U.S. patent application number 15/406535 was filed with the patent office on 2017-07-06 for peptide for protection of allergic respiratory disorders.
This patent application is currently assigned to Board of Supervisors of Louisiana State University and Agricultural and Mechanical College. The applicant listed for this patent is Board of Supervisors of Louisiana State University and Agricultural and Mechanical College. Invention is credited to Sudhirdas K. Prayaga, Changaram S. Venugopal.
Application Number | 20170190750 15/406535 |
Document ID | / |
Family ID | 47296766 |
Filed Date | 2017-07-06 |
United States Patent
Application |
20170190750 |
Kind Code |
A1 |
Venugopal; Changaram S. ; et
al. |
July 6, 2017 |
Peptide for Protection of Allergic Respiratory Disorders
Abstract
A peptide (Peptide-1) based on the C-terminal of Equine CC10 has
been discovered that can be used as a vaccine to protect horses
from respiratory airway obstruction (RAO), Antibodies to Peptide-1
may also be administered for short-term passive immunotherapy to
RAO-affected horses, and can be used to measure the level of CC10
protein in serum to identify potential RAO horses (horses with
reduced CC10). Due to similarities between equine RAO and human
asthma, this peptide or its antibodies may also be useful in
treatment or prevention of human asthma.
Inventors: |
Venugopal; Changaram S.;
(Prairieville, LA) ; Prayaga; Sudhirdas K.;
(Dardenne Prairie, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Supervisors of Louisiana State University and Agricultural
and Mechanical College |
Baton Rouge |
LA |
US |
|
|
Assignee: |
Board of Supervisors of Louisiana
State University and Agricultural and Mechanical College
Baton Rouge
LA
|
Family ID: |
47296766 |
Appl. No.: |
15/406535 |
Filed: |
January 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14124297 |
Dec 6, 2013 |
9546200 |
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PCT/US2012/041592 |
Jun 8, 2012 |
|
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15406535 |
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61494948 |
Jun 9, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/18 20130101;
A61K 38/00 20130101; C07K 14/47 20130101; A61K 2039/577 20130101;
G01N 2800/122 20130101; A61K 2039/552 20130101; C07K 2317/34
20130101; G01N 2333/435 20130101; G01N 33/6854 20130101; C07K
2317/33 20130101; A61K 2039/55566 20130101; A61P 31/00 20180101;
A61K 39/35 20130101; G01N 33/6893 20130101; A61K 2039/505 20130101;
A61K 39/0005 20130101; G01N 2800/50 20130101; A61K 2039/6081
20130101; A61K 2039/575 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61K 39/35 20060101 A61K039/35; G01N 33/68 20060101
G01N033/68; C07K 16/18 20060101 C07K016/18 |
Claims
1. An isolated, purified peptide with an amino acid sequence that
is at least 80% homologous to the sequence set forth in SEQ ID
NO:1.
2. The isolated peptide of claim 1, wherein said peptide has an
amino acid sequence that is at least 85% homologous to the sequence
set forth in SEQ ID NO:1.
3. The isolated peptide of claim 1, wherein said peptide has an
amino acid sequence that is at least 95% homologous to the sequence
set forth in SEQ ID NO:1.
4. The isolated peptide of claim 1, wherein said peptide has a
sequence as in SEQ ID NO:1.
5. A medical composition comprising the isolated peptide of claim 1
and a pharmaceutically acceptable carrier.
6. A vaccine for mammalian respiratory disorders comprising the
isolated peptide of claim 1.
7. The vaccine of claim 6, wherein the peptide is covalently or
non-covalently linked to a protein carrier.
8. The vaccine of claim 7, wherein the protein carrier is keyhole
limpet hemocyanin.
9. The vaccine of claim 6, wherein the peptide is administered with
an adjuvant or other pharmaceutically acceptable compound.
10. The vaccine of claim 9, wherein the adjuvant is incomplete
Feund's adjuvant.
11. An isolated antibody to the peptide of claim 1.
12. The antibody of claim 11, wherein the antibody is a polyclonal
antibody.
13. The antibody of claim 11, wherein the antibody is a monoclonal
antibody.
14. A method to treat or prevent a respiratory allergic disease in
a mammal, said method comprising administering to the mammal a
therapeutically effective amount of the vaccine of claim 4.
15. The method of claim 12, wherein the mammal is a horse.
16. The method of claim 12, wherein the mammal is a human.
17. A method to increase treat or prevent respiratory airway
disease in a mammal, said method comprising administering to the
mammal a therapeutically effective amount of the antibody of claim
6.
18. The method of claim 17, wherein the mammal is a horse.
19. The method of claim 17, wherein the mammal is a human.
20. A method to detect mammals that are susceptible to respiratory
airway disease, said method comprising measuring the levels of CC10
protein in the serum of a mammal using the antibody of claim
11.
21. The method of claim 20, wherein said mammal is a horse.
22. The method of claim 20, wherein said mammal is a human.
23. A method to detect mammals that are susceptible to respiratory
airway disease, said method comprising measuring the levels of
antibody to CC10 protein in the serum of a mammal using the
isolated peptide of claim 1.
24. The method of claim 23, wherein the mammal is a horse.
25. The method of claim 23, wherein the mammal is a human.
26. A DNA expression vector with a nucleic acid sequence encoding
for the peptide as in claim 1.
27. A method to treat or prevent a respiratory allergic disease in
a mammal, said method comprising administering to the mammal a
therapeutically effective amount of the DNA expression vector of
claim 26.
28. The method of claim 27, wherein the mammal is a horse.
29. The method of claim 27, wherein the mammal is a human.
Description
[0001] The benefit of the filing date of provisional U.S.
application Ser. No. 61/494,948, filed 9 Jun. 2011, is claimed
under 35 U.S.C. .sctn.119(e) in the United States, and is claimed
under applicable treaties and conventions in all countries.
TECHNICAL FIELD
[0002] This invention pertains to a unique peptide isolated from
horse CC10 that can be used as a vaccine to protect horses from
recurrent airway obstruction (RAO) and also to antibodies to this
peptide which can be used in passive immunotherapy for short term
protection against RAO or in assays to determine the level of CC10
protein in mammals. This new peptide can also be used to protect
humans from asthma, based on the similarity between horse RAO and
human asthma.
BACKGROUND ART
[0003] Recurrent Airway Obstruction
[0004] Recurrent Airway Obstruction (RAO), a respiratory disease
similar to human asthma, is one of the most commonly diagnosed
conditions affecting the lung of older horses all around world. It
is a type-1 immediate hypersensitivity allergic disease involving a
series of events that begin with reaginic antibodies, mainly IgE.
These antibodies bind with high affinity to Fc epsilon-RI
(Fc.epsilon.RI) receptors via the Fc portion that are found on the
membrane of mast cells and basophils. Once bound, the antibodies
can cross-link with environmental allergens, resulting in mast cell
degranulation leading to the production of histamine and other
chemical mediators that act together to induce airway inflammation.
At least one reaginic antibody against Faenia rectivirgula, a
common gram-positive bacteria, was found to be associated with lung
diseases in RAO-affected and unaffected horses, finding the
antibody present in about 15% in RAO-affected horses (28).
[0005] Most evidence suggests that RAO is the result of a pulmonary
hypersensitivity to inhaled antigens. Exposure of affected horses
to hay dust, pollens, and mold spores leads to neutrophil
accumulation in the lung and bronchospasm. The identification of
allergen-specific IgE in bronchoalveolar lavage (BAL) fluid and
sera of affected horses supports the involvement of a late phase,
IgE-mediated, hypersensitivity reaction in the pathogenesis of
equine RAO. The cytokine profiles of horse affected by RAO have
been measured, and a modified Th2-like immune response was thought
to be involved with elevated IL-4, IL-13 and IFNg levels
(6,22).
[0006] CC10 in RAO
[0007] Clara Cell 10 kDa protein (CC10) is one of the members of a
family of anti-inflammatory defense proteins produced predominantly
in the airway epithelium of man and animals. In humans it is also
called human uteroglobin (See U.S. Pat. Nos. 6,255,281 and
7,846,899). CC10 has been shown to inhibit the entry of eosinophils
and neutrophils into the human airways during inflammation in the
lung, and to inhibit enzymes that release inflammatory mediators
(23). Some of the members of this family have been directly linked
to protection from asthma, allergy and related inflammatory
responses. The gene that encodes the protein was found to be down
regulated during RAO in horses (34), and the production of the CC
10 protein was also reported to be reduced in RAO horses (24). The
amount of CC10 in the horse serum was measured using antibodies
produced in rabbits using a synthetic 21 amino acid peptide from
horse CC10 (EPSKPDADMKAATTQLKTLV; SEQ ID NO:2). This peptide
corresponded to amino acids 47-66 of horse CC10 protein (protein
accession no. NP 001075327.1) with two amino acid variations.
[0008] A hypo-allergic vaccine to an environmental antigen of
Timothy grass pollen Profilins was developed by engineering its
structure (35). The engineered protein was demonstrated to induce
IgG's and inhibit allergic patients' IgE antibody binding to
Profilins to a similar degree as those induced by immunization with
the wild type. IgG's inhibited Profilin-induced basophil
degranulation. Another environmental antigen, a novel Fel d1 cat
allergy vaccine, was developed that induced strong IgG response,
which abrogated the allergy response in mice (32). Allergen-induced
systemic basophil degranulation was shown inhibited in an Fc
gamma-RIIb-dependent manner (26). In addition, antibodies have been
found in healthy horses; elevated IgG response were found in
healthy horses to auto antigen (1). IgGb specific to KLH was found
elevated in healthy versus control horses. An inverse correlation
was reported between BCG vaccination and incidence of IgE mediated
allergic diseases (18). BCG immunization was demonstrated to
inhibit IgE production by B cells.
[0009] Receptors for the Fc fragment of immunoglobulin-G (Fc
gamma-R's) are important molecules not only to mediate and control
the effectors' functions of IgG antibodies, but they also control
the autoimmunity-tolerance balance in the periphery. In humans,
three different types of Fc gamma-R's, belonging to the
immunoglobulin gene super-family have been identified via Fc
gamma-RI (CD64), Fc gamma-RII (CD32) and Fc gamma-RIII (CD16). In
humans, myeloid cells express both activating and inhibitory
receptors of the Fc gamma-RII family. Fc gamma-RIIA mediates
processes associated with cell activation, while Fc gamma-RIIB down
regulates such signaling. Differences in affinity for IgG between
activating and inhibitory Fc gamma-R can result in substantial
local changes in their relative concentrations with important
functional consequences. Human airway smooth muscle cells express
IgG-Fc gamma-RIIb with the potential to suppress remodeling and
immune-modulation (36). A wide range of inflammatory and autoimmune
diseases, such as vasculitis, glomerulonephritis, and autoimmune
hemolytic anemia, seems to be mediated, in part, by Fc
gamma-R's.
[0010] Human Asthma and Animal Models
[0011] Human asthma is a multifactorial disease, and is usually a
chronic lung condition which can develop at any age and progress to
a devastating disease. Asthma is classified as a hypersensitivity
disease (5,30). An estimate shows that there are more than 150
million people with asthma worldwide, of which 20.3 million are in
America. It is also important to note that the trend of deaths and
hospitalization due to asthma is increasing in the industrialized
countries of the world. Despite extensive research into the
pathophysiology and treatment of asthma, high morbidity continues
to threaten the human population. Because asthma can be induced by
multiple stimuli, no single theory satisfactorily explains all
types and cases, thus making a therapeutic regimen cumbersome.
[0012] It is a well-established fact that the salient feature of
human asthma is the tendency of the affected airways to overreact
to a wide variety of nonspecific stimuli, which has led to the use
of bronchial provocation tests as a method to diagnose the
condition (19). This hyperreactivity is characterized by
hyperresponsiveness and hypersensitivity of airways to various
inflammatory mediators (13). This condition can result from
abnormal tissue reaction in the airways either due to intrinsic
stimuli arising from a biochemical, neurological, or humoral
physiological imbalance in the body or due to extrinsic stimuli
arising from the environment. The changes in the environment are
detected in the airway mucosa by sensory receptors, which are
richly innervated by sensory (afferent) fibers. Various respirable
stimuli, including charged particles, allergens, irritants, as well
as heat, cold and acid, activate sensory receptors.
[0013] Animal models of human asthma: To examine various factors
and mechanisms involved in pathogenesis of asthma, a number of
mammalian animal models have been identified and used to study
human airway hyperreactivity. For example, guinea pigs, which are
not naturally susceptible to asthma, are used because of the
ability to generate an experimentally-inducible model of asthma
with indices similar to human asthma (14,15). Another animal model
for asthma is mice which again are not naturally susceptible to
asthma, but asthma can be induced experimentally. One advantage to
mice is the ability to study the genetic aspects of the disease
(12).
[0014] Equine models of human asthma: A major advantage to an
equine model is that horses get asthma naturally like people.
Horses are important for the similarity between the signs and
symptoms of recurrent airway obstruction (RAO) to those of asthma,
for the similarity in the nature of disease progression to human
asthma, and for the ability in horses to induce or precipitate an
"asthmatic" episode (7,33). Histological findings such as
thickening of epithelial basement membrane, edema, inflammatory
infiltrate in bronchial walls, oversized submucosal glands,
hypertrophy of bronchial wall muscle, and emphysematous changes are
observed in both human asthma and in equine RAO (6,7,33). One
difference is that the cellular infiltrate in RAO is mostly
neutrophils, whereas esosinophils are more common with human
asthma. Recently, identification of persistent nuclear factor
kappa-B (NF-.sub..kappa.B) activity in asthma and RAO clearly
suggests that equine RAO is a better animal model of human asthma
than those of other mammalian species (8,20). Chronic obstructive
pulmonary disease (COPD) in horses, commonly called heaves, and
currently termed recurrent airway obstruction (RAO), is an airway
hyperreactivity disease comparable to human asthma (7,33). Horses
with heaves spontaneously develop acute bronchiolitis after
exposure to antigens such as hay or pasture mold. Heaves is a more
common condition in temperate climatic regions where horses are
confined in stables during winter seasons and where they are house
near stored hay. Another form of RAO seen in the southern regions
of the U.S. is summer pasture-associated obstructive pulmonary
disease (SPAOPD), caused by allowing horses to graze on lush
pasture in the humid summer months where molds and pollens in the
grass serve as antigen stimulation (4). Both forms of RAO share
common signs and symptoms (4,6,11). The clinically abnormal
condition of RAO is characterized by hyperreactivity of airways to
antigens, inflammatory mediators and various other external
stimuli. The condition also presents a decreased dynamic compliance
of lung and increased airflow resistance to histamine- or
methacholine-challenges, which are the common tests performed for
diagnosing either human asthma or equine RAO. When horses are
exposed to diverse stimuli such as allergens or respirable
irritants in the environment, their airways react with a specific
response that includes infiltration of inflammatory cells in the
lungs, damage of airway epithelium, and inflammatory mediator
release. This reaction primes the airways to respond excessively to
a broader range of stimuli. Continued stimulation leads to airway
hyperreactivity, which is characterized by hypersensitivity
(increased sensitivity of airway smooth muscles) and
hyperresponsiveness (increased magnitude of smooth muscle
contractility) to diverse stimuli as observed in human asthma
(3,6,7,11). Thus, RAO in horses shares several common features of
human asthma, such as airway hyperreactivity, severe
bronchoconstriction, increased mucus production, presence of airway
inflammatory mediators in bronchoalveolar lavage fluids,
infiltration of inflammatory cells, difficulty in breathing and
exercise intolerance. Horses then have several advantages as an
animal model for human asthma. Of course, the main disadvantages to
using horses are size and cost to obtain and house.
[0015] U.S. Pat. Nos. 7,846,899 and 6,255,281 and U.S. Patent
Application Publication No. 2005/0261180 describe the use of
recombinant human CC10 (also referred to as human uteroglobin) and
its use in various human inflammatory and respiratory
disorders.
[0016] U.S. Patent Application Publication No. 2011/0240012
describes the use of recombinant human CC10 and methods to
administer CC10 nasally, and its use in treatment of various nasal
inflammatory diseases, including rhinitis and sinusitis.
DISCLOSURE OF INVENTION
[0017] We have discovered a 21 amino acid (aa) peptide (Peptide-1;
SEQ ID NO: 1; KNTKDSILKLMDKIAKSPLCA) based on the C-terminal of
Equine CC10 that can be used as a vaccine to protect horses from
RAO, and protect humans from asthma. We have produced a rabbit
polyclonal antibody to this peptide, and validated the specificity
of antibody by ELISA. We tested the sera of healthy and RAO
affected horses for CC10 using this antibody in a competitive
inhibition ELISA, and surprisingly found that sera from some of the
healthy horses showed significantly higher titers than the maximum
titer expected with the control. This result could only be
explained if horse sera itself had antibodies to Peptide-1,
suggesting that healthy horses are protected from RAO by antibodies
similar to the polyclonal antibodies produced by Peptide-1. We
believe that such auto-antibodies may be the result of exposure to
environmental allergens that have `molecular mimicry` to the
sequence of Peptide-1. To confirm this belief, we performed a
sequence analysis using the BLAST algorithm and found that several
known natural antigens have a similar sequence to Peptide-1. We
have identified a unique isolated peptide sequence (Peptide-1)
which can be used as a vaccine for protection of horses from RAO
and seasonal allergy. The advantages to Peptide-1 are: (1) it may
be used in young animals as a vaccine to protect them from RAO
later in life; (2) antibodies to Peptide-1 may be administered for
short-term passive immunotherapy to RAO-affected horses; and (3)
antibodies to Peptide-1 can be used in measuring the level of CC10
protein in animals to identify potential RAO horses (horses with
reduced CC10). In addition due to the homology with the human
protein CC10 and since human seasonal allergy (or asthma) has the
same mechanism of induction as horse RAO, we believe that this
Peptide-1 or homologous sequences or its antibodies may be useful
therapeutically in humans for protection against or alleviating the
symptoms of asthma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 illustrates the results of an antibody capture
endpoint ELISA showing the Peptide-1 specific antibody titer with
Peptide-1 as the capture antigen, peroxidase-conjugated goat
anti-rabbit IgG as the detection antibody, and the isolated rabbit
antibody at several dilutions.
[0019] FIG. 2 illustrates the results of a competitive inhibition
ELISA assay using a series of concentrations of Peptide-1.
[0020] FIG. 3 illustrates the amount of CC10 IgG titer from five
healthy horses (without RAO) as compared to the control as measured
using competitive inhibition ELISA assay.
[0021] FIG. 4 illustrates the amount of Peptide-1 specific IgG
titer using a direct sandwich ELISA and measuring serum from eight
healthy horses standardizing the value by subtracting the control
value of UGRP peptide from the experimental values for CC10.
[0022] FIG. 5A illustrates the amount of Peptide-1 specific IgG
titer in serum from Foal #2 primed on day 0 and boosted on day 29
by subcutaneous injection of 0.1 mg/kg Peptide-1. Antibody titer
determined using a direct sandwich end point ELISA with Peptide-1
as a capture antigen and peroxidase conjugated goat anti-equine IgG
as a detection antibody.
[0023] FIG. 5B illustrates the amount of Peptide-1 specific IgG
titer in serum from Foal #3 primed on day 0 and boosted on day 29
by subcutaneous injection of 0.1 mg/kg Peptide-1. Antibody titer
determined using a direct sandwich end point ELISA with Peptide-1
as a capture antigen and peroxidase conjugated goat anti-equine IgG
as a detection antibody.
MODES FOR CARRYING OUT THE INVENTION
[0024] The term "Peptide-1" refers to a 21 amino acid (aa) peptide
(Peptide-1; SEQ ID NO: 1; KNTKDSILKLMDKIAKSPLCA) based on the
C-terminal of Equine CC10 that can be used as a vaccine to protect
mammals from a respiratory airway disorder. This invention relates
not only to a functional Peptide-1 as described in this
specification, but also to peptides having modifications to such a
sequence resulting in an amino acid sequence having the same
function (i.e., the ability to be used as a vaccine for protection
against antigen-caused respiratory disorders), and about 80%,
preferably 85%, and more preferably 90% homology to the sequence of
the amino acid sequence as described, and most preferably about 95%
or greater homology, particularly in conserved regions. "Homology"
as used here means identical amino acids or conservative
substitutions (e.g., acidic for acidic, basic for basic, polar for
polar, nonpolar for nonpolar, aromatic for aromatic). The degree of
homology can be determined by simple alignment based on programs
known in the art, such as, for example, GAP and PILEUP by GCG, or
the BLAST software available through the NIH interne site. Most
preferably, a certain percentage of "homology" would be that
percentage of identical amino acids.
[0025] The term "vaccine" refers to a composition or compound (an
antigen) used to stimulate an immune response in a mammal and so
confer resistance to the disease or infection in that mammal,
including an ability of the immune system to remember the
previously encountered antigen. Antibodies are produced as a result
of the first exposure to an antigen and stored in the event of
subsequent exposure.
[0026] The term "adjuvant" refers to non-antigenic substance that,
in combination with an antigen, enhances antibody production by
inducing an inflammatory or other non-defined response, which leads
to a local influx of antibody-forming cells. Adjuvants are used
therapeutically in the preparation of vaccines, since they increase
the production of antibodies against small quantities of antigen,
lengthen the period of antibody production, and tend to induce
memory cell responses. Such adjuvants could include, but are not
limited to, complete Freund's adjuvant, incomplete Freund's
adjuvant, aluminium hydroxide, dimethyldioctadecylammonium bromide,
Adjuvax (Alpha-Beta Technology), Imject Alum (Pierce),
Monophosphoryl Lipid A (Ribi Immunochem Research), MPL+TDM (Ribi
Immunochem Research), Titermax (CytRx), toxins, toxoids,
glycoproteins, lipids, glycolipids, bacterial cell walls, subunits
(bacterial or viral), carbohydrate moieties (mono-, di-, tri-
tetra-, oligo- and polysaccharide) various liposome formulations or
saponins. Alum is the adjuvant currently in use for human patients.
However, for horses, incomplete Freund's adjuvant may be used.
[0027] The term "immune response" refers to the reaction of the
body to an antigen, which is usually a foreign or potentially
dangerous substances (antigens), particularly disease-producing
microorganisms. However, in the current technology, a new peptide
sequence that is similar to an endogenously produced defense
protein (CC10) is used for the antigen. The response involves the
production by specialized white blood cells (lymphocytes) of
proteins known as antibodies, which react with the antigens to
render them harmless. The antibody-antigen reaction is highly
specific. Vaccines also stimulate immune responses.
[0028] The term "immunologically effective amount" refers to the
quantity of an immune response inducing substance required to
induce the necessary immunological memory required for an effective
vaccine. For example, Peptide-1 at a dose of 0.1 mg/kg body weight
when given subcutaneously has been found an immunologically
effective amount.
[0029] The pharmaceutical compositions of the present invention are
advantageously administered in the form of injectable compositions.
A typical composition for such purpose comprises a pharmaceutically
acceptable carrier. For instance, the composition may contain human
serum albumin in a phosphate buffer containing NaCl. Other
pharmaceutically acceptable carriers include aqueous solutions,
non-toxic excipients, including salts, preservatives, buffers and
the like (REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed., Easton
ed., Mack Publishing Co., pp 1405-1412 and 1461-1487 (1975) and THE
NATIONAL FORMULARY XIV, 14th Ed., American Pharmaceutical
Association, Washington, D.C. (1975), both hereby incorporated by
reference). Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oil and injectable organic esters
such as ethyloleate. Aqueous carriers include water,
alcoholic/aqueous solutions, saline solutions, parenteral vehicles
such as sodium chloride, Ringer's dextrose, etc. Intravenous
vehicles include fluid and nutrient replenishers. The pH and exact
concentration of the various components the pharmaceutical
composition are adjusted according to routine skills in the art.
Goodman and Gilman, THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th
ed.).
[0030] In addition for a small peptide, the peptide may be
covalently linked to an immunogenic protein carrier to increase its
recognition by the immune system as foreign. Common protein
carriers include keyhole limpet hemocyanin (KLH). Bovine serum
albumin (BSA), chicken egg albumin (OVA), and immunoglublin Fc
fragment. Recently, non-proteinic nanoparticles have been used as
carriers.
[0031] Typically, such vaccines are prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. The preparation also may be emulsified. The active
immunogenic ingredient is often mixed with an excipient that is
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like and combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents,
pH-buffering agents, adjuvants or immunopotentiators that enhance
the effectiveness of the vaccine.
[0032] The vaccines are conventionally administered
intraperitoneally, intramuscularly, intradermally, subcutaneously,
orally, nasally, or parenterally. Additional formulations are
suitable for other modes of administration and include oral
formulations. Oral formulations include such typical excipients as,
for example, pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. Additionally, the peptide can be
encapsulated in a sustained release formulations or a coating that
resist the acidic pH of the stomach. The compositions take the form
of solutions, suspensions, tablets, pills, capsules, sustained
release formulations or powders and contain 10%-95% of active
ingredient, preferably 25-70%. In a preferred solution, equal
volumes of Peptide-1 conjugated with KLH and Incomplete Freund's
adjuvant were used in a subcutaneous injection. Other possible
routes of administration is to use a DNA expression vector with a
nucleic acid sequence encoding the Peptide 1 (SEQ ID NO:1) for
immunization using intradermal or intramuscular techniques.
[0033] The dose to be administered depends on a predetermined
quantity of active material calculated to produce the desired
therapeutic effect in association with the required diluent, i.e.,
carrier or vehicle, and a particular treatment regimen. The
quantity to be administered, both according to number of treatments
and amount, depends on the subject to be treated, capacity of the
subject's immune system to synthesize antibodies, and degree of
protection desired. The precise amounts of active ingredient
required to be administered depend on the judgment of the
practitioner and are peculiar to each individual. However, suitable
dosage ranges are on the order of one to several hundred micrograms
of active ingredient per individual subject. Suitable regimes for
initial administration and booster shots also vary but are typified
by an initial administration followed in one or two week intervals
by one or more subsequent injections or other administration.
Annual boosters may be used for continued protection.
EXAMPLE 1 Development of CC10 Peptide Antibody
[0034] An isolated 21-amino acid peptide was designed and made
based on the `C` terminal 21 amino acid peptide of Equine CC10
protein (Accession no. NP_001075327.1) ("Peptide-1; SEQ ID NO:1;
KNTKDSILKLMDKIAKSPLCA). The sequence of this peptide corresponds to
amino acids 71-91 of equine CC10 and does not overlap with that of
the peptide (amino acids 47-66) previously used to make rabbit
antibodies (24). Peptide-1 was used to develop rabbit polyclonal
antibodies. Briefly, Peptide-1 was conjugated to the carrier
protein KLH (Keyhole Limpet Hemocyanin), and the conjugate used to
immunize naive rabbits to induce an immune response. Antibodies
were purified from serum of immunized rabbits by established
chromatographic techniques as described previously (21). The rabbit
antibody was verified in an antibody capture endpoint Enzyme-Linked
ImmunoSorbent Assay (ELISA) with Peptide-1 as the capture antigen
and peroxidase conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch Laboratories, Inc., West Grove, Pa.) as the
detection antibody as described previously (9). As shown in FIG. 1,
the rabbit antibody had a titer of >100,000 with an optimum
detection range between 1000 and 10,000 dilutions (FIG. 1).
EXAMPLE 2
[0035] Development of a Competitive Inhibition ELISA Assay
[0036] Based on the results in FIG. 1, a 1:5000 dilution of this
antibody was used for further CC10 detection in a competitive
ELISA. To validate the competitive inhibition assay, a series of
Peptide-1 dilutions starting at 1000 .mu.g/mL was prepared. From
each dilution, 50 .mu.L was mixed with 50 .mu.L of a solution of
1:5000 diluted rabbit polyclonal antibody. This mixture was
incubated for 30 min, and then used in ELISA with a Peptide-1
coated plate as described previously (9). As shown in FIG. 2, the
inhibition of antibody binding to peptide coated plated was
dependent on Peptide-1 concentration.
EXAMPLE 3
[0037] Determination of CC10 Specific IgG Titer--Elevated CC10 IgG
Titer
[0038] The competitive ELISA system as described above (Example 2)
was used to measure relative levels of CC10 in the serum of 5
healthy and 5 RAO horses. Briefly, 10 .mu.L of serum from each
horse was mixed with 50 .mu.L of a 1:5000 diluted rabbit antibody
to Peptide-1 and incubated at 37.degree. C. for 30 min. This
serum-antibody mixture was then added to wells on ELISA plate
pre-coated with Peptide-1 and blocked with 2% milk powder proteins
(Carnation Milk, Nestle). The amount of free peptide specific
rabbit antibody available to bind to Peptide-1 coated on the plate
varied, depending on the amount of native CC 10 present in the
scrum sample. Plates were washed, and the amount of rabbit IgG
bound to the plate was measured using Goat anti-rabbit IgG-HRP
(Jackson ImmunoResearch Laboratories, Inc.). Surprisingly, some of
the healthy horses had a significantly high titer than the control
samples which had only rabbit peptide IgG without the addition of
any horse sera. In FIG. 3, the absorbance titer data for healthy
horses was normalized to that of control as 100%. Two of the five
healthy horses, or 40%, showed significantly increased titer for
CC10 in sera over that of the controls.
[0039] The titer levels were too significant to consider the levels
as resulting from non-specific binding of IgG present in horse
serum. Without wishing to be bound by this theory, we believe that
at least some healthy horses have significant IgG antibody levels
already that would bind to the 21 amino acid peptide (Peptide-1) of
CC10. The goat anti-IgG to rabbit was used for detection, and it is
a pan specific antibody that is cross-reactive across many species
including horse IgG. Such auto-antibodies in the horse serum may be
produced due to exposure to environmental allergens that show
`molecular mimicry` to the sequence of Peptide-1. As described
below, a sequence analysis using the BLAST algorithm as previously
described (9) was conducted, and found that several known antigens
in nature have a similar sequence to Peptide-1.
EXAMPLE 4
[0040] BLAST Sequence Homology Analysis--Homology to Microbial
Antigens, Molecular Mimicry
[0041] The Peptide-1 sequence was used in a protein BLAST sequence
analysis against published bacterial and fungal proteins. High
homology to some bacterial and fungal proteins including to
Aspergillus, a known environmental allergen was found. (Table
1).
TABLE-US-00001 TABLE 1 No. Protein ID Species % Identity 1.
Q65R19_MANSM Mannheimia 90% AspA Protein succiniciproducens (Bovine
rumen bacteria) 2. D3V5R2_XENBV Xenorhabdus bovienii 71% (bacteria
found in nematodes) 3. C4VBG4_NOSCE Nosema ceranae 58%
(microsporidium usually affecting bees) 4. A2R1F5_ASPNC Aspergillus
niger 61% (common soil and plant fungus)
[0042] BLAST searches using the whole horse CC10 was also conducted
and found significant homology to a cat allergen Fel d 1 and to a
putative U1p1 protease protein in Aspergillus fumigatus (Table
2).
TABLE-US-00002 TABLE 2 No. Protein ID Species % Identity 1.
P30438.2 Fel d1 Felis catus 37% (domestic cat) 2. NC_007 198.1
Aspergillus fumigatus 51% U1 p1 protease (fungus commonly found in
soil)
EXAMPLE 5
[0043] Elevated Peptide-1 Specific IgG in Healthy Horses
[0044] To further validate if an elevated CC10 specific IgG
antibody titer existed in healthy horses, sera from eight healthy
horses with no sign of RAO were collected. To determine levels of
Peptide-1 specific IgG titer, a direct sandwich ELISA was used
according to published protocols (18). Diluted healthy horse serum
samples were added to a plate coated with Peptide-1. The peptide
bound horse IgG antibody was then detected using peroxidase
conjugated goat anti horse IgG antibody (Jackson ImmunoResearch
Laboratories, Inc.). As control, a 17 amino acid `C` terminal
peptide from UGRP1 protein (another member of the CC10 family) was
used. As shown in FIG. 4, elevated amounts of antibodies to
Peptide-1 was again found in healthy horses. This result further
supported that Peptide-1 specific IgG is present in many healthy
horses (FIG. 4).
[0045] These results confirm that elevated levels of Peptide-1
specific antibodies are found in healthy horses. Without wishing to
be bound by this theory, we believe that this elevated level is due
to molecular mimicry. Homology of a self-protein to a microbial
protein can induce antibody response to the self-antigen if exposed
to the microbial or fungal product early in life. Sequence analysis
as shown above indicated a high homology of Peptide-1 to some
microbial and fungal proteins including Aspergillus, a known
environmental allergen. It is possible that augmented immune
responses generated in these healthy horses early in life afforded
them protection subsequently in life.
[0046] The hygiene hypothesis suggests that a reduced frequency of
infections, less severe infection, and prevention of infection (for
example, by a frequent use of antibiotics) would prevent maturation
of Th-1 immunity and, therefore, would give rise to an
allergen-specific Th-2 immune response following natural exposure
to allergens. The immunological explanation for the "hygiene
hypothesis" has been proposed to be the induction of T helper 1
(Th1) responses by microbial products. However, the protective
results of hygiene hypothesis-linked microbial exposures are
currently shown to be unlikely to result from a Th1-skewed
response. The hygiene hypothesis has been proposed to be more
important in regulating the PMN-dominant inflammatory response than
in inducing a Th1-dominant response (10). Endotoxins, part of the
outer membrane of Gram negative bacteria, are a potent inducer of
neutrophilic airway inflammation. The risk of non-atopic asthma,
characterized by neutrophilic response, is enhanced in subjects
with higher endotoxin exposure. This information is in accordance
with the so-called hygiene hypothesis and have been supported by
animal studies and at the cellular level (11). Thus vaccinating the
horses at a young age with Peptide-1 will produce protective immune
response which will protect them from environmental allergens.
[0047] Auto-antibodies to equine CC10 `C` terminal 21 amino acid
peptide (Peptide-1) were observed in horses resistant to RAO.
Sequence analysis showed some homology of Peptide-1 to several
bacterial and fungal sequences. Therefore, it is possible that the
antibodies were developed due to molecular mimicry to microbial
antigens. Activation of B-cells carrying Fc gamma-RII receptor by
these autoantibodies may be protective, while activation of
Fc.di-elect cons.R1 receptor induces disease. Immunization of
horses with Peptide-1 early in life can provide protection from RAO
later in life.
[0048] Peptide-1 can be used as a vaccine for young horses to
protect them from developing the respiratory allergy later in their
life. In addition, the antibody produced to Peptide-1 can be used
in passive immunotherapy for short term therapy to decrease the
symptoms of a horse or other mammal with respiratory airway
disease. Antibodies to Peptide-1 can also be used to determine the
level of CC10 protein in horses. A decreased level of CC10 in the
serum could be a biomarker for detecting horses that are vulnerable
to RAO which will facilitate early selection and improved breeding.
In addition, Peptide-1 or similar peptide may also be useful in
developing a vaccine to protect people that are susceptible to
allergic diseases such as human asthma.
EXAMPLE 6
[0049] Vaccination of Foals with Peptide-1
[0050] A pilot study in foals was started in January 2012 to
determine whether an immune response could be generated by
vaccinating foals with Peptide-1, a newly identified peptide from
Clara Cell proteins of horses. The study was approved by the
Institutional Animal Care and Use Committee (IACUC) of Louisiana
State University to be conducted in nine Shetland foals. The foals
were obtained from the Equine Health Studies Program of the LSU
School of Veterinary Medicine, Baton Rouge, La. 70803. They ranged
in age from 4 to about 6 months. This study is still continuing. So
far Peptide-1 has been injected into 3 foals, but all nine foals
will be injected in a manner similar to that described below. The
body weights of the three foals injected ranged from about 101 to
about 112 kg.
[0051] The foals were selected based on a thorough physical
examination by a board certified equine clinician and a complete
blood count (CBC) was performed to determine any clinical
pathology. Since Peptide-1 is a small peptide, it was covalently
conjugated with a large carrier protein, Keyhole Limpet Hemocyanin
(KLH), for easy recognition by the lymphocytes to produce antibody
production. KLH is a commonly used as a carrier protein.
[0052] Two days before the injection of the peptide-1, a foal along
with the mare was brought from the pasture to a bedded stall of the
LSU School of Veterinary Medicine. They were fed hay and
concentrated horse chow (Horse Chow #200.sup.R, Purina Mills, St.
Louis, Mo.); water was provided ad libitum. A physical examination
was conducted on the day of injection. The foal's body temperature,
heart rate, respiratory rate, body weight, body score, sex, breed,
age and general physical conditions were recorded. After the
physical examination, 5 ml of blood was drawn from jugular vein to
determine pre-immune antibody titer (control value) and CBC.
[0053] Foal #1:
[0054] Since the peptide-1 has not been tried in any animals, the
first foal was used to test the injection method and dosage. A low
dose of 0.1 mg/kg body weight of conjugated peptide-1 with KLH was
used, but without an adjuvant to minimize any adverse reactions due
to either a high dose of peptide-1 or adjuvant. This protocol
allowed a determination of any reaction due to the conjugated
peptide-1 alone. A subcutaneous route of administration was chosen
to produce a consistent "slow and steady" absorption. The first
injection was given on the left side of the neck after cleaning
with alcohol. No major adverse reaction was observed at the site of
injection or on the animal as a whole. A small swelling was
observed at the site of injection which disappeared after two days.
The foal did not have any fever or any other clinical pathological
changes. After the injection, 5 ml blood was collected weekly. A
booster injection with the same dose and route was given on the
right side of the neck on the 15.sup.th day following the initial
injection. Weekly blood collections continued for 42 days. The
antibody titer did not change from the pre-immune values in this
foal (Data not shown). For the remaining foals, a new protocol was
used in that an adjuvant was added to the injection and the booster
injection was given on the 28.sup.th day after the initial
injection.
[0055] Foal #2
[0056] The same protocol as described above for Foal #1 was used in
this foal except for the addition of the adjuvant and a change in
booster injection time. An equal volume of Incomplete Freund's
Adjuvant (IFA; Sigma Chemicals, St. Louis, Mo.) and Peptide-1 was
mixed well and administered subcutaneously. The total volume of
injection did not exceed 2 ml, and the does of Peptide-1 remained
as in Foal #1. Prior to the first injection and weekly thereafter,
5 ml blood was drawn for analysis. A booster injection of
adjuvant/Peptide-1 was given on the 28.sup.th day following the
initial injection.
[0057] Immediately after injection (either the first injection or
the booster), a small swelling (size 1''.times.0.5'') at the site
of injection was observed. The foal was observed continuously for
the first hour and later every hour for eight hours. The foal
evinced pain upon touch at the injection site, but it was eating,
nursing, and drinking within 15 minutes after injection. The foal
showed no other adverse reaction, and it was bright, alert and
responsive. No sweating, fever, cough, seizure or difficulty in
breathing was observed.
[0058] By the end of 24 h, the swelling at the injection site had
spread to an area of 2.5''.times.2'' size, and had a thickness of
only 0.5''. The foal remained under close observation for another
two days, when the swelling began to subside. On the third day,
foal and mare were allowed to go to the pasture. A similar swelling
occurred at the booster injection time also. Therefore, in the
animal protocol, foals would be kept in the bedded stall with the
mare for a week after injection to observe the foals closely. In
addition, the protocol demanded that if the swollen area is more
than 2.5'' in diameter and more than 0.5'' in thickness, hot water
packs are to be applied to relieve pain and swelling. Veterinary
medical care is to be consulted if the condition worsens. After one
week in the stall, the Foal #2 was let out to pasture and continued
to do well for the remainder of the experimental period of 57
days.
[0059] The peptide-1 specific total IgG antibody titers were
determined in the serum samples by enzyme labeled immune sorbent
assay (ELISA). So far, eight samples have been tested in this foal
for a time period of 57 days. The results are shown in FIG. 5A. The
samples were taken on pre-injection serum collected on day 0, and
then every seven days thereafter. The results indicate that the
antibody titer to Peptide-1 increased exponentially after day 15 up
to day 22, when there was a drop. After the booster on day 29, the
antibody titer increased to a peak level at day 36, but remained at
elevated levels for the remaining period. As shown in FIG. 5A,
Peptide-1 at a dose of 0.1 mg/kg when injected subcutaneously with
an adjuvant can produce significant amounts of antibodies. We
believe that this increase in antibodies to Peptide-1 will immunize
these young horses and protect them from recurrent airway
obstruction (RAO). We also believe that the purified antibodies
produced in the foal can be isolated and given to adult horses to
protect horses with a history of RAO (susceptible horses).
[0060] Foal #3
[0061] The basic protocol is as described for Foal #2. However,
before injecting the peptide, extra care was taken in cleaning the
injection site by scrubbing the site with Septisol antiseptic foam
(Septisol.sup.R, Steris Corp., Ohio) and alcohol. This additional
step reduced the swelling considerably over what was seen in Foal
#2. There was a small swelling in this foal which lasted only for
24 hours, probably due to the adjuvant. The adjuvant has an oily
consistency, which would facilitate slow absorption of the injected
solution.
[0062] Foal #3 is still being followed. The results to date are
shown in FIG. 5B. As shown in FIG. 5B, the antibody titer did not
increase until about day 15, similar to Foal #2. It then peaked on
day 22, and then again after the booster on day 29. The response
curve for this foal closely follows the response curve of Foal #2,
confirming the effectiveness of Peptide-1 in producing
antibodies.
[0063] Future Studies in Foals
[0064] Thus far, only a single dose and route have been tried. In
future studies with foals, a lower (0.03 mg/kg) and a higher dose
(0.3 mg/kg) of the peptide-1 will be tried to show what difference
in response can be expected with one log dose difference.
Administration of a higher dose and observation of the foal for
adverse reactions will also demonstrate if any toxicity exists with
the peptide. With large animals such as horses, determination of
LD.sub.50 (lethal dose for 50% of the animals tested) is not
usually performed. In addition, a nasal route of administering
Peptide-1 will be tried because of its convenience to use.
EXAMPLE 7
[0065] Use of Peptide-1 or its Antibodies to Protect Adult
Horses
[0066] Peptide-1 will be used to vaccinate adult horses, or
antibodies to Peptide-1 will be used for short term protection for
adult horses. Initial experiments will determine the effectiveness
of use of the peptide or its antibody to protect adult horses from
recurrent airway obstruction (RAO). Experiments to achieve the
following three objectives will be conducted: (1) a determination
of the antibody levels in horses that receive two subcutaneous
injections of the peptide one month apart; (2) a determination of
whether the horses can maintain a sufficient titer of antibody for
immunity from an injection given during the remission period; and
(3) a determination of whether horses that receive the peptide
injection or an injection of antibodies are resistant to RAO or
develop lesser symptoms of RAO.
[0067] Following approval from the IACUC, this study will be
performed in 6 horses that are known to be susceptible to RAO (3
for injection of Peptide-1 (the "active immunity" group), and 3 for
injection of antibodies (the "immunotherapy" group)). Horses with a
history of RAO will be obtained by donation to the LSU School of
Veterinary Medicine. Susceptibility to RAO in the horses will be
determined on the basis of their history of the disease, physical
examination, clinical scoring based on signs of RAO, pulmonary
function testing (determining transpleural pressure (Ppl)), and
bronchoalveolar (BAL) fluid examination. None of the horses will
have received medications within 10 days prior to the assessment.
Three horses will be injected with the peptide, and 3 will be
injected with antibody to Peptide-1. The antibodies will be
extracted from the blood of foals that were injected with Peptide-1
as described above. A onetime collection of blood of 450 ml from
each foal will be collected. If adult horses, as expected, maintain
high antibody titer values after injection of Peptide-1, antibody
from the adult horses can also be used. The sera with high antibody
titers value will be pooled for purification and antibody
extraction.
[0068] Techniques for Selection of Horses, Antibody Titer
Determination & Antibody Extraction.
[0069] Transpleural pressure determination: The change in
transpleural pressure (Ppl) will be measured indirectly as
previously described (31,34). Briefly, a 10-cm long 3.5-cm
circumference esophageal balloon will be secured at one end of a
catheter (PE tubing 350). The catheter will be connected to a
pressure transducer (model PESO) interfaced with a polygraph (Grass
model 7D). The balloon will be inserted through a lubricated
nasogastric tube (bigger tube) placed in the rostral esophagus.
Once the balloon is located between the heart and the diaphragm,
the nasogastric tube will be removed. The balloon will be inflated
with 1.5 ml of water, and 10 measurements will be recorded. Changes
in esophageal pressure (peak inspiratory pressure minus peak
expiratory pressure) during tidal breathing will reflect the change
in Ppl (measures as cm H.sub.2O). Horses with a Ppl of about 15 or
greater will be selected for the study. This measurement will help
confirm the horse has RAO and will get episodes of the disease
seasonally.
[0070] Bronchoalveolar lavage (BAL) fluid collection: Horses will
be sedated using a combination of xylazine hydrochloride (0.5
mg/kg, given intravenously (IV)) and butorphanol tartarate (11
mg/kg, IV). After placement of a nose twitch, a 244-cm (11-mm outer
diameter, 3-mm inner diameter) flexible silicone tube will be
passed through the nasal passage into the trachea to wedge in the
distal airway. As the silicone tube is advanced through the trachea
and carina, 36 mL of 1% lidocaine will be injected through the tube
and followed by 30 mL of air. The cuff will be inflated with 4 mL
of air, and five 60-mL aliquots of sterile 0.9% saline will be
infused manually with 60 mL syringes. Immediately after infusion,
bronchoalveolar lavage fluid (BALF) will be collected and pooled
into a sterile flask. Cytological analysis of BAL fluid will
demonstrate whether the horses have any other respiratory problem
(10,31)
[0071] Clinical Scoring: The horses will be assigned a clinical
score (CS). The CS is determined by the following algorithm (31,
34.
CS = ( Medial nostril flare + lateral nostril flare ) 2 + Abdominal
lift ##EQU00001##
[0072] Each of these variables in the formula is scored from 0 to
4. The zero score indicates the nostril has little movement, and
the ventral flank shows little or no movement. A score of 4
indicates the nostril remains maximally flared throughout the
respiratory cycle and abdominal lift resulting in a "heave line"
extending cranially to the 5.sup.th intercostal space. Thus, the
maximum CS is 8. Clinically healthy horses will have clinical
scores <4.0. RAO horses will have a score of >5.0. This
non-invasive procedure will show whether the animal has RAO.
[0073] Blood Collection: Foals that have previously injected with
Peptide-1 will be used to supply antibodies to Peptide-1 as
discussed above. A one-time sample of 450 ml blood will be
collected from each foal, all of whom are currently above 200 lb
body weight. For adult horses, 10 ml blood will be collected from
the adult horses before the injection (peptide or antibody) and
weekly thereafter until the season (June, July, August) is
over.
[0074] Antibody extraction: Total IgG antibody from hyper-immune
sera collected from foals will be purified by Protein-A/G affinity
chromatography, by established procedures (2). Briefly, IgG from
sera will be allowed to bind to Protein-A/G conjugated agarose
beads in a chromatography column. After washing off all unbound
serum proteins, Protein-A/G bound IgG will be eluted out by
lowering pH to 2.5. Antibodies will stored in -80.degree. C.
[0075] Antibody titer determination by ELISA: Relative antibody
titer will be determined by sandwich ELISA by a well-established
protocol (17), and as described above. Briefly, ELISA plate will be
coated with Peptide-1. Serial 10 fold dilutions of sera up to
100,000 will be used as a sample. After washing all unbound
proteins from the plate, the amount of bound IgG specific to
Peptide-1, will be determined by a secondary antibody,
peroxidase-conjugated goat anti-horse IgG (17).
[0076] Experimental Protocol: Eight adult horses that have a
history of RAO will be clinically scored and pulmonary function
tested during the active RAO period (June, July, August & may
be September). This will confirm their susceptibility to RAO. The
horses will remain in the pasture for the complete RAO season.
After the RAO season, the horses will begin to undergo remission,
and by November, will not show active signs of RAO. Without
treatment, the RAO will return in the next RAO season, starting in
June. This initial season of monitoring will allow each horse to be
used as its own control.
[0077] One month before the beginning of the second RAO season, 3
horses will be given antibodies at a dose of 1 mg/kg body weight
subcutaneously. A day before the injection, a horse will be brought
to a bedded stall. The horse will be fed hay and concentrates; and
water will be given ad libitum. A 10-ml blood sample will be drawn
for determining CBC and antibody titer. A thorough physical
examination including body temperature, heart rate, respiratory
rate, body weight, body score, sex, breed age and general physical
conditions will be recorded.
[0078] The following day, purified antibody at a dose of 1 mg/kg
body weight will be administered subcutaneously. The horse will be
monitored for any abnormal reactions for 4 h continuously. The
horse will be checked for pain and swelling at the site of
injection. If the animal shows abnormal reactions such as sweating,
high fever, cough, seizure or difficulty in breathing, it will be
treated symptomatically. After 4 h, it will be observed once every
hour until 8 h. The horse will be housed in the stall for 3 days.
If everything is normal, the horse will be released back to the
pasture. The remaining two horses will be treated and monitored
similarly and released after 3 day if everything goes well. Blood
(10 ml) will be collected weekly from each horse. Any horse
reacting to the antibody will be treated with analgesics,
corticosteroids and hot packs while in the stall.
[0079] The remaining three horses will be given Peptide-1/KLH and
adjuvant subcutaneously at a dose of 0.1 mg/kg, the same
composition and dose used for foals as described above. In foals,
this dose induced antibody production and did not produce severe
reactions. Blood (10 ml) will be collected before the injection
(for control value). After the peptide injection, 10 ml blood will
be collected every 7.sup.th day for the RAO season to determine
antibody titer. Peptide injection will be performed in a stall, and
the horses will be closely monitored for 3 days. If everything goes
well with the horses, they will be released to the pasture. Their
ability to resist RAO will be weekly observed during the
period.
[0080] Analysis of results: The data will be analyzed for
statistical difference between the groups and between the treated
and untreated horse using a two-tailed repeated ANOVA for the titer
values. If the significance is indicated, a post-hoc analysis will
be performed using Tukey's test. Significance will be set at a "p"
value of <0.05 for all tests. It is expected that the horses
given either Peptide-1 or its antibodies will have fewer symptoms
and less severe symptoms of RAO during the RAO season.
EXAMPLE 8
[0081] Vaccination of Guinea Pigs with Peptide-1
[0082] We believe that other mammals can be treated with Peptide-1
or its antibodies to decrease the symptoms of asthma, including
humans. Guinea pigs, a common animal model for human asthma, will
be used. Guinea pigs will be vaccinated with Peptide-1 to see if
Peptide-1 can help protect guinea pigs from getting asthma
(experimentally induced asthma). This study will determine the
antibody levels in guinea pigs injected twice (an initial injection
and a 4-week booster injection) with Peptide-1 at a dose of 0.1
mg/kg subcutaneously, will determine the effectiveness of
antibodies to Peptide-1 in protecting guinea pigs from
experimentally induced asthma, and will determine the protection of
guinea pigs from experimentally induced asthma by vaccination with
Peptide-1.
[0083] Description of Investigation: Following approval from the
IACUC, this study will be performed in 3 groups of healthy guinea
pigs (10 guinea pigs in each group). In group #1, guinea pigs will
be injected with Peptide-1/KLH and adjuvant initially and then
again 4 weeks later (the booster injection. Blood (2 ml) will be
drawn from the jugular vein to determine antibody titer. Blood
samples will be collected before the initial injection of the
peptide, and then once every week for two months. The blood samples
will be used to determine the level of antibody titer. If the
guinea pigs have sufficient level of antibody, they will be used
for antibody extraction.
[0084] The second group of guinea pigs will be used for injecting
antibodies to Peptide-1 at a dose of 1 mg/kg body weight. These
animals will be observed for asthma signs. It is expected that the
asthma signs from experimentally induced asthma will be reduced
from the injection of the antibodies. The third group will be used
to vaccinate with the peptide (first injection and booster
injections). This group will be tested for protection from the
experimental asthma as did with the second group.
[0085] Experimental Induction of asthma: Asthma will be induced in
guinea pigs by a method previously described (16). Healthy Guinea
pigs will be procured from a USDA licensed dealer and housed in a
regulated animal facility. Animals will be weighed and prepared for
sterile injection of ovalbumin. Blood (2 ml) will be collected from
the jugular vein.
[0086] The animals will be sensitized by an intraperitoneal
injection of ovalbumin at a dose of 100 mg/Kg body weight. The
sensitized animals will be housed in separate cages and
continuously observed for any clinical, physiological or behavioral
alterations. On the 10.sup.th day after injection, the animals will
be weighed. Then the animals will be challenged with ovalbumin at a
lower dose of 100 ug/kg body weight (challenging dose)
intraperitoneally (25). In sensitized animals, this challenging
dose will cause an asthmatic response such as difficulty in
breathing, increased effort to exhale air, gasping for air, rough
coat, and curving of body. If administration of Peptide-1 can
produce antibodies (active immunotherapy) or of antibodies to
Peptide-1 (passive immunotherapy) can prevent guinea pigs from
developing asthma signs, it will support the therapeutic use for
asthma in mammals of the peptide or the antibody to the Peptide,
including use in humans.
[0087] Blood Collection: Blood (2 ml) will be collected from
jugular vein from guinea pigs sedated with barbiturates.
[0088] Antibody extraction: Total IgG antibody from hyper-immune
sera collected from guinea pigs will be purified by Protein-A/G
affinity chromatography, by established procedures (2). Briefly,
IgG from sera will be allowed to bind to Protein-A/G conjugated
agarose beads in a chromatography column. After washing off all
unbound serum proteins, Protein-A/G bound IgG will be eluted out by
lowering pH to 2.5. Antibodies will stored in -80.degree. C.
[0089] Antibody titer determination by ELISA: Relative antibody
titer will be determined by sandwich ELISA by a previously
described method (17), and as described above. Briefly, ELISA plate
will be coated with peptide. Serial 10-fold dilutions of sera up to
100,000 will be used as sample. After washing all unbound proteins,
the amount of bound IgG specific to the peptide, will be determined
by a secondary antibody, peroxidase-conjugated goat anti-guinea pig
IgG (17).
[0090] Analysis of results: The data will be analyzed for
statistical difference between the experimental groups and the
control group using a two-tailed repeated ANOVA for the titer
values. If the significance is indicated, a post-hoc analysis will
be performed using Tukey's test. Significance will be set at a "p"
value of <0.05 for all tests.
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M. A., Aviza G. IgG antibody responses to an inhaled antigen in
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B., Bjorck L. (1986) A physicochemical study of protein G, a
molecule with unique immunoglobulin G-binding properties. J. Biol.
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Inflammatory mediators of asthma. Pharmacol. Rev., 40(1):49-84
(1988). [0094] 4. Beadle R. E. Summer pasture-associated
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[0128] The complete disclosures of all references cited in this
application are hereby incorporated by reference. In the event of
an otherwise irreconcilable conflict, however, the present
specification shall control.
Sequence CWU 1
1
2121PRTArtificial sequenceArtificial peptide 1Lys Asn Thr Lys Asp
Ser Ile Leu Lys Leu Met Asp Lys Ile Ala Lys 1 5 10 15 Ser Pro Leu
Cys Ala 20 220PRTArtificial sequenceSynthetic peptide 2Glu Pro Ser
Lys Pro Asp Ala Asp Met Lys Ala Ala Thr Thr Gln Leu 1 5 10 15 Lys
Thr Leu Val 20
* * * * *