U.S. patent application number 16/801696 was filed with the patent office on 2020-09-24 for compositions and methods for treatment in broad-spectrum, undifferentiated or mixed clinical applications.
The applicant listed for this patent is PANTHERYX, INC.. Invention is credited to Timothy W. Starzl.
Application Number | 20200299360 16/801696 |
Document ID | / |
Family ID | 1000004882085 |
Filed Date | 2020-09-24 |
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United States Patent
Application |
20200299360 |
Kind Code |
A1 |
Starzl; Timothy W. |
September 24, 2020 |
COMPOSITIONS AND METHODS FOR TREATMENT IN BROAD-SPECTRUM,
UNDIFFERENTIATED OR MIXED CLINICAL APPLICATIONS
Abstract
The disclosure provides improved compositions and methods for
passive immunization. In embodiments, a composition comprising a
synergistic combination of specific polyclonal antibodies in a
carrier matrix is provided. The disclosure provides effective,
economical compositions and methods for the treatment of diarrhea
and enteric infections in broad-spectrum, undifferentiated, or
mixed clinical applications.
Inventors: |
Starzl; Timothy W.;
(Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PANTHERYX, INC. |
Boulder |
CO |
US |
|
|
Family ID: |
1000004882085 |
Appl. No.: |
16/801696 |
Filed: |
February 26, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15616724 |
Jun 7, 2017 |
10611828 |
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16801696 |
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13302836 |
Nov 22, 2011 |
9701735 |
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15616724 |
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61416667 |
Nov 23, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/70 20130101;
C07K 16/1232 20130101; C07K 2317/11 20130101; C07K 16/121 20130101;
C07K 16/10 20130101; C07K 16/1282 20130101; A61K 2039/545 20130101;
A61K 2039/507 20130101 |
International
Class: |
C07K 16/10 20060101
C07K016/10; C07K 16/12 20060101 C07K016/12 |
Claims
1. A dosage form comprising a composition for administration to a
non-neonate human in need thereof, the composition comprising: a) a
non-neonate effective amount of at least one antigen specific
antibody, or antigen-binding fragment thereof, obtained from a
first nonhuman animal; and, b) a carrier matrix comprising
colostrum, or at least two components thereof obtained from a
second nonhuman animal, wherein the at least two components are
selected from the group consisting of enzymes, lactoferrin,
transferrin, nonspecific immunoglobulins, cytokines, white blood
cells, complement components, interferons, growth factors, and
fibronectin, wherein the at least one antigen-specific antibody or
antigen-binding fragment thereof and the colostrum or the at least
two components of the carrier matrix are obtained from different
non-human animals.
2. The dosage form of claim 1, wherein the carrier matrix comprises
bovine colostrum.
3. The composition of claim 1, wherein the at least two components
of the carrier matrix are selected from the group consisting of
lysozyme, phospholipase, defensins, opsonins, nonspecific
immunoglobulins, proline-rich polypeptides (PRPs), components of
the complement system, beta-lysin, lactoferrin, lactoperoxidase,
transferrin, cytokines, interleukins, chemokines, interferons,
TNF-alpha, fibronectin, leukocytes, white blood cells, phagocytes,
macrophages, monocytes, neutrophils, polymorphonuclear cells,
dendritic cells, mast cells, eosinophils, basophils, natural killer
(NK) cells, lymphokine activated killer (LAK) cells, elastase,
cathepsin G, myeloperoxidase, NADPH oxidase, insulin-like growth
factor I, insulin-like growth factor II, transforming growth factor
alpha, transforming growth factor beta 1, transforming growth
factor beta 2, fibroblast growth factors, epidermal growth factor,
granulocyte-macrophage stimulating growth factor, platelet-derived
growth factor, vascular endothelial growth factor,
colony-stimulating factor-I, leptin, hepatocyte growth factor, and
combinations thereof.
4. The dosage form of claim 1, wherein the antigen is present in or
is derived from a bacterial or viral pathogen, a pathogen related
toxin, a pathogen related adhesion element, undesirable strain, or
a combination thereof.
5. The dosage form of claim 4, wherein the bacterial or viral
pathogen is a human or veterinary, enteric or gastrointestinal,
pathogen capable of causing gastroenteritis.
6. The dosage form of claim 5, wherein the bacterial or viral
pathogen is selected from the group consisting of: Campylobacter
jejuni, Salmonella, Salmonella typhimurium, Salmonella enterica
serovar Typhi, Shigella dystenteriae, Plesiomonas shigelloides,
Escherichia coli, enteropathogenic E. coli, enterotoxigenic E.
coli, enteroaggregative E. coli, enteroinvasive E. coli,
haemorrhagic E. coli, Clostridium difficile, Yersinia
enterocolitica, Vibrio cholerae O1, Vibrio O139, Non-O1 Vibrios,
Vibrio parahaemolyticus, Aeromonas hydrophila, Clostridium
perfringens, enterohepatic Helicobacter, Helicobacter pylori,
Staphylococcus aureus, Klebsiella, Gardnerella spp., Neisseria
gonorrhoeae, Chlamydiaceae trachomatis, Mycoplasma spp.,
Campylobacter jejuni, Trichomonas vaginalis, herpes virus type 1,
herpes virus type 2, Candida albicans, Candida glabrata, Candida
tropicalis, Candida parapsilosis and Candida krusei, Group A
Streptococcus spp., rotavirus, coronavirus, norovirus, calicivirus,
enteric adenovirus, cytomegalovirus, astrovirus, S. pneumoniae, H.
influenzae, herpes zoster virus, Fusarium spp., and Acanthamoeba
spp.
7. The dosage form of claim 6, wherein the at least one
antigen-specific antibody, or antigen-binding fragment thereof
comprise a mixture of polyclonal antibodies that are specific for
one or more antigens present in the bacterial and/or viral
pathogen, pathogen related toxin, or pathogen related adhesion
element, derived from one, two, three, four, five, six, seven, or
eight, or more, different pathogenic microorganisms.
8. The dosage form of claim 4, wherein the pathogen related toxin
comprises an endotoxin or exotoxin.
9. The dosage form of claim 4, wherein the pathogen related
adhesion element comprises adhesins, cadherins, cilia, fimbrillae,
a viral adhesion structure, or a combination thereof.
10. (canceled)
11. The dosage form of claim 1, wherein the at least one antigen
specific antibody or antigen-binding fragment thereof is selected
from a mixture of polyclonal antibodies or a monoclonal
antibody.
12. The dosage form of claim 11, wherein the at least one antibody
is an IgG.
13. The dosage form of claim 11, wherein the at least one antibody
is an IgY.
14. The dosage form of claim 11, wherein the at least one antibody
is a mixture of polyclonal antibodies.
15. The dosage form of claim 2, wherein the bovine colostrum is
non-hyperimmune bovine colostrum.
16. The dosage form of claim 2, wherein the bovine colostrum is
full fat bovine colostrum.
17. The dosage form of claim 1, wherein the antigen specific
antibody or antigen-binding fragment thereof is in a solid
form.
18. The dosage form of claim 17, wherein the antigen specific
antibody or antigen-binding fragment thereof is crystalline.
19. The dosage form of claim 1, wherein the carrier matrix is in a
solid form.
20. The dosage form of claim 1 further comprising a
pharmaceutically acceptable diluent, binder, excipient, lubricant,
sweetening agent, flavoring agent, wetting agent, absorbent, and/or
retarding agent.
21.-23. (canceled)
24. A method for the treatment or prevention of a pathogenic
infection or undesirable strain of microorganisms in a non-neonate
human in need thereof; the method comprising administration of a
composition comprising: a) a non-neonate effective amount of at
least one antigen specific antibody, or antigen-binding fragment
thereof, obtained from a first nonhuman animal; and, b) a carrier
matrix comprising colostrum or at least two components thereof
obtained from a second nonhuman animal, wherein the at least two
components are selected from the group consisting of enzymes,
lactoferrin, transferrin, nonspecific immunoglobulins, cytokines,
white blood cells, complement components, interferons, growth
factors, and fibronectin, wherein the at least one antigen specific
antibody or antigen-binding fragment thereof and the colostrum or
at least two components of the carrier matrix are obtained from
non-human different animals.
25. The method of claim 24, wherein the pathogenic infection is
selected from the group consisting of undifferentiated diarrhea,
traveler's diarrhea, rotavirus diarrhea, toxin-mediated diarrhea,
cholera, C. difficile infection, dysentery, typhoid fever, and
peptic ulcers.
26. The method of claim 25, wherein the undifferentiated diarrhea
is pediatric undifferentiated diarrhea.
27. The method of claim 24, wherein the composition is administered
in an amount effective for conferring passive immunity to a
subject.
28. The method of claim 24, wherein the treatment or prevention of
an undesirable strain of microorganisms is used for
gastrointestinal flora management.
29. The method of claim 24, wherein the pathogenic infection is
caused by a microorganism selected from the group consisting of:
Campylobacter jejuni, Salmonella, Salmonella typhimurium,
Salmonella enterica serovar Typhi, Shigella dystenteriae,
Plesiomonas shigelloides, Escherichia coli, enteropathogenic E.
coli, enterotoxigenic E. coli, enteroaggregative E. coli,
enteroinvasive E. coli, haemorrhagic E. coli, Clostridium
difficile, Yersinia enterocolitica, Vibrio cholerae O1, Vibrio
O139, Non-O1 Vibrios, Vibrio parahaemolyticus, Aeromonas
hydrophila, Clostridium perfringens, enterohepatic Helicobacter,
Helicobacter pylori, Staphylococcus aureus, Klebsiella, Gardnerella
spp., Neisseria gonorrhoeae, Chlamydiaceae trachomatis, Mycoplasma
spp., Campylobacter jejuni, Trichomonas vaginalis, herpes virus
type 1, herpes virus type 2, Candida albicans, Candida glabrata,
Candida tropicalis, Candida parapsilosis and Candida krusei, Group
A Streptococcus spp., rotavirus, coronavirus, norovirus,
calicivirus, enteric adenovirus, cytomegalovirus, astrovirus, S.
pneumoniae, H. influenzae, Neisseria gonorrhoeae, herpes zoster
virus, Fusarium spp., and Acanthamoeba spp.
30. (canceled)
31. The method of claim 24, wherein the colostrum is full fat
bovine colostrum.
32. The method of claim 24, wherein the colostrum is
non-hyperimmune bovine colostrum.
33. The method of claim 24, wherein the at least two components of
the carrier matrix are selected from the group consisting of
lysozyme, phospholipase, defensins, opsonins, nonspecific
immunoglobulins, proline-rich polypeptides (PRPs), components of
the complement system, beta-lysin, lactoferrin, lactoperoxidase,
transferrin, cytokines, interleukins, chemokines, interferons,
TNF-alpha, fibronectin, leukocytes, white blood cells, phagocytes,
macrophages, monocytes, neutrophils, polymorphonuclear cells,
dendritic cells, mast cells, eosinophils, basophils, natural killer
(NK) cells, lymphokine activated killer (LAK) cells, elastase,
cathepsin G, myeloperoxidase, NADPH oxidase, insulin-like growth
factor I, insulin-like growth factor II, transforming growth factor
alpha, transforming growth factor beta 1, transforming growth
factor beta 2, fibroblast growth factors, epidermal growth factor,
granulocyte-macrophage stimulating growth factor, platelet-derived
growth factor, vascular endothelial growth factor,
colony-stimulating factor-I, leptin, hepatocyte growth factor, and
combinations thereof.
34. The method of claim 33, wherein the at least two components of
the carrier matrix include a growth factor and an antimicrobial
factor.
35. The method of claim 24, wherein the antigen is present in or is
derived from a bacterial or viral pathogen, a pathogen related
toxin, a pathogen related adhesion element, undesirable strain, or
a combination thereof.
36. The method of claim 35, wherein the bacterial or viral pathogen
is a human or veterinary, enteric or gastrointestinal, pathogen
capable of causing gastroenteritis.
37. The method of claim 36, wherein the bacterial or viral pathogen
is selected from the group consisting of: Campylobacter jejuni,
Salmonella, Salmonella typhimurium, Salmonella enterica serovar
Typhi, Shigella dystenteriae, Plesiomonas shigelloides, Escherichia
coli, enteropathogenic E. coli, enterotoxigenic E. coli,
enteroaggregative E. coli, enteroinvasive E. coli, haemorrhagic E.
coli, Clostridium difficile, Yersinia enterocolitica, Vibrio
cholerae O1, Vibrio O139, Non-O1 Vibrios, Vibrio parahaemolyticus,
Aeromonas hydrophila, Clostridium perfringens, enterohepatic
Helicobacter, Helicobacter pylori, Staphylococcus aureus,
Klebsiella, Gardnerella spp., Neisseria gonorrhoeae, Chlamydiaceae
trachomatis, Mycoplasma spp., Trichomonas vaginalis, herpes virus
type 1, herpes virus type 2, Group A Streptococcus spp., rotavirus,
coronavirus, norovirus, calicivirus, enteric adenovirus,
cytomegalovirus, astrovirus, S. pneumoniae, H. influenzae, herpes
zoster virus.
38. The method of claim 37, wherein the bacterial or viral pathogen
is selected from the group consisting of E. coli, rotavirus, and
coronavirus.
39. The method of claim 37, wherein the at least one
antigen-specific antibody, or antigen-binding fragment thereof
comprise a mixture of polyclonal antibodies that are specific for
one or more antigens present in the bacterial and/or viral
pathogen, pathogen related toxin, or pathogen related adhesion
element, derived from one, two, three, four, five, six, seven, or
eight, or more, different pathogenic microorganisms.
40. The method of claim 39, wherein the mixture of polyclonal
antibodies comprise IgY antibodies specific for at least
enterotoxigenic E. coli spp., E. coli K99 pili adherence factor,
Clostridium perfringens toxoid, Salmonella typhimurium, rotavirus,
and coronavirus.
41. The dosage form of claim 1, wherein the dosage form is in a
form selected from the group consisting of powder, tablet, capsule,
troche, or liquid.
42. The dosage form of claim 1, wherein the dosage form is a solid
dosage form.
43. The dosage form of claim 1, wherein one dose of the composition
comprises from 1 g to 7 g dried immune egg and from 1 g to 7 g
dried bovine colostrum.
44. The dosage form of claim 1, that is an oral dosage form.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/616,724, filed Jun. 7, 2017, which is a continuation of U.S.
application Ser. No. 13/302,836, filed Nov. 22, 2011, now U.S. Pat.
No. 9,701,735, issued Jul. 11, 2017, which claims the benefit of
U.S. Provisional Application No. 61/416,667, filed Nov. 23, 2010,
to Timothy W. Starzl, of Boulder, Colo., entitled "Compositions and
Methods for Treatment in Broad-Spectrum, Undifferentiated or Mixed
Clinical Applications", the entire contents of each of which is
incorporated herein by reference.
FIELD OF THE DISCLOSURE
[0002] The disclosure provides compositions and methods for passive
immunization. In embodiments, compositions comprising a synergistic
combination of specific polyclonal antibodies with a carrier matrix
are provided. The disclosure provides effective and economical
compositions and methods for the treatment of pathogenic infections
in broad-spectrum, undifferentiated, or mixed clinical
applications. In one embodiment, compositions and methods for the
treatment of diarrhea and enteric infections are provided.
BACKGROUND OF THE DISCLOSURE
[0003] Antibodies, immunoglobulins, and other biological immune
factors (referred to here collectively as antibodies), both natural
and their synthetic analogues, are known therapeutic agents in
humans and animals. Antibodies operate by binding (via non-covalent
forces) between the antigen combining site on the antibody and a
portion of the antigen called the antigenic determinant or epitope.
Antibodies are capable of high degrees of specificity. For example,
the field of monoclonal antibodies has developed largely under the
impetus of producing ever more specific and precise binding
characteristics. However, this high specificity can lead to
excessively limited binding attributes, where agents or antigens
that are functionally identical do not react identically with the
immunoreagent or immunotherapeutic. Cross-reactivity on the other
hand, usually considered an error or failure to achieve binding
specificity., is the reaction between an antigen and an antibody
that was generated against a similar but different antigen.
Controlled cross-reactivity may constructively be used to broaden
the binding range of the antibody.
[0004] Colostrum has evolved naturally in mammals specifically to
deliver its components to neonates to and through the
gastrointestinal tract in a very concentrated low-volume form.
Colostrum is known to contain antibodies such as IgA, IgG, and IgM.
Other components of colostrum include lactoferrin, lysozyme,
lactoperoxidase, complement, and proline-rich polypeptides (PRP). A
number of cytokines (small messenger peptides that control the
functioning of the immune system) are found in colostrum as well,
including interleukins, tumor necrosis factor, chemokines, and
others. Colostrum also contains a number of growth factors, such as
insulin-like growth factors I, and II, transforming growth factors
alpha, beta 1 and beta 2, fibroblast growth factors, epidermal
growth factor, granulocyte-macrophage stimulating growth factor,
platelet-derived growth factor, vascular endothelial growth factor,
and colony-stimulating factor-I.
[0005] The antibodies and cofactors in colostrum can, through
breast feeding provide a passive immunity to the recipient.
Normally antibodies and cofactors are passed to the neonate from
the mother and provide the first protection against pathogens.
Growth factors also stimulate the development and repair of the
gut.
[0006] One condition that could be addressed by using passive
immunity is diarrhea. Diarrhea is caused mainly by the ingestion of
pathogens. According to the World Health Organization (WHO),
eighty-eight percent of cases diarrhea worldwide are attributable
to unsafe water, inadequate sanitation or insufficient hygiene.
These cases result in about 1.5 million deaths each year, most
being the deaths of children. (Pruss-Urstun et al., Safer water,
better health: costs, benefits and sustainability of interventions
to protect and promote world health. World Health Organization,
Geneva, 2008. ISBN 978 92 4 1596435).
[0007] Of particular global concern are the instances of infectious
diarrhea in the developing world, which are a cause of tremendous
ongoing morbidity and mortality, particularly in the pediatric
population. For example, India has one of the highest infant
mortality rates in the world according to a 2009 United Nations
Human Development report. For example, Save the Children, a global
non-profit, reports that one child dies every 15 seconds in India,
and 90% of these deaths are due to preventable diseases, such as
diarrhea. Rotavirus and measles vaccines, handwashing with soap,
improved drinking water supply and community-wide sanitation are
recommended by WHO for the prevention of diarrhea; however, these
measures are not effective to treat the disease.
[0008] Standard treatment protocol in much of the world for
pediatric diarrhea includes a concomitant administration of
antibiotics and oral rehydrative therapy. For many reasons,
antibiotics are a prescription drug. Antibiotics are not effective
in the treatment of viral infection. For example, rotavirus is
estimated to cause about 40 percent of all hospital admissions due
to diarrhea among children under five years of age worldwide.
(Weekly Epidemiological Record, vol. 83, no. 47, 21 Nov. 2008). The
inappropriate use of antibiotics can promote resistant strains of
bacteria. Conversely, the infection may be caused by a resistant
strain of bacteria. Even under the best of circumstances, use of an
appropriate antibiotic may take several days to reduce the severity
of the symptoms of diarrhea.
[0009] Another disadvantage of antibiotics is that administration
can induce the destruction of both pathogenic and benign bacteria
found in the GI tract which can further result in release of
endotoxic lipopolysaccharides. (Holzheimer, The significance of
endotoxin release in experimental and clinical sepsis in surgical
patients--evidence for antibiotic-induced endotoxin release?
Infection. 1998 March-April; 26(2):77-84). These endotoxins have a
host of adverse systemic effects including fever, changes in white
blood cell counts, disseminated intravascular coagulation,
hypotension, shock and death, malabsorption; in fact, the direct
injection of fairly small doses of endotoxin results in death in
most mammals. Todar K. Bacterial Endotoxin. Textbook of
Bacteriology. 2008. textbookofbacteriology.net.
[0010] According to WHO, oral rehydration therapy and zinc with
continued feeding, including breastfeeding, is recommended for
treatment of childhood diarrhea. Zinc syrup or zinc-fortified oral
rehydration solution (ORS, 40 mg/L) is typically employed at a dose
of about 15 to 30 mg per day. Zinc is inexpensive, but has modest
efficacy. Zinc syrup results in only about a 25 percent reduction
in duration of acute diarrhea, and a 40 percent reduction in
treatment failure or death. (Bhutta et al. Therapeutic effects of
oral zinc in acute and persistent diarrhea in children in
developing countries: pooled analysis of randomized controlled
trials. The American Journal of Clinical Nutrition. 2000;
72(6):1516-22). One study evaluated the efficacy and safety of a
zinc-fortified (40 mg/L) ORS among 1,219 children with acute
diarrhea. Clinical outcomes among the zinc-fortified ORS group were
modestly improved, compared with those for the control group, who
received standard ORS only. In that study, the total number of
stools was lower among the zinc-ORS group compared with the total
number for the control group. No substantial effect on duration of
diarrhea or risk for prolonged diarrhea was noted. (Bahl R,
Bhandari N, Saksena M, et al. Efficacy of zinc-fortified oral
rehydration solution in 6- to 35-month-old children with acute
diarrhea. J Pediatr 2002; 141:677-82).
[0011] It is known that antibiotics are ineffective to treat a
viral infection, such as a rotavirus infection. Other interventions
have limited effectiveness. Additionally, appropriate diagnostic
tools to distinguish the cause of diarrhea are not always readily
available or affordable.
[0012] Clearly a rapid, effective and economical alternative for
the treatment of undifferentiated diarrhea is desirable. There
remains a need for effective, economical compositions and methods
for treatment of diarrhea and enteric infections in broad-spectrum,
undifferentiated, or mixed clinical applications.
SUMMARY OF THE DISCLOSURE
[0013] The disclosure provides compositions and methods of passive
immunization wherein a specific binding molecule, such as a
specific immunoglobulin, is combined with a carrier matrix to
provide a composition for oral or mucosal administration for
management of microorganisms; including treatment or prophylaxis of
a pathogenic infection or undesirable strain. In embodiments, the
compositions are administered to a non-neonatal subject.
[0014] In one embodiment, the disclosure provides a composition for
administration to a non-neonate human for the management of
microorganisms, the composition comprising at least one specific
binding molecule, or fragment thereof, derived from the adaptive
immune system of an animal, wherein the specific binding molecule
is selected from an immunoglobulin, antibody, peptide, variable
lymphocyte receptor, transfer factor, or a mixture thereof; and a
carrier matrix comprising two or more components of the innate
immune system of a non-human mammal, wherein the matrix can be
selected from, or derived from the components of, colostrum, milk,
serum, plasma, saliva, lymph fluid, mucous, or lachrymal fluid;
wherein the matrix and the specific binding molecule are derived
from different species.
[0015] In a preferred embodiment, the carrier matrix comprises
bovine colostrum. In another embodiment, the matrix comprises the
components of the innate immune system that are selected from
lysozyme, phospholipase, defensins, opsonins, components of the
complement system, beta-lysin, protein-rich peptides (PRP), (PRPs),
lactoferrin, transferrin, cytokines, interleukins, chemokines,
interferons, TNF-alpha, fibronectin, leukocytes, white blood cells,
phagocytes, macrophages, monocytes, neutrophils, polymorphonuclear
cells, dendritic cells, mast cells, eosinophils, basophils, natural
killer (NK) cells, lymphokine activated killer (LAK) cells,
defensins, elastase, cathepsin G, myeloperoxidase, and NADPH
oxidase.
[0016] In various embodiments, the composition includes a
pharmaceutically acceptable carrier. In other embodiments, the
composition includes a food grade carrier. In embodiments, the
compositions can be administered via oral delivery, nasal delivery,
ocular delivery or combinations thereof.
[0017] In other embodiments, the composition does not include an
exogenously added polymer, copolymer, liposome, hydrogel or fibrin.
In other embodiments, the composition does not include microspheres
or microcapsules. In yet a further embodiment, the composition does
not include an exogenously added antigen.
[0018] In a further embodiment, the specific binding molecules
specifically bind to a pathogen, a pathogen related toxin, a
pathogen related adhesion element, undesirable strain, or a
combination thereof. In one aspect, the pathogen comprises a human
or veterinary, enteric or gastrointestinal, pathogen causing
gastroenteritis.
[0019] In various aspects, the pathogen or undesirable strain is
selected from the group consisting of: Campylobacter jejuni,
Salmonella, Salmonella enterica serovar Typhi, Shigella
dystenteriae, Plesiomonas shigelloides, Escherichia coli [including
(EPEC) enteropathogenic E. coli, (ETEC) enterotoxigenic E. coli,
(EaggEC) enteroaggregative E. coli, (EIEC) enteroinvasive E. coli,
and (EHEC) haemorrhagic E. coli], Clostridium difficile, Yersinia
enterocolitica, Vibrio cholerae O1, Vibrio O139, Non-O1 Vibrios,
Vibrio parahaemolyticus, Aeromonas hydrophila, Clostridium
perfringens, Clostridium difficile, enterohepatic Helicobacter
(including Helicobacter pylori), Staphylococcus aureus, Klebsiella,
rotavirus, coronavirus, norovirus, calicivirus, enteric adenovirus,
cytomegalovirus, astrovirus, S. pneumoniae, H. influenzae,
Neisseria gonorrhoeae, herpes zoster virus, Fusarium spp., and
Acanthamoeba spp.
[0020] In a specific aspect, the pathogen related toxin comprises
an endotoxin or exotoxin.
[0021] In another specific aspect, the pathogen related adhesion
element comprises adhesins, cadherins, cilia, fimbrillae, a viral
adhesion structure, or a combination thereof.
[0022] In various embodiments, the composition is administered
orally in an amount effective for the treatment or prevention of
undifferentiated diarrhea, traveler's diarrhea, rotavirus diarrhea,
toxin-mediated diarrhea, cholera, C. difficile infection,
dysentery, typhoid fever, peptic ulcers, or for gastrointestinal
flora management. In another aspect, an effective amount of the
composition confers passive immunity to a subject.
[0023] In another embodiment, the disclosure provides a method for
preparing the composition of the disclosure by the steps of: (a)
obtaining from an animal at least one specific binding molecule or
fragment thereof that binds to a specific antigen, wherein the
specific binding molecule is selected from an immunoglobulin,
antibody, peptide, variable lymphocyte receptor, transfer factor,
and a mixture thereof; (b) obtaining at least one carrier matrix,
comprising at least two components obtained from a nonhuman animal
selected from the group consisting of enzymes, lactoferrin,
transferrin, nonspecific immunoglobulins, cytokines, white blood
cells, complement components, interferons, and fibronectin; (c)
preparing a solid form of the carrier matrix and of the specific
binding molecule or fragment thereof; and (d) mixing the solid form
of the carrier matrix with the solid form of the specific binding
molecule or fragment thereof.
[0024] In another embodiment, the present invention provides a
method for preparing an immunity conferring composition. The method
includes (a) obtaining at least one exogenously sourced
specifically targeted immune factor; (b) preparing a powderized
form of the at least one exogenously sourced specifically targeted
immune factor; (c) obtaining at least one exogenously sourced
carrier matrix, optionally mixed the exogenously sourced carrier
matrix with a mixture of agents to support and interact with the
exogenously sourced specifically targeted immune factor; (d)
preparing a powderized form of the at least one exogenously sourced
carrier matrix; and (e) mixing the powderized form of step (b) with
the powderized form of step (d), thereby obtaining the passive
immunity conferring composition. In one aspect, the passive
immunity conferring composition includes a dose controlled
formulation. In various aspects, the passive immunity conferring
composition includes a pharmaceutically acceptable carrier. In
various aspects, the passive immunity conferring composition does
not include a polymer, copolymer, liposome, hydrogel, or fibrin. In
various aspects, the passive immunity conferring composition does
not include microspheres or microcapsules. In various aspects, the
passive immunity conferring composition does not include an
immunogen or antigen.
[0025] The present invention includes at least one of the following
distinguishing attributes: (a) it enables customized design of the
matrix, specific factors, and the activating events for specified
or targeted diseases; (b) it enables dose controlled formulation of
a variety of mixtures of components, which may be tuned or adjusted
for effect; (c) it enables dose controlled formulation that
provides specified components in excess of normal physiological
levels that can be achieved in natural systems; (d) it uses complex
multi-component multi-pathway interactions to create a systems
effect that emulates a native immune system response; (e) it
enables creation of a preconditioned or potentiated passive immune
response that can be administered in its potentiated state, and
subsequently activated by the presence of the target pathogens,
toxins, disease state, or syndrome; (f) it enables the creation of
formulations that have a defined specificity or broad-spectrum
effect, to match the needs of the specific target disease state or
syndrome, or of the practice environment within which the product
is to be used; and (g) it enables the creation of formulations that
can be targeted for prophylaxis as well as for therapeutic
intervention.
[0026] In another aspect, by adjusting the amounts of the specific
binding molecules, such as polyclonal antibodies, in the
composition a dose controlled formulation can be prepared.
[0027] In a preferred embodiment, the at least one specific binding
molecule comprises IgY derived from immunized chickens In other
specific aspects, the IgY comprises a pool of IgY specific for at
least enterotoxigenic E. coli spp., E. coli K99 pili adherence
factor, Clostridium perfringens toxoid, Salmonella typhimurium,
rotavirus, and coronavirus.
[0028] In another embodiment, the composition is topically
administered to a mucosal membrane.
[0029] In another embodiment, the pathogen comprises a pathogen
causing vaginitis. In various aspects, the pathogen is selected
from the group consisting of: Gardnerella spp., Neisseria
gonorrhoeae, Chlamydiaceae trachomatis, Mycoplasma spp.,
Campylobacter jejuni, Trichomonas vaginalis, herpes virus type 1,
herpes virus type 2, Candida albicans, Candida glabrata, Candida
tropicalis, Candida parapsilosis and Candida krusei.
[0030] In another embodiment, the pathogen is Group A Streptococcus
bacteria.
[0031] In another embodiment, the pathogen comprises a pathogen
causing conjunctivitis selected from the group consisting of S.
aureus, S. pneumoniae, H. influenzae, Neisseria gonorrhoeae,
Chlamydia trachomatis, adenovirus, herpes simplex, herpes zoster
virus, enteroviruses, Fusarium spp, Candida spp. and Acanthamoeba
spp.
[0032] In another embodiment, the compositions of the disclosure
are useful as nutritional compositions for administering to a
subject in need thereof who is afflicted with a disease that
creates special dietary needs such as pediatric diarrhea, Crohn's
disease, and ulcerative colitis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows average daily stool frequency over a five day
period for two field trial test groups compared to negative control
for the composition of Example 1A. In Trial 1 (n=29) and Trial 2
(n=31), the composition Example 1A is administered once daily for
three days with antibiotic and oral rehydration salts (ORS). In the
Negative Control (n=28), only antibiotic and ORS are administered
without a composition of the disclosure.
[0034] FIG. 2 shows average daily stool consistency on a 1-5 scale
(1=normal and 5=liquid) over the same five day period for the same
three groups from FIG. 1.
[0035] FIG. 3 shows average physician assessed wellbeing on a 1-5
scale (1=normal and 5=severely ill) over the same five day period
for the same three groups from FIG. 1.
[0036] FIG. 4 shows average daily stool frequency over a five day
period for three field study (trial) test groups. Trials 1 and 2,
and the negative control, are as described for FIG. 1. In Trial 3
(n=140), patients were administered the composition of Example 1B
with antibiotic and ORS.
[0037] FIG. 5 shows average daily stool consistency on a 1-5 scale
(1=normal and 5=liquid) over the same five day period for the same
four groups from FIG. 4.
[0038] FIG. 6 shows average physician assessed wellbeing on a 1-5
scale (1=normal and 5=severely ill) over the same five day period
for the same four groups from FIG. 1.
[0039] FIG. 7 shows average daily stool frequency over a five day
period for Trials 1 and 2, negative control, Trial 3 is broken into
6 subgroups (ES204A): 2 g spray dried egg with 4 g colostrum
administered for 3 days; (ES204B): 2 g spray dried egg with 4 g
colostrum administered for 2 days; (MT204A) 2 g thermal dried egg
with 4 g colostrum for 3 days; (MT304A) 3 g thermal dried egg with
4 g colostrum for 3 days; (MS204A) 2 g spray dried egg with 4 g
colostrum for 3 days; (MS304A) 3 g spray dried egg with 4 g
colostrum for 3 days.
[0040] FIG. 8 shows average daily stool consistency on a 1-5 scale
(1=normal and 5=liquid) over the same five day period for the same
groups from FIG. 7.
[0041] FIG. 9 shows average physician assessed well-being on a 1-5
scale (1=normal and 5=severely ill) over the same five day period
for the same groups from FIG. 7.
DETAILED DESCRIPTION
Definitions
[0042] The terms "prevention", "prevent", "preventing",
"prophylaxis" and as used herein refer to a course of action (such
as administering a compound or pharmaceutical composition of the
present disclosure) initiated prior to the onset of a clinical
manifestation of a disease state or condition so as to prevent or
reduce such clinical manifestation of the disease state or
condition. Such preventing and suppressing need not be absolute to
be useful.
[0043] The terms "treatment", "treat" and "treating" as used herein
refers a course of action (such as administering a compound or
pharmaceutical composition) initiated after the onset of a clinical
manifestation of a disease state or condition so as to eliminate or
reduce such clinical manifestation of the disease state or
condition. Such treating need not be absolute to be useful.
[0044] The term "in need of treatment" as used herein refers to a
judgment made by a caregiver that a patient requires or will
benefit from treatment. This judgment is made based on a variety of
factors that are in the realm of a caregiver's expertise, but that
includes the knowledge that the patient is ill, or will be ill, as
the result of a condition that is treatable by a method, compound
or pharmaceutical composition of the disclosure.
[0045] The term "in need of prevention" as used herein refers to a
judgment made by a caregiver that a patient requires or will
benefit from prevention. This judgment is made based on a variety
of factors that are in the realm of a caregiver's expertise, but
that includes the knowledge that the patient will be ill or may
become ill, as the result of a condition that is preventable by a
method, compound or pharmaceutical composition of the
disclosure.
[0046] The term "individual", "subject" or "patient" as used herein
refers to any animal, including birds or mammals, such as mice,
Norway rats, cotton rats, gerbils, cavies, hamsters, other rodents,
rabbits, dogs, cats, swine, cattle, sheep, goat, horses, or
primates, and humans. The term may specify male or female or both,
or exclude male or female. In one aspect, the patient is an adult
human. In another aspect, the patient is a non-neonate human
infant. In another aspect, the patient is a human toddler, child,
or adolescent.
[0047] The term "neonate", or newborn, refers to an infant in the
first 28 days after birth. The term "non-neonate" refers to an
animal older than 28 days.
[0048] The term "effective amount" as used herein refers to an
amount of an agent, either alone or as a part of a pharmaceutical
composition, that is capable of having any detectable, positive
effect on any symptom, aspect, or characteristics of a disease
state or condition. Such effect need not be absolute to be
beneficial.
[0049] The term "including" as used herein is non-limiting in
scope, such that additional elements are contemplated as being
possible in addition to those listed; this term may be read in any
instance as "including, but not limited to."
[0050] The term "immunize", "actively immunize", "actively
immunizing", and "active immunization" means to purposefully
immunize a subject by exposing a subject to an antigen, for
example, an antigen derived from a microorganism, such as but not
limited to, a virus or a bacteria; such exposure may be carried out
by exposing the subject to an intact organism, an attenuated
organism, a portion of the organism, one or more antigens present
on the organism or a combination of the foregoing.
[0051] The term "passively immunize", "passively immunizing", and
"passive immunization" means to provide antibodies against an
antigen, for example, an antigen derived from a microorganism, such
as but not limited to, a virus or a bacteria, to a subject without
necessarily eliciting an immune response to the organism in the
subject. Passive immunization provides immediate protection but the
subject does not develop memory cells as a result.
[0052] The term "passive immunity" as used herein refers to
artificially acquired immunity achieved by the transfer of
antibodies to the subject. The terms "egg" or "egg product" each
mean an avian sourced whole shell egg (conventional, immunized or
otherwise) or any product or fraction derived therefrom.
[0053] The terms "immune egg" or "immune egg product" each mean
whole egg or any product or fraction derived therefrom, obtained
from an egg producing animal maintained in a immunized state.
[0054] The term "antigen" refers to an entity or fragment thereof
which can induce an immune response in an organism, particularly an
animal. The term includes immunogens and regions thereof
responsible for antigenicity or antigenic determinants.
[0055] The term "polyclonal antibody" refers to antibodies that are
heterogeneous populations of antibody molecules derived from the
sera of animals immunized with an antigen or an antigenic
functional derivative thereof. For the production of polyclonal
antibodies, various host animals may be immunized by injection with
the antigen. Various adjuvants may be used to increase the
immunological response, depending on the host species.
[0056] The term "monoclonal antibody" is well recognized in the art
and refers to an antibody that is mass produced in the laboratory
from a single clone and that recognizes only one antigen.
Monoclonal antibodies are typically made by fusing a normally
short-lived, antibody-producing B cell to a fast-growing cell, such
as a cancer cell (sometimes referred to as an "immortal" cell). The
resulting hybrid cell, or hybridoma, multiplies rapidly, creating a
clone that produces large quantities of the antibody. "Monoclonal
antibodies" are substantially homogenous populations of antibodies
directed to a particular antigen or epitope. They may be obtained
by any technique which provides for the production of antibody
molecules by continuous cell lines in culture. Monoclonal
antibodies may be obtained by methods known to those skilled in the
art. See, for example, Kohler, et al., Nature 256:495-497, 1975,
and U.S. Pat. No. 4,376,110.
[0057] The term "crystalline" refers to an antibody, such as a
monoclonal antibody that has been purified by crystallization, such
as by batch crystallization. Crystalline antibodies can be used in
order to generate a small volume, highly concentrated forms. (Yang
et al., 2003, Crystallline antibodies for subcutaneous delivery.
PNAS 100(12):6934-6939).
[0058] The term "undifferentiated diarrhea" means that the
causative agent of the diarrhea is undiagnosed.
[0059] The term "antibody fragment" encompasses any synthetic or
genetically engineered protein that acts like an antibody by
binding to a specific antigen to form a complex. For example,
antibody fragments include isolated fragments, "Fv" fragments,
consisting of the variable regions of the heavy and light chains,
recombinant single chain polypeptide molecules in which light and
heavy chain variable regions are connected by a peptide linker
("scFv proteins"), and minimal recognition units consisting of the
amino acid residues that mimic the hypervariable region. Antibody
fragments include a portion of an antibody such as F(ab')2, F(ab)2,
Fab', Fab, Fv, sFv and the like. Regardless of structure, an
antibody fragment binds with the same antigen that is recognized by
the intact antibody.
[0060] The term "transfer factor" refers to an immune molecule of
approximately 5000 Daltons, made up of amino acids, that cause
antigen-specific cell-mediated immunity, primarily delayed
hypersensitivity and the production of lymphokines, as well as
binding to the antigens themselves. (Kirkpatrick 1993, Structural
nature and functions of transfer factors. Ann. N.Y. Acad. Sci.
685:362-368.)
[0061] The term "variable lymphocyte receptors" refers to
lymphocyte-derived molecules discovered in jawless vertebrates such
as the lamprey and hagfish. These animals possess a large array of
variable lymphocyte receptors that are produced from only a small
number of genes and that bind to pathogenic antigens in a similar
way to antibodies, and with the same degree of specificity. (Alder
et al., 2005, Diversity and function of adaptive immune receptors
in a jawless vertebrate. Science, 310(5756):1970-1973).
[0062] The term "cell receptor" refers to the ligand binding moiety
of the B-cell receptor; a membrane bound immunoglobulin molecule of
one isotype (e.g., IgD, IgM, IgE). With the exception of the
presence of an integral membrane domain, these are identical to
their secreted forms.
[0063] The term "specific binding" in the context of the
characteristics of specific binding molecules, also known as
specific targeted immune factors, such as an antibody, antibody
fragment, variable lymphocyte receptor, or transfer factor, refers
to the ability to preferentially bind to a particular antigen that
is present in a homogeneous mixture of different antigens. In
certain embodiments, a specific binding interaction will
discriminate between desirable and undesirable antigens (e.g.,
"target" and "non-target" antigens) in a sample, in some
embodiments more than about 10 to 100-fold or more (e.g., more than
about 1000- or 10,000-fold). In some embodiments, the specific
binding molecule may specifically bind to an epitope shared among
different species or strains of a microorganism as compared to
non-shared epitopes. In certain embodiments, the affinity between
an antibody and antigen when they are specifically bound in an
antibody-antigen complex is characterized by a K.sub.D
(dissociation constant) of less than 10.sup.-6 M, less than
10.sup.-7 M, less than 10.sup.-8M, less than 10.sup.-9M, less than
10.sup.-10 M, less than 10.sup.-11 M, or less than about
10.sup.-12M or less.
[0064] The term "innate immune system", or non-specific immune
system, refers to the cells, molecular components and mechanisms
that defend the host from infection by other organisms in a
non-specific manner. The cells and molecular components of the
innate immune system recognize and respond to pathogens in a
generic way, but unlike the adaptive immune system, it does not
confer long-lasting or protective immunity to the subject. Innate
immune systems provide immediate defense against infection.
Vertebrates possess a second layer of protection, the adaptive
immune system, which is activated by the innate response.
[0065] The term "adaptive immune system" refers to highly
specialized, systemic cells and processes that recognize and
respond to an antigen, for example, to eliminate, neutralize or
prevent pathogenic growth. The system is highly adaptable due to
somatic hypermutation (a process of accelerated somatic mutation)
and V(D)J recombination (an irreversible genetic recombination of
antigen receptor gene segments). Adaptive immunity is also referred
to as acquired immunity and creates an immunological memory. An
adaptive immune response is pathogen and antigen specific and there
is a lag time between exposure and maximal response. An adaptive
immune response is based on the principle of clonal recognition,
such that upon first exposure to an antigen, primed lymphocytes
either differentiate into immune effector cells or form an expanded
pool of memory cells that respond to secondary exposure to the same
antigen by mounting an amplified and more rapid response.
[0066] The term "animal" refers to the animal kingdom
definition.
[0067] All pronouns are intended to be given their broadest
meaning. Unless stated otherwise, female pronouns encompass the
male, male pronouns encompass the female, singular pronouns
encompass the plural, and plural pronouns encompass the
singular.
[0068] Numerical ranges as used herein are intended to include
every number and subset of numbers contained within that range,
whether specifically disclosed or not. Further, these numerical
ranges should be construed as providing support for a claim
directed to any number or subset of numbers in that range. For
example, a disclosure of from 1 to 10 should be construed as
supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1
to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.
[0069] All patents, patent publications, and peer-reviewed
publications (i.e., "references") cited herein are expressly
incorporated by reference to the same extent as if each individual
reference were specifically and individually indicated as being
incorporated by reference. In case of conflict between the present
disclosure and the incorporated references, the present disclosure
controls.
Modes of the Disclosure
[0070] The disclosure provides compositions and methods useful in
the management of undesirable strains or pathogenic
microorganisms.
[0071] One embodiment of the present invention is based on a method
to create a targeted antibody-based formulation embedded or
subsumed within a carrier matrix, where the antibodies use a
controlled form of cross-reactivity to multiple clusters of related
target antigens, and where the carrier matrix contains support and
cofactors that enhance the effect of the antibodies. The utility of
such antibody/matrix formulations may include providing
broad-spectrum therapeutic interventions under conditions where the
class of causative agent, but not the precise or specific causative
agent, is known or suspected or under circumstances where multiple
(mixed) causative agents are active.
[0072] A novel approach to the use of antibodies in this manner has
been developed, that takes advantage of both the specificity and
cross-reactive attributes of antibodies, and then further utilizes
the components within the carrier matrix to generate a
multi-component in situ immune response. In this embodiment,
antibodies are designed to bind to all of several closely related
epitopes that represent a structurally related cluster of antigens.
These antigens may differ markedly in other respects, and may
originate from diverse sources, organisms, or species.
[0073] One embodiment of the invention involves the method of
specific binding molecules (immune factors, for example
antibodies), within the carrier matrix, where the specific binding
molecules have specificity to a class of related antigens, and are
specifically cross-reactive to different instances of members of
that class. There exists a degree of structural similarity in
related clusters of target antigens, without regard to the organism
or pathogen that is the source of the antigen. Similarity in
structure can result in a phenomenon known as "cross-reactivity"
(the steric binding of a reactive molecule to an antigen other than
the antigen intended). Cross-reactivity is often unintentional, and
in most cases is considered a source of error and non-specificity.
However, in this embodiment the extent and degree of
cross-reactivity is controlled by various means to limit and
channel its expression so as to provide desired
characteristics.
[0074] This treatment confers passive immunity to patients. The
nature of the treatment makes the associated risk factors
comparable to that of eating food from the source where the
antibodies were harvested (e.g., risk factors would be similar to
that of eating an egg and a glass of milk). This is an effective
treatment with less toxicity than the currently available
alternative interventions.
[0075] The present invention is based on the seminal discovery that
the use of an exogenously sourced (containing components obtained
from an animal different from the animal to be treated) carrier
matrix in conjunction with exogenously sourced (obtained or
corresponding to immune factors obtained from an animal different
form the animal to be treated) specifically targeted immune factors
can be used to transport and introduce an effective multi-parameter
immunity to a subject in need thereof.
[0076] In one aspect, the disclosure provides a composition
comprising: a) a non-neonate human effective amount of at least one
specific binding molecule, or fragment thereof obtained from an
animal and that specifically binds to an antigen, wherein the
specific binding molecule is selected from an immunoglobulin,
antibody, peptide, variable lymphocyte receptor, transfer factor,
and a mixture thereof; and, b) a carrier matrix comprising at least
two components obtained from a nonhuman animal selected from the
group consisting of enzymes, lactoferrin, trnasferrin, nonspecific
immunoglobulins, cytokines, white blood cells, complement
components, interferons, and fibronectin, wherein the at least one
specific binding molecule and the at least two components of the
carrier matrix are obtained from different animals.
[0077] In another aspect, the disclosure provides a method for
preparing the composition comprising: (a) obtaining at least one
specific binding molecule or fragment thereof from an animal that
binds to a specific antigen, wherein the specific binding molecule
is selected from an immunoglobulin, antibody, peptide, variable
lymphocyte receptor, transfer factor, and a mixture thereof; (b)
obtaining at least one carrier matrix, comprising at least two
components obtained from a nonhuman animal selected from the group
consisting of enzymes, lactoferrin, trnasferrin, nonspecific
immunoglobulins, cytokines, white blood cells, complement
components, interferons, and fibronectin; (c) preparing a solid
form of the carrier matrix and of the specific binding molecule or
fragment thereof; and (d) mixing the solid form of the carrier
matrix with the solid form of the specific binding molecule or
fragment thereof.
[0078] In yet another aspect, the compositions of the disclosure
are useful in the treatment or prevention of microbial infections.
In embodiments, microbial infections include those caused by
Campylobacter jejuni, Salmonella, Salmonella enterica serovar
Typhi, Shigella dystenteriae, Plesiomonas shigelloides, Escherichia
coli, enteropathogenic E. coli, enterotoxigenic E. coli,
enteroaggregative E. coli, enteroinvasive E. coli, haemorrhagic E.
coli, Clostridium difficile, Yersinia enterocolitica, Vibrio
cholerae O1, Vibrio O139, Non-O1 Vibrios, Vibrio parahaemolyticus,
Aeromonas hydrophila, Clostridium perfringens, enterohepatic
Helicobacter, Helicobacter pylori, Staphylococcus aureus,
Klebsiella, rotavirus, coronavirus, norovirus, calicivirus, enteric
adenovirus, cytomegalovirus, and astrovirus. In embodiments, the
compositions are useful to treat or prevent conditions such as
undifferentiated diarrhea, traveler's diarrhea, rotavirus diarrhea,
toxin-mediated diarrhea, cholera, C. difficile infection,
dysentery, typhoid fever, peptic ulcers, vaginitis, or for
gastrointestinal flora management.
[0079] In a specific embodiment, the compositions and methods of
the disclosure are employed in the treatment or prevention of
diarrhea. There are multiple diarrhea causing organisms including
viruses, bacteria, parasites and protozoa. The primary causes of
bacterial infection, for example in India, include Escherichia coli
spp., Enterotoxigenic E. coli, Entero-adherent E. coli. Auromonas
spp., Camphylobacter jejuni, Shigella spp., Vibrio spp., Vibrio
cholera O1, Vibrio parahaemolyticus, Salmonella spp.,
Staphylococcus aureus, Clostridium difficile, Clostridium
perfringens, and Yersinia enterocolitica. Secondary causes include
Clostridium difficile (toxin A or B), The primary cause of viral
diarrhea is infection by Rotavirus; although Calcivirus,
Astrovirus, Norwalk virus, and Adenovirus are also known to cause
diarrhea. Secondary causes of viral diarrhea include enteric
adenovirus, herpes simplex virus and viral hepatitis. (John B.
Sullivan and Gary R. Krieger, Clinical Environmental Health and
Toxic Exposures, 2nd Ed., Lippincott Williams & Wilkins, 2001,
page 1040).
[0080] There are also known to be regional and seasonal differences
in prevalence. For example, in Pranam, India, one study reported
rotavirus accounted for an average 15-25% of childhood cases of
diarrhea. Enterotoxigenic E. coli was responsible for 10 to 20% of
total diarrhea cases, with Enteropathogenic E. coli causing about 1
to 5% of cases. Camphylobacter jejuni infection caused about 10 to
15%, and Shigella caused an estimated 5 to 15% of cases of
childhood diarrhea. Vibrio cholera caused about 5 to 10% of cases.
Salmonella (non-typhoid) caused about 1 to 5% of cases. Protozoan
infection was caused by primarily by Cryptosporidium (5-15%). No
pathogenic cause was identified in about 20 to 30% of cases.
(Fricker, Children in the Tropics, Putting an end to diarrheal
diseases, 1993-No. 204: 1-66).
[0081] Different regions within India ascribe bacterial cases of
childhood diarrhea to different pathogens with different degree of
prevalence. For example a study in Orissa, India found, among 866
culture-positive samples that E. coli sp. (75.5%), pathogenic E.
coli (13.2%), Aeromonas spp. (2%), Shigella spp. (4.5%), Vibrio
cholera O1 (17.3%), V. cholera O139 (1%) and Salmonella spp.
(0.7%), find-health-articles.com/rec_pub_18806340-incidence.
[0082] Due to the wide variety of etiology, an effective, broad
spectrum, economical and safe method of treating undifferentiated
diarrhea is desired. A majority of childhood diarrhea cases seem to
be caused by bacterial and viral infection, but an alternative to
antibiotics and antiviral agents is desirable.
[0083] A. Compositions
[0084] One aspect of the disclosure involves composition useful in
the treatment, prevention or management of microbial flora. In
embodiments, the compositions are useful for treating pathogenic
infections, in particular of the gastrointestinal tract.
[0085] In embodiments, the disclosure provides a composition
comprising:
[0086] a) a non-neonate effective amount of at least one specific
binding molecule, or fragment thereof obtained from an animal and
that specifically binds to an antigen, wherein the specific binding
molecule is selected from an immunoglobulin, antibody, peptide,
variable lymphocyte receptor, transfer factor, and a mixture
thereof; and,
[0087] b) a carrier matrix comprising at least two components
obtained from a nonhuman animal selected from the group consisting
of enzymes, lactoferrin, trnasferrin, nonspecific immunoglobulins,
cytokines, white blood cells, complement components, interferons,
and fibronectin, wherein the at least one specific binding molecule
and the at least two components of the carrier matrix are obtained
from different animals.
[0088] Specific Binding Molecules
[0089] The compositions and methods of the disclosure provide
specific binding molecules or fragments thereof obtained from an
animal and that specifically bind to an antigen. A specific binding
molecule includes an antibody, an antibody fragment, a peptide, a
variable lymphocyte receptor, a transfer factor, and a mixture
thereof.
[0090] Antibodies
[0091] Antibodies, immunoglobulins, and other biological immune
factors (referred to collectively as antibodies), both natural and
their synthetic analogues, are known therapeutic agents in humans
and animals.
[0092] Antibodies operate by binding (via non-covalent forces)
between the antigen-combining site on the antibody and a portion of
the antigen called the antigenic determinant or epitope. Antibodies
are capable of high degrees of specificity. For example, the field
of monoclonal antibodies has developed largely under the impetus of
producing ever more specific and precise binding characteristics.
However, this high specificity can lead to excessively limited
binding attributes, where agents or antigens that are functionally
identical do not react identically with the immunoreagent or
immunotherapeutic. Cross-reactivity on the other hand, usually
considered an error or failure, is the reaction between an antigen
and an antibody that was generated against a similar but different
antigen. Controlled cross-reactivity may constructively be used to
broaden the binding range of the antibody.
[0093] One embodiment of the present disclosure is based on a
method to create a targeted antibody-based formulation embedded or
subsumed within a carrier matrix, where the antibodies use a
controlled form of cross-reactivity to multiple clusters of related
target antigens, and where the carrier matrix contains support and
cofactors that enhance the effect of the antibodies. The utility of
such antibody/matrix formulations may include providing
broad-spectrum therapeutic interventions under conditions where the
class of causative agent, but not the precise or specific causative
agent is known or suspected or under circumstances where multiple
(mixed) causative agents are active. A novel approach to the use of
antibodies in this manner has been developed, that takes advantage
of both the specificity and cross-reactive attributes of
antibodies, and then further utilizes the components within the
carrier matrix to generate a multi-component in situ immune
response. In this embodiment, antibodies are designed to bind to
all of several closely related epitopes that represent a
structurally related cluster of antigens. These antigens may differ
markedly in other respects, and may originate from diverse sources,
organisms, or species.
[0094] For the purposes of this disclosure, antibodies may be
either monoclonal, polyclonal derived from any animal, fragments,
chimeric, humanized or any other form, and antibodies may be of any
isotype: for example IgA, IgD, IgE, IgG and IgM (placental
mammals), IgY (chicken), or others, may be a bispecific or
bifunctional, or multispecific or multifunctional antibody or
fragment thereof. In embodiments, the specific binding molecule can
be selected from one of three main categories: mammalian monoclonal
antibodies, mammalian polyclonal antibodies, and avian polyclonal
antibodies; or any fragments derived therefrom that retain the
ability to bind to the pathogenic component.
[0095] One embodiment of this invention is its use in the
production of a broad spectrum therapeutic. One method for
producing this type of reactive formulation involves the production
of polyclonal antibodies harvested from an appropriately immunized
animal, and where such antibodies are then embedded in a carrier
matrix. Polyclonal antibodies (or antisera) are antibodies that are
derived from from different B cell lines. They are typically
harvested en-mass from the serum, milk, colostrum, eggs, or
biological fluids of an immunized animal. They are a mixture of
immunoglobulin molecules secreted against a specific antigen, or
group of antigens, recognizing a range of different epitopes. It is
possible to have multiple antibodies for a single antigen (binding
to different epitopes) or for a single antibody to bind to multiple
antigens due to cross-reactivity. The polyclonal antibodies can be
obtained from animals, such as cattle, sheep, horses, goats, swine,
rabbits, chickens, ducks, geese, or turkeys that have been
vaccinated or inoculated with antigens derived from target
components. The antibodies can be harvested from, for example,
tissue, serum, milk or eggs produced by, or derived, from the
inoculated animal. This is in contrast to monoclonal antibodies,
which are identical and monospecific; being produced by one type of
immune cell that are all clones of a single parent cell.
[0096] The antibodies used in this invention may be collected from
serum, plasma, colostrum, milk, eggs, or other suitable
biologically derived fluid, or from cell culture media,
supernatant, etc. The antibodies used in this invention may be
treated in any suitable manner to prepare for formulation and use,
including but not limited to separations, plasmapheresis, drying
processes, lyophilization, pasteurization, and preservation
methods. The antibodies used in this invention may be treated,
concentrated, separated, or purified in various ways depending upon
their final intended use.
[0097] By altering the mix of antibodies to those appropriate to
various embodiments, the disclosure provides compositions and
methods appropriate for treating or preventing other
gastrointestinal infections such as cholera, C. difficile,
dysentery, Salmonella typhi (typhoid fever), and H. pylori (peptic
ulcers).
[0098] In one embodiment, antibodies are preferably raised in
animals by, e.g., multiple subcutaneous (sc) or intraperitoneal
(ip) injections of the relevant antigen and optionally an adjuvant.
In one aspect, it may be useful to conjugate the relevant antigen
(especially when synthetic peptides are used) to a protein that is
immunogenic in the species to be immunized. For example, the
antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using
a bifunctional or derivatizing agent, e.g., maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R N.dbd.C.dbd.NR, where R and R
are different alkyl groups. Animals are immunized against the
antigen, immunogenic conjugates, or derivatives as described
herein. In other embodiments, the antibodies may be synthetic or
semisynthetic, for example, as are obtained in a phage display
library, or prepared as humanized or chimeric antibodies.
[0099] Birds (such as laying-hens) are highly cost-effective as
producers of antibodies compared with mammals traditionally used
for such production. Avian antibodies have biochemical advantages
over mammalian antibodies. Immunologic differences between mammals
and birds result in increased sensitivity and decreased background
in immunological assays; as well as high specificity and lack of
complementary immune effects when administered to mammalian
subjects. In contrast to mammalian antibodies, avian antibodies do
not activate the human complement system through the primary or
classical pathway nor will they react with rheumatoid factors,
human anti-mouse IgG antibodies, staphylococcal proteins A or G, or
bacterial and human Fc receptors. Avian antibodies can however
activate the non-inflammatory alternative pathway. Thus avian
antibodies offer many advantages over mammalian antibodies.
[0100] In a preferred embodiment, the specific molecules are
polyclonal antibodies prepared in eggs of hens inoculated with one
of or a mixture of pathogenic components. Various preparations of
specific antigens can also be employed for inoculation. After
inoculation, the hen produces eggs containing substantial
quantities of specific IgY immunoglobulin in the yolk, as well as
small amounts of IgM and IgA immunoglobulins in the albumin.
Therefore eggs are an excellent source for large quantities of
economically produced, highly specific and stable antibodies. In
one embodiment, chickens are used to produce avian antibody;
however, turkeys, ducks, geese, ostriches, etc. may alternatively
be used. In one aspect, hens are inoculated by any method known in
the art, as described herein. For example, the antigen may be
injected intramuscularly or subcutaneously. The preferred muscle
for injection in an avian is the breast muscle. Other methods of
administration that can be used include subcutaneous injection,
intravenous injection, intraperitoneal injection, intradermal,
rectal suppository, aerosol or oral administration.
[0101] The specific immune state is preferably induced and
maintained in the target animal by immunization and repeated
booster administrations of an appropriate dosage at fixed time
intervals. The time intervals are preferably 1-8 week intervals
over a period of 1-12 months. Dosage is selected between about
0.01-5 milligrams of the antigen. In one aspect, the dosage is 0.01
mg to 1.0 mg of antigen per inoculation, preferably 100 mg, 200 mg,
250 mg, 300 mg, 400 mg, 500 mg or 750 mg antigen per inoculation of
a hen chicken. The total number of vaccinations can be selected
from 1, 2, 3, 4, 5, or 6 in a 12 month period. Typically, a first
inoculation is performed on day 1, with booster vaccinations on day
10, and day 20. The hen chicken can be re-vaccinated as needed by
monitoring the specific antibody concentration, or titer, in the
eggs by, e.g., ELISA. A typical subcutaneous dosage volume for a
hen chicken is selected from between about 0.2 to 1.0 mL, 0.3 to
0.7 mL, or 0.5 mL. However, it is essential that the booster
administrations do not lead to immune tolerance. Such processes are
well known in the art.
[0102] It is possible to use other inoculation maintenance
procedures or combination of procedures, such as, for example,
intramuscular injection for primary immunization and intravenous
injection for booster injections. Further procedures include
simultaneously administering microencapsulated and liquid
immunogen, or intramuscular injection for primary immunization, and
booster dosages by oral administration or parenteral administration
by microencapsulation means. Several combinations of primary and
booster immunization are known to those skilled in the art.
[0103] Adjuvants, also known as pharmaceutical carriers, or
functional equivalents hereof may be included in the immunization
solution/vaccine composition to enhance the specific immune
response of the animal. A large number of adjuvants have been
described and used for the generation of antibodies in laboratory
animals, such as mouse, rats, rabbits and chickens. In such setting
the tolerance of side effects is rather high as the main aim is to
obtain a strong antibody response.
[0104] Adjuvants pertaining to the present disclosure may be
grouped according to their origin, be it mineral, bacterial, plant,
synthetic, or host product. The first group under this
classification is the mineral adjuvants, such as aluminum
compounds. Antigens precipitated with aluminum salts or antigens
mixed with or adsorbed to performed aluminum compounds have been
used extensively to augment immune responses in animals and humans.
In one embodiment, the adjuvant in the immunization composition is
from a bacterial origin. Adjuvants with bacterial origins can be
purified and synthesized (e.g. muramyl dipeptides, lipid A) and
host mediators have been cloned (Interleukin 1 and 2). Known
chemical purification of several adjuvants of active components of
bacterial origin includes: Bordetella pertussis, Mycobacterium
tuberculosis, lipopoly-saccharide, Freund's Complete Adjuvant (FCA)
and Freund's Incomplete Adjuvant (Difco Laboratories, Detroit,
Mich.) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway,
N.J.). In a specific aspect, Freund's Complete Adjuvant or Freund's
Incomplete Adjuvant is employed in the immunization compositions of
the disclosure. Additionally suitable adjuvants in accordance with
the present invention are e.g., Titermax Classical adjuvant
(SIGMA-ALDRICH), ISCOMS, Quil A, ALUN, see U.S. Pat. Nos. 5,876,735
and 5,554,372, Lipid A derivatives, choleratoxin derivatives, HSP
derivatives, LPS derivatives, synthetic peptide matrixes, GMDP, and
other as well as combined with immunostimulants (U.S. Pat. No.
5,876,735). B. pertussis is of interest as an adjuvant in the
context of the present invention due to its ability to modulate
cell-mediated immunity through action on T-lymphocyte populations.
Freund's Complete Adjuvant is the standard in most experimental
studies. Mineral oil may be added to the vaccination composition in
order to protect the antigen from rapid catabolism.
[0105] Many other types of materials can be used as adjuvants in
immunogenic or immunization compositions according to the present
disclosure. They include plant products such as saponin, animal
products such as chitin and numerous synthetic chemicals.
[0106] Chickens immunized by the intramuscular route can produce
high specific antibody levels in their eggs by day 28 after
immunization and continue producing specific antibodies during more
than 200 days making antibody preparations available in a short
period of time, e.g. less than 4-5 weeks. Eggs contain IgY antibody
concentrations of from up to about 50 to about 100 mg per egg. Over
100 mg of purified IgY can be obtained from a single egg. The
percentage of antigen specific antibodies in one egg yolk can be up
to about 2% to 10%. (daSilva et al., IgY: A promising antibody for
use in immunodiagnostic and in immunotherapy. Veterinary Immunol.
Immunopath., 135(2010):173-180). One chicken of a high egg-laying
strain can produce around 20 eggs per month. Eggs weigh from about
33 to about 77 grams, with about 10.5% of the whole egg due to
shell. The yolk is about 31% of the weight of the whole egg. Upon
drying, about 1 kg of dried whole egg powder can be produced from
72 eggs. Therefore, in this calculation, one egg can return about
13.9 g dried whole egg. In another aspect, one immune egg can
return from 10 g to about 15 g dried whole egg. In another aspect,
the immune eggs of the disclosure are from 40 to 55 mL per egg with
about 1-2 mg/mL total IgY per egg. In another aspect, immune eggs
of the disclosure contain about 0.01 mg/mL to 0.05 mg/mL specific
IgY per egg. Therefore, in one aspect after processing, one dried
whole immune egg contains about 80 to 110 mg total IgY and about 6
to 10 mg of total mixed antigen-specific IgY, e.g., from a chicken
immunized with, for example a mixed antigen preparation.
[0107] It can be determined whether the vaccine has elicited an
immune response in the egg-producing animal through a number of
methods known to those having skill in the art of immunology.
Examples of these include enzyme-linked immunosorbent assays
(ELISA), tests for the presence of antibodies to the stimulating
antigens, and tests designed to evaluate the ability of immune
cells from the host to respond to the antigen. The minimum dosage
of immunogen necessary to induce an immune response depends on the
vaccination procedure used, including the type of adjuvants and
formulation of immunogen(s) used as well as the type of
egg-producing animal used as the host.
[0108] In one embodiment, hen chickens suitable for the commercial
production of eggs are employed in the production of polyclonal
antibodies. Any breed of chicken appropriate for egg production can
be employed. For example, Rhode Island Reds, White Leghorns, Brown
Leghorns, Lohmann Brown hens, sex-linked hybrid crosses, or other
breeds suited to large egg size, high volume egg production and
ease of handling can be selected. In one aspect, chickens are
inoculated as chicks as for standard diseases (e.g. Salmonella,
avian influenza, or Newcastle virus etc.). In one aspect, chickens
of any age can be inoculated. Hens which are about to reach laying
age, about 15-19 weeks for chickens, or any preselected time before
or thereafter, are inoculated on a schedule predetermined by the
amount and timing of final product to result in a steady continuous
production stream. Typically, after a suitable period of isolation
and acclimatization of about 2 to 4 weeks, each group will enter
into an inoculation program using various antigens or immunization
compositions comprising specific antigens to which an antibody is
desired.
[0109] In one embodiment, the eggs are collected from inoculated
chickens and processed as whole eggs. Eggs are stored under
refrigeration conditions until enough are collected to prepare a
batch. Batches of eggs from predetermined groups of chickens are
cracked, the contents are separated from the shells and mixed and
preferably pasteurized to eliminate potential contamination from
pathogenic microorganisms from the chicken.
[0110] In one aspect, the immune egg products are pasteurized. Egg
products are processed in sanitary facilities. Shell eggs are
processed into immune egg product by automated equipment that
removes the shell eggs from flats, washes and sanitizes the shells,
breaks the eggs. Optionally, the whites are separated from the
yolks. The liquid egg product is optionally filtered, optionally
mixed with other ingredients, and is then chilled prior to
additional processing. The resulting egg products liquid then
receives a lethality treatment such as pasteurization or is heated
in the dried form. In the U.S., the 1970 Egg Products Inspection
Act (EPIA) requires that all egg products distributed for
consumption be pasteurized.
[0111] Following pasteurization, the total egg content is dried
using standard commercial methods, such as spray drying using
ambient or hot air, thermal drying, freeze drying, or
lyophilization. In one aspect, an appropriate method of drying the
pasteurized liquid egg minimizes damage to the antibodies and
molecular components in the egg, resulting in a product that has a
high nutrient value and is capable of conferring passive
protection.
[0112] In one aspect, the dried egg is tested to determine overall
titer or antibody level. Standard test procedures are used, such as
ELISA, FIA (fluorescent immunoassay), RIA (radioimmunoassay), or
the like. In another aspect, the batch is blended with batches from
groups of chickens at other average production levels resulting in
a lot containing a standardized amount of antibodies. The dried egg
containing specific polyclonal antibodies may be stored in an
airtight container at room temperature prior to formulation into
the compositions of the disclosure. In embodiments, the dried egg
material is used as a whole egg and is not separated out. In
embodiments, the whole dried egg material contains at least 5 mg
per egg of specific IgY.
[0113] In another embodiment, IgY is isolated. The first step in
the isolation of IgY is to separate the water-soluble proteins from
lipoproteins. Water-soluble proteins constitute 42.4% of the total
proteins in egg yolk (Osuga et al., "Egg Proteins: In Food
Proteins, J. R. Whitaker and S. R. Tannenbaum eds., AVI Pub. Co.,
Westport, Conn. (1977)).
[0114] Many methods have been used for the isolation and
purification of immunoglobulins from egg yolk (Martin et al., Can
J. Biochem. Physiol. 35:241 (1957); Martin et al., Can. J. Biochem
Physiol. 36:153 (1958); Jensenius et al., J. Immunol. Methods 46:63
(1981); Bade et al., J. Immunol. Methods 72:421 (1984); Polson et
al., Immunol. Invest. 14:323 (1985); Hassl et al., J. Immunol.
Methods 110:225 (1988)). Hatta et al. (Agric. Biol. Chem. 54:2531
(1990)) used food-grade natural gums (e.g., carrageenan) to remove
yolk lipoprotein as a precipitate and to recover IgY in the
water-soluble fraction from egg yolk. Methods for recovering
antibodies from chicken egg yolk are well known in the art. Several
methods can be used for the extraction of IgY from egg yolk, and
commercial extraction kits are available (van Regenmortel, M. H. V.
(1993). Eggs as protein and antibody factories. In Proceedings of
the European Symposium on the Quality of Poultry Meat, pp. 257-263.
Tours, France: INRA).
[0115] In another embodiment, the steric specific binding molecule
may be a monoclonal antibody specific for a pathogenic
component.
[0116] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature. 256:495 (1975), or may be
made by recombinant DNA methods (U.S. Pat. No. 4,816,567). In the
hybridoma method, a mouse or other appropriate host animal, such as
a hamster, is immunized as described above to elicit lymphocytes
that produce or are capable of producing antibodies that will
specifically bind to the protein used for immunization.
Alternatively, lymphocytes may be immunized in vitro. After
immunization, lymphocytes are isolated and then fused with a
myeloma cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59-103 (Academic Press,
1986)).
[0117] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (MA) or enzyme-linked immunosorbent assay (ELISA).
The binding affinity of the monoclonal antibody can, for example,
be determined by the Scatchard analysis described in Munson et al.,
Anal. Biochem. 107:220 (1980).
[0118] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, affinity chromatography (e.g., using protein A or protein
G-Sepharose) or ion-exchange chromatography, hydroxylapatite
chromatography, gel electrophoresis, dialysis, etc.
[0119] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce antibody protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells.
[0120] Review articles on recombinant expression in bacteria of DNA
encoding the antibody include Skerra et al., Curr. Opinion in
Immunol., 5:256-262 (1993) and Pl{acute over (.upsilon.)}ckthun.
Immunol. Revs. 130:151-188 (1992).
[0121] In a further embodiment, monoclonal antibodies or antibody
fragments can be isolated from antibody phage libraries generated
using the techniques described in McCafferty et al. Nature.
348:552-554 (1990). Clackson et al. Nature. 352:624-628 (1991) and
Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology. 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0122] The DNA that encodes the antibody may be modified to produce
chimeric or fusion antibody polypeptides, for example, by
substituting human heavy chain and light chain constant domain
(C.sub.H and C.sub.L) sequences for the homologous murine sequences
(U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl Acad.
Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding
sequence with all or part of the coding sequence for a
non-immunoglobulin polypeptide (heterologous polypeptide). The
non-immunoglobulin polypeptide sequences can substitute for the
constant domains of an antibody, or they are substituted for the
variable domains of one antigen-combining site of an antibody to
create a chimeric bivalent antibody comprising one
antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
Antigens for Immunization to Prepare a Specific Binding Protein
[0123] The antigens selected for immunization can be bacterial,
viral, protozoal, fungal, parasitic, cellular, or any other
substances to which the immune system of an animal will respond. In
one aspect, the immunogenicity of the antigens is enhanced by use
of an adjuvant.
[0124] In one aspect, the animal is inoculated with the pathogenic
components, antigens, or immunogens in a vaccination composition,
inoculant or vaccine. In one aspect, the pathogenic components or
specific antigens can be obtained or derived from commercial
sources such as the American Type Culture Collection (ATCC). In
another aspect, the pathogenic components can be isolated from a
wild type strain. In another aspect, the pathogenic components or
undesirable strains are present in a mixed antigen preparation. Any
antigens or combination of antigens derived from various
undesirable strains or pathogenic components can be employed in the
immunization composition.
[0125] In one aspect, the inoculant, antigen or immunogen is
selected to a common or preserved component or region of the
targeted antigen cluster, while ignoring the variable or
distinguishing components or regions of the individual members of
the cluster of related antigens. The method involves the
preparation of an appropriate immunogen with characteristics that
elicit the production of antibodies that are cross-reactive to
desired instances of that epitope, but which are not reactive to
other epitopes, and the inoculation or exposure of the producing
cells or organism to that immunogen so as to cause the production
of antibodies, with the resultant antibodies being embedded within
the suitable carrier matrix for administration. Formulations of
this type may be developed that use admixtures of antibodies
produced according to this method to provide broad coverage of more
than one cluster of target antigens. For example, in the case where
two clusters of unrelated antigens are associated with a disease or
condition, and it is desirable to create a single formulation to
address this disease or condition, an admixture of two antibodies,
immunoglobulins, or biological immune factors may be prepared using
this method that simultaneously provides two broad domains of
reactivity. One example of this embodiment is for the production of
antitoxin antibodies that are specifically reactive to clusters of
structurally related toxins.
[0126] In one embodiment, this approach is used to make a broadly
reactive antibody to lipopolysaccharide (LPS) (endotoxin) from any
Gram-negative bacteria (Escherichia coli, Salmonella, Shigella, and
other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter,
Stenotrophomonas, and others), or for example a broadly reactive
antibody to AB5 toxins (including Campylobacter enterotoxin,
cholera toxin, heat-labile enterotoxins (LT and LT-II) (E. coli),
pertussis toxin, shiga toxin (Shigella), shiga-like toxin (or
verotoxin)).
[0127] In a preferred aspect, these example anti-toxin antibodies
have effect without regard to the species originating the toxin. In
another aspect, the antibodies produced are neutralizing
antibodies, capable of neutralizing or inactivating the biological
activity of the target toxins. Such a broad-spectrum neutralizing
antibody could be used to intervene in pathology cases (for example
certain types of diarrhea) where the toxin mediating the symptoms
was one of the cluster targeted (in these examples, AB5 or LPS),
without requiring knowledge of which organism was causative.
Further, if an admixture was prepared containing both the anti AB5
antibody and the anti LPS antibody in clinically effective amounts,
the formulation could be used to intervene in case where the active
toxin was either AB5 or LPS or both.
[0128] This method can be extended to include any number of (in
this example) toxin clusters, and to include broad-spectrum
neutralizing antibodies to mediators of other toxin-like reactions
(for example viral toxin-like phenomena), to create a broadly
applicable intervention (in this example to) diarrhea where
symptoms and pathology can be managed without knowledge of the
infectious causes, or where there are multiple infectious causes.
In one aspect, the disclosure provides a composition comprising a
synergistic combination of anti-toxin antibodies combined in a
carrier matrix.
[0129] In some embodiments, the methods and compositions of the
invention are used for a variety of pathogens or agents including,
without limitation, cholera toxin (Vibrio cholera), E. coli
(including enterotoxigenic (ETEC)), Shigella, Salmonella,
Campylobacter, Clostridium difficile, parasites (e.g., Giardia,
Entamoeba histolytica, Cryptosporidiosis, Cyclospora), and
diarrheal viruses (e.g., rotavirus).
[0130] After entering the gastrointestinal tract many pathogens,
including but not limited to bacteria such as E. coli, bind
(adhere) to epithelial, mucosal, or other tissue and become
embedded in gastrointestinal tract tissue, such as the wall of the
intestine. After binding to tissue in the gastrointestinal tract
the pathogens replicate, causing an increase in toxin
concentrations, either directly from production or indirectly from
increased lysing of pathogen cells by immune system action.
Inhibiting the ability of pathogens to bind to the gastrointestinal
tract tissue promotes a more effective mobilization of the
pathogens, digestion and excretion before colonies of sufficient
size to cause lesions and other symptoms are formed. By blocking
the class of receptors and ligands on the pathogen that would be
used to adhere to the gastrointestinal tract, including but not
limited to adhesins, cadherins, cilia, fimbrillae, and/or viral
adhesion structures, adhesion to gastrointestinal tract tissue can
be prevented or minimized, ultimately resulting in substantially
decreased pathology from pathogens that utilize this mode of
action.
[0131] In various embodiments, the pathogen is selected from one or
a combination of human or veterinary, enteric or gastrointestinal,
pathogens causing gastroenteritis. In various aspect, the pathogen
is selected from the group consisting of: Campylobacter jejuni,
Salmonella, Salmonella typhimurium, Salmonella enterica serovar
Typhi, Shigella dystenteriae, Plesiomonas shigelloides, Escherichia
coli [including (EPEC) enteropathogenic E. coli, (ETEC)
enterotoxigenic E. coli, (EaggEC) enteroaggregative E. coli, (EIEC)
enteroinvasive E. coli, and (EHEC) haemorrhagic E. coli], Yersinia
enterocolitica, Vibrio cholerae O1, Vibrio O139, Non-O1 Vibrios,
Vibrio parahaemolyticus, Aeromonas hydrophila, Clostridium
perfiingens, Clostridium difficile, enterohepatic Helicobacter
(including Helicobacter pylori), Staphylococcus aureus, Klebsiella,
rotavirus, coronavirus, norovirus, calicivirus, enteric adenovirus,
cytomegalovirus, and astrovirus. In another aspect, the pathogen
related toxin includes an endotoxin or exotoxin. In another aspect,
the pathogen related adhesion element includes adhesins, cadherins,
cilia, fimbrillae, a viral adhesion structure, or a combination
thereof.
[0132] In various specific aspects the pathogenic components,
immunogens or antigens can be derived from, e.g., Rotavirus, Corona
virus; Clostridium Perfringens Type C; Escherichia coli (cellular);
Enterotoxigenic strains of, and Enterotoxins from, E. coli; any
bacteria having K99, K88, 987P, or F41 pili adherence factor
antigen; endotoxin (or LPS) caused by E. coli and Salmonella
typhimurium (gram negative bacteria, generally). In a particular
aspect, hens are inoculated with antigens or toxins derived from
one, two, three, four, five, six, seven, or eight, or a number of
pathogenic microorganisms.
[0133] In one aspect, the immune response is more potent when the
distance between the antigen source and the immune system of the
vaccinated animal increases.
[0134] In a specific embodiment, a first flock of chickens is
inoculated with a first one mixed antigenic preparation. In one
aspect, a second flock of chickens is inoculated with a second
mixed antigenic preparation containing a different set of antigens
than the first. In another aspect, a third flock of chickens is
inoculated with a third mixed antigenic preparation. In a further
aspect, a fourth flock of chickens is inoculated with a fourth
mixed antigenic preparation. While not meant to limit the scope of
the invention, it is believed to be advantageous to immunize
different flocks with different antigens in order to avoid antigen
overload.
[0135] Eggs from each flock are collected, optionally titered as to
specific and/or total IgY, optionally isolated and/or purified, and
processed separately to prepare a dry powder. In another aspect,
dry powdered eggs from the first and second; first, second and
third; or first, second, third and fourth flocks are blended, or
packaged, with a carrier matrix to prepare a composition of the
disclosure. In one aspect, a first antigenic preparation comprises
bovine rotavirus (serotypes G6 and G10), bovine coronavirus,
enterotoxigenic strains of Escherichia coli having the K99 pili
adherence factor, and Clostridium perfringens type C. The mixed
antigenic preparation can is optionally adjuvanted to enhance the
immune response.
[0136] In another aspect, a second antigenic preparation comprises
beta toxin produced by Clostridium perfringens type C and
enterotoxigenic strains of Escherichia coli producing heat-labile
toxin or having the K99, K88, 987P, or F41 adherence factors.
[0137] In one aspect, a third antigenic preparation comprises E.
coli and Salmonella typhimurium. JVAC reduces the incidence and
severity of endotoxemia caused by E. coli and Salmonella
typhimurium. Commonly associated with their endotoxins are Coliform
Mastitis and other gram-negative diseases associated with
Endotoxemia.
[0138] In another aspect, the antigens are prepared by any means
known in the art. For example, cells from a wild type source, such
as an animal suffering from, e.g., E. coli diarrhea. The isolate
cells can be cultured in, e.g., Trypticase Soy Broth (TSB) at
37.degree. C. overnight and concentrated by centrifugation. The
resulting pellet can be re-suspended with 0.4% formaldehyde in PBS
buffer and incubated at 37.degree. C. for inactivation.
Formaldehyde can be removed by centrifugation. The pellet can be
resuspended in PBS and used as antigen. In one aspect, the antigens
are emulsified with an equal volume of adjuvant prior to
inoculation.
[0139] In another embodiment, the antigens are selected from those
pathogenic organisms causing conjunctivitis. Known causative
pathogens are described in US 2008/0031903, Gambotto et al., which
is incorporated herein by reference.
[0140] Epidemic Keratoconjuctivitis (EKC) is a debilitating
infectious disease of the eye that is seen all over the world. The
disease is caused mostly by adenoviruses especially serotype 8, 19
and 37. Serotype 3, 4 and 11 were also implicated in some EKC
epidemics. The disease affects all age groups, is highly contagious
and spreads quickly in schools, schools, swimming pools, pediatric
unit and camps. Treatment is presently symptomatic as there is no
effective treatment. Development of effective anti-viral topical
agent is desirable to treat the disease and prevent epidemic.
[0141] Conjunctivitis also can be caused by a number of additional
bacterial, viral, fungal and protozoa agents, including, but not
limited to: S. aureus, S. pneumoniae, H. influenzae, Neisseria
gonorrhoeae, Chlamydia trachomatis, Adenovirus, Herpes Simplex,
Herpes zoster virus, Enteroviruses, Fusarium species, Candida
species and Acanthamoeba species. Certain viral infections, such as
adenoviral infections may be treated with antiviral drug products,
such as cidofovir. Typically, drug products have side effects, such
as the ocular and renal side effects associated with cidofovir.
Other logistical issues arise with drug products, including
stability, cost of production, etc. As such, an inexpensive,
readily-available, well-accepted and stable drug product for
treatment of ocular infections is desirable.
[0142] In one aspect, the disclosure provides a composition for the
treatment of conjunctivitis, or pink eye, comprising polyclonal
antibodies to these pathogens combined in a carrier matrix as
described below. The antibodies are produced as described
herein.
[0143] In another embodiment, the antigens are selected from those
pathogenic organisms causing vaginitis. The infection may be
bacterial, fungal (yeast), or parasitic. Bacterial vaginitis can be
caused, for example, by Gardnerella spp., Neisseria gonorrhoeae,
Chlamydiaceae trachomatis, Mycoplasma spp., Campylobacter jejuni.
Parasitic vaginitis can be caused by, e.g., Trichomonas vaginalis.
Viral vaginitis can be caused by e.g., herpes virus type 1 or type
2. Candidal vaginitis is caused by yeastlike fungi Candida. There
are more than 170 species of yeastlike fungi is described. C.
albicans is the most frequent causative agent of a candidal
vaginitis in 85-90% of women. C. glabrata (5-10%), C. tropicalis
(3-5%), C. parapsilosis (3-5%) and C. krusei (1-3%) are also
clinically significant among other species of Candida. Any of these
pathogens may be selected as the antigenic source for polyclonal
antibody production as described herein.
[0144] Candidal vulvovaginitis is frequently caused by a number of
predisposing factors, such as long and uncontrolled using of
antibiotics, corticosteroids, cytostatics, oral contraceptives,
radiation therapy, serious infectious disease, endocrine disorder,
immunodeficiency state, etc. Prescription of broad spectrum
antibiotics suppresses not only pathogenic bacteria, but also
mucous vaginas saprophytes: lactobacilli and bifidobacteria. As a
result vaginal pH raises (towards to alkaline range), and
disturbance of self-cleaning processes occurs. Besides, Candida is
able to use some antibiotics as nutrient substrates. Thus favorable
conditions for active overgrowth of Candida arises in female
genital organs. In one aspect, the disclosure provides a
composition for the treatment of vaginitis comprising polyclonal
antibodies to one or more of the described pathogens combined in a
carrier matrix as described below.
[0145] In a specific aspect, the composition of the disclosure
comprising a mixture of specific polyclonal antibodies in a carrier
matrix provides a broad spectrum method of treating bacterial,
viral, fungal or parasitic vaginitis. In another aspect, the
compositions of the disclosure can be used to treat
undifferentiated vaginitis in a subject in need thereof
[0146] Other Specific Binding Molecules
[0147] The compositions and methods of the disclosure include other
specific binding molecules including transfer factors, variable
lymphocyte receptors and cell receptors. A transfer factor is an
immune molecule of approximately 5000 Daltons, made up of amino
acids, that cause antigen-specific cell-mediated immunity,
primarily delayed hypersensitivity and the production of
lymphokines, as well as binding to the antigens themselves.
(Kirkpatrick 1993, Structural nature and functions of transfer
factors. Ann. N.Y. Acad. Sci. 685:362-368.) Variable lymphocyte
receptors are lymphocyte-derived molecules discovered in jawless
vertebrates such as the lamprey and hagfish. These animals possess
a large array of variable lymphocyte receptors that are produced
from only a small number of genes and that bind to pathogenic
antigens in a similar way to antibodies, and with the same degree
of specificity. (Alder et al., 2005, Diversity and function of
adaptive immune receptors in a jawless vertebrate. Science,
310(5756):1970-1973).
Carrier Matrix
[0148] The disclosure provides compositions for the treatment or
prophylaxis of pathogenic infection in a subject. The compositions
comprise specific binding molecules, such as polyclonal antibodies,
combined with a carrier matrix. While not meant to limit the scope
of the invention, the carrier matrix serves a dual purpose. First,
to protect the antibodies in their intended functional environment,
for example, upon oral administration, and within the
gastrointestinal tract of the non-neonate subject; and further to
provide components, e.g., components of the innate immune system,
to react synergistically with the antibodies in the management of
an infection.
[0149] The term "carrier matrix", or protective/reactive matrix,
refers to any substrate, compound, formulation, or supplemental
admixture (whether natural or synthetic) containing elements,
co-factors, or other components in appropriate ratios and
concentrations so as to supply elements required to propagate,
promote, support, or enhance an in situ immune-type response,
cascade, or reaction. These elements may variously promote cleavage
and maturation reactions, the formation of assemblies and
complexes, depletion and adsorption functions, supply essential
elements, biologics, or compounds, and provide protective functions
for active elements or components. A carrier matrix may or may not
contain endogenous antibodies (immune factors), which may or may
not be specific to targeted antigens.
[0150] In one embodiment, the carrier matrix the matrix is selected
from, or derived from, serum, plasma, colostrum, milk, saliva,
lymph fluid, mucous, or lachrymal fluid derived from a non-human
mammal.
[0151] An example of a naturally occurring carrier matrix is
colostrum. Colostrum has evolved naturally in mammals specifically
to deliver its components to neonates to and through the
gastrointestinal tract in a very concentrated low-volume form.
Colostrum, or "first milk", is produced by mammals immediately
postpartum. The antibodies and cofactors are passed to the neonate
from the mother and provide the first protection against pathogens.
Growth factors also stimulate the development and repair of the
gut.
[0152] Colostrum contains a host of immuno-complimentary factors.
They include interferons, immunglobulins (including IgG and
secretory IgA), polymorphonuclear leukocytes, macrophages, and
lymphocytes. Colostrum also contains proline-rich polypeptide, or
PRP, a T-cell activator. Colostrum is known to be high in
immunoglobulin content compared to milk. Colostrum is known to
contain antibodies such as IgA, IgG, and IgM in mammals. IgA is
absorbed through the intestinal epithelium, travels through the
blood, and is secreted onto other Type 1 mucosal surfaces. Bovine
Colostrum is noted to be anywhere from 6% to 20% immunoglobulin;
primarily IgG.sub.1 and IgG.sub.2. In one aspect, whole bovine
colostrum is used as the carrier matrix.
[0153] Colostrum also helps to regulate the intestinal environment,
rendering it hostile to foreign pathogens. Colostrum contains
lactoferrin, an iron-binding protein that prevents bacteria and
viruses from obtaining iron necessary for replication. Colostrum
also selectively fertilizes certain probiotic species that in turn
help to ward off infection. It is the only natural source of two
major growth factors, Transforming Growth Factors (TGF) alpha and
beta, as well as a source of Insulin-Growth Factors 1 and 2. These
factors promote tissue repair and development. Colostrum is also a
source of Hepatocyte Growth Factor, which stimulates the growth and
expansion of intestinal wall cells. Colostrum is naturally designed
to serve as a carrier matrix within a gastrointestinal environment.
Synthetic versions of a carrier matrix are also included in this
disclosure. Carrier matrices that are composed of both natural and
synthetic components are also included within the disclosure.
[0154] Colostrum is very rich in proteins, vitamin A, and sodium
chloride, but contains lower amounts of carbohydrates, lipids, and
potassium than normal milk. The most pertinent bioactive components
in colostrum are growth factors and antimicrobial factors. The
antibodies in colostrum provide passive immunity, while growth
factors stimulate the development of the gut. They are passed to
the neonate and provide the first protection against pathogens. The
passive immunity from the mother gets transferred to the
newborn.
[0155] Newborns have very small digestive systems, and colostrum
delivers its nutrients in a very concentrated low-volume form. The
gastrointestinal tract of the neonate is particularly receptive to
passive transfer of immunity from colostrum. At birth gastric pH
ranges vary from 6-8 due to residual amniotic fluid in the stomach.
Gastric pH then falls to a pH of 1.5 to 3 in 24 to 48 hours.
Therefore, The GI conditions of the newborn are conducive to
passive immunization. In addition, gastric emptying time in
neonates and premature infants is prolonged, with adult values
being reached at 6-8 months of age. The antibodies and cofactors in
colostrum can, under certain circumstances (e.g., breastfeeding)
provide a passive immunity to the recipient; this is particularly
true for the neonate. The gastrointestinal tract of non-neonatal
babies, children, adolescents and healthy adults is a more hostile
environment with respect to oral administration of
immunoglobulins.
[0156] Other immune components of colostrum include the major
components of the innate immune system, such as lactoferrin,
transferrin, lysozyme, lactoperoxidase, complement, and
proline-rich polypeptides (PRP). A number of cytokines (small
messenger peptides that control the functioning of the immune
system) are found in colostrum as well, including interleukins,
tumor necrosis factor, chemokines, and others. Colostrum also
contains a number of growth factors, such as insulin-like growth
factors I, and II, transforming growth factors alpha, beta 1 and
beta 2, fibroblast growth factors, epidermal growth factor,
granulocyte-macrophage-stimulating growth factor, platelet-derived
growth factor, vascular endothelial growth factor, and
colony-stimulating factor-1.
[0157] In one aspect, the carrier matrix is comprised of two or
more, three or more, four or more, five or more, or six or more, or
seven or more non-immunoglobulin components of colostrum. In
another aspect, the carrier matrix comprises colostrum that has
been processed to remove the majority of immunoglobulins. In
embodiments, a carrier matrix comprises at least two components
obtained from a nonhuman animal selected from the group consisting
of enzymes, lactoferrin, transferrin, nonspecific immunoglobulins,
components of the complement system, cytokines, white blood cells,
complement components, interferons, and fibronectin, wherein the at
least one specific binding molecule and the at least two components
of the carrier matrix are obtained from different animals. In
another aspect, the matrix is comprised of two or more agents
selected from lysozyme, phospholipase, defensins, opsonins,
proline-rich polypeptides (PRP), beta-lysin, lactoferrin,
transferrin, cytokines, interleukins, chemokines, interferons,
TNF-alpha, fibronectin, proline-rich polypeptides, insulin growth
factor type 1, insulin Growth Factor type 2, derived platelet
growth factor, epidermal growth factor, fibroblast platelet growth
factor, transforming growth factor alpha, transforming growth
factor beta, nerve growth factor, leptin, leukocytes, white blood
cells, phagocytes, macrophages, monocytes, neutrophils,
polymorphonuclear cells, and dendritic cells, mast cells,
eosinophils, basophils, natural killer (NK) cells, lymphokine
activated killer (LAK) cells, cationic proteins including
defensins, proteolytic enzymes including elastase, cathepsin G,
myeloperoxidase, NADPH oxidase components, or a combination
thereof. In another aspect, the matrix includes a mixture of agents
from the innate immune system. In a preferred aspect, the carrier
matrix is comprised of non-hyperimmune bovine colostrum.
[0158] Bovine colostrum is produced by cows for their newborn
calves. In many dairy cow herds the calves are not permitted to
nurse; rather, they are fed colostrum and later milk from a bottle
then a bucket. The colostrum is collected and processed for
commercial uses. Various compositions including colostrum and
processes for preparing colostrum have been disclosed in U.S. Pat.
Nos. 5,846,569, 6,410,058, 6,475,511, and 6,521,277, the contents
of which are incorporated by reference in their entireties. Dried
bovine colostrum is commercially available. In one specific aspect,
the carrier matrix is commercial dried bovine colostrum.
[0159] Livestock husbands/breeders commonly bank colostrum from
their animals. Colostrum produced on their own premises is
considered to be superior to colostrum from other sources, because
it is produced by animals already exposed to (and thus making
antibodies to) pathogens occurring on the premises. Generally,
colostrum from animals exposed to relevant pathogens will have
superior immunological characteristics.
[0160] Bovine colostrum and its components are safe for human
consumption, except in the context of intolerance or allergy to
lactose or other components. Bovine colostrum from pasture-fed cows
contains immunoglobulins specific to many human pathogens,
including Escherichia coli, Cryptosporidium parvum, Shigella
flexneri, Salmonella, Staphylococcus, and rotavirus, depending upon
their natural exposure to these pathogens. Before the development
of antibiotics, colostrum was the main source of immunoglobulins
used to fight infections.
[0161] Hyperimmune colostrum represents an attempt to boost the
effectiveness of natural bovine colostrum by immunizing cows with a
specific pathogen. This approach is promising as antibodies are
produced to the specific pathogens or antigens used in the original
challenge. However, varying response to antigens, biological
variability, and low production yield of colostrum have limited its
clinical and commercial utility.
[0162] In one aspect, the disclosure provides a composition
comprising colostrum that is not hyperimmune colostrum or that does
not contain a measurable or significant amount of antibodies
specific for the pathogenic or target antigen components. In
another aspect, the disclosure provides a composition in which the
carrier matrix contains various components of the innate immune
system without a significant amount of either specific or
non-specific antibodies.
[0163] In one embodiment, the colostrum can be processed to remove
the majority of immunoglobulin, e.g., by absorbing the antibodies
onto an affinity resin (e.g. Protein G or Protein A Sepharose; or
Protein A or Protein G Agarose) in a batch or column format and
retaining the eluate for further processing. Immunoglobulin can
also be removed by gel filtration chromatography on Sephadex G-200
or DEAE Sephadex A-25 ion exchange chromatography. (Lloyd and
Soulsby, Immunology, The role of IgA immunoglobulin in the passive
transfer of protection to Taenia taeniaeformis in the mouse. 34,
939-945) These processes can be run on a column or a batch format
by various methods and techniques known in the art.
[0164] In one specific embodiment, the carrier matrix includes
colostrum. In one aspect, commercial colostrum is employed as the
supportive/reactive matrix. In a preferred aspect, the commercial
bovine colostrum is an agglomerated and instantized, pasteurized,
full cream, whole colostrum powder produced from first milking
colostrum only. In another aspect, the colostrum is processed at
low pressures and low temperatures and is spray dried using
indirect steam to maintain maximum bioactivity. In another aspect
the commercial colostrum is from antibiotic free sources. In
another aspect, the colostrum is subjected to microbiological
analysis and is found to be negative, or below acceptable levels
with respect to a variety of pathogens. In various other aspects,
the colostrum is assayed for other contaminants such as nitrates,
aflatoxin, nitrofuran, dioxins, melamine, and heavy metals and
found to be negative or below specified levels.
[0165] In one embodiment, the invention may be composed of
colostrums of several hyperimmunized sources, each targeting a
different cluster or class of antigen, where the colostrums are
admixed to provide a broad-spectrum antibody formulation.
[0166] In another embodiment, the carrier matrix is comprised of a
reconstituted or artificial mucosal secretion such as tear fluid,
nasal or bronchial mucous, cervical mucous, seminal plasma, sweat,
blood plasma or saliva. Body fluids are known to contain several
components in varying amounts. (Schenkels et al., Biochemical
composition of human saliva in relation to other fluids, Crit. Rev.
Oral Biol. Med., 1995, 6(2):161-175). Saliva contain mucins, acidic
PRPs, alpha-amylase, basic PRPs, basic PRG, secretory IgA,
cystatins, statherin, IgG, extra-parotid glycoprotein (EP-GP), VEGh
(a lipocalin), histatins, lysozyme, kallikrein, lactoferrin,
lactoperoxidase, haptocorrin, beta-microseminoprotein, IgM,
albumin, and Zn-alpha2-glycoprotein. In one aspect, the carrier
matrix comprises two or more, three or more, four or more, five or
more, six or more, or seven or more of the components of body
fluids.
[0167] Tear fluid, or lachrymal fluid, has many of the same
components as saliva and has a particularly high concentration of
secretory IgA, VEGh, lysozyme, and lactoferrin. In one aspect,
artificial lacrimal fluids containing salts such as sodium chloride
and the like as a main ingredient, or eye drops containing
hydroxyethylcellulose, chondroitin sulfate or hyaluronic acid or
xanthan gum (U.S. Pat. No. 7,875,271, which is incorporated herein
by reference) as known in the art are fortified with two or more,
three or more, four or more, five or more components of the body
fluids as described and used as a carrier matrix for purified
polyclonal antibodies, as described herein. In one aspect, a
composition could be used to treat microbial infections of the eye,
such as pink eye.
[0168] Cervical mucous contains mucins, alpha-amylase, lysozyme,
lactoperoxidase, albumin, and beta-microseminoprotein. The matrix
is formed by combination of two or more, three or more, four or
more, five or more of these components as a carrier matrix in a
composition with a steric specific binding molecule, such as
anti-bacterial or anti-fungal polyclonal antibodies, prepared by
the methods of the disclosure.
[0169] In one aspect, the disclosure provides a composition
comprising a gum capable of fixing water or swellable in water,
containing carboxymethylstarch combined with a cellulose as a
permeating agent, which when put into contact with water form
almost instantaneously gels and is readily applicable for vaginal
application. Tablets comprising the antibody/matrix composition of
the disclosure could for example comprise carboxymethylstarch and
cellulose as described in U.S. Pat. No. 4,808,415, which is
incorporated herein by reference. In a particular aspect, the
antibacterial and antifungal polyclonal antibodies are combined in
the matrix and formulated to provide a broad spectrum treatment for
vaginosis. In one aspect, the composition is used to treat a
vaginal bacterial infection, such as trichomonas infection, or
fungal vaginosis, such as a candida infection.
[0170] Saliva is a mucosal secretion present in the oral cavity and
produced by salivary glands. Saliva serves protective functions
such as tissue coating, lubrication, humidification, and
remineralization of the teeth. Saliva also serves host defense
functions with immunological activity, anti-bacterial, anti-viral
and anti-fungal activity. Saliva also serves digestive activity
with digestive enzymes, bolus formation and taste. Saliva contains
various proteins such as histatins, and acidic proline-rich
proteins that are unique to saliva. Saliva also contains proteins
present in other body fluids such as lysozyme, mucins, statherins
and immunoglobulins. Saliva contains proteins such as albumin and
Zn-alpha-2-glycoprotein that originate in blood plasma. There is a
known therapeutic value of bovine saliva. (Varshney et al., 1997,
Therapeutic value of bovine saliva in wound healing: a
histomorphological study., Indian J. Biol. May 1997, 35(5):535-7).
In one aspect, components of saliva could be useful, for example,
in toothpaste or mouthwash, or other preparations for oral mucosal
administration.
[0171] Bronchial mucous contains mucins, alpha-amylase, basic
proline-rich polypeptides (PRPs), cystatins, statherin, EP-GP,
lysozyme, beta-microseminoprotein, and albumin. In one aspect, the
disclosure provides a composition comprising a steric specific
binding molecule and a carrier matrix comprising two or more, three
or more, four or more of the components of saliva or bronchial
secretions. In one aspect, the composition with the carrier matrix
is to be packaged in a dry format with the steric specific binding
molecule, such as anti-Group A Streptococcus polyclonal antibodies
prepared according to the disclosure. In one aspect, the dry
formulation is reconstituted, for example in a saline solution, and
administered as a throat spray for treatment of strep throat.
[0172] Other carrier matrices may be prepared to function in other
use environments, for example for aerosolized (inhaled), ocular,
topical, or other preparations.
[0173] In a specific embodiment, the specific binding molecule and
the carrier matrix are derived from different species. In a further
aspect, both the specific binding molecule and the carrier matrix
are derived from non-human species. In another aspect, the specific
binding molecule is derived from a non-mammalian animal. In another
aspect, the carrier matrix is derived from a non-human mammal.
Formulations and Compositions
[0174] In one embodiment antibodies are harvested from the plasma,
serum, or blood, colostrum, eggs, or other component of an
inoculated animal or artificial production system (such as cell
culture), then purified or treated, and added to a carrier matrix
such as colostrum. The compositions allow are used as a delivery
medium for, e.g., oral administration of the antibody formulation.
This approach may provide an effective way of reliably scaling
antibody production for formulation in this manner, so as to
control titer, consistency, and continuous availability, for
commercial use. In one embodiment antibodies are harvested from the
eggs of an inoculated animal, and may be purified or treated or
retained in the egg material, and added to bovine colostrums.
[0175] There is a clear need for low cost and effective treatments
for many gastrointestinal pathogens, and orally administered
antibodies are candidates for this role. In addition to having
demonstrated efficacy, orally administered antibodies are typically
non-immunogenic. They are considered typically well tolerated with
no adverse side effects reported and comparatively no different
reactions than a comparable ingested food product. Notably several
products containing orally administered antibody have received GRAS
(Generally Recognized as Safe) certification by the FDA.
[0176] One embodiment of this invention is a broad spectrum
therapeutic or prophylactic antitoxin formulation composed of an
admixture of broad-spectrum neutralizing antibodies, embedded
within a carrier matrix, produced according to this method, for the
purposes of allowing for effective administration across a wide
range of unknown or undiagnosed conditions resulting in toxin
mediated diarrhea.
[0177] One embodiment of this invention is a broad spectrum
therapeutic or prophylactic anti-pathogen formulation, embedded
within a carrier matrix, containing an admixture of broad-spectrum
anti-pathogen antibodies produced according to this method.
[0178] One embodiment of this invention is a broad spectrum
therapeutic or prophylactic anti-adhesin formulation embedded
within a carrier matrix, containing an admixture of broad-spectrum
anti-adhesin antibodies produced according to this method.
[0179] One embodiment of this invention is a broad spectrum
therapeutic or prophylactic formulation embedded within a carrier
matrix, containing an admixture of broad-spectrum antitoxin,
anti-pathogen, and anti-adhesin antibodies produced according to
this method.
[0180] One important limitation of using natural food based
products is that preparations are limited to the results allowed by
natural processes. The present invention allows for the selective
addition of levels of specific antibodies and general immune
factors (formulation) that are significantly higher than
physiological levels that can normally be achieved in nature. The
present invention also allows for a weighting of various factors in
a manner so as to create greater specificity to targeted diseases,
pathogens, or substances.
[0181] In one embodiment, the formulation comprising the specific
binding molecule is a dry solid (egg powder) formulation. The
powdered formulation is sealed in airtight packets, optionally
layered with an inert gas. The formulation can be stored for
extended periods of time at room temperature, under refrigeration,
or frozen temperatures. In other embodiments, the dried composition
is formulated into capsules or tablets for oral administration. In
another embodiment, the formulation is compressed into chewable
tablets.
[0182] Another embodiment of the present invention relates to the
pharmaceutical acceptable diluents for formulating the composition,
wherein said pharmaceutical acceptable diluents are selected from
the group consisting of a lactose, mannitol, sorbitol,
microcrystalline cellulose, sucrose, sodium citrate, dicalcium
phosphate, or any other ingredient of the similar nature alone or
in a suitable combination thereof; binder selected from the group
consisting of gum tragacanth, gum acacia, methyl cellulose,
gelatin, polyvinyl pyrrolidone, starch or any other ingredient of
the similar nature alone or in a suitable combination thereof;
excipients selected from the group consisting of agar-agar, calcium
carbonate, sodium carbonate, silicates, alginic acid, corn starch,
potato tapioca starch, primogel or any other ingredient of the
similar nature alone or in a suitable combination thereof;
lubricants selected from the group consisting of a magnesium
stearate, calcium stearate or steorotes, talc, solid polyethylene
glycols, sodium lauryl sulfate or any other ingredient of the
similar nature alone; glidants selected from the group consisting
of colloidal silicon dioxide or any other ingredient of the similar
nature alone or in a suitable combination thereof; a sweetening
agent selected from the group consisting of such as sucrose,
saccharin or any other ingredient of the similar nature alone or in
a suitable combination thereof; a flavoring agent selected from the
group consisting of peppermint, methyl salicylate, orange flavor,
vanilla flavor, or any other pharmaceutically acceptable flavor
alone or in a suitable combination thereof; wetting agents selected
from the group consisting of acetyl alcohol, glyceryl monostearate
or any other pharmaceutically acceptable wetting agent alone or in
a suitable combination thereof; absorbents selected from the group
consisting of kaolin, bentonite clay or any other pharmaceutically
acceptable absorbents alone or in a suitable combination thereof;
retarding agents selected from the group consisting of wax,
paraffin, or any other pharmaceutically acceptable retarding agent
alone or in a suitable combination thereof.
[0183] In another aspect, the daily dose for the non-neonate human
is standardized by any method of quantifying the specific
antibodies. In one aspect, the dose of the composition is
standardized by use of an ELISA to evaluate the concentration of
specific anti-antigen antibody in the formulation. In one aspect,
one dose of the oral composition effective to treat a pathogenic
infection contains antigen-specific binding molecule in an amount
from about 0.0001 mg to 20 mg; from 0.001 mg to 15 mg; from 0.01 to
10 mg; from 0.05 to 5 mg; from 0.1 to 1 mg of mixed antigen
specific binding molecule.
[0184] The term "solid form" refers to a dried form of a specific
binding molecule, or a dried form of a carrier matrix, or a solid
dosage form comprising both the dried specific binding molecule and
the carrier matrix as a powder, compressed tablet, troche, or
capsule. In one aspect, the solid dosage form is intended for oral
administration. In one aspect, the powder is a formulation for
suspension. In one aspect, powdered dried immune egg and powdered
dried colostrum are packaged in an airtight packet. Immediately
prior to oral administration, the contents of the packet are
suspended, or dissolved, in about a liquid and administered
orally.
[0185] In one aspect, the composition may also be provided in a
liquid form for administration.
[0186] In one aspect, one dose contains 1 g, 2 g, 3 g, 4 g, 5 g, 5
g, 6 g, or 7 g of dried immune egg and 1 g, 2 g, 3 g, 4 g, 5 g, 5
g, 6 g, or 7 g dried bovine colostrum. In one aspect, one dose of
the dried dosage form contains 3 g dried immune egg product and 4 g
dried bovine colostrum. In one aspect, one dose of the dried dosage
form contains 2 g dried immune egg product and 4 g dried bovine
colostrum. In one aspect, one dose of the dried dosage form
contains 4 g dried immune egg product and 4 g dried bovine
colostrum. In another aspect, the contents of a single dose packet
are dissolved in about 2 ounces of water and administered
orally.
[0187] Formulations for oral use may also be prepared as troches,
chewable tablets, or as hard gelatin capsules wherein the active
ingredient is mixed with an inert solid diluent (e.g., potato
starch, lactose, microcrystalline cellulose, calcium carbonate,
calcium phosphate or kaolin), or as soft gelatin capsules wherein
the active ingredient is mixed with water or an oil medium, for
example, peanut oil, liquid paraffin, or olive oil. Powders and
granulates may be prepared using the ingredients mentioned above
under tablets and capsules in a conventional manner using, e.g., a
mixer, a fluid bed apparatus or a spray drying equipment.
[0188] In various embodiments, the formulations of the disclosure
provide a variety of advantages with respect to the prior art. In
one aspect, the formulations of the disclosure comprising
antigen-specific IgY and a carrier matrix of bovine colostrum have
the advantage of being prepared in a rapid time period of within
about 6 weeks, once the antigens of interest are identified. This
allows easy reproducibility and standardization of the chicken
vaccination protocol. In one specific aspect, different flocks of
chickens are vaccinated with a single, mixed antigen preparation
each, and then combined for a broad spectrum composition for the
treatment of a pathogenic infection. In one specific aspect, three
flocks of chickens are vaccinated with separate mixed antigen
preparations then pooled to prepare a broad-spectrum composition
for the treatment of undifferentiated diarrhea without knowledge of
the causative microbial pathogens. This method has the advantage
that the mix of antigen-specific antibodies in the composition can
be tailored for a particular outbreak, region, or season, if
desired. Finally, in embodiments, the specific binding molecule
need not be separated from the whole dried egg for rapid
preparation and long term storage.
[0189] In another aspect, the compositions of the disclosure are
effective for oral administration in the treatment of a pathogenic
infection in non-neonates. The gastrointestinal tract of the
non-neonate is very acidic and less absorptive than the neonate, as
described herein. In the examples of the disclosure, the
compositions were effective for treating undifferentiated diarrhea
in non-neonatal children of 6 months to 5 years of age. In another
aspect, the compositions of the disclosure are effective to treat
or prevent traveler's diarrhea in adults. The carrier matrix is a
protective and reactive matrix for combination with the
antigen-specific binding molecules. In another aspect, the
compositions of the disclosure are provided in a powdered, solid
form for suspension immediately prior to administration. In one
aspect, the suspended, or reconstituted, dosage form has the
advantage of being very palatable to infants and children, even
when suffering from the symptoms of a pathogenic infection. This
has the advantage that the full dose is easily administered and
ingested by the subject suffering from the pathogenic
infection.
[0190] In another aspect, the compositions of the invention can be
used for administering broad-spectrum passive immunity in either
treatment, or prophylaxis of pathogenic infection. In one aspect, a
low level of immunization of chickens can be sufficient to prepare
a composition with an effective amount of anti-antigen specific
binding molecule to result in an effective, broad-spectrum
formulation when administered with a carrier matrix.
Treatment or Prophylaxis of Pathogenic Infection
[0191] The compositions of the disclosure comprise a specific
binding protein embedded within a carrier matrix. The compositions
can be administered in any method suitable to their particular
immunogenic or biologically or immunologically reactive
characteristics, including oral, intravenous, buccal, nasal,
mucosal, dermal or other method, within an appropriate carrier
matrix. A specific embodiment involves the oral administration of
the composition of the disclosure.
[0192] In various embodiments, the composition is administered as a
prophylactic or therapeutic composition. In various aspects, the
composition includes a pharmaceutically acceptable carrier. In
various aspects, the composition does not include a polymer,
copolymer, liposome, hydrogel, or fibrin. In various aspects, the
composition does not include microspheres or microcapsules. In
various aspects, the composition does not include an immunogen or
antigen. The composition of the invention can be administered via
oral delivery, nasal deliver, ophthalmic delivery, ocular delivery,
mucosal delivery, or a combination thereof.
[0193] One embodiment of this invention uses oral administration.
It has been demonstrated in both human and animal systems that oral
(ingested) administration of antibodies, immunoglobulins, and other
biological immune factors can have measurable effects on the
course, severity and duration on diseases of, in, associated with,
or influenced by, the gastrointestinal system.
[0194] The admixture of broad-spectrum antibodies is embedded in a
within a carrier matrix, such as for example colostrums for oral
administration. Colostrum serves to provide synergistic protective
and efficacious attributes to the antibody formulation. Any
combination of antibodies can be used in within a carrier,
including but not limited to a combination of anti-pathogen,
anti-toxin, and anti-adhesin antibodies.
[0195] In one aspect, the compositions of the disclosure are used
to treat patients suffering from various pathogenic infections. The
compositions and formulations for oral administration can be
administered once, twice, three times, or four times a day for two,
three, four, five, six, seven, eight, nine, 10, 11, or 12
consecutive days for the treatment of a pathogenic infection. In
one aspect, the composition is administered twice per day for five
days for the treatment of a pathogenic infection. In another
specific aspect, the composition is administered once per day for
three consecutive days for the effective treatment of
undifferentiated diarrhea in non-neonatal children, or in the
treatment of traveler's diarrhea in non-neonatal children or
adults. In another aspect, the composition may be regularly
administered for the prophylaxis of a pathogenic infection.
[0196] In the case of a composition for the treatment of a
pathogenic infection of a mucosal membrane by topical
administration to a mucosal membrane, the composition can be
administered two to six times per day for a period of three to 12
days.
[0197] In a preferred embodiment, the disclosure provides a
composition effective for treating undifferentiated diarrhea in
non-neonate humans. The composition takes advantage of an effective
polyclonal antibody production strategy (chicken innoculation, with
antibody harvesting through eggs) to generate high specificity
antibodies targeted to several of the causes of diarrhea pathology.
In a specific embodiment, the composition comprises specific
polyclonal antibodies in a carrier matrix that is commercial bovine
colostrum.
[0198] In a preferred embodiment, the disclosure provides an
economical composition for the effective treatment of
undifferentiated pediatric diarrhea. The composition comprises a
mixture of polyclonal antibodies, primarily IgY, specific for E.
coli, Salmonella spp., rotavirus, gram negative bacteria, toxins
produced by pathogens, and adhesins that enable pathogen attachment
and colonization in the gastrointestinal tract.
[0199] In a specific aspect, the composition comprises an
equivalent weight amount of dried immune egg product from each of
three flocks inoculated with different antigens or different mixed
antigen preparations is co-packaged with a specific weight amount
of commercial dried non-hyperimmune bovine colostrum. In one
aspect, 0.5 to 3 g. 0.7 to 2.0 g, 1.0 g, 1.3 g, or 1.5 g of dried
immune egg product from each flock is added to a single dose
packet. Preferably either 1.0 g or 1.3 g each immune egg product is
added to a one dose packet. In another aspect, 1 to 5 g, 2 g to 4
g, 1.5 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 4.5 g or 5 g dried
colostrum is added to the same packet.
[0200] Prior to use, the contents of the packet, or sachet, are
mixed into approximately 2 ounces of purified water, or some other
oral liquid. The entire reconstituted formulation is administered
orally to the subject. The composition can be administered one to
four times per day for two to ten days. In a specific embodiment,
the composition is administered once per day for 3 consecutive
days. The disclosure provides a method of treating undifferentiated
pediatric diarrhea, by administration of the composition of the
disclosure once per day for two, three or four days.
[0201] In one aspect, the composition of the disclosure is
administered as an adjunct therapy to antibiotic treatment. In this
aspect, the composition may be administered once per day for the
first three days of treatment. In another aspect, the composition
of the disclosure is administered with oral rehydration solution
(ORS). In another aspect, the composition of the disclosure is
co-administered with an oral zinc formulation. In another aspect,
the composition of the disclosure is administered as an adjunct to
antibiotic treatment to prevent overgrowth of a particular
pathogenic organism that is resistant to the antibiotic. As
described in detail in the examples, the composition and method is
effective to rapidly resolve the symptoms of undifferentiated
pediatric diarrhea, resulting in significantly decreased stool
volume, stool frequency and duration of diarrhea, as well as
significantly improved physician reported well-being.
[0202] In one alternative embodiment, the compositions of the
disclosure are used to treat traveler's diarrhea. The onset of TD
usually occurs within the first week of travel but may occur at any
time while traveling, and even after returning home. The most
important determinant of risk is the traveler's destination.
High-risk destinations are the developing countries of Latin
America, Africa, the Middle East, and Asia. Persons at particular
high-risk include young adults, immunosuppressed persons, persons
with inflammatory-bowel disease or diabetes, and persons taking H-2
blockers or antacids. Most TD cases begin abruptly. The illness
usually results in increased frequency, volume, and weight of
stool. Altered stool consistency also is common. Typically, a
traveler experiences four to five loose or watery bowel movements
each day. Other commonly associated symptoms are nausea, vomiting,
diarrhea, abdominal cramping, bloating, fever, urgency, and
malaise.
[0203] Infectious agents are the primary cause of TD. Bacterial
enteropathogens cause approximately 80% of TD cases. The most
common causative agent isolated in countries surveyed has been
enterotoxigenic Escherichia coli (ETEC). ETEC produce watery
diarrhea with associated cramps and low-grade or no fever. Besides
ETEC and other bacterial pathogens, a variety of viral and
parasitic enteric pathogens also are potential causative
agents.
[0204] In one aspect, the composition of the disclosure is
administered to the subject once per day for three consecutive days
as an alternative or adjunct to antibiotic treatment of traveler's
diarrhea. Limited field study evidence suggests improvement in
diarrheal symptoms within 24 or 48 hours of the first dose.
Alternatively, two doses per day of the composition of the
disclosure are administered on day 1, followed by a single dose on
days 2 and 3. In one aspect, the composition of the disclosure is
administered on an alternate daily or weekly schedule, or on a
reduced dosage schedule to for prophylaxis of traveler's
diarrhea.
[0205] In another alternative embodiment, the compositions of the
disclosure are used to as a "prebiotic" for gastrointestinal flora
management of a subject, for example, prior to administration of a
probiotic. As used herein, the term "prebiotic" refers to a
composition that allows specific changes, both in the composition
and/or the activity of the gastrointestinal microflora that confers
benefits upon the subject's well-being and health. In one aspect,
the composition is useful to manage gastrointestinal flora so as to
reduce or eliminate one or more undesirable strains of bacteria. In
one aspect, the anti-antigenic immunoglobulin composition is
tailored to manage gastrointestinal flora so as to reduce or
eliminate one or more undesirable strains of bacteria. In another
aspect, the compositions are used as an adjunct to traditional
prebiotics. In a further aspect, the composition of the disclosure
further comprises a soluble fiber. In a further aspect the
composition is used alone for flora management.
[0206] In another aspect, the disclosure provides a method of
gastrointestinal flora management in a subject comprising the steps
of administering the composition of the disclosure to reduce or
eliminate one or more undesirable strains of bacteria, followed by
administering a probiotic to introduce one or more desirable
strains of bacteria. In another aspect, the composition of the
disclosure is administered as an adjunct to antibiotic treatment to
prevent overgrowth of a particular pathogenic organism that is
resistant to the antibiotic.
Example 1. Compositions for the Treatment of Diarrhea
[0207] Diarrhea is a symptom of a broad range of causes including
bacterial, viral, protozoal and parasitic infections. Bacterial
diarrhea is induced by multiple organisms, including various forms
of Escherichia coli, Salmonella, Vibrio cholerae and
parahemolyticus, Shigella, Campylobacter, Yersinia and others.
Viral pediatric diarrhea is often caused by Rotavirus, but also may
be caused by several other viruses.
[0208] There are known to be multiple causative organisms in
diarrhea. These causative organisms can be organized into common
clusters that produce structurally related toxins, to which a
series of broad-spectrum neutralizing antibodies can be created
that, when admixed into a formulation with clinically effective
titers, can be used as a broad-spectrum organism-independent
therapeutic intervention for toxin-mediated diarrhea.
[0209] Briefly, antibodies specific to causative organisms of
diarrhea are generated by inoculation of chickens with antigen.
Immune eggs are collected and whole egg is pasteurized and spray
dried to obtain a powderized form. Commercial bovine colostrum is
mixed in a powderized form. The two powders are added sequentially
to a single dose packet and sealed, and distributed in dried form
for an oral formulation. Before administration, the powdered oral
formulation is mixed with a small quantity of water prior to oral
consumption.
[0210] This treatment confers passive immunity to patients, as
demonstrated in the Examples herein. The nature of the treatment
makes the associated risk factors comparable to that of eating food
from the source where the antibodies were harvested (e.g., risk
factors would be similar to that of eating an egg and a glass of
milk). This is an effective treatment with less toxicity than the
currently available alternative medicines.
Example 1A
[0211] Chickens were individually inoculated with purified antigens
derived from 5 E. coli strains: four ATCC strains, containing E.
coli adherence pili antigens F41, 97P, F19 and K99, and one wild
type E. coli strain derived from milk. Each chicken was inoculated
with only one antigen. Chickens were inoculated once per week for
three weeks. Freund's adjuvant was employed for the first
inoculation, followed by Freund's incomplete adjuvant for the
second and third inoculations. Two shots, left and right breast
were used per inoculation. Eggs were housed separately; eggs were
collected, flash pasteurized and spray dried. Each of the five
antibody preparations were mixed in equal parts. The dried egg
powder anti E. coli antibody preparation was stored frozen for
about 2 years.
[0212] A second flock of chickens was inoculated with a mixed
antigen preparation containing rotavirus, coronavirus and E. coli
antigens. The same inoculation, collection and egg processing
protocols were employed as above. The dried egg powder anti-scours
antibody preparation was stored frozen for 1.5 years. ELISA was
used to characterize the antibody preparations.
[0213] One gram each of the dried anti-E. coli antibody preparation
and the dried anti-scours antibody preparation were added with 3
grams or 4 grams of commercial dried full-fat bovine colostrum in a
single dose packet.
Example 1B
[0214] Three flocks of chickens were individually inoculated with
one each of different mixerd antigen preparations: a first antigen
preparation containing rotavirus (serotypes G6 and G10),
coronavirus, enterotoxigenic E. coli stains with K99 pili adherence
factor, and Clostridium perfringens type C toxoid with adjuvant); a
second preparation containing enterotoxigenic strains of E. coli
having K99, K88, 987P or F41 adherence factors); and a third mixed
antigen preparation containing various E. coli endotoxin; with
adjuvant). Each of three flocks only received a single mixed
antigen preparation. Eggs were collected, cleaned, broken,
pasteurized and spray dried or thermal dried to create three dried
immune egg products. Dried egg product was optionally evaluated by
ELISA for specific IgY activity. An equal weight of each of the
three dried immune egg products was combined with 3 g or 4 g of
dried colostrum in a single dose packet. Either 2 g, 3 g, or 4 g of
combined weight of dried immune egg product was employed per single
dose packet, as described below. In one aspect, the commercial
dried colostrum did not exhibit specific activity toward the
antigens of the vaccines.
Example 1C
[0215] Immunization of chickens for IgY Production.
[0216] The following immunization protocol was adapted from a
Gallus Immunotech, Inc. protocol and can be utilized for generation
of IgY polyclonal antibodies. A few eggs are optionally collected
prior to immunization to serve as a baseline control. If a mixed
antigen preparation for cattle or hogs is employed, it is diluted
at 1:2, 1:4, 1:8, or 1:16 prior to administration. On day 0,
chickens are injected with between 0.02 and 0.5 mg antigen with
Freund's complete adjuvant. Injections can be either subcutaneous
or intramuscularly into the breast tissue of the hen at multiple
sites. The total volume of antigen/adjuvant mixture can be about 1
mL with adjuvant from one-half to two-thirds of the volume.
Immunizations are repeated, typically, on days 14, 21 and 28, using
Freund's incomplete adjuvant, with about half the initial amount of
antigen. Typically, specific antibody can be detected at about day
30 in eggs. For prolonged antibody production, hens are boosted
every couple of months. Eggs can be stored in cold storage prior to
processing and/or purification of IgY. In one aspect, eggs can be
held in cold storage for up to one month, or up to two months,
prior to processing or purification. In another aspect, IgY can be
generated in a similar fashion in duck, goose, ostrich, quail, or
turkey eggs, with use of appropriate amounts of antigen.
Example 2. Ingested Antibody Treatment for Clostridium
difficile
[0217] In one embodiment, the invention methods and compositions
are used to treat Clostridium difficile (C. difficile), a bacterium
that is naturally present in most people. The population levels of
C. difficile are kept under control by the other natural flora of
the bowel. Patients often develop C. difficile infections when
antibiotics administered for another medical condition deplete the
natural flora of the bowel, allowing C. difficile populations to
multiply unchecked. While many strains of C. difficile can be
treated by specialized antibiotics an increasingly large number of
C. difficile strains are resistant to antibiotic treatment. This
leads to a lengthy and difficult recovery for patients, and may
even become life-threatening in certain circumstances. A process
neutralizing C. difficile populations with an ingested antibody
that confers passive immunity is capable of controlling C.
difficile population levels to allow a natural bowel flora balance
to be restored.
[0218] As is the case in anti-diarrhea formulations caused by
rotavirus and gram-negative bacteria, an antibody embedded in a
carrier matrix formulated specifically to bind to C. difficile or
its toxins is an effective therapeutic approach. This formulation
can be used to either treat an ongoing infection, or to prevent
such an infection from occurring. Therefore, the treatment can be
administered alone, or concurrently with an antibiotic. This
treatment not only benefits patients recovering from a C. difficile
episode, but can be administered to patients at high risk of
developing C. difficile as a prophylactic.
[0219] The antibodies that neutralize C. difficile are ingested in
a carrier matrix (a mixture of proteins and enzymes that are
intended to "activate" the antibody in the bowel, as well as
provide useful secondary immunity, protection or nutrition). In one
embodiment, the antibodies are produced by injecting, or
inoculating, an animal with an antigen, or a combination of
antigens, which may or may not be contained in a mixed antigen
preparation, (potentially combined with an adjuvant to elicit a
stronger immune response).
[0220] In one aspect, the antigen is obtained from, or derived
from, a C. difficile antigen or toxin. In another aspect, the
combination of antigens contains one of more antigens or toxins
derived from C. difficile, and one or more additional viral
antigens. In another aspect, the combination of antigens contains
one or more antigens or toxins derived from C. difficile, and one
or more additional bacterial antigens or toxins. In another aspect,
the combination of antigens contains one or more antigens derived
from C. difficile, and one or more additional protozoal antigens.
In another aspect, the combination of antigens contains one or more
antigens derived from C. difficile, and one or more additional
fungal antigens.
[0221] The antibodies are then either obtained from, isolated from,
or derived from, an animal product, such as milk, eggs, or
colostrum from the animal or harvested directly from, the animal,
e.g. serum, plasma. In a particular aspect, hens are inoculated
with the antigen, combination of antigens, or vaccine, and the
antibodies are obtained from whole eggs, or egg yolks, or derived
from, or purified from whole eggs or egg yolks of the inoculated
chickens. In another aspect, the antibodies are polyclonal
antibodies.
[0222] This composition is intended to help treat C. difficile
infections, or be a prophylactic against C. difficile infection.
For example, the substance is comprised of antibodies targeted
specifically to C. difficile, embedded within a carrier matrix (for
example, colostrum). After harvesting, the antibodies may be
powderized. The carrier matrix may also be powderized. The two
powders may then be mixed thoroughly, or added separately to a
single dose packet, or vial, and distributed in dried form. In a
preferred method of administration, the substance will be
administered orally, by ingestion. To consume, the powdered
substance will be mixed with a small quantity of a liquid, such as
water, milk, juice, or electrolyte solution, immediately prior to
consumption, and will be taken as directed by a physician. Other
methods of delivery are also contemplated.
[0223] Current treatment for C. difficile infection focuses on
antibiotic therapy. However, in cases where strong antibiotics were
the cause of infection, and in cases where resistance to
antibiotics has been developed, few alternative treatments are
currently available. The present embodiment seeks to neutralize C.
difficile by utilizing natural immune mechanisms, rather than toxic
antibiotics. It has the advantage of allowing the growth of
naturally occurring flora in the bowel while reducing C. difficile
population levels.
[0224] The combination of antibodies embedded within a carrier
matrix to enhance the effectiveness of the antibodies is not
currently used by any C. difficile disease treatment. The invention
methods confer passive immunity to patients. The nature of the
treatment makes the associated risk factors comparable to that of
eating food from the source where the antibodies were harvested
(e.g., risk factors would be similar to that of eating an egg and a
glass of milk). This is an effective treatment with less toxicity
than the currently available alternative medicines.
[0225] In one aspect, selected antibodies are obtained, purified
and isolated and prepared in a powderized form. In another aspect,
the selected antibodies are not purified, or isolated, but
processed as a whole product. For example, the contents of the
whole egg obtained from the inoculated chicken is processed, e.g.
pasteurized, and prepared in a powderized form, without additional
purification steps. An activating enzyme/protein mixture (for
example, including colostrum) is also prepared in a powderized
form. The two powders are mixed thoroughly and distributed in dried
form for an oral formulation. Before administration, the powdered
oral formulation is mixed with a small quantity of water prior to
consumption.
[0226] This treatment confers passive immunity to patients. The
nature of the treatment makes the associated risk factors
comparable to that of eating food from the source where the
antibodies were harvested (e.g., risk factors would be similar to
that of eating an egg and a glass of milk). This is an effective
treatment with less toxicity than the currently available
alternative medicines.
Example 3. Ingested Antibody Treatment for Helicobacter avlori
[0227] Helicobacter pylori (H. pylori) is a gram-negative bacterium
which can inhabit areas of the stomach. It is generally believed
that H. pylori is associated duodenal and gastric ulcers and
possibly stomach cancer. H. pylori can escape the acidic
environment of the stomach lumen by burrowing into the mucus layer
of the epithelial cell surface which has a more neutral pH
environment. H. pylori can produce adhesins for binding to membrane
associated lipids or carbohydrates of epithelial cells.
Colonization of H. pylori inside areas of the stomach can results
in chronic gastritis, a long-lasting inflammation of the stomach. A
major cause of peptic ulcer is H. pylori infection.
[0228] Selected antibodies against Helicobacter pylori are obtained
and prepared in a powderized form. An activating enzyme/protein
mixture (for example, including colostrum) is also prepared in a
powderized form. The two powders are mixed thoroughly, or added
separately to single dose packets or vials, and distributed in
dried form for an oral formulation. Before administration, the
powdered oral formulation is mixed with a small quantity of water
prior to consumption.
[0229] This treatment confers passive immunity to patients. The
nature of the treatment makes the associated risk factors
comparable to that of eating food from the source where the
antibodies were harvested (e.g., risk factors would be similar to
that of eating an egg and a glass of milk). This is an effective
treatment with less toxicity than the currently available
alternative medicines.
Example 4. Clinical Studies--Efficacy in Undifferentiated
Diarrhea
[0230] Effective broad-spectrum treatment of diarrhea is a
significant challenge due to the wide range of causative organisms,
the limited availability of diagnostic testing for directing
treatment regimes. Current standard intervention for cases of
severe diarrhea includes ubiquitous administration of antibiotics
and oral rehydration salts (ORS). However, this approach has shown
limited effectiveness, and has promoted the development of
antibiotic resistant bacteria strains.
Example 4A
[0231] Field study (trial)s 1 and 2.
[0232] Clinical studies were performed to evaluate the tolerability
and efficacy of the formulation of Example 1A in treating, or
accelerating the resolution of undifferentiated diarrhea. A first
open, single-center, non-comparative study enrolled a total of 63
pediatric patients with pediatric diarrhea of both genders between
six months and five years of age. The study compared clinical
outcomes of Test Group A, "Trial 1", receiving the oral formulation
of Example 1A, administered with antibiotic and ORS, to a Control
Group B, receiving only antibiotic and ORS. A second Test Group AA,
"Trial 2", enrolled 33 patients in a follow up study to test the
formulation of Example 1A under different seasonal conditions.
[0233] All participating pediatric patients presented a "serious"
or "severe" diarrhea profile (level 4 or 5) on a 5 point scale (see
Table 1), as assessed by attending physician. No diagnostic
differentiation was made as to causative agent or etiology of the
pediatric diarrhea. Patients with rice water stool of bloody stool
were excluded. Additionally, patients with known allergies to milk,
chicken, or egg products were excluded.
TABLE-US-00001 TABLE 1 The 5-point scale Level 1 Level 5 Stool
Frequency 1-2 per day 10 or more per day Stool Consistency 1 =
normal 5 = fully liquid Physician Assessed Well-being 1 = normal 5
= severe (typically inpatient)
[0234] Enrolled children (n=63) were divided into two groups, an
experimental group Study 1, "Group A" (34 enrolled children; 29
completing trial), negative control "Group B", (29 enrolled
children; 28 completing trial), and Study 2 "Group AA" (31
enrolled). A second control group "Group BB" receiving antibiotic
and ORS was used as a negative control concurrently with the Group
AA, however, the results are omitted from the figures.
[0235] Each test group received 2 g combined egg powder and 4 g
colostrum, mixed in water, administered orally once per day for
three consecutive days. Each group was observed and the data are
collected for five days. Group A received the composition from
Example 1 in addition to a standard regiment of antibiotics and
oral rehydration supplements (ORS), as determined by the attending
pediatrician. Group B is treated with a standard regimen of
antibiotics and ORS. A six month window of time between Study 1 and
Study 2 was allowed elapse in order to test seasonality. Both
trials were conducted in the same study center. In each group,
antibiotic and ORS prescriptions were determined on a case-by-case
basis by the attending pediatrician (Table 2).
TABLE-US-00002 TABLE 2 Study Groups with Numbers of Cases Completed
Therapy Group administered Completed Treatment Period Observation A
Composition 29 Composition 5 days from Example 1 + from Example
antibiotic + ORS 1: days 1-4 Antibiotic + ORS: days 1-6 AA
Composition 31 Composition 5 days from Example 1 + from Example
Antibiotic + ORS 1: days 1-3 Antibiotic + ORS: days 1-6 B
Antibiotic + ORS 28 Antibiotic + 5 days ORS: days 1-5
[0236] The composition from Example 1A was packaged in 5 gram
powder single dose sachets. The composition was administered
orally, with one packet re-suspended in approximately 2 ounces of
drinking water. Patients were required to drink the entire
suspension in one setting, immediately after re-suspension was
complete, and this protocol was followed in all cases.
[0237] Parameters covered in this example, as measured for each
patient, included stool frequency, stool consistency, and physician
assessed well-being. Stool frequency is the guardian or hospital
reported number of diarrheal bowel movements per 24 hour period.
Stool consistency is a 1-5 scale of consistency with 1 indicating
normal and 5 indicating liquid. Physician assessed well-being is a
1-5 scale of overall condition with 1 indicating normal parameters
for a healthy child and 5 indicating a severely ill child.
[0238] Physicians participating in the trial were asked to provide
their experience of the typically patient progression, as measured
by the three parameters described, over the course of six days. The
reported values were aggregated into a single expected patient
progression baseline for each parameter. Patients were evaluated
both in terms of improvement relative to expected outcomes based on
doctor experience, and against the concurrent negative
controls.
[0239] Data analysis was conducted with MS Excel and Matlab.
Statistical significance was computed by a Chi-square test with
p-value of <0.05 considered significant. Results are shown in
FIGS. 1 to 3.
[0240] Dramatic improvements in patients receiving the composition
from Example 1A were observed within 24 hours of the initial dose
administration. Within 48 hours after initial dose administration
patients were generally stabilized at normal or near normal
levels.
[0241] As shown in FIG. 1, average number of diarrheal bowel
movements in a 24 hour period decreased from 9 to 2 in Group A
(Trial 1) after the initial dose of the composition from Example 1.
Group AA (Trial 2) exhibited a similar reduction from 10 to 3. In
contrast, average number of episodes in Group B (Negative control)
decreased from 11 to 10 in the same time period. The average number
of episodes in Groups A and AA (Trials 1 and 2) remained constant
at 2 from day 3 onward, while Group B diminished gradually
eventually exhibiting 6 episodes per 24 hours by day five. In Group
A, within 24 hours of the treatment with the composition from
Example 1, stool frequency rates returned to near normal levels,
2.32.+-.2.48, an over 86% reduction in the duration of
gastroenteric symptoms when compared to the control population
(P<0.001). Within 48 hours stool frequency rates improve to
2.14.+-.2.19. In Group AA, frequency rates showed similar
stabilization rates, improving to 2.56+/-0.68 within 24 hours and
2.00+/-0.45 within 48 hours, a marked improvement compared to
control (P<0.001)
[0242] As shown in FIG. 2, iinitial stool consistency was liquid in
all patients. Group A and Group AA stool consistency improved to
near-normal levels in 24 hours, after the first dose of the
composition, after the first dose of the composition from Example
1. Control Group B consistency improved but was still liquid in 24
hours, with symptoms not fully resolving the entire observation
period. Group A obtained normal stool consistency in 48 hours and
for the remainder of the study, while Group B, by day 3, improved
to mostly liquid. Group B eventually reached near-normal levels of
stool consistency by day 6, while Group A stool consistency
remained normal throughout days 3-6.
[0243] Surprisingly, within 24 hours of the treatment with the
composition from Example 1A, stool consistency rates returned to
mild levels, 2.05.+-.1.02 for Group A and 1.96+/-0.61 for Group AA,
an over 86% reduction in the duration of gastroenteric symptoms
when compared to the control population (P<0.001). Within 48
hours, Group A stool consistency dropped further to near normal
levels 1.41.+-.0.9, and Group AA levels were 1.17+/-0.37.
[0244] As shown in FIG. 3, all patients enrolled in the study were
rated as severely ill by attending physicians, up to and including
serious dehydration, vomiting and low responsiveness. Patients in
Group A and Group AA improved significantly overnight (P<0.001),
after the first dose of the composition from Example 1, to an
average well-being assessment level of approximately two. At 24
hours, Group B patients remained severely ill. Group A and Group AA
patients, at 48 hours, improved to near normal and continued to
improve on day three obtaining normal condition, while Group B
patients improved but remained very ill. Throughout days 4-6,
patients in Group A remain fully recovered while patients in Group
B improved in a linear manner; however they remained moderately ill
at the end of the study.
[0245] Overall physician reported well-being retuned to near
healthy level within one day, with Group A dropping from an initial
value of 4.46.+-.0.5 1 to 1.9.+-.0.9, a level considered within
normal parameters for this population. Group AA displayed similar
results, falling from an initial level of 4.3+/-0.46 to
2.03+/-0.49. This collectively represents an 86% reduction in the
duration of illness when compared the control population
(p<0.001). Within 48 hours, physician reported well-being
improved further to 1.26.+-.0.83 in Group A and 1.4+/-0.49 for
Group AA.
[0246] A check to confirm the normal distribution of trial cases
against expected prevalence of Rotavirus was made within Group A
(Trial 1), independent of the primary trial evaluation. Stool
samples were collected for 26 of the 29 experimental patients in
Group A and 24 of the 31 patients from Group AA, and were tested at
an independent reference lab using an established commercial
agglutination assay (Slidex Rota-kit, bioMerieux, France). Seven of
the 26 patients sampled in Group A test positive for Rotavirus (27%
of the tested population). This pediatric Rotavirus infection
prevalence is in line with expected results for the season and the
degree of severity of diarrhea cases admitted to the study. Four of
the 24 tested positive in Group AA (17% of the tested population).
The prevalence of rotavirus fro Group AA was somewhat lower than
expected. Therefore, the composition of Example 1A was deemed
effective as administered to treat undifferentiated diarrhea,
including that caused by rotavirus infection.
[0247] To further determine the similarity of response to the
composition from Example 1 between the Rotavirus positive group
(RV) and the non-Rotavirus positive group (Non-RV), the Pearson's
Product-Moment Correlation Coefficient was used (represented as
"R), the strength of which is represented in the range -1 to 1.
Calculation of R used the average of the Non-RV and RV group for
each time point, with calculation of the R-value from the average
value of each group over the 6 days.
[0248] The R value of the RV group's association with the Non-RV
group for the Physician Assessed Well-being dataset is 0.99029,
showing a very strong linear dependence and covariance between the
two groups. The behaviors of Non-RV and RV patients are strongly
predictive of each other, and showed very similar responses to the
treatment over the six day treatment and observation period. These
results confirm the efficacy of the composition from Example 1 in
Rotavirus mediated diarrhea cases (Table 3).
TABLE-US-00003 TABLE 3 RV/Non-RV Average Values Day RV Non-RV 1
4.71 4.38 2 1.71 1.95 3 1.33 1.24 4 1.28 1 5 1.14 1 6 1 1
[0249] Of the 96 patients enrolled in the studies, 88 completed the
full six day study period. Four patients were withdrawn from Group
A, and two from Group AA by the physician when it was determined
that their enteritis was co-morbid with, or the result of, other
conditions; as shown in Table 4. One patient from Group A was lost
to the trial when his guardian decided that the patient was well
enough to withdraw after the second dose of the composition from
Example 1. And, one patient was withdrawn from Group B due to
record keeping error (Table 4).
TABLE-US-00004 TABLE 4 Patients Withdrawn from Study Patient #
Group Reason Withdrawn by A 12 Experimental (A) Measles Study
doctor A 19 Experimental (A) Meningitis 1 A26 Experimental (A)
Patient deemed well Guardian A28 Experimental (A) Measles Study
doctor A33 Experimental (A) Meningitis Study doctor B08 Control (B)
Record keeping error Study doctor
[0250] These results suggest that the composition from Example 1
may provide a safe and effective treatment for undifferentiated
pediatric diarrhea. Reducing the duration and severity of diarrhea
will prevent a significant amount of morbidity and mortality
associated with pediatric diarrhea and may also help prevent
diarrhea-associated co-morbidities from developing in pediatric
patients.
[0251] After one day of the treatment with the composition from
Example 1, pediatricians report substantial improvement in overall
well-being in 100% of the patients completing the trial.
Surprisingly, significant reduction in both the duration and
severity of illness provided an 86% reduction in length of diarrhea
episode after two days of the treatment with the composition from
Example 1. Independent Rotavirus testing confirmed efficacy of the
composition from Example 1A in these cases.
[0252] The composition from Example 1A was shown to be highly
effective in the treatment of undifferentiated diarrhea, greatly
reducing the length and severity of illness when compared to
conventional therapies alone. The composition from Example 1A is
well-tolerated with no adverse side-effects reported. The results
of this study represent an important and robust improvement in the
treatment of pediatric diarrhea within demanding field
environments. These results provide an opportunity for additionally
investigation of the mechanisms and biochemistry by which the
composition of the invention protects patients from the most severe
symptoms of undifferentiated diarrhea.
Example 4B
[0253] Field study (trial) 3.
[0254] A third study trial was conducted with 140 treated patients
and 30 negative control patients enrolled in Trial 3. The daily
dose of the composition in the treated arm contained either 2 g
total of equal portions of dried whole egg from each of three
flocks, each inoculated separately with one commercial scours or
mastitis vaccine; and 4 grams of dried bovine colostrum (ES204A;
MS204A); or 3 grams of equal portions by weight of dried whole egg
from each of three flocks, each inoculated separately with one
commercial scours or mastitis vaccine and four grams of dried
bovine colostrum (MS304A). In addition, egg was processed either by
spray drying (S) or thermal drying (T). The flocks were housed at
two different geographic locations within the United States (M) or
(E).
[0255] Trial 3 was conducted as described above; patients were
treated with the compositions once per day, for three consecutive
days. Average results for Trial 3 compared to Trials 1 and 2 are
shown in FIGS. 4-9. A small arm of Trial 3 with 15 patients was
treated with 2 g dried egg and 4 g colostrum once per day for two
days and exhibited significant improvement at days one and two in
each measured parameter. This group exhibited a slight average
relapse effect in symptom scoring in physician reported well-being
on day 4, stool consistency on days 3 and 4 (ES204B). However,
these values were still significantly improved compared to the
negative control group.
[0256] These results show that a solid formulation for suspension
comprising specific binding molecules which are antigen-specific
IgY antibodies in whole dried egg and a carrier matrix, which is
non-immune dried bovine colostrum is economical and effective. The
matrix of the disclosure, dried bovine colostrum, is easily
commercially available and can provide higher levels of various
matrix components than milk. This is in contrast to, for example,
the prior art teachings of Larrson et al, US 2010/0233162. Larsson
provides a method for local administration of isolated chicken yolk
immune globulins (IgY) in human breast milk to treat and prevent
fungal infections. At the very least, the use of human breast milk
makes the Larsson composition less economical and difficult to
rapidly produce and store. Further, after three days of treatment,
the compositions of the present disclosure are shown to
significantly decrease the duration of undifferentiated diarrhea in
non-neonatal babies and children; where the conditions of the
gastrointestinal tract are harsher than in the neonate. This is in
contrast to Larsson et al., US 2006/0134101, which provides a
method for the use of avian antibodies for treatment and
prophylaxis of enteric infections in newborn infants. This is also
contrast to, Sarker et al., 2001, who reported a clinical trial of
hyperimmunized chicken egg yolk immunoglobulin in non-neonate
children with rotavirus diarrhea that showed little or no
difference in the duration of diarrhea. (Sarker et al., 2001,
Randomized, placebo-controlled, clinical trial of hyperimmunized
chicken egg yolk immunoglobulin in children with rotavirus
diarrhea. J. Pediatr. Gastroenterol Nutr. 32: 19-25).
[0257] In addition, the present compositions utilize dried whole
egg containing antigen-specific IgY with a protective and reactive
carrier matrix such as bovine colostrum to both (1) protect the
antibodies during oral administration, and (2) to further activate
passive immunity as described. This is in contrast to Lee et al.,
US 2003/0185856, which provides a method for the production of egg
containing anti-pathogenic bacteria specific IgY and compositions
in the form of yogurt or ice cream containing the IgY; however, a
protective and reactive carrier matrix is not described. Yogurt and
ice cream generally do not have a high enough concentration of the
matrix components present in the matrix derived from colostrum.
[0258] Unlike an immunoregulatory response, the effects of
administration of the composition could generally be observed
within 6-12 hours of the first administration. The compositions of
the disclosure are effective without reliance on the subject's
immune response.
Example 5. Clinical Study--Unexpected Efficacy in Typhoid Fever
[0259] Evidence for the efficacy of the claimed composition was
provided through an unplanned and unexpected demonstration of
clinical efficacy caused by an unknown prior inoculation.
[0260] During one field study in India a small number of children
were brought forward for treatment who had been clinically
diagnosed with "Typhoid Fever". Typhoid fever is an infection most
commonly caused by a type of bacteria called Salmonella typhi (S.
typhi). Classical symptoms of this disease, beyond diarrhea, are
caused by its systemic infection phase. The bacteria typically
first travel into the intestines, and then into the bloodstream,
where they can migrate to the lymph nodes, gallbladder, liver,
spleen, and other parts of the body. These patients displayed
classical symptoms of advanced disease, including high fever,
general ill-feeling, and abdominal pain, and significantly, a
classical rash--"rose spots," which are small red spots on the
abdomen and chest.
[0261] As is typical for this practice environment, no diagnostic
testing was performed on these patients beyond gross physical
examination. Although these patients did not fit into the inclusion
criteria for the field study they were provided with the
composition of Example 1B, at the request of the attending
physicians, on compassionate grounds.
[0262] The standard inoculation protocol for chickens with three
commercially available vaccines did not specifically include
antigens for Salmonella, so only a limited clinical response was
expected. A mild improvement due to endotoxin neutralization was
predicted, with some associated relief of the major diarrheal
symptoms, but no effect on the course of the disease itself.
[0263] Surprisingly, all of the typhoid fever patients receiving
the composition of Example 1B showed dramatic improvement in
diarrhea symptoms within 24 to 48 hours. This improvement appeared
to be beyond what might be expected for endotoxin neutralization
alone. More surprising however was the fact that the systemic
symptoms of typhoid fever in all cases also disappeared within the
following 24 hour period, yielding a time period to normal or near
normal status of 48 to 72 hours. In typhoid fever symptoms usually
improve in 2 to 4 weeks with treatment.
[0264] None of the patients exhibited any rebound or recurrence of
disease during the field trial observation period (5 days). It is
well established that symptoms may return rapidly if the treatment
has not completely cured the infection.
[0265] Treatment with the composition of Example 1B, once per day
for three consecutive was sufficient to cause (in conjunction with
standard of care) a dramatic reduction in symptoms of the disease,
both GI and systemically, within a remarkably short period of time.
The timeframe of response could not be explained by natural or
"standard of care" effects alone.
[0266] In an attempt to discover the source of this unexpected
efficacy, the entire history of the production process for that lot
was carefully reviewed. It was discovered that as part of an
ordinary, but discretionary, inoculation protocol for commercial
laying hens the chickens we used were inoculated with salmonella
vaccine.
[0267] Although the birds were vaccinated as chicks, the
formulation was found to be highly efficacious against Salmonella
typhi. Salmonella was not one of the antigens in the inoculation
protocol used for the chickens in the Example 1 preparations. The
unexpected response of these typhoid fever patients to the
composition of the disclosure was noted to be very surprising to
the attending physicians during the field trial.
Example 6. Quantitative ELISA for Egg Powder Specific IgY and Total
IgY
[0268] The antibody activity of total IgY and specific anti-antigen
IgY can be determined using Enzyme-Linked Immunosorbant Assay
(ELISA) by a modification of the method of Liou et al., 2011, J.
Anim. Vet. Adv., 10(18):2349-2356, as described below.
[0269] Microtiter plates are coated with either 100 uL mixed
antigen preparation (10 ug per well) or coated with 100 uL rabbit
anti-chicken IgY antibody (10 ug/mL, Sigma-Aldrich), for control
wells. The plate is incubated overnight at 4.degree. C. After
washing with PBS-Tween 20 buffer, plates are blocked with 2% BSA
and incubated overnight at 4.degree. C. The wells are then washed
with PBS-Tween 20 buffer and once with PBS. Thereafter, diluted
dried egg powder stock (10 mg/mL) is serially diluted with 1% BSA
and added to sample wells at 100 uL per well. Wells for standard
curve are filled with 100 uL serial dilutions of standard IgY at,
e.g., at concentration ranges of, e.g., 0.015-1 ug/mL and incubated
overnight at 4.degree. C. After washing with PBS-Tween 20 buffer,
100 uL of alkaline phosphatase-conjugated goat anti-chicken IgY is
added to the wells and incubated 2 hours at 37.degree. C. After
washing with PBS-Tween 20 buffer, 100 uL disodium p-nitrophenyl
phosphate as substrate is added to each well and allowed to react
for 10 min at 37.degree. C. The absorbance is measured at 405 nm
using a plate reader. The absorbance of standard curves provides a
relative measurement of specific anti-antigen IgY
concentration.
[0270] For measurement of total IgY, each well of the microtiter
plate is coated with rabbit anti-chicken IgY antibody (10 ug/mL).
After incubation and washing as above, 100 uL of diluted dried egg
powder is added and assay is performed as above.
[0271] Although the invention has been described with reference to
the above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
* * * * *