0057] In some embodiments, the methods are methods of increasing
gut health of the subject. As the methods of these embodiments are
methods of increasing the gut health of a subject, embodiments of
such methods result in the subject having an enhanced maintenance
of healthy microbiota or improving the resilience of microbiota,
for instance, by reducing the numbers or colonization of pathogenic
bacteria or viruses and by maintaining and improving the intestinal
integrity and barrier function. Gut health or gut balance may be
characterized as a state in the body where there is not an
imbalance of inflammation. For example, when T cell proliferation
is decreased (e.g., as described in greater detail below in the
Examples section and accompanying figures). The magnitude of gut
health enhancement may vary, where in some instances the magnitude
is 2-fold or greater, e.g., 5-fold or greater, including 10-fold or
greater, e.g., as compared to a suitable control.
[0058] In some embodiments, the methods are methods of enhancing
wellness or maintaining immune balance of the subject. As the
methods of these embodiments are methods of enhancing wellness of a
subject, embodiments of such methods result in the subject having a
quality or state of being healthy in body and mind. Wellness may be
characterized as a state of the body where there is not an
imbalance of immune problems (for example, less IgE, more IgG4,
less cell inflammation (e.g., as described in greater detail below
in the Examples section and accompanying figures). The magnitude of
wellness enhancement may vary, where in some instances the
magnitude is 2-fold or greater, e.g., 5-fold or greater, including
10-fold or greater, e.g., as compared to a suitable control.
[0059] In some embodiments, the methods are methods of at least
decreasing the potential of the subject for developing an immune
mediated condition, such as an immune-mediated inflammatory disease
condition. By at least decreasing the potential of the subject for
developing an immune mediated condition is meant that the
probability of the subject for developing the immune mediated
condition is reduced, such that the risk of the subject for
developing the immune mediated condition is reduced. For example,
to determine risk reduction, if 100 different individuals were
administered the composition, 20% or more of the individuals would
show a decrease in their immune markers, e.g., IgE, as compared to
the control group. The magnitude of the decrease in potential may
vary, where in some instances the magnitude is 2-fold or greater,
e.g., 5-fold or greater, including 10-fold or greater, e.g., as
compared to a suitable control. In some instances, the methods are
methods of preventing the subject from developing an immune
mediated condition. As such, the methods of the invention include
administering a composition of the invention to a subject that is
not known to have or does not have an immune mediated condition.
While the subject may not have or may not be known to have the
immune mediated condition, the subject may be one that is suspected
to be or known to be at risk of developing the immune mediated
condition.
[0060] Aspects of the invention further include methods of treating
a subject for an immune mediated condition. By treating or
treatment is meant at least an amelioration of one or more symptoms
associated with the disease condition, e.g., immune mediated
condition, afflicting the subject, where amelioration is used in a
broad sense to refer to at least a reduction in the magnitude of a
parameter, e.g., symptom, associated with the pathological
condition being treated, etc. As such, treatment also includes
situations where the pathological condition, or at least symptoms
associated therewith, are completely inhibited, e.g., prevented
from happening, or stopped, e.g., terminated, such that the subject
no longer suffers from the pathological condition, or at least the
symptoms that characterize the pathological condition.
[0061] Treatment may also manifest in the form of a modulation of a
surrogate marker of the disease condition. For example, where the
target condition is an allergy, e.g., as described below, Th2 cell
proliferation may be reduced, e.g., as determined using the assay
described in the Examples Section, below. The magnitude of Th2 cell
proliferation reduction may vary, and in certain instances may
range from 1.2.times. to 10.times., such as 2.times. to 4.times..
Where the target condition is an allergy, e.g., as described below,
specific IgG4 levels may be increased, e.g., as determined using
the assay described in the Examples Section, below. The magnitude
of IgG4 level increase may vary, and in certain instances may range
from 1.2.times. to 100.times., such as 2.times. to 6.times.. Where
the target condition is an allergy, e.g., as described below,
specific IgE levels may be reduced, e.g., as determined using the
assay described in the Examples Section, below. The magnitude of
IgE level reduction may vary, and in certain instances may range
from 1.1.times. to 7.times., such as 2.times. to 6.times..
[0062] As summarized above, immune mediated conditions that are the
targets of methods of the invention include immune-mediated
inflammatory conditions, where such conditions include, but are not
limited to conditions characterized by common inflammatory pathways
leading to inflammation, and which may result from, or be triggered
by, a dysregulation of the normal immune response. Examples of
immune-mediated inflammatory conditions include, but are not
limited to: allergy, autoimmune diseases, ankylosing spondylitis,
psoriasis, psoriatic arthritis, Behcet's disease, arthritis,
inflammatory bowel disease (IBD), cardiovascular disease,
neuromuscular disease, and infectious disease, etc.
[0063] Also provided are methods of treating an autoimmune disease
or inflammatory disease in a human subject in need thereof, which,
e.g., may comprise administering a composition comprising 1 to 20
or more (e.g., 1 to 100 more) different protein allergens, and
optionally a vitamin, such as a vitamin selected from vitamin D and
vitamin C. In certain embodiments, the method of treating of an
autoimmune disease or inflammatory disease is prophylactic, e.g.,
the treating reduces the incidence of one or more autoimmune
diseases or inflammatory diseases or reduces the probability of
developing one or more autoimmune diseases or inflammatory diseases
in a subject. In certain embodiments, the method of treating an
autoimmune disease or inflammatory disease in a subject, e.g.,
comprising administrating a composition comprising 1 to 20 or more
(e.g., 1 to 100 more) different protein allergens, and optionally a
vitamin, such as a vitamin selected from vitamin D and vitamin C,
results in improved immune protection against autoimmune
inflammation as measured by the subject's Th1 proliferation to the
allergen, as compared to levels before administration.
[0064] In certain embodiments, the autoimmune disease is selected
from a group consisting of postmyocardial infarction syndrome,
postpericardiotomy syndrome, subacute bacterial endocarditis (SBE),
anti-glomerular basement membrane nephritis, interstitial cystitis,
lupus nephritis, autoimmune hepatitis, primary biliary cirrhosis
(PBC), primary sclerosing cholangitis, antisynthetase syndrome,
alopecia areata, autoimmune angioedema, autoimmune progesterone
dermatitis, autoimmune urticarial, bullous pemphigoid, cicatricial
pemphigoid, dermatitis herpetiformis, discoid lupus erythematosus,
epidermolysis bullosa acquisita, erythema nodosum, gestational
pemphigoid, hidradenitis suppurativa, Lichen planus, Lichen
sclerosus, linear IgA disease (LAD), morphea, Pemphigus vulgaris,
Pityriasis lichenoides et varioliformis acuta, Mucha-Habermann
disease, psoriasis, systemic scleroderma, vitiligo, Addison's
disease, autoimmune polyendocrine syndrome (APS) type 1, autoimmune
polyendocrine syndrome (APS) type 2, autoimmune polyendocrine
syndrome (APS) type 3, autoimmune pancreatitis (AIP), diabetes
mellitus type 1, autoimmune thyroiditis, Ord's thyroiditis, Graves'
disease, autoimmune oophoritis, endometriosis, autoimmune orchitis,
Sjogren's syndrome, autoimmune enteropathy, coeliac disease,
Crohn's disease, microscopic colitis, ulcerative colitis,
antiphospholipid syndrome (APS, APLS), aplastic anemia, autoimmune
hemolytic anemia, autoimmune lymphoproliferative syndrome,
autoimmune neutropenia, autoimmune thrombocytopenic purpura, cold
agglutinin disease, essential mixed cryoglobulinemia, Evans
syndrome, IgG4-related disease, paroxysmal nocturnal
hemoglobinuria, pernicious anemia, pure red cell aplasia,
thrombocytopenia, adiposis dolorosa, adult-onset Still's disease,
ankylosing spondylitis, CREST syndrome, drug-induced lupus,
enthesitis-related arthritis, eosinophilic fasciitis, Felty
syndrome, juvenile arthritis, Lyme disease (chronic), mixed
connective tissue disease (MCTD), palindromic rheumatism, Parry
Romberg syndrome, Parsonage-Turner syndrome, psoriatic arthritis,
reactive arthritis, relapsing polychondritis, retroperitoneal
fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis,
Schnitzler syndrome, systemic lupus erythematosus (SLE),
undifferentiated connective tissue disease (UCTD), dermatomyositis,
fibromyalgia, inclusion body myositis, myositis, myasthenia gravis,
neuromyotonia, paraneoplastic cerebellar degeneration,
polymyositis, acute disseminated encephalomyelitis (ADEM), acute
motor axonal neuropathy, anti-N-Methyl-D-Aspartate (anti-NMDA)
receptor encephalitis, balo concentric sclerosis, Bickerstaffs
encephalitis, chronic inflammatory demyelinating polyneuropathy,
Guillain-Barre syndrome, Hashimoto's encephalopathy, idiopathic
inflammatory demyelinating diseases, Lambert-Eaton myasthenic
syndrome, multiple sclerosis, pattern II, Oshtoran syndrome,
pediatric autoimmune neuropsychiatric disorder associated with
streptococcus (PANDAS), progressive inflammatory neuropathy,
restless leg syndrome, stiff person syndrome, sydenham chorea,
transverse myelitis, autoimmune retinopathy, autoimmune uveitis,
Cogan syndrome, Graves ophthalmopathy, intermediate uveitis,
ligneous conjunctivitis, Mooren's ulcer, neuromyelitis optica,
opsoclonus myoclonus syndrome, optic neuritis, scleritis, Susac's
syndrome, sympathetic ophthalmia, Tolosa-Hunt syndrome, autoimmune
inner ear disease (AIED), Mdniere's disease, Behget's disease,
eosinophilic granulomatosis with polyangiitis (EGPA), giant cell
arteritis, granulomatosis with polyangiitis (GPA), IgA vasculitis
(IgAV), Kawasaki's disease, leukocytoclastic vasculitis, lupus
vasculitis, rheumatoid vasculitis, microscopic polyangiitis (MPA),
polyarteritis nodosa (PAN), polymyalgia rheumatic, urticarial
vasculitis, vasculitis, chronic fatigue syndrome, complex regional
pain syndrome, eosinophilic esophagitis, gastritis, interstitial
lung disease, POEMS syndrome, Raynaud's phenomenon, primary
immunodeficiency, and pyoderma gangrenosum.
[0065] In certain embodiments, a disclosed method comprises
inhibiting symptoms of one or more food allergies in a human
subject, comprising orally administering to the subject a
composition comprising 2 to 20 different protein allergens. In
certain embodiments, administration of the composition
significantly inhibits the symptoms of a food allergy in the
subject as compared to administering to the subject a composition
that includes only one protein allergen. The one or more food
allergies may be a single food allergy or a multiple food allergy.
The target allergy may vary widely, where in some instances the
target allergy is food allergy, drug allergy, environmental
allergy, animal allergy, and insect and/or bee allergy. As such,
aspects of the invention include methods of reducing the risk of a
subject for developing an allergy. In certain embodiments, the
methods result in the subject having a reduced risk of developing a
food allergy to a food allergen that is not present in the mixed
allergen composition that is administered to the subject. For
example, the administered composition may provide to the subject
allergen protection for a nut protein in the formulation as well as
a nut protein not found in the formulation. In certain embodiments,
the reduced risk for a first food allergen is reduced to a greater
extent using the mixed formulation than would be achieved using a
formulation with only a single allergen. For example, feeding a
mixed allergen formulation as described herein can result in lower
risk of allergy development for an allergen (e.g., with respect to
Th2/IgE/IgG4 analyses, as shown below) than if the formulation only
included that single allergen. In some cases, this phenomenon is
referred to as "synergy" (e.g., cashew synergizes with pistachio,
walnut with pecan, shrimp with lobster (and other crustacean), and
vice-versa). Where the method is a method of treating the subject
for the allergy, as reviewed above the method may result in at
least an amelioration of one or more symptoms associated with the
allergy, e.g., as described above. Allergy symptoms that may be
ameliorated, but are not limited to: eczema, asthma, atopic
dermatitis, bronchospasm, cough, rhinorrhea, angioedema, gastric
hypermotility, urticaria (hives), pruritis, fatigue, bradycardia,
allergic rhinitis, allergic conjunctivitis, and/or hypotension. The
magnitude of the symptom reduction may vary, where in some
instances the magnitude is 2-fold or greater, e.g., 5-fold or
greater, including 10-fold or greater, e.g., as compared to a
suitable control. In some instances, treatment of an allergy
results the subject being cured of the allergy, such that the
subject no longer suffers from the allergy. In some embodiments of
allergy treatment methods, the methods include administering to a
subject a mixed allergen composition that includes a nut allergen;
an animal allergen; and at least one of: a non-nut plant allergen a
biotic agent; and a vitamin, such as described above.
[0066] Also provided are methods of significantly reducing the
probability of developing one or more food allergies in a subject.
In certain embodiments, a subject is an unborn child of a pregnant
woman or a child of nursing mother, and the method comprises
administering to the pregnant or nursing mother a disclosed
composition. In certain embodiments, the subject is a child having
a developing immune system. Further provided are methods for
desensitizing a human to one major allergen or to two or more major
allergens comprising administering to the human a food product
comprising two or more allergens, wherein the composition is
administered according to a schedule of treatment.
[0067] In some instances, the methods are methods of treating a
subject for an eosinophilic disorder. Eosinophilic disorders are
disorders characterized by the occurrence of eosinophils in
above-normal amounts in various parts of the body. Eosinophilic
disorders of interest include, but are not limited to: eosinophilic
esophagitis (esophagus); eosinophilic gastritis (stomach);
eosinophilic enteritis (small intestine); eosinophilic colitis
(large intestine); hypereosinophilic syndrome (blood and any
organ); and the like. In some instances the methods are methods of
reducing the risk of a subject for developing an eosinophilic
disorder. The magnitude of the risk reduction may vary, where in
some instances the magnitude is 2-fold or greater, e.g., 5-fold or
greater, including 10-fold or greater, e.g., as compared to a
suitable control.
[0068] In some instances the methods are methods of treating a
subject for inflammation, where the inflammation may be a symptom
of a variety of different disease conditions. Disease conditions in
which the inflammation thereof may be treated according to
embodiments of the invention include, but are not limited celiac
disease, multiple sclerosis, inflammatory bowel disease,
eosinophilic diseases, allergy, food allergy, etc.
[0069] Aspects of the invention further include methods of at least
decreasing the potential of a fetus or suckling infant for
developing an immune mediated condition, such as an immune-mediated
inflammatory disease condition, e.g., allergy, such as described
above. By at least decreasing the potential of the fetus or
suckling infant for developing an immune mediated condition is
meant that the probability of the fetus or suckling infant for
developing the immune mediated condition is reduced, such that the
risk of the fetus or suckling infant for developing the immune
mediated condition is reduced. The magnitude of the decrease in
potential may vary, where in some instances the magnitude is 2-fold
or greater, e.g., 5-fold or greater, including 10-fold or greater,
e.g., as compared to a suitable control. In some instances, the
methods are methods of preventing the fetus or suckling infant from
developing an immune mediated condition. As such, the methods of
the invention include administering a composition of the invention
to a mother of a fetus or suckling infant that is not known to have
or does not have an immune mediated condition. While the fetus or
suckling infant may not have or may not be known to have the immune
mediated condition, the fetus or suckling infant may be one that is
suspected to be or known to be at risk of developing the immune
mediated condition.
[0070] In certain embodiments, the mixed allergen composition or
physiologically acceptable composition that includes the same is
administered to a pregnant or nursing mother of a pediatric
subject, fetus or suckling infant. For example, the mixed allergen
composition or physiologically acceptable composition that includes
the same may be administered to a mother pregnant with the fetus.
In such instances, the mixed allergen composition or
physiologically acceptable composition that includes the same may
be administered to the mother using any convenient dosing schedule,
e.g., as described above, starting at any convenient time during
the pregnancy, e.g., at the start of the second trimester, at the
start of the third trimester, etc. Where the method is a method of
reducing risk in a suckling infant, the mixed allergen composition
or physiologically acceptable composition that includes the same
may be administered to a mother that is breast feeding the infant.
In such instances, the mixed allergen composition or
physiologically acceptable composition that includes the same may
be administered to the breast feeding mother using any convenient
dosing schedule, e.g., as described above, starting at any
convenient time during the breast feeding, e.g., at the start of
lactation, 1 week after lactation commencement, etc.
[0071] Practice of the invention will be more fully understood from
the foregoing examples, which are presented herein for illustrative
purposes only, and should not be construed as limiting the
invention in any way.
EXAMPLES
Example 1--Mixed Allergen Assay #1
Study Design and Population/Allergen Treatment
[0072] For each allergen or allergen mix listed below (the allergen
groups), five subjects from each of five different age groups were
fed a total of 300 mg of the allergen or allergen mix daily for one
year. The only exceptions are for Formula 1 at 3.times. (i.e.,
3.times. mix) and Formula 1 at 10.times. (i.e., 10.times. mix), in
which the subjects were fed 900 mg and 3,000 mg of the formula on a
daily basis, respectively. Allergen mixes were formulated at a 1:1
ratio. Thus, for an allergen mix with 2 allergens, the mix would
include 150 mg of each. Five subjects were included in each age
group that were not fed an allergen (Not Treated, or NT group,
listed last in FIGS. 1-3).
[0073] The following 18 groups were randomized equally. Children
ate (daily) either 1) cashew alone, 2) cashew and pistachio, 3)
walnut alone, 4) walnut and pecan, 5) a protein mix containing
equal parts walnut, hazelnut, shrimp, salmon, hen's egg, cow's
milk, peanut, cashew, almond, and wheat (1.times. mix supplement at
equal protein ratios), 6) a 3.times. mix supplement of the same
proteins as the 1.times. mix, 7) a 10.times. mix supplement of the
same proteins as the 1.times. mix, 8) control group that followed
AAP guidelines prior to October 2015, 9) white fish alone 10) white
fish and pink fish, 11) shrimp alone, 12) shrimp and crab, 13)
peanut alone, 14) peanut and soy, 15) hazelnut alone, 16) almond
alone, 17) cow's milk, 18) hen's egg, Supplement mixes were added
to breast milk or to relevant food items eaten (i.e. apple sauce,
pudding, shakes, yogurt) by the different age groups, eaten
freshly, and parents were told to watch and document the full
intake of the supplement daily. The protein mix was approximately a
teaspoon daily.
[0074] Each treatment group consisted of five children from each of
the following five age groups: 4 to 6 months, 7 to 12 months, 1 to
3 years, 3 to 5 years, and 5 years or older. The subjects were not
selected based on observed or suspected food allergy (or other
allergy) profile, and thus the cohort of subjects tested included
those who may have, or have the propensity to develop, a food
allergy as well as those who do not. Children were both low- and
high-risk (defined by a first degree relative with atopic
dermatitis or two first degree relatives with atopy) for developing
allergies, but none had a doctor's diagnosis of food allergy.
Allergen or Allergen Mix/Protein Powders
[0075] The allergens included in the study were flours/dry powders
from peanuts, almonds, cashews, walnut, pecan, hazelnut, wheat,
soy, shrimp, salmon, hen's egg (sodium lauryl sulphate was added as
an anti-caking agent), cow's milk, white fish, pink fish, sesame,
and crab. The details of the allergens and allergen mixes were as
follows: [0076] 1. Cashew: Finely ground cashew from "Wellbee's",
Spring Valley, N.Y. 10977. [0077] 2. Cashew and pistachio (pis):
Finely ground cashew from "Wellbee's", Spring Valley, N.Y. 10977.
Pistachio flour from "nuts.com", Cranford, N.J. 07016. [0078] 3.
Walnut: Roasted walnut from "Holmquist Hazelnut Orchards", Lynden
Wash. 98264. [0079] 4. Walnut and pecan: Roasted walnut from
"Holmquist Hazelnut Orchards", Lynden Wash. 98264. Ground pecan
from "King Arthur Flour", Norwich, Vt., 05055. [0080] 5. Formula 1
at 1.times. (1.times. mix): equal parts walnut (Roasted walnut from
"Holmquist Hazelnut Orchards", Lynden Wash. 98264), hazelnut
(Natural hazelnuts from "Holmquist Hazelnut Orchards", Lynden Wash.
98264), shrimp (White shrimp from "Seattle Seafoods", Bellevue,
Wash. 98008), salmon (Sockeye salmon from "Seattle Seafoods",
Bellevue, Wash. 98008), hen's egg (Powdered egg whites with sodium
lauryl sulphate as an anti-caking agent from "Honeyville Food
Products", Honeyville, Utah 84314), cow's milk (Organic non-fat dry
milk powder from "Now Foods" Bloomingdale, Ill. 60108), peanut
(Defatted peanuts from "Byrd Mill Company", Ashland, Va. 23005),
cashew (Finely ground cashew from "Wellbee's", Spring Valley, N.Y.
10977), almond (Blanched almond flour from "Honeyville" Rancho
Cucamonga, Calif. 91730), and wheat (Wheat flour from "Arrowhead
Mills", USA. Boulder, Colo. 80301) (30 mg of protein each). [0081]
6. Formula 1 at 3.times. (3.times. mix): equal parts walnut,
hazelnut, shrimp, salmon, hen's egg, cow's milk, peanut, cashew,
almond, and wheat as above (90 mg of each protein) [0082] 7.
Formula 1 at 10.times. (10.times. mix): equal parts walnut,
hazelnut, shrimp, salmon, hen's egg, cow's milk, peanut, cashew,
almond, and wheat as above (300 mg of each protein) [0083] 8.
Non-treated control (NT) [0084] 9. White fish: Pacific cod from
"Seattle Seafoods", Bellevue, Wash. 98008. [0085] 10. White fish
and pink fish: Pacific cod and sockeye salmon from "Seattle
Seafoods", Bellevue, Wash. 98008. [0086] 11. Shrimp: White shrimp
from "Seattle Seafoods", Bellevue, Wash. 98008. [0087] 12. Shrimp
and crab: White shrimp and crab from "Seattle Seafoods", Bellevue,
Wash. 98008. [0088] 13. Peanut: Defatted peanuts from "Byrd Mill
Company", Ashland, Va. 23005. [0089] 14. Peanut and soy: Defatted
peanuts from "Byrd Mill Company", Ashland, Va. 23005. Stone ground
soy from "Bob's Red Mill", Milwaukie, Oreg. 97222. [0090] 15.
Hazelnut: Natural hazelnuts from "Holmquist Hazelnut Orchards",
Lynden Wash. 98264. [0091] 16. Almond: Blanched almond flour from
"Honeyville" Rancho Cucamanga, Calif. 91730. [0092] 17. Milk:
Organic non-fat dry milk powder from "Now Foods" Bloomingdale, Ill.
60108. [0093] 18. Egg: Powdered egg whites with sodium lauryl
sulphate as an anti-caking agent from "Honeyville Food Products",
Honeyville, Utah 84314.
[0094] The total dose of allergens used was 300 mg for single
allergens and for allergen mixes unless specified otherwise. A
3.times. and 10.times. formulation containing 10 allergens each
containing 900 mg and 3000 mg total allergen, respectively, were
also used in the study. Allergen mixes were formulated so that
equal amounts of each allergen were present in the mix, so for a 2
and 10 allergen-mix formulation with 300 mg total allergen, the
amount of each allergen was 150 mg and 30 mg, respectively. Mixing
occurred prior to ingestion.
Blood Sample Collection
[0095] Peripheral blood mononuclear cells (PBMCs) and plasma were
extracted from blood via ficoll procedure and stored in liquid
nitrogen and at -80.degree. C., respectively. Allergen-specific IgE
and IgG4 were measured using a standard ImmunoCAP assay (Phadia,
Uppsala, Sweden).
Stimulation and Enumeration of Th2 Cell Subsets
[0096] PBMCs from subjects were labeled with carboxyfluorescein
diacetate succinimidyl ester (CFSE) and cultured with the same food
allergen or food allergen mix that was fed to the participant at
100 .mu.g/mL or anti-CD3/CD28 (to test for nonspecific
proliferation capacity) for 7 days to identify T cell subsets. At
day 7, cells were washed and stained for surface CD4, CD25, CD127,
CD45RO, CD45RA, CD40L, and CD69 and intracellular Foxp3 and IL-10
along with Live/Dead staining (Invitrogen) (see T-cell flow
cytometry method below). Th2 cells were defined as the cells that
proliferated in response to food allergen (CFSElo) and were CD41
IL-4, IL-13 cells. Antigen-induced T cells were also identified by
isolating CD40L and CD69 double-positive cells after antigen
stimulation.
T-Cell Flow Cytometry
[0097] Cells were fixed with Lyse/Fix PhosFlow buffer (BD
Biosciences). For intracellular staining, fixed cells were
permeabilized with Perm Buffer III (BD Biosciences) at 48.degree.
C. for 30 minutes, followed by staining at 48.degree. C. for 20
minutes. Flow cytometry was performed with an LSRII flow cytometer
(BD Biosciences). Viable cells were identified with a Live/Dead
probe (Invitrogen). Phenotypes of T cells were detected with
antibodies against surface CD3 (UCHT1), CD4 (SK3), CD25 (4E3),
CD127 (SB199), CD45RO (UCHL1), CD45RA (HI100), CD62L (DREG-56),
CCR4 (1G1), and CCR8 from BD Biosciences; CCR7, CD69, and CD40L and
intracellular IL-10 (JES3-19F1), IL-4 (MP4-25D2), and IL-13
(JES10-52A2) from BD Biosciences; Helios (22F6) from BioLegend;
anti-CD49b from BioLegend; anti-LAG3 from R&D Systems
(Minneapolis, Minn.); and Foxp3 (150D) from BioLegend and stained
per the manufacturer-recommended protocol.
Measurement of Antibody Titers
[0098] Total and allergen-specific blood IgE and IgG4 levels were
measured in all subjects in the Clinical Laboratories at Stanford
Hospital and Clinics using a standard ImmunoCAP assay (Phadia,
Uppsala, Sweden).
Statistical Analysis
[0099] Comparisons between cohorts, and between baseline and 1 year
and control groups were evaluated with the nonparametric
Mann-Whitney test, paired Wilcoxon test, and 1-way and 2-way ANOVA
(GraphPad Prism Software 5.0; GraphPad Software, La Jolla, Calif),
as appropriate. A P value of less than 0.05 was considered
statistically significant.
Compliance and Safety
[0100] Compliance was excellent with no dropouts in control arm and
fifteen drop outs in ingested arms total. Of those who completed
the study, there were only 90 missed doses (out of a total of
240,810) as determined by reviews of electronic dietary records
(see Table 2). Adverse events were minimal in the active with only
21 cases of mild skin rash (21/660; 0.03%). Adverse events in the
control group consisted of 3 cases of mild skin rash (3/30; 10%).
15 dropped out of the study at about 1 year in the active arm due
to non-compliance, not due to safety issues.
TABLE-US-00001 TABLE 1 Summary of adverse reactions in ingestion
and control groups Reaction Rates Fed Control Participants (ITT
analysis) N = 660 N = 30 Ingestions completed 240,810 (90 missed)
n/a Reactions Total 21 (0.03%) 3 (10%) Mild 21 (skin rash) 3 (skin
rash) Moderate 0 0 Severe 0 0
Th2 Cell Proliferation
[0101] PBMCs from each subject in allergen groups 1 to 18 were
stimulated ex vivo with the allergen or allergen mix fed to the
subject for 7 days and Th2 cell proliferation was analyzed (as
described above). It is noted that for Formula 1 3.times. and
Formula 1 10.times. groups, the PBMCs were stimulated with 3.times.
and 10.times. the amount of allergen ex vivo, respectively (i.e.,
300 .mu.g/mL and 1000 .mu.g/mL, respectively).
[0102] As shown in FIG. 1, stimulation of PBMCs from cohorts fed
single allergens or allergen mixes had a lower degree of Th2
proliferation than controls (non-treated 1-year and non-treated
baseline), indicating decreases in allergic response on exposure to
allergens or allergen mixes. Additionally, the allergen mixes
containing 10 allergens had the lowest degree of Th2 proliferation,
significantly more than that of cohorts containing allergen mixes
of 2 allergens or single allergens, indicating that diversity of
food proteins further decreases allergic response.
[0103] Specifically, subjects in allergen groups 1 to 4 and 9 to 18
showed a somewhat reduced Th2 proliferation in response to allergen
stimulation as compared to the non-treated control (Group 8;
Baseline-NT). However, Th2 cell proliferation in PBMCs from
subjects in allergen groups 5 to 7 (fed increasing amounts of
Formula 1), who were fed formulations having 10 separate allergens,
as described above, had significantly reduced Th2 cell
proliferation as compared to both the control group (NT) as well as
all other antigen groups (1 to 4 and 9 to 18). This phenomenon was
seen for all subjects in all allergen groups at all ages tested,
including for the different doses (1.times., 3.times., and
10.times. allergen mix).
Immunoglobulin Analyses
[0104] In addition to the Th2 proliferation assay above, plasma
from subjects in allergen groups 1 to 18 were analyzed for the
presence of IgG4 and IgE antibodies (using standard methods, as
described above), the former being an indicator of a non-allergenic
or non-inflammatory (or anti-allergenic or non-inflammatory) state
and the latter an indicator of pro-allergenic or pro-inflammatory a
sate.
[0105] Increased IgG4 levels indicate a protective
anti-inflammatory state. As shown in FIG. 2, when compared to
baseline, samples from participants from all cohorts showed an
increase in IgG4, with the 10-allergen mix formula showing greater
IgG4 levels than from those from cohorts fed single allergens or
2-allergen mixes. No significant difference in IgG4 was observed
between the 1.times., 3.times., and 10.times. allergen mixes
containing 10 allergens.
[0106] Specifically, subjects in allergenic groups 1 to 4 and 9 to
18 showed moderately increased levels of IgG4 as compared to the
baseline group (Group 8; NT). However, allergen groups 5 to 7 (fed
increasing amounts of Formula 1) showed significantly increased
levels of IgG4 as compared to both the baseline group as well as
all other antigen groups (1 to 4 and 8 to 18). This result is
consistent with the results for Th2 cell proliferation as described
above. Specifically, increased levels of IgG4 in the plasma and
reduced Th2 cell proliferation are indicators of a reduced or
non-allergenic or non-inflammatory state in a subject.
[0107] Increased specific IgE is indicative of an inflammatory
allergic state. As shown in FIG. 3, samples from participants from
all cohorts fed allergens or allergen mixes showed decreases in
IgE, with the plasma of those fed the 10-allergen mix (1.times.,
3.times., and 10.times. formulations) having IgE levels below the
assay detection level. In particular, subjects in allergenic groups
1 to 4 and 9 to 17 showed significantly higher levels of IgE in the
plasma as compared to allergen groups 5 to 7 (fed increasing
amounts of Formula 1). Indeed, IgE levels in the allergen groups 5
to 7 were below the level of detection of the assay.
[0108] These results are consistent with the results for Th2 cell
proliferation and the IgG4 levels as described above. Specifically,
low levels of IgE and increased levels of IgG4 in the plasma
coupled with reduced Th2 cell proliferation are indicators of a
reduced or non-allergenic or non-inflammatory state in a
subject.
[0109] It is clear from the results above that continual feeding of
a complex mixture of food allergens to subjects (e.g., in the form
of Formula 1 described above) at a very young age can
prophylactically protect a subject from developing an allergenic or
inflammatory immune profile to a wide variety of antigens. In other
words, this process can induce in a subject a non-allergenic or
non-inflammatory (or anti-allergenic or anti-inflammatory) state.
Of particular interest in the results shown herein is the
indication that feeding antigens in a complex mixture provides
broad spectrum protection against developing allergies/inflammation
that is superior to single or even double allergen formulas that
target a single allergen (as in allergen groups 1, 3, 11, 13 and 15
to 18 above) or multiple related allergens (e.g., as in allergen
groups 2, 4, 10, 12, and 14 above).
[0110] The above shows that feeding subjects complex antigen
mixtures will not only protect against the development of allergies
and/or inflammation to the allergens/antigens in the complex
mixture, but also to allergens or antigens that are not present in
the complex mixture. In essence, feeding complex food antigen
mixtures creates a general anti-allergenic or anti-inflammatory
state in a subject that broadly prevents the development of
allergies or inflammatory state, even to allergens/antigens not yet
fed to the subject.
Mixed Allergen Compositions (+ Probiotics and/or Vitamin)
[0111] Subjects were also fed compositions comprising allergen
mixes in a 1:1 blend with probiotics and/or vitamins. The details
of the compositions that were used are shown below: [0112] 1.
Formula 1 at 1.times. (equal parts walnut, hazelnut, shrimp,
salmon, hen's egg, cow's milk, peanut, cashew, almond, and wheat)
[0113] 2. Non-treated control (NT) [0114] 3. 1 part Formula 1 at
1.times. (as above) with 1 part vitamin D (400 IU; Vitamin in this
instance was Poly Vi Sol brand for children). [0115] 4. 1 part
Formula 1 at 1.times. (as above) with 1 part probiotics (Probiotic
in this instance was Lactobacillus; Culturelle brand). [0116] 5. 1
part Formula 1 at 1.times. (as above) with 1 part vitamin D (400
IU; Vitamin in this instance was Poly Vi Sol brand for children)
and 1 part probiotics (Lactobacillus; Culturelle brand). [0117] 6.
1 part Formula 1 at 1.times. (as above) after baking (15 min at
350+ F.). [0118] 7. 1 part Formula 1 at 1.times. (as above) after
incubation at pH=2 for 15 min.
[0119] Each composition had 300 mg each of the dietary supplement
mixture, probiotics, and/or vitamins when present. Mixtures at pH 2
were incubated in vinegar for 40 min at room temperature before
using. Mixtures that were baked were heated for 40 min at
350.degree. F. Control received no dietary supplement.
T-Cell Proliferation
[0120] PBMCs from each subject were stimulated ex vivo with the
allergen or mixed compositions described above and Th2 cell
proliferation was analyzed as described above. As shown in FIG. 4,
PBMCs on stimulation ex vivo with the allergen or allergen mix
under the following conditions showed similar Th2 proliferation:
lower pH, addition of probiotics, addition of vitamin D, or after
baking.
[0121] Subject samples showed similar Th2 proliferation decreases
with the mixture or under different conditions with the mixture.
This phenomenon was seen for all subjects in all allergen groups
and at all ages tested.
Immunoglobulin Analyses
[0122] In addition to the Th2 proliferation assay above, plasma
from subjects in allergen groups were analyzed for the presence of
IgG4 and IgE antibodies (using standard methods, as described
above), the former being an indicator of a non-allergenic or
non-inflammatory (or anti-allergenic or anti-inflammatory) state
and the latter an indicator of pro-allergenic or pro-inflammatory
state.
[0123] As shown in FIGS. 5 and 6, subjects showed moderately
increased levels of IgG4 as compared to control group and
significantly higher levels of IgE in the plasma. As shown in FIG.
5, similar IgG4 levels were observed under the following different
conditions with the allergen-mix: lower pH, addition of probiotics,
addition of vitamin D, or after baking. As shown in FIG. 6, no IgE
levels were observed in response to the different tested mixed
allergen compositions. There were similar effects seen with the
mixture or under different conditions with the mixture, with
similar IgE levels observed under the following different
conditions with the allergen-mix: lower pH, addition of probiotics,
addition of vitamin D, or after baking.
T-Cell Proliferation
[0124] T cell proliferation was performed (please see methods
section above) and data presented in FIG. 7. The data show
anti-inflammatory properties to gluten (i.e., celiac agent), and
insulin (i.e., diabetes agent) and bacterial flagellin
(inflammatory bowel disease agent). This demonstrates that the
mixture could also decrease inflammatory states involved in
diseases like celiac, diabetes and inflammatory bowel diseases.
Example 2--Mixed Allergen Assay #2
Study Design and Population/Allergen Treatment
[0125] Infants and children were seen in a research setting from 4
weeks old to 3 years of age. A single allergen or allergen mix
supplement was consumed orally daily for one year. Subjects were
fed a total of 300 mg of a single allergen or allergen mix daily
for one year. The only exceptions are for Formula 1 at 3.times.
(i.e., 3.times. mix) and Formula 1 at 10.times. (i.e., 10.times.
mix), in which the subjects were fed 900 mg and 3,000 mg of the
formula on a daily basis, respectively. Allergen mixes were
formulated at a 1:1 ratio. Thus, for an allergen mix with 2
allergens, the mix would include 150 mg of each. Thirty subjects
were included that were not fed an allergen (control group
listed).
[0126] There was similar distribution of age and sex, and all
children were breast fed for at least the first 4 months of life.
There was similar distribution of high risk and low risk for food
allergy (i.e. high risk are those infants born to a family with one
first degree relative with food allergy or one first degree
relative with atopic dermatitis or two first degree relatives with
atopic disease). None had a doctor's diagnosis of food allergy.
[0127] The subjects were not selected based on observed or
suspected food allergy (or other allergy) profile, and thus the
cohort of subjects tested included those who may have, or have the
propensity to develop, a food allergy as well as those who do not.
The total number of subjects was 60 including control.
TABLE-US-00002 TABLE 2 Demographics Age at screening Control (n =
30) Fed Allergen (n = 30) 1 mo-3 yrs, median (SD) 8 months (5 mo) 9
months (6 mo) High risk* 17/30 18/30 *Defined by first degree
relative with atopic dermatitis or two first degree relatives with
atopy
Allergen or Allergen Mix/Protein Powders
[0128] The allergens included in the study were flours/dry powders
from peanuts, almonds, cashews, walnut, pecan, hazelnut, wheat,
soy, shrimp, salmon, hen's egg (sodium lauryl sulphate was added as
an anti-caking agent), cow's milk, white fish, pink fish, and
sesame. The details of the allergens and allergen mixes were as
follows: [0129] 1. Formula 1 at 1.times. (1.times. mix): equal
parts peanuts (Defatted peanuts from "Byrd Mill Company", Ashland,
Va. 23005), almonds (Blanched almond flour from "Honeyville" Rancho
Cucamonga, Calif. 91730), cashews (Finely ground cashew from
"Wellbee's", Spring Valley, N.Y. 10977), walnut (Roasted walnut
from "Holmquist Hazelnut Orchards", Lynden Wash. 98264), pecan
(Ground pecan from "King Arthur Flour", Norwich, Vt., 05055),
hazelnut (Natural hazelnuts from "Holmquist Hazelnut Orchards",
Lynden Wash. 98264), wheat (Wheat flour from "Arrowhead Mills",
Boulder, Colo. 80301), soy (Stone ground soy from "Bob's Red Mill",
Milwaukie, Oreg. 97222), shrimp (White shrimp from "Seattle
Seafoods", Bellevue, Wash. 98008), hen's egg (Powdered egg whites
with sodium lauryl sulphate as an anti-caking agent from
"Honeyville Food Products", Honeyville, Utah 84314), cow's milk
(Organic non-fat dry milk powder from "Now Foods" Bloomingdale,
Ill. 60108, white fish (Pacific cod from "Seattle Seafoods",
Bellevue, Wash. 98008), pink fish (Pacific salmon from "Seattle
Seafoods", Bellevue, Wash. 98008), and sesame (Sesame flour from
"Dipasa", Brownsville, Tex. 78526), (30 mg of protein each) with or
without vitamin D3 (400 IU). (i.e. Early Adaptive Tolerance Blend)
[0130] 2. Formula 1 at 3.times. (3.times. mix): equal parts as
above (90 mg of each protein) with or without vitamin D3 (400 IU).
(i.e. Early Adaptive Tolerance Blend) [0131] 3. Formula 1 at
10.times. (10.times. mix): equal parts as above (300 mg of each
protein) with or without vitamin D3 (400 IU). (i.e. Early Adaptive
Tolerance Blend) [0132] 4. Peanut: Defatted peanuts from "Byrd Mill
Company", Ashland, Va. 23005. [0133] 5. Peanut and egg: Defatted
peanuts from "Byrd Mill Company", Ashland, Va. 23005, Powdered egg
whites with sodium lauryl sulphate as an anti-caking agent from
"Honeyville Food Products", Honeyville, Utah 84314. [0134] 6. Milk:
Organic non-fat dry milk powder from "Now Foods" Bloomingdale, Ill.
60108. [0135] 7. Control
[0136] The total dose of allergens used was about 300 mg total for
single allergens and for allergen mixes unless specified otherwise.
A 3.times. and 10.times. formulation containing allergens each
containing about 900 mg and 3000 mg total allergen, respectively,
were also used in the study. Allergen mixes were formulated so that
equal amounts of each allergen were present in the mix. Mixing
occurred prior to ingestion.
Blood Sample Collection
[0137] Peripheral blood mononuclear cells (PBMCs) and plasma were
extracted from blood via ficoll procedure and stored in liquid
nitrogen and at -80.degree. C., respectively. Allergen-specific IgE
and IgG4 were measured using a standard ImmunoCAP assay (Phadia,
Uppsala, Sweden).
Stimulation and Enumeration of Th2 Cell Subsets
[0138] PBMCs were labeled with carboxyfluorescein diacetate
succinimidyl ester (CFSE) and cultured with the same food allergen
or food allergen mix at 100 .mu.g/mL or anti-CD3/CD28 (to test for
nonspecific proliferation capacity) for 7 days to identify T cell
subsets. At day 7, cells were washed and stained for surface CD4,
CD25, CD127, CD45RO, CD45RA, CD40L, and CD69 and intracellular
Foxp3 and IL-10 along with Live/Dead staining (Invitrogen). Th2
cells were defined as the cells that proliferated in response to
food allergen (CFSElo) and were CD41 IL-4, IL-13 cells.
Antigen-induced T cells were also identified by isolating CD40L and
CD69 double-positive cells after antigen stimulation.
Measurement of Antibody Titers
[0139] Total and allergen-specific blood IgE and IgG4 levels were
measured using a standard ImmunoCAP assay (Phadia, Uppsala,
Sweden).
Statistical Analysis
[0140] Comparisons between cohorts, and between baseline and 1 year
and control groups were evaluated with the nonparametric
Mann-Whitney test, paired Wilcoxon test, and 1-way ANOVA (GraphPad
Prism Software 5.0; GraphPad Software, La Jolla, Calif.), as
appropriate. A P value of less than 0.05 was considered
statistically significant.
Compliance and Safety
[0141] Compliance was excellent with no dropouts in control arm or
fed arm. Of those who completed the study, there were only 3 missed
doses (out of a total of approximately 10,950) as determined by
reviews of electronic dietary records (see Table 3). Adverse events
were minimal in the fed arm with only 2 cases of mild skin rash
(2/30; 7%). Adverse events in the control group consisted of 5
cases of mild skin rash (5/30; 17%).
TABLE-US-00003 TABLE 3 Summary of adverse reactions in ingestion
and control groups Reaction Rates Fed Control Participants N = 30 N
= 30 (ITT analysis) Ingestions completed 10,950 (3 missed doses)
n/a Reactions Total 2 (7%) 5 (17%) Mild 2 (skin rash) 5 (skin rash)
Moderate 0 0 Severe 0 0
Th2 Cell Proliferation
[0142] PBMCs were stimulated ex vivo with the an allergen or
allergen mix fed to the subject for 7 days and Th2 cell
proliferation was analyzed (as described in the Methods section
above). It is noted that for Formula 1 3.times. and Formula 1
10.times., the PBMCs were stimulated with 3.times. and 10.times.
the amount of allergen ex vivo, respectively (i.e., 300 .mu.g/mL
and 1000 .mu.g/mL, respectively).
[0143] As shown in FIG. 8, PBMCs from fed subjects stimulated with
single allergens or allergen mixes had a lower degree of Th2
proliferation than stimulated PBMCs from control subjects,
indicating decreases in allergic response on exposure to the
allergen mixes.
Immunoglobulin Analyses
[0144] In addition to the Th2 proliferation assay above, plasma
from subjects were analyzed for the presence of IgG4 and IgE
specific antibodies (using standard methods, as described above),
the former being an indicator of a non-allergenic or
non-inflammatory (or anti-allergenic or non-inflammatory) state and
the latter an indicator of pro-allergenic or pro-inflammatory
state.
[0145] Increased IgG4 levels indicate a protective
anti-inflammatory state. As shown in FIG. 9, when compared to
baseline, samples from fed participants showed an increase in IgG4,
with samples from participants fed the allergen mix formula showing
greater IgG4 levels than those from cohorts fed single or double
food compositions. No significant difference in IgG4 was observed
between samples from participants fed the 1.times., 3.times., and
10.times. allergen mixes or with vitamin D3.
[0146] Increased specific IgE is indicative of an inflammatory
allergic state. As shown in FIG. 10, samples from participants fed
single food and food mixes showed decreases in IgE, with the lowest
levels seen for samples from participants fed the 1.times.,
3.times., and 10.times. allergen mixes.
[0147] These results are consistent with the results for Th2 cell
proliferation and the IgG4 levels as described above. Specifically,
low levels of IgE and increased levels of IgG4 in the plasma
coupled with reduced Th2 cell proliferation are indicators of a
reduced or non-allergenic or non-inflammatory state in a subject.
It is clear from the results above that continual feeding of a
simple or complex mixture of food allergens to subjects (e.g., in
the form of Formula 1) at a very young age can prophylactically
protect a subject from developing an allergenic or inflammatory
immune profile to a wide variety of antigens. In other words, this
process can induce in a subject a non-allergenic or
non-inflammatory (or anti-allergenic or anti-inflammatory) state.
Although a single allergen product showed evidence of effect, of
particular interest in the results shown herein is the indication
that feeding antigens in a complex mixture provides broad spectrum
protection against developing allergies/inflammation that is
superior to single or even double allergen formulas that that
target a single allergen.
[0148] The above shows that feeding subjects simple or complex
antigen mixtures will not only protect against the development of
allergies and/or inflammation to the allergens/antigens in the
complex mixture, but also to allergens or antigens that are not
present in the complex mixture. In essence, feeding complex food
antigen mixtures creates a general anti-allergenic or
anti-inflammatory state in a subject that broadly prevents the
development of allergies or inflammatory state, even to
allergens/antigens not yet fed to the subject.
Example 3--Mixed Allergen Compositions in the Treatment of
Autoimmune Disease
[0149] The mixed allergen compositions disclosed herein will be
further tested for their ability to treat or prevent a range of
autoimmune diseases or inflammatory diseases.
[0150] The mixed allergen compositions will be given to subjects
via different routes, e.g., orally, mucosal, subcutaneously, or
cutaneously. Therapy will be daily or weekly, starting at small
amounts (about 1 mg or less). As appropriate, it will be possible
to stay on the same amount or increase by 5-100% each week, over
time, to larger amounts (about 300 mg or higher). Blood from
subjects will be collected at baseline and then during and after
therapy. Biomarkers would be predicted to show improved immune
balance and immune protection against autoimmune inflammation. For
example, Th1 proliferation to the antigen would decrease over time
during the exposure to the mixed allergen compositions.
[0151] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
[0152] Accordingly, the preceding merely illustrates the principles
of the invention. It will be appreciated that those skilled in the
art will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
* * * * *