U.S. patent application number 10/103671 was filed with the patent office on 2003-02-13 for methods for obtaining transfer factor from eggs, compositions including egg-derived transfer factor, and methods of use.
Invention is credited to Hennen, William J., Lisonbee, David T..
Application Number | 20030031686 10/103671 |
Document ID | / |
Family ID | 24676991 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030031686 |
Kind Code |
A1 |
Hennen, William J. ; et
al. |
February 13, 2003 |
Methods for obtaining transfer factor from eggs, compositions
including egg-derived transfer factor, and methods of use
Abstract
An egg-derived transfer factor, compositions including the
egg-derived transfer factor, and methods for generating and
preparing the egg-derived transfer factor. The egg-derived transfer
factor may have specificity for one or more antigens. A method of
using the egg-derived transfer factor includes administering either
antigen-specific egg-derived transfer factor or antigen
non-specific egg-derived transfer factor to animals, such as
mammals, to treat or prevent pathogenic infections in the
animals.
Inventors: |
Hennen, William J.;
(Springville, UT) ; Lisonbee, David T.; (Orem,
UT) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Family ID: |
24676991 |
Appl. No.: |
10/103671 |
Filed: |
March 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10103671 |
Mar 21, 2002 |
|
|
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09667147 |
Sep 21, 2000 |
|
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Current U.S.
Class: |
424/278.1 ;
424/157.1 |
Current CPC
Class: |
C07K 14/465 20130101;
A61K 38/00 20130101; A61K 39/245 20130101; A61P 31/12 20180101;
A61K 39/292 20130101; C07K 14/52 20130101; A61K 39/165 20130101;
A61P 37/02 20180101; A61K 39/00 20130101 |
Class at
Publication: |
424/278.1 ;
424/157.1 |
International
Class: |
A61K 039/395; A61K
039/42; A61K 047/00; A61K 039/40; A61K 045/00 |
Claims
What is claimed is:
1. A method for obtaining transfer factor, comprising: exposing a
source animal to at least one antigenic agent that will cause said
source animal to elicit a T-cell mediated immune response;
permitting said source animal to elicit a T-cell mediated immune
response to said at least one antigenic agent; collecting at least
one egg from said source animal following said T-cell mediated
immune response, said at least one egg including transfer factor
that transfers cellular immunity to another animal in vivo.
2. The method of claim 1, wherein said collecting comprises
collecting at least one egg that includes transfer factor molecules
having molecular weights of about 3,000 Da to about 5,000 Da.
3. The method of claim 1, wherein said exposing said source animal
comprises exposing an avian source animal to said at least one
antigenic agent.
4. The method of claim 3, wherein said exposing said avian source
animal comprises exposing a hen to said at least one antigenic
agent.
5. The method of claim 1, wherein said exposing said source animal
to at least one antigenic agent comprises permitting said source
animal to be exposed to its natural environment.
6. The method of claim 1, wherein said exposing comprises at least
one of injecting said source animal with said at least one
antigenic agent, generating an aerosol comprising said at least one
antigentic agent in the presence of said source animal, and
administering a composition comprising said at least one antigenic
agent to said source animal orally.
7. The method of claim 1, wherein said exposing is conducted in the
presence of an adjuvant.
8. The method of claim 1, wherein said exposing is conducted with
substantially no adjuvant.
9. The method of claim 1, wherein said exposing comprises exposing
said source animal to at. least one of Newcastle Virus,
measles-mumps-rubella vaccine, a hepatitis B antigen or vaccine, an
antigen or vaccine of Epstein-Barr Virus, and an antigen or vaccine
of H. pylori.
10. The method of claim 1, wherein said exposing comprises exposing
said source animal substantially concurrently to a plurality of
antigens.
11. The method of claim 1, wherein said exposing comprises exposing
said source animal to at least one of a live vaccine, an attenuated
vaccine, a killed vaccine, a recombinant antigen, a synthetic
antigen, and a natural antigen.
12. The method of claim 1, wherein said collecting said at least
one egg is effected at least about seven days after said
exposing.
13. The method of claim 1, wherein said collecting said at least
one egg is effected at least about fourteen days after said
exposing.
14. The method of claim 1, further comprising collecting a water
soluble fraction of said at least one egg.
15. The method of claim 14, wherein said collecting said water
soluble fraction comprises collecting a water soluble fraction of a
yolk of said at least one egg.
16. The method of claim 14, further comprising removing
substantially all antibodies from said water soluble fraction.
17. The method of claim 1, wherein said collecting includes
substantially purifying said transfer factor from other proteins or
peptides of said at least one egg having molecular weights of
greater than about 8,000 Da.
18. The method of claim 17, wherein said substantially purifying
transfer factor comprises causing said other proteins or peptides
having molecular weights of greater than about 8,000 Da to
precipitate from a solution including said transfer factor.
19. A method for eliciting a T-cell mediated immune response in an
animal, comprising administering to the animal a quantity of a
composition including an extract of an egg obtained from a source
animal, said extract comprising transfer factor generated by said
source animal in a T-cell mediated immune response to at least one
antigenic agent.
20. The method of claim 19, wherein said administering comprises
administering to the animal a quantity of said composition with
said extract comprising transfer factor molecules having molecular
weights of about 3,000 Da to about 5,000 Da.
21. A composition for eliciting a T-cell mediated immune response
by a treated animal, comprising at least an extract of an egg of a
source animal, said egg including at least one type of transfer
factor for transferring cellular immunity to the treated animal in
vivo and being generated by way of a T-cell mediated immune
response of said source animal to at least one antigenic agent.
22. The composition of claim 21, wherein said at least one type of
transfer factor includes transfer factor molecules having molecular
weights of about 3,000 Da to about 5,000 Da.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of application
Ser. No. 09/667,147, filed Sep. 21, 2000, pending.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to methods for
generating antigen-specific transfer factor, compositions including
such antigen-specific transfer factor, and uses of these
compositions. In particular, the present invention relates to
methods for generating antigen-specific transfer factor in an avian
host and obtaining the antigen-specific transfer factor from
eggs.
[0004] 2. Background of Related Art
[0005] Many deadly pathogens are passed to humans from the animal
kingdom. For example, monkeys are the sources of the type I human
immunodeficiency virus (HIV-I), which causes acquired immune
deficiency syndrome (AIDS) and monkeypox, which is similar to
smallpox; ground-dwelling mammals are believed to be the source of
the Ebola virus; fruit bats and pigs are the source of the Nipah
virus; the Hendra virus comes from horses; the virus responsible
for the "Hong Kong Flu" originated in chickens; and wild birds,
especially ducks, are the sources of many of the deadly influenza
viruses. Many diseases also have animal reservoirs. By way of
example, mice carry Hanta virus, rats carry the Black Plague, and
deer carry Lyme disease.
[0006] The Immune System
[0007] The immune systems of vertebrates are equipped to recognize
and defend the body from invading pathogenic organisms, such as
parasites, bacteria, fungi, and viruses. Vertebrate immune systems
typically include a cellular component and a noncellular
component.
[0008] The cellular component of an immune system includes the
so-called "lymphocytes", or white blood cells, of which there are
several types. It is the cellular component of a mature immune
system that typically mounts a primary, nonspecific response to
invading pathogens, as well as being involved in a secondary,
specific response to pathogens.
[0009] In the primary, or initial, response to an infection by a
pathogen, white blood cells that are known as phagocytes locate and
attack the invading pathogens. Typically, a phagocyte will
internalize, or "eat" a pathogen, then digest the pathogen. In
addition, white blood cells produce and excrete chemicals in
response to pathogenic infections that are intended to attack the
pathogens or assist in directing the attack on pathogens.
[0010] Only if an infection by invading pathogens continues to
elude the primary immune response is a specific, secondary immune
response to the pathogen needed. As this secondary immune response
is typically delayed, it is also known as "delayed-type
hypersensitivity". A mammal, on its own, will typically not elicit
a secondary immune response to a pathogen until about seven (7) to
about fourteen (14) days after becoming infected with the pathogen.
The secondary immune response is also referred to as an acquired
immunity to specific pathogens. Pathogens have one or more
characteristic proteins, which are referred to as "antigens". In a
secondary immune response, white blood cells known as B
lymphocytes, or "B-cells", and T lymphocytes, or "T-cells", "learn"
to recognize one or more of the antigens of a pathogen. The B-cells
and T-cells work together to generate proteins called "antibodies",
which are specific for one or more certain antigens on a
pathogen.
[0011] The T-cells are primarily responsible for the secondary, or
delayed-type hypersensitivity, immune response to a pathogen or
antigenic agent. There are three types of T-cells: T-helper cells,
T-suppressor cells, and antigen-specific T-cells, which are also
referred to as cytotoxic (meaning "cell-killing") T-lymphocytes
("CTLs"), or T-killer cells. The T-helper and T-suppressor cells,
while not specific for certain antigens, perform conditioning
functions (e.g., the inflammation that typically accompanies an
infection) that assist in the removal of pathogens or antigenic
agents from an infected host.
[0012] Antibodies, which make up only a part of the noncellular
component of an immune system, recognize specific antigens and,
thus, are said to be "antigen-specific". The generated antibodies
then basically assist the white blood cells in locating and
eliminating the pathogen from the body. Typically, once a white
blood cell has generated an antibody against a pathogen, the white
blood cell and all of its progenitors continue to produce the
antibody. After an infection is eliminated, a small number of
T-cells and B-cells that correspond to the recognized antigens are
retained in a "resting" state. When the corresponding pathogenic or
antigenic agents again infect the host, the "resting" T-cells and
B-cells activate and, within about forty-eight (48) hours, induce a
rapid immune response. By responding in this manner, the immune
system mounts a secondary immune response to a pathogen, the immune
system is said to have a "memory" for that pathogen.
[0013] Mammalian immune systems are also known to produce smaller
proteins, known as "transfer factors," as part of a secondary
immune response to infecting pathogens. Transfer factors are
another noncellular part of a mammalian immune system.
Antigen-specific transfer factors are believed to be structurally
analogous to antibodies, but on a much smaller molecular scale.
Both antigen-specific transfer factors and antibodies include
antigen-specific cites. In addition, both transfer factors and
antibodies include highly conserved regions that interact with
receptor sites on their respective effector cells. In transfer
factor and antibody molecules, a third, "linker", region connects
the antigen-specific cites and the highly conserved regions.
[0014] The Role of Transfer Factor in the Immune System
[0015] Transfer factor is a low molecular weight isolate of
lymphocytes. Narrowly, transfer factors may have specificity for
single antigens. U.S. Pat. Nos. 5,840,700 and 5,470,835, both of
which issued to Kirkpatrick et al. (hereinafter collectively
referred to as "the Kirkpatrick Patents"), disclose the isolation
of transfer factors that are specific for certain antigens. More
broadly, "specific" transfer factors have been generated from cell
cultures of monoclonal lymphocytes. Even if these transfer factors
are generated against a single pathogen, they have specificity for
a variety of antigenic sites of that pathogen. Thus, these transfer
factors are said to be "pathogen-specific" rather than
antigen-specific. Similarly, transfer factors that are obtained
from a host that has been infected with a certain pathogen are
pathogen-specific. Although such preparations are often referred to
in the art as being "antigen-specific" due to their ability to
elicit a secondary immune response when a particular antigen is
present, transfer factors having different specificities may also
be present. Thus, even the so-called "antigen-specific",
pathogen-specific transfer factor preparations may be specific for
a variety of antigens.
[0016] Additionally, it is believed that antigen-specific and
pathogen-specific transfer factors may cause a host to elicit a
delayed-type hypersensitivity immune response to pathogens or
antigens for which such transfer factor molecules are not specific.
Transfer factor "draws" at least the non-specific T-cells, the
T-inducer and T-suppressor cells, to an infecting pathogen or
antigenic agent to facilitate a secondary, or delayed-type
hypersensitivity, immune response to the infecting pathogen or
antigenic agent.
[0017] Typically, transfer factor includes an isolate of proteins
having molecular weights of less than about 10,000 daltons (D) that
have been obtained from immunologically active mammalian sources.
It is known that transfer factor, when added either in vitro or in
vivo to mammalian immune cell systems, improves or normalizes the
response of the recipient mammalian immune system.
[0018] The immune systems of newborns have typically not developed,
or "matured", enough to effectively defend the newborn from
invading pathogens. Moreover, prior to birth, many mammals are
protected from a wide range of pathogens by their mothers. Thus,
many newborn mammals cannot immediately elicit a secondary response
to a variety of pathogens. Rather, newborn mammals are typically
given secondary immunity to pathogens by their mothers. One way in
which mothers are known to boost the immune systems of newborns is
by providing the newborn with a set of transfer factors. In
mammals, transfer factor is provided by a mother to a newborn in
colostrum, which is typically replaced by the mother's milk after a
day or two. Transfer factor basically transfers the mother's
acquired, specific (i.e., delayed-type hypersensitive) immunity to
the newborn. This transferred immunity typically conditions the
cells of the newborn's immune system to react against pathogens in
an antigen-specific manner, as well as in an antigen- or
pathogen-nonspecific fashion, until the newborn's immune system is
able on its own to defend the newborn from pathogens. Thus, when
transfer factor is present, the immune system of the newborn is
conditioned to react to pathogens with a hypersensitive response,
such as that which occurs with a typical delayed-type
hypersensitivity response. Accordingly, transfer factor is said to
"jump start" the responsiveness of immune systems to pathogens.
[0019] Much of the research involving transfer factor has been
conducted in recent years. Currently, it is believed that transfer
factor is a protein with a length of about forty-four (44) amino
acids. Transfer factor typically has a molecular weight in the
range of about 3,000 to about 5,000 Daltons (Da), or about 3 kDa to
about 5 kDa, but it may be possible for transfer factor molecules
to have molecular weights outside of this range. Transfer factor is
also believed to include three functional fractions: an inducer
fraction; an immune suppressor fraction; and an antigen-specific
fraction. Many in the art believe that transfer factor also
includes a nucleoside portion, which could be connected to the
protein molecule or separate therefrom, that may enhance the
ability of transfer factor to cause a mammalian immune system to
elicit a secondary immune response. The nucleoside portion may be
part of the inducer or suppressor fractions of transfer factor.
[0020] The antigen-specific region of the antigen-specific transfer
factors is believed to comprise about eight (8) to about twelve
(12) amino acids. A second highly-conserved region of about ten
(10) amino acids is thought to be a very high-affinity T-cell
receptor binding region. The remaining amino acids may serve to
link the two active regions or may have additional, as yet
undiscovered properties. The antigen-specific region of a transfer
factor molecule, which is analogous to the known antigen-specific
structure of antibodies, but on a much smaller molecular weight
scale, appears to be hyper-variable and is adapted to recognize a
characteristic protein on one or more pathogens. The inducer and
immune suppressor fractions are believed to impart transfer factor
with its ability to condition the various cells of the immune
system so that the cells are more fully responsive to the
pathogenic stimuli in their environment.
[0021] Sources of Noncellular Immune System Components
[0022] Conventionally, transfer factor has been obtained from the
colostrum of milk cows. While milk cows typically produce large
amounts of colostrum and, thus, large amounts of transfer factor
over a relatively short period of time, milk cows only produce
colostrum for about a day or a day-and-a-half every year. Thus,
milk cows are neither a constant source of transfer factor nor an
efficient source of transfer factor.
[0023] Transfer factor has also been obtained from a wide variety
of other mammalian sources. For example, in researching transfer
factor, mice have been used as a source for transfer factor.
Antigens are typically introduced subcutaneously into mice, which
are then sacrificed following a delayed-type hypersensitivity
reaction to the antigens. Transfer factor is then obtained from
spleen cells of the mice.
[0024] While different mechanisms are typically used to generate
the production of antibodies, the original source for antibodies
may also be mammalian. For example, monoclonal antibodies may be
obtained by injecting a mouse, a rabbit, or another mammal with an
antigen, obtaining antibody-producing cells from the mammal, then
fusing the antibody-producing cells with immortalized cells to
produce a hybridoma cell line, which will continue to produce the
monoclonal antibodies throughout several generations of cells and,
thus, for long periods of time.
[0025] Antibodies against mammalian pathogens have been obtained
from a wide variety of sources, including mice, rabbits, pigs,
cows, and other mammals. In addition, the pathogens that cause some
human diseases, such as the common cold, are known to originate in
birds. As it has become recognized that avian (i.e., bird) immune
systems and mammalian immune systems are very similar, some
researchers have turned to birds as a source for generating
antibodies.
[0026] U.S. Pat. No. 5,080,895, issued to Tokoro on Jan. 14, 1992
(hereinafter "the '895 patent"), discloses a method that includes
injecting hens with pathogens that cause intestinal infectious
diseases in neonatal mammals. The hens then produce antibodies that
are specific for these pathogens, which are present in eggs laid by
the hens. The '895 patent discloses compositions that include these
pathogen-specific antibodies and use thereof to treat and prevent
intestinal diseases in neonatal piglets and calves. In addition,
the '895 patent assumes that a pathogen-specific "transfer
factor-like" substance is passed from a hen to her eggs.
Nonetheless, the '895 patent does not disclose that such a
"transfer factor-like" substance was in fact present in the eggs,
or that an antibody-free composition derived from eggs that were
assumed to contain this transfer factor-like substance actually
treated or prevented intestinal diseases in neonatal mammals. In
fact, the '895 patent discloses the use of a filter with about 0.45
.mu.m diameter holes to isolate transfer factor from antibodies. As
those of skill in the art are aware, however, antibodies, larger
molecules, viruses, and even some bacteria will pass through the
pores of a 0.45 .mu.m filter. In reality, it is not likely that any
individual protein molecules (including those having molecular
weights of less than about 12,000 D) were separated by such a
filter. Based on the pore size of the filter used, however, it is
more likely that no individual protein molecules, including
antibodies, were removed by the filter.
[0027] Avian antibodies that are specific for mammalian pathogens
have also been obtained by introducing antigens into eggs.
[0028] Treatment of pathogenic infections in mammals with avian
antibodies is typically not desirable, however, since the immune
systems of mammals are likely to respond negatively to the large
avian antibody molecules by eliciting an immune response to the
antibodies themselves. Moreover, as mammalian immune systems do not
recognize avian antibodies as useful for their abilities to
recognize certain pathogens, or the specificities of avian
antibodies for antigens of such pathogens, avian antibodies do not
even elicit the desired immune responses in mammals.
[0029] The inventors are not aware of any art that teaches a method
for deriving transfer factor from eggs, an efficient method for
obtaining transfer factor from eggs, or a method for using such
egg-derived transfer factor in treating or preventing infections by
pathogens.
SUMMARY OF THE INVENTION
[0030] The present invention includes a method for generating the
production of transfer factor by a source animal, as well as a
method for obtaining transfer factor from eggs of the source
animal. In addition, compositions including egg-derived transfer
factor are also within the scope of the present invention, as are
methods of using these compositions.
[0031] The transfer factor generated, obtained, and used in
accordance with the present invention may either be antigen
non-specific or antigen-specific (i.e., configured to bind or
recognize one or more antigens). Unless otherwise indicated, the
term "transfer factor", as used herein, includes the previously
discussed broad definition, which includes each of the various
types of transfer factors, including pathogen-specific,
antigen-specific, and transfer factors that are not specific for
particular pathogens or antigenic agents. The term "non-specific",
when used herein with respect to transfer factors, refers to both
transfer factors that are not specific for particular antigens and
to mixtures that include transfer factors with different antigen
specificities.
[0032] Non-specific transfer factor includes transfer factor that
the source animal already produces. Individual non-specific
transfer factor molecules that are produced by the source animal
may have specificity for various antigenic agents, including
pathogens, that are present in the source animal's environment.
Nonetheless, for purposes of the present invention, transfer factor
that is generated merely by a source animal's reaction to its
environment is referred to as "non-specific".
[0033] On the other hand, antigen-specific transfer factor is
generated by exposing a source animal to one or more antigens. The
antigens of various types of pathogens, including, but not limited
to, bacteria, viruses, fungi, and parasites, may induce the
production of non-specific transfer factor in source animals.
Antigen-specific transfer factor has been generated by source
animals by both natural antigens (including from live, inactivated,
and attenuated sources) and synthetic antigens, and is present in
the eggs of such source animals.
[0034] The production of transfer factor in a source animal may be
induced by introducing an antigen characteristic of a certain
pathogen into a female source animal, such as by injection, by
aerosol, through the skin, or otherwise, as known in the art.
Exemplary types of source animals that may be used include, without
limiting the scope of the present invention, birds, reptiles,
amphibians, fish, and mammals. The source animal may produce eggs
on a frequent basis, as do hens, or, as in the case of many
mammals, have a limited number of eggs that are internally
maintained. These source animals produce transfer factor, which
then appears in the eggs of these source animals. The transfer
factor may be obtained from laid eggs or from eggs that are
surgically or otherwise extracted from the source animal.
Alternatively, an egg of a source animal may be exposed to the
antigenic agent (e.g., by injection of the antigenic agent into the
egg) to induce production of transfer factor by the egg itself.
[0035] The transfer factor generated by a source animal or by the
egg of a source animal may be recovered from the egg. Such recovery
may, but need not, include separation of transfer factor from other
constituents of the egg, including proteins of larger molecular
weight, such as antibodies. Alternatively, a crude preparation
including transfer factor may be obtained from one or more eggs of
a source animal.
[0036] The transfer factor may then be incorporated into a
composition or apparatus for administration to a mammalian or
non-mammalian subject or administered directly to the subject. The
transfer factor or compositions including the transfer factor may
be administered enterally (i.e., orally), or parenterally (i.e., by
a non-oral route, such as by injection, topically, as described in
U.S. Pat. No. 4,435,384 to Warren, the disclosure of which is
hereby incorporated in its entirety by this reference, or otherwise
through the skin, by exposing the source animal to an aerosol
including the transfer factor, etc.). Administration of both
non-specific and specific egg-derived transfer factors have been
found to initiate an early, specific (i.e., secondary) immune
response in mammals to various invading pathogens. Thus,
egg-derived transfer factor has been found to be useful in treating
and preventing diseases that may be caused by these various
pathogens.
[0037] Other features and advantages of the present invention will
become apparent to those of skill in the art through consideration
of the ensuing description, the accompanying drawings, and the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] In the drawings, which illustrate exemplary embodiments of
the present invention:
[0039] FIG. 1 is a schematic representation of an exemplary method
for generating the production of transfer factor by a source
animal;
[0040] FIG. 2 is a schematic representation of an exemplary method
for generating transfer factor directly in the eggs of a source
animal;
[0041] FIG. 3 is a schematic representation of an exemplary method
for obtaining transfer factor from eggs; and
[0042] FIG. 4 is a schematic representation of an exemplary method
for testing for the presence of transfer factor in a solution and
for using transfer factor to prevent infection by pathogens or to
treat pathogenic infections.
DETAILED DESCRIPTION OF THE INVENTION
[0043] As explained previously herein, mammalian mothers pass
transfer factor to their newborn children in the colostrum, which
is replaced by mother's milk after about a day or two. The transfer
factor present in colostrum transfers delayed-type hypersensitivity
for certain antigens to the child, thus "jump-starting" the ability
of the immune system of the newborn child to respond to certain
pathogens, if the child becomes infected with these pathogens.
[0044] Over recent years, it has been discovered that avian (i.e.,
bird) immune systems are very similar to those of mammals. In fact,
early studies of the components of immune systems were performed on
birds. As a result of these early studies of immune systems,
B-cells, one of the types of white blood cells discussed previously
herein, was so named due to its origin in the bursa of birds. In
addition, it is known that various infectious agents, including
some viruses that cause the common cold and influenza A virus,
originate in birds and are passed onto humans.
[0045] As avian immune systems bear some resemblances to the immune
systems of mammals, the inventors believe that transfer factor is
also a component of avian immune systems, as well as of the immune
systems of other non-mammalian vertebrates. In addition, the
inventors believe that although non-mammalian mothers do not
provide colostrum to their newborn children, these animals could
still transfer immunity to their children by way of transfer
factor. In birds and other egg-laying vertebrates, the mother's
primary opportunity to provide transfer factor to her children is
in the egg-yolk, which supplies the growing embryo with the
necessary nutrients during growth. Likewise, transfer factors may
be present in the eggs of female animals that are capable of live
birth, as are most female mammals. Thus, the inventors have long
believed that antigen non-specific and antigen-specific transfer
factor could be obtained from eggs.
[0046] FIG. 1 schematically illustrates a method for obtaining
desired transfer factor from a source 10 of transfer factor, in
this case a hen. Source 10 may be exposed to environmental
antigenic agents 12a or exposed to specific antigenic agents 12b.
Source 10 may be exposed to specific antigenic agents 12b,
including, but not limited to, live vaccine, an attenuated vaccine,
a killed vaccine, a recombinant antigen, a synthetic antigen, a
natural antigen, and the like, by injection, orally, by exposing
the source animal to an aerosol including antigenic agents 12b, or
otherwise, as known in the art. Source 10 may be exposed to
antigenic agents 12b either with or without an adjuvant present.
Such exposure to specific antigenic agents 12b may occur once or be
repeated. For simplicity, antigenic agents 12a and 12b are also
referred to herein as antigenic agents 12 or simply as
antigens.
[0047] Alternatively, with reference to FIG. 2, an egg 14' of an
animal may be directly exposed to one or more antigenic agents 12,
such as by injection or otherwise, as known in the art.
[0048] With reference to FIG. 3, after source 10 or eggs 14' that
were directly exposed to one or more antigenic agents 12 have been
given an adequate opportunity to elicit a secondary, or
delayed-type hypersensitivity, immune response to antigenic agents
12, eggs 14 are collected. Of course, in the case of source animals
that do not lay eggs, the eggs may be collected by surgical
extraction or otherwise. The yolks 16 and whites 18 of eggs 14 are
then separated from one another, and various filtration processes
are conducted on yolks 16 to obtain a water soluble fraction 20
thereof that includes transfer factor. Larger molecular weight
proteins, such as antibodies, may also be removed from water
soluble fraction 20 of yolks 16 by known processes, such as by
filtering on the basis of molecular weight or by causing these
larger molecular weight proteins to precipitate out of solution
(e.g., in cold ethyl alcohol), then removing the precipitate 21
from water soluble fraction 20 (e.g., by filtration) to provide a
substantially antibody-free, transfer factor-containing solution
22. Alternatively, the yolks 16 and whites 18 need not be
separated.
[0049] In addition, antigen-specific, egg-derived transfer factor
present in water soluble fraction 20 of yolks 16 or in or in
solution 22 may be substantially purified from other constituents
of water soluble fraction 20 or solution 22 by known techniques,
such as by use of the gel permeation and affinity chromatography
techniques disclosed in U.S. Pat. Nos. 5,840,700 and 5,470,835,
both of which issued to Kirkpatrick et al. (hereinafter
collectively referred to as "the Kirkpatrick Patents"), the
disclosures of both of which are hereby incorporated by this
reference in their entireties. The technique disclosed in the
Kirkpatrick Patents is used to isolate biomolecules, such as
transfer factor and antibodies, from the other constituents of a
solution on the basis of the specificity of these biomolecules for
one or more antigens or other specific binding agents. Thus, when
the technique disclosed in the Kirkpatrick Patents is used on the
antibody- and transfer factor-containing water soluble fraction 20
of egg yolk 16, both transfer factor and antibody may be isolated
from the remainder of water soluble fraction 20 with the resulting
solution 24 including both antibody and transfer factor. If, on the
other hand, the technique disclosed in the Kirkpatrick Patents is
conducted on a substantially antibody-free, transfer
factor-containing solution 22, the product will be a substantially
pure solution 26 of transfer factor specific for one or more
antigens. Of course, other methods for obtaining transfer factor
from eggs are also within the scope of the present invention,
including methods for obtaining transfer factor from various egg
preparations, including powdered or freeze-dried whole eggs or egg
yolks.
[0050] Of course, the use of other methods to obtain transfer
factor from eggs is also within the scope of the present invention.
Further, in the case of eggs that do not include separate or
distinct "whites" and "yolks", transfer factor may be obtained
without the types of separation describe herein, without departing
from the scope of the present invention.
[0051] Alternatively, crude preparations that include transfer
factor may be obtained from any type of egg, without use of
separation and/or isolation processes, are also within the scope of
the present invention, as are methods for obtaining and forming
such crude preparations.
[0052] In any event, transfer factor obtained from eggs in
accordance with teachings of the present invention may have a
molecular weight in the range of about 3,000 to about 5,000 Daltons
(Da), or about 3 kDa to about 5 kDa. The present invention is not,
however, limited to egg-derived transfer factor molecules having
molecular weights within this range.
[0053] Referring now to FIG. 4, an exemplary method for testing for
the presence of egg-derived transfer factor specific for one or
more antigens in a solution, known as a mouse footpad assay, is
schematically depicted.
[0054] About seven (7) days prior to testing the effectiveness of
avian transfer factor in causing mice to elicit a secondary immune
response to a particular antigen or pathogen for which the avian
transfer factor was specific, a positive control population of six
female BALB/c mice is prepared. Each mouse 30 of the positive
control population, having ages of about nine (9) weeks to about
ten (10) weeks, is anesthetized with isoflurane. About 0.02 ml of a
50/50 (wt/wt) mixture of Freund's adjuvant and the particular
antigen 36 against which the avian transfer factor to be tested is
specific is administered to each mouse 30 by way of two
intramuscular injections, one injection at each side of the base 39
of the tail 38. As these injections are conducted about seven (7)
days prior to conducting the mouse footpad assay, the mice of the
positive control population are permitted to generate their own
secondary, or delayed-type hypersensitivity response to antigen
36.
[0055] About twenty-four (24) hours prior to the mouse footpad
test, the mice of a first test population, which also includes six
female BALB/c mice that are about nine (9) to about ten (10) weeks
old (i.e., about the same age as the mice of the positive control
population), are also anesthetized with isoflurane. About 0.5 ml of
a solution 20, 24 including a preparation containing both avian
transfer factor and avian antibody, reconstituted in distilled
water, is then administered by subcutaneous injection at the back
of the neck 40 of each mouse 30 of the first test population. By
comparing the results obtained from these mice with the results
obtained from mice of a second test population that had been
treated with a substantially antibody-free preparation, the
relative contributions of transfer factor and antibody to the
swelling could be determined. As antibodies do not elicit a
secondary immune response, it was believed prior to conducting the
experiments described herein that the measure of the secondary
immune response in the first and second test populations of mice
would be very similar.
[0056] Each mouse of the second test population that includes six
female BALB/c mice, having ages of about nine (9) to about ten (10)
weeks old (i.e., about the same age as the mice of the positive
control and first test populations), is also anesthetized with
isoflurane. Each of the six mice 30 is given, by subcutaneous
injection in the back of the neck 40, about 0.5 ml of a solution
22, 26 including, reconstituted in distilled water, a lyophilized
antigen-specific avian transfer factor preparation with
substantially no antibodies.
[0057] A negative control population also includes six female
BALB/c mice of about nine (9) to about ten (10) weeks in age (i.e.,
about the same ages as the other three populations of mice).
[0058] In order to conduct the mouse footpad assay, the mice of
each of the four populations are anesthetized and the distances
across each of the largest right hind footpad 32 and the largest
left hind footpad 34 of each mouse 30 are measured, such as with a
Starrett gauge. Right hind footpad 32 is then subcutaneously
injected with an antigen 36-containing solution. Left hind footpad
34, which is used as a control, is injected with about the same
volume of a control solution 37, such as a sterile saline diluent,
as the volume of solution that is injected into right hind footpad
32.
[0059] After a sufficient amount of time (e.g., about sixteen (16)
to about twenty-four (24) hours) has elapsed, each mouse 30 is
again anesthetized and the distances across right and left hind
footpads 32, 34 are again measured. A significant amount of
swelling, determined by an increase in the distances across a right
hind footpad 32 of mouse 30, is indicative of the occurrence of a
delayed-type hypersensitivity reaction in that footpad 32.
[0060] Of course, different solutions 24, 26 including transfer
factors with specificities for different antigens may be tested on
different sets of mice to detect any differences in the abilities
of these solutions to transfer delayed-type hypersensitivity
immunity to the mice. In addition, the results for each solution
may be compared to those obtained from positive control and
negative control populations of mice 30. If significant swelling
occurs in the right hind footpads 34 of mice 30 to which a
substantially antibody-free solution, such as solution 22 or
solution 26 of FIG. 3, was administered, the delayed-type
hypersensitivity that causes such swelling is attributed to the
administered transfer factor.
[0061] The following examples are merely illustrative of
embodiments of methods for generating, obtaining, and using
transfer factor that incorporate teachings of the present
invention:
EXAMPLE 1
[0062] Transfer factor specific for Newcastle Virus was generated
by exposing day-old chicks to a coarse spray of infectious
bronchitis/Newcastle virus (IBNC) vaccine, as known in the art, at
zero (0) days, forty-two (42) days, and eighty-four (84) days. Eggs
laid by these five hens at about one-hundred seventy-five (175)
days following the first IBNC vaccine injection were collected.
EXAMPLE 2
[0063] The yolks from a first sampling of the antigen specific
transfer factor-containing eggs generated in EXAMPLE 1 were
separated from the whites, diluted about six (6) to about nine (9)
times, by volume, in deionized water (i.e., about one (1) part egg
white mixed with about five (5) parts water to about eight (8)
parts water) and frozen. The lipid layer from these frozen egg
yolks was mechanically separated from the water-soluble fraction of
the egg yolks. This water-soluble fraction was then permitted to
thaw to a temperature of about 4.degree. C. to about 6.degree. C.
and vacuum filtered by use of Whatman qualitative filter paper
using a 55 mm diameter porcelain Buchner funnel. The filtrate was
then vacuum filtered through a glass microfiber filter, again using
a 55 mm diameter Buchner funnel.
[0064] A third filtration was then conducted to collect proteins
and to remove lipids and lipoproteins from the solution. The third
filtration was effected by way of a DURAPORE hydrophilic membrane.
The protein-containing fraction, which included both transfer
factor and antibody specific for the infectious bronchitis pathogen
and Newcastle Virus was collected, frozen, and lyophilized, or
freeze-dried, as known in the art.
EXAMPLE 3
[0065] The water-soluble fractions of diluted yolk preparations
from a second sampling of the eggs collected in EXAMPLE 1 were
again mechanically separated from the lipid portions thereof and
filtered, as explained previously herein in EXAMPLE 2.
[0066] In accordance with the method disclosed in U.S. Pat. No.
4,180,627, which issued to Klesius et al., the disclosure of which
is hereby incorporated by this reference in its entirety, an
adequate volume of ethyl alcohol (EtOH), or ethanol, was added to
the protein-containing fraction to dilute the ethyl alcohol to a
concentration of about 60% of the total volume of the
alcohol-protein fraction solution. This solution was then cooled to
a temperature of about 4 to about 6.degree. C. for a long enough
period of time (e.g., overnight, or for about 10-12 hours) for
larger molecular weight proteins, including antibodies, present in
the solution to precipitate from the solution. Smaller molecular
weight proteins (e.g., proteins having molecular weights of about
8,000 Da to 10,000 Da or less), including any transfer factor from
the egg yolks, remained in solution.
[0067] The larger molecular weight protein-containing precipitate
was then removed from the solution by filtering the solution
through a Whatman glass microfiber filter in a 55 mm diameter
Buchner funnel. CELITE.RTM., a diatomite, or diatomaceous earth,
filtration aid available from Celite Corporation of Lompoc,
California, was used to prevent the precipitate from clogging the
filter during filtration of the solution. This substantially
precipitate-free solution was then collected, frozen, and
lyophilized, as known in the art.
EXAMPLE 4
[0068] Each mouse of a test population that included three BALB/c
mice, each having an age in the range of about nine (9) to about
ten (10) weeks, was tested to determine whether the IBNV-specific
avian transfer factor would impart an early secondary, or
delayed-type hypersensitivity, immune response to the mice. Each
mouse was anesthetized with isoflurane. The distances across the
largest footpads of both the left and right hind feet of each mouse
were then measured with a Starrett gauge. Each mouse was then given
a subcutaneous injection in the back of the neck of about 0.5 ml of
a solution that included about 16%, by weight, of the IBNV-specific
avian transfer factor reconstituted in distilled water.
[0069] After about twenty-four (24) hours, each of the mice was
again anesthetized with isoflurane. About 0.01 ml of a sterile
saline diluent was then injected into the largest footpad of the
left hind foot of each mouse, which footpad served as a control,
while the largest footpad of the right hind foot of each mouse was
injected with about 0.01 ml of a solution including about 10,000
doses of Newcastle-Bronchitis vaccine reconstituted in about 250 ml
of distilled water.
[0070] Before another twenty-four (24) hours had elapsed, one of
the mice (Mouse #1) died. The two remaining mice were again
anesthetized with isoflurane and the largest footpads on their hind
feet were again measured. The results follow:
1TABLE 1 Newcastle Virus--Test Population Footpad size (.mu.m):
Before Sample Injection Final Difference Mouse #1 Left Foot
(Control) 2150 Right Foot (Test) 2151 Mouse #2 Left Foot (Control)
2180 2350 50 Right Foot (Test) 2165 2440 85 Mouse #3 Left Foot
(Control) 2145 2160 15 Right Foot (Test) 2110 2200 90
[0071] The greater increase in size, or swelling, of the right
footpad (increases of 85 .mu.m and 90 .mu.m) over that of the left
footpad (increases of 50 .mu.m and 15 .mu.m, respectively)
indicates that the IBNV-specific avian transfer factor-containing
solution induced a delayed-type hypersensitivity reaction in the
right feet of Mouse #2 and Mouse #3 within about twenty-four hours
following the introduction of the Newcastle-Bronchitis vaccine.
[0072] In the remaining examples, substantially the same methods as
those disclosed in EXAMPLES 1-3 were used to generate avian
transfer factors specific for different types of antigens,
including measles, mumps, rubella, Hepatitis B, Epstein-Barr Virus
(EBV), and H. pylori.
[0073] The effectiveness of each of these various types of
antigen-specific avian transfer factors in inducing early
secondary, or delayed-type hypersensitivity, immune responses in
mammals was then tested by way of mouse footpad assays. Each type
of antigen-specific avian transfer factor was tested using four
different populations of mice, including a positive control
population, a first test population, a second test population, and
a negative control population, which were prepared as described
previously herein with reference to FIG. 4. The mouse footpad assay
for each type of antigen-specific transfer factor was conducted in
accordance with the teachings of Petersen E A, Greenberg L E,
Manzara T, and Kirkpatrick C H, "Murine transfer factor," I.
Description of the model and evidence for specificity, J. Immunol.,
126: 2480-84 (1981), the disclosure of which is hereby incorporated
by this reference in its entirety.
[0074] In each mouse footpad assay, four populations of mice were
prepared in the manner described in reference to FIG. 4.
[0075] In conducting the various mouse footpad assays on each of a
positive control, a first and a second test, and a negative control
populations, each mouse was anesthetized with isofluorane, the
largest footpad of the left hind footpad of each mouse, which
served as a control, was injected with about 0.01 ml of sterile
saline diluent, and the largest footpad of the right hind foot of
each mouse was injected with about 0.01 ml of a solution including
the antigen or pathogen for which the avian transfer factor was
specific.
[0076] About sixteen (16) to about twenty-four (24) hours following
the hind footpad injections, each of the mice of the positive
control, test, and negative control populations was again
anesthetized with isoflurane and the sizes of the left and right
hind footpads of each of the mice were again measured, for example,
with a Starrett Gauge.
EXAMPLE 5
[0077] Using the same procedures described in EXAMPLES 1-3, avian
transfer factor and avian antibodies specific for measles, mumps,
and rubella (MMR) vaccine were generated in hens. Each hen received
one dose of Merck MMR II vaccine, as described in EXAMPLE 1, at 150
days, 163 days, 190 days, 221 days, and 249 days. Eggs were
collected from these hens just after the third
innoculation-sometime in the period of about day 192 to about day
223 and prepared as described in EXAMPLE 1. This was done in this
EXAMPLE and in the following EXAMPLES to ensure that a high level
of transfer factor was present in the eggs. It is believed that
transfer factor will be present in eggs about seven (7) days
following the first innoculation.
[0078] A positive control population of mice was prepared about
seven (7) days prior to the beginning of the mouse footpad assay by
injecting each mouse of the positive control population with Merck
MMR II vaccine, as described previously herein in reference to FIG.
4.
[0079] A solution containing both avian antibody and avian transfer
factor specific for MMR vaccine was made by reconstituting in
distilled water a lyophylized preparation similar to that described
in EXAMPLE 2 to a concentration of about 8%, by weight. This
transfer factor- and antibody-containing solution was administered
to the first test population of mice in the manner described in
reference to FIG. 4.
[0080] Lyophilized avian transfer factor specific for measles,
mumps, and rubella, prepared by a method similar to that described
in EXAMPLE 3, was reconstituted in distilled water to a
concentration of about 8%, by weight. This reconstituted
MMR-specific avian transfer factor was then administered to a
second test population of mice in the manner described previously
herein in reference to FIG. 4.
[0081] About 0.1 ml of a dose of Merck MMR II Vaccine was then
administered to the largest footpad of the right hind foot of each
mouse of each of positive control, first test, second test, and
negative control populations, while substantially the same amount
of sterile saline diluent was administered to the largest footpad
of the left hind foot of each mouse, as described in reference to
FIG. 4.
[0082] About sixteen (16) to about twenty-four (24) hours later,
the mice were again anesthetized and the sizes of the largest
footpads of both hind feet of each mouse measured, as previously
described. The results follow:
2TABLE 2 MMR Vaccine--First Test Population (Antibody and Transfer
Factor Administered) Footpad size (.mu.m): Before Sample Injection
Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control)
2159.00 2235.20 76.20 Right Foot (Test) 2133.60 2387.60 254.00
Mouse #2 Left Foot (Control) 2133.60 2159.00 25.40 Right Foot
(Test) 2133.60 2184.40 50.80 Mouse #3 Left Foot (Control) 2159.00
2159.00 0.00 Right Foot (Test) 2159.00 2184.40 25.40 Mouse #4 Left
Foot (Control) 2209.80 2235.20 25.40 Right Foot (Test) 2286.00
2311.40 25.40 Mouse #5 Left Foot (Control) 2184.40 2184.40 0.00
Right Foot (Test) 2209.80 2260.60 50.80 Mouse #6 Left Foot
(Control) 2260.60 2336.80 76.20 Right Foot (Test) 2235.20 2438.40
203.20
[0083] The data for Mouse #6 may have been inaccurate since the
scabs from bite marks were present on one or both hind footpads of
this mouse at the time the second measurements were taken (i.e., at
about sixteen (16) to about twenty-four (24) hours). Nonetheless,
with the exception of Mouse #4, each of the remaining mice of the
first test population exhibited greater swelling at the time the
second footpad measurements were taken in the footpads that were
injected with the MMR II vaccine than in the footpads that were
injected with the control solution. In Mouse #4, the amount of
swelling was about the same in both the left and right
footpads.
[0084] Overall, as can be seen from the data of TABLE 2, the
largest footpads of the right feet of the first test population of
mice represented exhibited an average of about 67.73 .mu.m more
swelling than the amount of swelling of the largest footpad of the
left feet of these mice.
3TABLE 3 MMR Vaccine--Second Test Population (Only Transfer Factor
Administered) Footpad size (.mu.m): Before Sample Injection Final
(0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2082.80
2133.60 50.80 Right Foot (Test) 2108.20 2235.20 127.00 Mouse #2
Left Foot (Control) 2336.80 2387.60 50.80 Right Foot (Test) 2387.60
2641.60 254.00 Mouse #3 Left Foot (Control) 2184.40 2184.40 0.00
Right Foot (Test) 2184.40 2311.40 127.00 Mouse #4 Left Foot
(Control) 2133.60 2133.60 0.00 Right Foot (Test) 2133.60 2133.60
0.00 Mouse #5 Left Foot (Control) 2082.80 2540.00 457.20 Right Foot
(Test) 2108.20 2235.20 127.00 Mouse #6 Left Foot (Control) 2260.60
2286.00 25.40 Right Foot (Test) 2286.00 2362.20 76.20
[0085] As scabs from bite marks were visible on the footpads of
Mouse #2 and Mouse #5 at about twenty-four hours following the
injection of antigen and sample, the data form these mice may have
been inaccurate. In addition, the largest footpad on the left foot
of Mouse #5 was swollen more than three times as much as the
corresponding footpad on the left foot of Mouse #5 and several
times more than the swelling that occurred in any of the footpads
of the other tested mice. Accordingly, the swelling data obtained
from Mouse #5 were also omitted as this swelling in the footpad of
the left foot was excessive. No increase in swelling in either
footpad was measured in Mouse #4. Nonetheless, each of Mouse #1,
Mouse #3, and Mouse #6 exhibited greater swelling in the (right)
footpad that was injected with the second, substantially
antibody-free, transfer factor-containing solution than in the
(left) footpad that was injected with the control solution.
[0086] Based on the data presented in TABLE 3, on average, the
largest footpads on the right feet of Mice ##1, 3, and 6 were
swollen about 91.4 .mu.m more than the largest footpads on the left
feet of these mice.
4TABLE 4 MMR Vaccine--Positive Control Footpad size (.mu.m): Before
Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left
Foot (Control) 2184.40 2235.20 50.80 Right Foot (Test) 2184.40
2260.60 76.20 Mouse #2 Left Foot (Control) 2184.40 2209.80 25.40
Right Foot (Test) 2184.40 2209.80 25.40 Mouse #3 Left Foot
(Control) 2006.60 2133.60 127.00 Right Foot (Test) 1981.20 2108.20
127.00 Mouse #4 Left Foot (Control) 2133.60 2184.40 50.80 Right
Foot (Test) 2133.60 2260.60 127.00 Mouse #5 Left Foot (Control)
2108.20 2133.60 25.40 Right Foot (Test) 2108.20 2286.00 177.80
Mouse #6 Left Foot (Control) 2082.80 2133.60 50.80 Right Foot
(Test) 2057.40 2209.80 152.40
[0087] While Mouse #2 and Mouse #3 of the positive control
population both exhibited substantially the same amount of swelling
in the largest footpads of both the left and right hind feet, each
of the other mice had a greater amount of swelling in the largest
footpads of their right hind feet and, thus, displayed a secondary
immune response to the MMR vaccine that was introduced into the
largest footpads of their right hind feet, than the amount of
swelling in the largest footpads of the left hind feet of these
mice, which were much less swollen.
[0088] Based on the data in TABLE 4, it is apparent that the
average amount of swelling in the largest footpads of the right
hind feet of these mice was about 59.27 .mu.m greater than the
swelling of the largest footpads on the left hind feet of these
mice.
5TABLE 5 MMR Vaccine--Negative Control Footpad size (.mu.m): Before
Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left
Foot (Control) 2159.00 2159.00 0.00 Right Foot (Test) 2159.00
2209.80 50.80 Mouse #2 Left Foot (Control) 2159.00 2159.00 0.00
Right Foot (Test) 2108.20 2133.60 25.40 Mouse #3 Left Foot
(Control) 2133.60 2133.60 0.00 Right Foot (Test) 2133.60 2133.60
0.00 Mouse #4 Left Foot (Control) 2108.20 2133.60 25.40 Right Foot
(Test) 2108.20 2108.20 0.00 Mouse #5 Left Foot (Control) 2057.40
2057.40 0.00 Right Foot (Test) 2032.00 2032.00 0.00 Mouse #6 Left
Foot (Control) 2082.80 2133.60 50.80 Right Foot (Test) 2032.00
2082.80 50.80
[0089] Two of the mice, Mouse #3 and Mouse #5, of the negative
control population exhibited no swelling in the largest footpad of
either hind foot. The largest footpads on both hind feet of Mouse
#6 were swollen by about the same amount. While the largest
footpads on the left hind feet of Mouse #1 and Mouse #4 were not
swollen and the footpads on the right hind feet of these two mice
were slightly swollen, the largest footpad on the right hind foot
of Mouse #4 was not swollen and the largest left hind footpad was
only slightly swollen. In fact, the average amount of swelling in
the largest footpads of the right hind feet of these mice was only
about 8.47 .mu.m greater than the amount of swelling measured in
the largest footpads of the left hind feet of the negative control
population of mice. Consequently, the data in TABLE 5 indicate that
the mice of the negative control population did not elicit a
secondary immune response to the MMR vaccine.
[0090] Collectively, the data of TABLES 2-5 indicate that a
secondary, or delayed-type hypersensitivity, immune response
occurred in the majority of mice in each of the first test
population, the second test population, and the positive control
population, while no such secondary immune response appeared to be
present in the negative control population. Accordingly, the data
in TABLES 2 and 3 indicate that avian transfer factor specific for
MMR vaccine, as well as avian antibody specific for MMR vaccine,
are capable of inducing an early secondary immune response in
mammals.
EXAMPLE 6
[0091] Repeating the procedures described previously herein in
EXAMPLES 1-3, avian transfer factor and avian antibodies specific
for the Hepatitis B virus were generated by use of a synthetic
Hepatitis B antigen vaccine sold under the tradename ENGERIX-B.
Each hen received one dose of the Hepatitis B vaccine, as described
in EXAMPLE 1, at 150 days, 163 days, 190 days, 221 days, and 249
days. Eggs were collected from these hens sometime in the period of
about day 193 and about day 223, as described in EXAMPLE 1 above,
and prepared as described in EXAMPLE 1.
[0092] A positive control population of mice was prepared about
seven (7) days prior to conducting the mouse footpad assay by
injecting each mouse of the positive control population with the
synthetic Hepatitis B vaccine, ENGERIX-B in the manner described in
reference to FIG. 4.
[0093] A first solution, which included both avian antibody and
avian transfer factor that were specific for Hepatitis B vaccine,
was made by reconstituting in distilled water a lyophilized
preparation similar to that described in EXAMPLE 2 to a
concentration of about 16%, by weight. This transfer factor- and
antibody-containing solution was administered to a first test
population of mice in the manner described in reference to FIG.
4.
[0094] In addition, lyophilized avian transfer factor specific for
Hepatitis B vaccine, which was prepared in a similar manner to that
described in EXAMPLE 3, was reconstituted in distilled water to a
concentration of about 16%, by weight. The reconstituted transfer
factor-containing solution was then administered to each of the
mice of a second test population, as explained previously herein in
reference to FIG. 4.
[0095] At the appropriate time, the synthetic Hepatitis B vaccine
was administered to the largest footpad of the right foot of each
mouse of each of the positive control, first test, second test, and
negative control populations, as described previously herein in
reference to FIG. 4. The largest footpad of the left foot of each
mouse of the four populations was substantially concurrently
injected with the same amount of sterile saline diluent, also as
described previously herein.
[0096] About sixteen (16) to about twenty-four (24) hours later,
each of the mice of the four populations was again anesthetized and
the sizes of the largest footpads of both hind feet of each mouse
were again measured, as described previously herein. The results
follow:
6TABLE 6 Hepatitis B Vaccine--First Test Population (Antibody and
Transfer Factor Administered) Footpad size (.mu.m): Before Sample
Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot
(Control) 2032.00 2108.20 76.20 Right Foot (Test) 2032.00 2082.80
50.80 Mouse #2 Left Foot (Control) 2260.60 2362.20 101.60 Right
Foot (Test) 2209.80 2336.80 127.00 Mouse #3 Left Foot (Control)
2159.00 2184.40 25.40 Right Foot (Test) 2159.00 2235.20 76.20 Mouse
#4 Left Foot (Control) 2108.20 2184.40 76.20 Right Foot (Test)
2108.20 2260.60 152.40 Mouse #5 Left Foot (Control) 1930.40 2032.00
101.60 Right Foot (Test) 1930.40 2108.20 177.80 Mouse #6 Left Foot
(Control) 2184.40 2184.40 0.00 Right Foot (Test) 2184.40 2235.20
50.80
[0097] Each of the mice of the first test population, with the
exception of Mouse #1, exhibited greater swelling in the largest
footpad of the right hind foot. On average, the largest footpads of
the right hind feet of the mice of the first test population were
about 42.17 .mu.m more swollen than the largest footpads of the
left hind feet of these mice. Thus, the data of TABLE 6 indicate
that the avian transfer factor in the preparation that included
transfer factor and antibody specific for the synthetic Hepatitis B
vaccine induced an early secondary immune response in each of these
mice.
7TABLE 7 Hepatitis B Vaccine--Second Test Population (Only Transfer
Factor Administered) Footpad size (.mu.m): Before Sample Injection
Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control)
1981.20 2032.00 50.80 Right Foot (Test) 2006.60 2159.00 152.40
Mouse #2 Left Foot (Control) 1981.20 1981.20 0.00 Right Foot (Test)
1981.20 2006.60 25.40 Mouse #3 Left Foot (Control) 2006.60 2032.00
25.40 Right Foot (Test) 2032.00 2082.80 50.80 Mouse #4 Left Foot
(Control) 1955.80 2133.60 177.80 Right Foot (Test) 1981.20 2108.20
127.00 Mouse #5 Left Foot (Control) 1930.40 2006.60 76.20 Right
Foot (Test) 1930.40 2057.40 127.00 Mouse #6 Left Foot (Control)
2032.00 2057.40 25.40 Right Foot (Test) 2006.60 2108.20 101.60
[0098] On average the largest footpads on the right hind feet of
the second test population of mice were about 38.10 .mu.m more
swollen than the largest footpads on the left hind feet of these
mice. With the exception of Mouse #4, the data of TABLE 7
illustrate that the administration of avian transfer factor
specific for Hepatitis B vaccine induced an early secondary, or
delayed-type hypersensitivity, immune response in the largest
footpad of the right hind foot of each mouse.
8TABLE 8 Hepatitis B Vaccine--Positive Control Footpad size
(.mu.m): Before Sample Injection Final (0 hrs.) (24 hrs.)
Difference Mouse #1 Left Foot (Control) 2108.20 2133.60 25.40 Right
Foot (Test) 2108.20 2159.00 50.80 Mouse #2 Left Foot (Control)
2032.00 2082.80 50.80 Right Foot (Test) 2006.60 2108.20 101.60
Mouse #3 Left Foot (Control) 1854.20 1930.40 76.20 Right Foot
(Test) 1879.60 2032.00 152.40 Mouse #4 Left Foot (Control) 2006.60
2108.20 101.60 Right Foot (Test) 2057.40 2209.80 152.40 Mouse #5
Left Foot (Control) 2133.60 2159.00 25.40 Right Foot (Test) 2133.60
2159.00 25.40 Mouse #6 Left Foot (Control) 2006.60 2133.60 127.00
Right Foot (Test) 2006.60 2184.40 177.80
[0099] In the positive control population of mice, only Mouse #5
failed to elicit a secondary immune response to the synthetic
Hepatitis B vaccine. The largest footpads on the right hind feet of
each of the other mice of the positive control population exhibited
an average of about 42.33 .mu.m increased swelling over that of the
largest footpads on the left hind feet of these mice.
9TABLE 9 Hepatitis B Vaccine--Negative Control Footpad size
(.mu.m): Before Sample Injection Final (0 hrs.) (24 hrs.)
Difference Mouse #1 Left Foot (Control) 2159.00 2159.00 0.00 Right
Foot (Test) 2133.60 2133.60 0.00 Mouse #2 Left Foot (Control)
2057.40 2057.40 0.00 Right Foot (Test) 2082.80 2082.80 0.00 Mouse
#3 Left Foot (Control) 2006.60 2032.00 25.40 Right Foot (Test)
1955.80 2032.00 72.60 Mouse #4 Left Foot (Control) 2057.40 2082.80
25.40 Right Foot (Test) 2057.40 2108.20 50.80 Mouse #5 Left Foot
(Control) 2133.60 2133.60 0.00 Right Foot (Test) 2133.60 2159.00
25.40 Mouse #6 Left Foot (Control) 2082.80 2133.60 50.80 Right Foot
(Test) 2082.80 2133.60 50.80
[0100] Three mice of the negative control population exhibited
substantially the same amount of swelling in the largest footpads
of both the left and right hind feet. Of the remaining three mice,
only mouse #3 exhibited a significantly greater amount of swelling
in the largest food pad of her right hind foot than in her left
hind foot. On average, the difference in swelling between the
largest footpads on the right and left hind feet of the mice of the
negative control population was only about 16.33 .mu.m.
[0101] Collectively, the data presented in TABLES 6-9 indicate the
result of EXAMPLE 6 to be that both avian antibody and avian
transfer factor specific for synthetic Hepatitis B vaccine cause
mammals to elicit an early secondary immune response to the antigen
of the synthetic Hepatitis B vaccine, which is also presented by
the Hepatitis B virus.
EXAMPLE 7
[0102] Again employing substantially the same procedures outlined
above in EXAMPLES 1-3, avian transfer factor and avian antibody
specific for the H. pylori bacteria were generated in hens. Each of
the hens was infected with the H. pylori EIA antigen, in a manner
similar to that described in EXAMPLE 1, at day 150, day 163, day
190, day 221, and day 249. Eggs were collected from these hens
during the period of about day 193 to about day 223, as described
in EXAMPLE 1, and prepared, as described in EXAMPLE 1.
[0103] As in the previous EXAMPLES, a positive control population
of mice was prepared about seven (7) days prior to conducting the
mouse footpad assay by injecting each of the mice of the positive
control population with the recombinant, or synthetic, H. pylori
EIA antigen, as described in reference to FIG. 4.
[0104] A solution including both avian antibody and avian transfer
factor specific for the H. pylori EIA antigen was made by
reconstituting in distilled water a lyophilized preparation
including such avian antibody and avian transfer factor, similar to
the preparation described above in EXAMPLE 2, to a concentration of
about 16%, by weight. This solution was administered to a first
test population of mice, as described previously herein in
reference to FIG. 4.
[0105] A substantially antibody-free solution including avian
transfer factor specific for H. pylori was prepared by
reconstituting a lyophilized preparation, obtained in a manner
similar to that described in EXAMPLE 3, in distilled water to a
concentration of about 16%, by weight. This substantially
antibody-free avian transfer factor-containing solution was then
administered to each of the mice of a second test population, as
described previously herein in reference to FIG. 4.
[0106] The largest footpad of the right foot of each mouse of each
of the positive control, first test, second test, and negative
control populations, was infected with H. pylori EIA antigen, while
the same amount of sterile saline diluent was administered to the
largest footpad of the left foot of each of these mice in the
manner detailed previously herein in reference to FIG. 4.
[0107] At the appropriate time, about sixteen (16) to about
twenty-four (24) hours following the infection of the largest
footpads of the right feet of the mice with H. pylon, the mice were
again anesthetized and the sizes of the largest footpads of both
hind feet of each mouse was measured, as described previously
herein. The results follow:
10TABLE 10 H. Pylori--First Test Population (Antibody and Transfer
Factor Administered) Footpad size (.mu.m): Before Sample Injection
Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control)
1955.80 1981.20 25.40 Right Foot (Test) 1930.40 1955.80 25.40 Mouse
#2 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test)
2108.20 2260.60 152.40 Mouse #3 Left Foot (Control) 2082.80 2082.80
0.00 Right Foot (Test) 2108.20 2133.60 25.40 Mouse #4 Left Foot
(Control) 2082.80 2184.40 101.60 Right Foot (Test) 2082.80 2286.00
203.20 Mouse #5 Left Foot (Control) 2108.20 2133.60 25.40 Right
Foot (Test) 2133.60 2133.60 0.00 Mouse #6 Left Foot (Control)
1955.80 2032.00 76.20 Right Foot (Test) 1930.40 2108.20 177.80
[0108] The data of TABLE 10 and, particularly those of Mouse #2,
Mouse #4, and Mouse #6, indicate that administration of the
solution containing both avian antibody and avian transfer factor
specific for H. pylori induced an early secondary immune response
in the mice of the first test population. While Mouse #1 exhibited
substantially equal amounts of swelling in the largest footpads of
both her left and right hind feet, Mouse #3 exhibited slightly
greater swelling in the largest footpad of her right hind foot than
in that of her left hind foot and Mouse #5 exhibited a slightly
greater amount of swelling in the largest footpad of her left hind
foot than in the largest footpad of her right hind foot. On
average, the largest footpads of the right hind feet of the mice of
the first test population were about 59.27 .mu.m more swollen than
the largest footpads of the left hind feet of these mice.
11TABLE 11 H. Pylori--Second Test Population (Only Transfer Factor
Administered) Footpad size (.mu.m): Before Sample Injection Final
(0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2235.20
2235.20 0.00 Right Foot (Test) 2184.40 2209.80 25.40 Mouse #2 Left
Foot (Control) 2006.60 2006.60 0.00 Right Foot (Test) 2006.60
2032.00 25.40 Mouse #3 Left Foot (Control) 2082.80 2184.40 101.60
Right Foot (Test) 2133.60 2209.80 76.20 Mouse #4 Left Foot
(Control) 2133.60 2133.60 0.00 Right Foot (Test) 2133.60 2159.00
25.40 Mouse #5 Left Foot (Control) 2159.00 2184.40 25.40 Right Foot
(Test) 2159.00 2235.20 76.20 Mouse #6 Left Foot (Control) 2057.40
2082.80 25.40 Right Foot (Test) 2032.00 2260.60 228.60
[0109] The results shown in TABLE 11 were similar to those in TABLE
10. Two of the mice, Mouse #5 and Mouse #6, exhibited much more
swelling in the largest footpads of their right hind feet than in
the largest footpads of their left hind feet. While the amount of
swelling in the largest footpads of the right hind feet of Mouse
#1, Mouse #2, and Mouse #4 was greater than that of the largest
footpads of the left hind feet of these mice, the difference was
only slight. Mouse #3 actually exhibited a slightly greater amount
of swelling in the largest footpad of her left hind foot than in
the largest footpad of her right hind foot. Nonetheless, as the
average swelling in the largest footpads of the right hind feet of
these mice is, on average, about 50.80 .mu.m greater than that of
the largest footpads on the left hind feet of these mice, the data
of TABLE 11 indicate that avian transfer factor specific for H.
pylori caused the increased swelling.
12TABLE 12 H. Pylori--Positive Control Footpad size (.mu.m): Before
Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left
Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test) 2108.20
2184.40 76.20 Mouse #2 Left Foot (Control) 2133.60 2133.60 0.00
Right Foot (Test) 2133.60 2209.80 76.20 Mouse #3 Left Foot
(Control) 2032.00 2108.20 76.20 Right Foot (Test) 2082.80 2209.80
127.00 Mouse #4 Left Foot (Control) 1981.20 2082.80 101.60 Right
Foot (Test) 1879.60 2133.60 254.00 Mouse #5 Left Foot (Control)
2133.60 2159.00 25.40 Right Foot (Test) 2184.40 2336.80 152.40
Mouse #6 Left Foot (Control) 2133.60 2133.60 0.00 Right Foot (Test)
2082.80 2260.60 177.80
[0110] Each of the mice of the positive control population in
EXAMPLE 7 elicited at delayed-type hypersensitivity immune response
to H. pylori, as indicated by the significant differences in the
amount of swelling in the largest footpads of the right hind feet
of these mice relative to that in the largest footpads of the left
hind feet of these mice. On average, the difference in swelling was
about 110.07 .mu.m.
13TABLE 13 H. Pylori--Negative Control Footpad size (.mu.m): Before
Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left
Foot (Control) 2006.60 2082.80 76.20 Right Foot (Test) 2514.60
2514.60 0.00 Mouse #2 Left Foot (Control) 2032.00 2082.80 50.80
Right Foot (Test) 2032.00 2133.60 101.60 Mouse #3 Left Foot
(Control) 2082.80 2108.20 25.40 Right Foot (Test) 2082.80 2108.20
25.40 Mouse #4 Left Foot (Control) 2006.60 2032.00 25.40 Right Foot
(Test) 1955.80 2032.00 76.20 Mouse #5 Left Foot (Control) 1930.40
1981.20 50.80 Right Foot (Test) 1955.80 2006.60 50.80 Mouse #6 Left
Foot (Control) 2133.60 2159.00 25.40 Right Foot (Test) 2133.60
2159.00 25.40
[0111] As indicated by the data of TABLE 13, the amount of swelling
in the largest footpads of both the left and right hind feet of
Mouse #3, Mouse #5, and Mouse #6, were substantially the same.
While the amount of swelling in the largest footpad of the right
hind foot of Mouse #2 was greater than the amount of swelling in
the largest footpad of the left hind foot of that mouse, the
largest footpad of the left hind foot of Mouse #1 was significantly
more swollen than the largest footpad of the right hind foot of
Mouse #1. The largest footpad of the right hind foot of Mouse #4
was only slightly more swollen than the largest footpad of the left
hind foot of Mouse #4. The average difference in swelling of the
largest footpads of the right and left hind feet of the mice of the
negative control population was only about 4.23 .mu.m.
[0112] The data of TABLES 10-13 indicate that avian transfer factor
specific for H. pylori facilitates an early secondary immune
response in mammals.
EXAMPLE 8
[0113] Again, employing substantially the same procedures described
previously herein in EXAMPLES 1-3, avian transfer factor and avian
antibody specific for the EBNA-1 antigen, a recombinant nuclear
antigen of the Epstein-Barr virus (EBV), were generated in hens.
Each hen received one dose of EBNA-1, such as described in EXAMPLE
1, at 150 days, 163 days, 190 days, and 249 days. Eggs were
collected from these hens during the period of about day 193 to
about day 223, as described above in EXAMPLE 1, and prepared as
described above in EXAMPLE 1.
[0114] A solution with both avian antibody and avian transfer
factor specific for EBNA-1 was formed by reconstituting in
distilled water a lyophilized preparation similar to that described
in EXAMPLE 2. The lyophilized preparation including both avian
antibody and avian transfer factor specific for EBNA-1 antigen was
diluted to a concentration of about 16%, by weight. This solution
was then administered to a first test population of mice in the
manner described in reference to FIG. 4.
[0115] In addition, a solution containing avian transfer factor
specific for EBNA-1, with substantially no avian antibody specific
for EBNA-1, was also reconstituted in distilled water to a
concentration of about 16%, by weight. This solution was
administered to the mice of a second test population in the manner
described previously herein in reference to FIG. 4.
[0116] A positive control population of mice was prepared by
injecting mice with EBNA-1 about seven (7) days before conducting
the mouse footpad assay.
[0117] Recombinant EBNA-1 antigen was then administered to the
largest footpad of the right hind foot of each mouse of each of
four populations, including a first test population, a second test
population, a positive control population, and a negative control
population. Substantially the same amount of sterile saline diluent
was administered to the largest footpad of the left hind foot of
each mouse. The method of administration was conducted in the same
manner as that described previously herein.
[0118] About sixteen (16) to about twenty-four (24) hours later,
the mice were again anesthetized and the sizes of the largest
footpads of both hind feet of each mouse measured, as previously
described. The results follow:
14TABLE 14 EBV EBNA-1--First Test Population (Antibody and Transfer
Factor Administered) Footpad size (.mu.m): Before Sample Injection
Final (0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control)
2032.00 2057.40 25.40 Right Foot (Test) 2032.00 2057.40 25.40 Mouse
#2 Left Foot (Control) 2159.00 2159.00 0.00 Right Foot (Test)
2159.00 2184.40 25.40 Mouse #3 Left Foot (Control) 2159.00 2159.00
0.00 Right Foot (Test) 2133.60 2286.00 152.40 Mouse #4 Left Foot
(Control) 2108.20 2108.20 0.00 Right Foot (Test) 2108.20 2209.80
101.60 Mouse #5 Left Foot (Control) 2108.20 2235.20 127.00 Right
Foot (Test) 2082.80 2260.60 177.80 Mouse #6 Left Foot (Control)
1981.20 2032.00 50.80 Right Foot (Test) 1981.20 2032.00 50.80
[0119] In TABLE 14, it is seen that three of the mice exhibited
significantly greater swelling in the largest footpads of their
right hind feet than in the largest footpads of their left hind
feet. While Mouse #2 also had a greater amount of swelling in the
largest footpad of her right hind foot than that in the largest
footpad of her left hind foot, the difference was only slight. Two
of the mice, Mouse #1 and Mouse #6, had substantially the same
amount of swelling in the largest footpads of both their left and
right hind feet. Nonetheless, as the amount of swelling in the
largest footpads of the right hind feet of the mice of the first
test population exceeded that of the largest footpads of the left
hind feet of these mice by an average of about 55.03 .mu.m, the
data presented in TABLE 14 tend to show that the avian transfer
factor in the solution containing both avian antibody and transfer
factor specific for EBNA-1 caused the mice of the first test
population to elicit an early secondary immune response to the
recombinant EBNA-1. As is known in the art, antibodies are passive
with respect to secondary immune responses and typically contribute
very little to swelling.
15TABLE 15 EBV EBNA-1--Second Test Population (Only Transfer Factor
Administered) Footpad size (.mu.m): Before Sample Injection Final
(0 hrs.) (24 hrs.) Difference Mouse #1 Left Foot (Control) 2133.60
2159.00 25.40 Right Foot (Test) 2108.20 2159.00 50.80 Mouse #2 Left
Foot (Control) 2006.60 2032.00 25.40 Right Foot (Test) 1955.80
1955.80 0.00 Mouse #3 Left Foot (Control) 2032.00 2133.60 101.60
Right Foot (Test) 2006.60 2159.00 152.40 Mouse #4 Left Foot
(Control) 2108.20 2133.60 25.40 Right Foot (Test) 2159.00 2159.00
0.00 Mouse #5 Left Foot (Control) 2184.40 2209.80 25.40 Right Foot
(Test) 2159.00 2260.60 101.60 Mouse #6 Left Foot (Control) 2057.40
2108.20 50.80 Right Foot (Test) 2082.80 2133.60 50.80
[0120] The mice of the second test population, which were treated
with the avian transfer factor-containing solution also exhibited
an early secondary immune response to recombinant EBNA-1. This
result was particularly evident in Mouse #3 and Mouse #5, which
exhibited significantly greater swelling in the largest footpads of
their right hind feet than that measured in the largest footpads of
their left hind feet. While the amount of swelling in the largest
footpad of the right hind foot of Mouse #1 was also greater than
the amount of swelling in the largest footpad of the left hind foot
of Mouse #1, the difference appears to be slight. Moreover, while
Mouse #2 and Mouse #4 displayed a greater amount of swelling in the
largest footpads of their left hind feet, the amounts of swelling
measured therein were only slightly greater than that measured in
the largest footpads of the right hind feet of these mice. On
average, the largest footpads of the right hand feet of these mice
was about 16.93 .mu.m greater than that measured in the largest
footpads of the left hind feet of these mice.
[0121] It is believed that transfer factor specific for EBNA-1 may
have become unstable when isolated from the corresponding antibody,
resulting in the lower measured secondary immune response in the
second test population relative to the overall secondary immune
response measured in the first test population of mice.
16TABLE 16 EBV EBNA-1--Positive Control Footpad size (.mu.m):
Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse
#1 Left Foot (Control) 2209.80 2209.80 0.00 Right Foot (Test)
2235.20 2286.00 50.80 Mouse #2 Left Foot (Control) 2184.40 2184.40
0.00 Right Foot (Test) 2209.80 2260.60 50.80 Mouse #3 Left Foot
(Control) 2159.00 2159.00 0.00 Right Foot (Test) 2133.60 2209.80
76.20 Mouse #4 Left Foot (Control) 2159.00 2336.80 177.80 Right
Foot (Test) 2133.60 2362.20 228.60 Mouse #5 Left Foot (Control)
2133.60 2133.60 0.00 Right Foot (Test) 2082.80 2260.60 177.80 Mouse
#6 Left Foot (Control) 2082.80 2082.80 0.00 Right Foot (Test)
2057.40 2209.80 152.40
[0122] As indicated by the greater amounts of swelling in the
largest footpads of the right hind feet of each mouse of the
positive control population than that of the largest footpads of
the left hind feet of these mice, all six of the mice of the
positive control population exhibited a delayed-type
hypersensitivity immune response to the recombinant EBNA-1 antigen.
The measured amount of swelling in the largest footpads of the
right hind feet of each of these mice was, on average, about 93.13
.mu.m greater than the measured amount of swelling in the largest
footpads of the left hind feet of these mice.
17TABLE 17 EBV EBNA-1--Negative Control Footpad size (.mu.m):
Before Sample Injection Final (0 hrs.) (24 hrs.) Difference Mouse
#1 Left Foot (Control) 2133.60 2184.40 50.80 Right Foot (Test)
2082.80 2133.60 50.80 Mouse #2 Left Foot (Control) 2133.60 2133.60
0.00 Right Foot (Test) 2159.00 2184.40 25.40 Mouse #3 Left Foot
(Control) 2108.20 2108.20 0.00 Right Foot (Test) 2133.60 2133.60
0.00 Mouse #4 Left Foot (Control) 2082.80 2133.60 50.80 Right Foot
(Test) 2159.00 2159.00 0.00 Mouse #5 Left Foot (Control) 2057.40
2082.80 25.40 Right Foot (Test) 1955.80 1981.20 25.40 Mouse #6 Left
Foot (Control) 2108.20 2133.60 25.40 Right Foot (Test) 2108.20
2133.60 25.40
[0123] In the negative control population, only two of the mice,
Mouse #2 and Mouse #4, exhibited different amounts of swelling in
the largest footpads of their hind feet. While the amount of
swelling in the largest footpad of the right hind foot of Mouse #2
was greater than that exhibited in the largest footpad of the left
hind foot, the largest footpad of the left hind foot of Mouse #4
was more swollen than the largest footpad of the right hind foot of
Mouse #4. In fact, on average, the largest footpads of the right
hind feet of the mice of the negative control population were about
4.23 .mu.m less swollen than the largest footpads of the left hind
feet of these mice.
[0124] Again, the data of TABLES 14-17 illustrate that avian
transfer factor specific for EBNA-1 cause mammals to elicit an
early secondary immune response (i.e., within about twenty-four
(24) hours as compared to the typical seven (7) to fourteen (14)
day time period it takes a mammal to elicit a secondary immune
response on its own) to EBNA-1 and viruses and other pathogens that
present this antigen.
[0125] The foregoing EXAMPLES illustrate that, by way of contrast
with the seven (7) to fourteen (14) day time period that it
typically takes a mammalian host to elicit a secondary immune
response to a pathogen or antigenic agent on its own, when an avian
transfer factor incorporating teachings of the present invention
has been administered, the mammalian host may elicit a secondary
immune response within about twenty-four (24) hours.
[0126] The similarities of the differences between the measurements
taken at the test and control footpads of each mouse in first and
second test groups of each assay indicate that the secondary, or
delayed-type hypersensitivity, immune response, was elicited
primarily by the transfer factor, not the antibody, which is
passive with respect to secondary immune responses and which
typically contributes very little to swelling.
[0127] It is apparent from EXAMPLES 1-8 and the data generated
thereby that avian transfer factor has the ability to generate an
early secondary immune response in mammals. As one of skill in the
art would readily recognize, avian transfer factor would also
generate an early secondary immune response in various types of
birds, as well as in reptiles, amphibians, and other species of
animals, including both mammals and non-mammals.
[0128] As avian transfer factor initiates an early delayed-type
hypersensitivity immune reaction in mice, it is reasonable for
those of ordinary skill in the art to assume that transfer factor
has the same effect in other mammals, including humans, as well as
in other types of animals, including birds, reptiles, amphibians,
fish, etc.
[0129] Although transfer factor was administered to mice in the
preceding EXAMPLES by way of injection, it is also within the scope
of the present invention to administer avian transfer factor to
mammals by other routes. For example, avian transfer factor could
be administered orally, by parenteral injection, or by parenteral
methods other than injection, such as transdermally, or through the
skin, by aerosol via the lungs, or by other methods known in the
art. Oral administration of avian transfer factor to mammals is
supported by the fact that mammalian mothers supply transfer factor
to their newborn children by way of colostrum, which the newborns
ingest orally. Transfer factor survives the conditions of both the
stomach and the small intestine, where transfer factor is absorbed
into the bloodstream of the mammalian newborn. Thus, transfer
factor is known to survive the intestinal tracts of mammals. The
ability of transfer factor to withstand the conditions of the
digestive tracts of mammals was demonstrated in Kirkpatrick C H,
"Activities and characteristics of transfer factors," Biotherapy,
9: 13-16 (1996), the disclosure of which is hereby incorporated by
this reference in its entirety.
[0130] Egg-derived transfer factor according to the present
invention may also be useful for preventing or treating various
other disease states, a prophetic example of which is discussed in
EXAMPLE 9.
EXAMPLE 9
[0131] As an example of a use of egg-derived transfer factor in
preventing or treating a disease state in an animal, egg-derived
transfer factor may be useful for preventing or treating "Shipping
Fever" in cattle, which is caused by a so-called "Bovine
Respiratory Disease" (BRD) complex of pathogens.
[0132] The BRD complex may include, among other pathogens, viruses
and bacteria. Viruses that may be part of the BRD complex include,
without limitation, bovine herpesvirus, parainfluenza 3 virus,
bovine viral diarrhea virus, and bovine respiratory syncytial
virus. Bacteria that have been associated with the BRD complex
include, but are not limited to, Pasteurella haemolytica,
Pasteurella multocida, Haemophilus somnus, various species of
Mycoplasma, and various species of Chlamydia.
[0133] A preparation according to the present invention that is
useful for preventing or treating Shipping Fever may include
non-specific egg-derived transfer factor, egg-derived transfer
factor molecules that are specific for one or more pathogens that
are associated with the BRD complex, such as those identified in
the preceding paragraph, or a combination of specific and
non-specific egg-derived transfer factor molecules. Egg-derived
transfer factor molecules that are specific for pathogens that have
been associated with the BRD complex may be obtained from eggs by
any suitable, known process, such as that described herein. The
preparation may then be made by using the egg-derived transfer
factor molecules that are to be administered in suitable, known
processes. In addition to the types of preparations described
above, the transfer factor molecules may be included in a
nutritional supplement that is to be administered to the cattle,
either independently or with their feed. Other known processes may
also be used to administer the egg-derived transfer factor
molecules to the cattle.
[0134] Egg-derived transfer factor against other pathogens or their
antigens may also be produced and obtained by the methods described
herein and administered to animals to prevent or treat various
types of disease states. By way of example only, the methods
described herein may be used to generate or to obtain egg-derived
transfer factors with specificity for Human Immunodeficiency Virus
(HIV) or variants (e.g., feline, simian, etc.) thereof or their
antigens, small pox, anthrax, and chlamydia.
[0135] Although the foregoing description contains many specifics,
these should not be construed as limiting the scope of the present
invention, but merely as providing illustrations of some of the
presently preferred embodiments. Similarly, other embodiments of
the invention may be devised which do not depart from the spirit or
scope of the present invention. Features from different embodiments
may be employed in combination. The scope of the invention is,
therefore, indicated and limited only by the appended claims and
their legal equivalents, rather than by the foregoing description.
All additions, deletions and modifications to the invention as
disclosed herein which fall within the meaning and scope of the
claims are to be embraced thereby.
* * * * *