U.S. patent application number 13/923496 was filed with the patent office on 2014-01-09 for enhanced immune response in bovine species.
The applicant listed for this patent is Bayer Animal Health GmbH. Invention is credited to Albert Abraham, Daniel Keil, Jason Nickell, Christian Weiss.
Application Number | 20140010865 13/923496 |
Document ID | / |
Family ID | 45444595 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140010865 |
Kind Code |
A1 |
Abraham; Albert ; et
al. |
January 9, 2014 |
ENHANCED IMMUNE RESPONSE IN BOVINE SPECIES
Abstract
The present invention relates to a method of immune activation
which is effective for eliciting a non-antigen-specific immune
response in a member of the bovine species. The method is
particularly effective for protecting a member of the bovine
species from infectious disease and treating animals inflicted with
infectious disease.
Inventors: |
Abraham; Albert; (Shawnee,
KS) ; Keil; Daniel; (Spring Hill, KS) ;
Nickell; Jason; (Parkville, MO) ; Weiss;
Christian; (Leverkusen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bayer Animal Health GmbH |
Leverkusen |
|
DE |
|
|
Family ID: |
45444595 |
Appl. No.: |
13/923496 |
Filed: |
June 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/EP2011/073414 |
Dec 20, 2011 |
|
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13923496 |
|
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61426255 |
Dec 22, 2010 |
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Current U.S.
Class: |
424/450 ;
424/184.1; 424/278.1 |
Current CPC
Class: |
A61K 9/1272 20130101;
A61K 39/39 20130101; A61K 45/06 20130101; A61P 31/12 20180101; A61K
2039/552 20130101; A61K 2039/55555 20130101; A61K 31/711 20130101;
A61K 2039/55566 20130101; A61K 39/102 20130101; A61P 37/02
20180101; A61P 31/04 20180101; A61P 11/00 20180101; A61P 43/00
20180101; A61P 37/04 20180101; A61K 9/0019 20130101; A61K 9/1271
20130101 |
Class at
Publication: |
424/450 ;
424/278.1; 424/184.1 |
International
Class: |
A61K 31/711 20060101
A61K031/711; A61K 45/06 20060101 A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 2011 |
UY |
033821 |
Claims
1. A method for treating bovine respiratory disease in cattle
comprising administering an immunomodulator composition comprising:
a. a cationic liposome delivery vehicle; and b. a nucleic acid
molecule
2. The method of claim 1, wherein the liposome delivery vehicle
comprises lipids selected from the group consisting of
multilamellar vesicle lipids and extruded lipids.
3. The method of claim 1, wherein the liposome delivery vehicle
comprises pairs of lipids selected from the group consisting of
DTMA and cholesterol; DOTAP and cholesterol; DOTIM and cholesterol;
and DDAB and cholesterol.
4. The method of claim 1, wherein the nucleic acid molecule is a
bacterially-derived nucleic acid vector or a fragment thereof that
does not code for an immunogen.
5. The method of claim 1 wherein administration is selected from
the group consisting of intravenously, intramuscularly,
intradermal, intraperitoneal, subcutaneously, by spray/aerosol,
orally, intraocularly, intratracheally, and intranasal.
6. The method of claim 1, wherein the immunomodulator composition
further comprises a biological agent selected from the group
consisting of an immune enhancer proteins, immunogens, vaccines,
antimicrobials and any combination thereof.
7. The method of claim 1, wherein the bovine respiratory disease is
caused by a viral infection and/or bacterial infection.
8. A method for reducing clinical signs caused by Mannheimia
haemolytica in cattle comprising administering an immunomodulator
composition comprising: a. a DOTIM and a cholesterol lipid
combination; and b. a bacterially-derived nucleic acid vector or a
fragment thereof that does not code for an immunogen.
9. The method of claim 8, wherein the immunomodulator composition
further comprises a biological agent.
10. (canceled)
11. (canceled)
12. The method of claim 9, wherein the biological agent is selected
from the group consisting of an immune enhancer proteins,
immunogens, vaccines, antimicrobials and any combination thereof.
Description
REFERENCE TO CORRESPONDING APPLICATIONS
[0001] The present application is a continuation of International
Application No. PCT/EP2011/073414, with an international filing
date of Dec. 20, 2011, and claims the benefit of Uruguayan Patent
Application No. 033821, filed Dec. 20, 2011, and U.S. Provisional
Patent Application Ser. No. 61/426,255, filed Dec. 22, 2010, the
entire contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a method of immune
activation in a member of the bovine species. In particular, the
present invention includes methods for eliciting systemic,
non-specific and antigen-specific immune responses, which are
useful for animal administration and protection against infectious
disease.
BACKGROUND OF THE INVENTION
[0003] Cattle are prime targets for many types of viral, bacterial,
and parasite infections. Modern production practices, such as
weaning, shipment of cattle, inclement weather, and nutritional
needs within the beef and dairy industries may also serve as risk
factors that potentiate the incidence of disease. Bovine
respiratory disease (BRD), or bovine respiratory diseases complex,
as it is often referred to, occurs in both dairy and beef cattle
and is one of the leading causes of economic loss to the cattle
industry throughout the world. These losses are due to morbidity,
mortality, reduced weight gains, treatment and prevention costs,
loss of milk production, and negative impacts on carcass
characteristics.
[0004] The pathogenesis of BRD is thought to arise from numerous
environmental and physiological stressors, mentioned above, coupled
with infectious agents. Mannheimia (Pasteurella) haemolytica,
Pasteurella multocida and Histophilus somni (formerly Haemophilus
somnus) are considered part of the normal flora of the bovine
upper-respiratory tract. Conversely, the lower respiratory tract is
a relatively sterile environment that is maintained by numerous
immunological pathways aimed at the prevention of microbial entry.
When cattle are subjected to environmental and physiological
stressors, the animal's innate and acquired immune functions are
compromised thereby allowing these aforementioned organisms to
proliferate and subsequently colonize the lower respiratory tract.
Various bovine viruses are known to have immunosuppressive effects
in the lung, such as infectious bovine rhinotracheitis virus (IBRV,
IBR, or BHV 1), bovine viral diarrhea virus (BVDV), bovine
respiratory syncytial virus (BRSV), and parainfluenza type 3 virus
(PI3). However, Mannheimia haemolytica is by far the most prevalent
bacterial pathogen among cases of BRD.
[0005] Current prevention and treatment of BRD consists of
antibiotic administration to populations of cattle upon arrival at
feedlots (i.e. metaphylaxis), antibiotic therapy for sick cattle,
and vaccination against BRD viruses and bacteria including M.
haemolytica.
[0006] There are different reasons why current vaccination programs
and pharmaceutical therapies are not optimal to control BRD in
cattle today. First, the host defense system plays a major role in
combating infectious disease in cattle. Conventional treatments
include the administration of antibiotics to treat or control
bacterial infections. However, there are no approved pharmaceutical
treatments available against viral infections. With BRD, in most
cases not only is there a bacterial infection but also a viral
infection. Second, timing of vaccination is often sub-optimal. For
a respiratory vaccine to be optimally effective the product should
be administered 2-4 weeks prior to stress or shipment and this is
typically not feasible in commercial cattle production. The
vaccines are either administered too early or too late to be
optimally effective.
[0007] Therefore a need exists for a method to stimulate the immune
system and build an offensive response to reduce or eliminate
disease causing organisms. It is important that this method is easy
to administer, works alone or in combination with vaccines or helps
to make such vaccines more effective, has a longer duration or that
does not require added injections to maximize immunity. The present
invention provides a method of eliciting a non-antigen-specific
immune response in the bovine species that is easy to administer,
works alone or in combination with vaccines, induces a protective
response against one or more infectious agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1.1 graphically depicts average rectal temperature data
according to dose of immunomodulator administered as described in
Example 1.
[0009] FIG. 1.2 graphically depicts average daily weight gain data
according to dose of immunomodulator administered as described in
Example 1.
[0010] FIG. 1.3 graphically depicts the model-adjusted lung lesion
scores with respect to dose of immunomodulator administered as
described in Example 1.
[0011] FIG. 2.1 graphically depicts average rectal temperature data
according to dose of immunomodulator administered as described in
Example 2.
[0012] FIG. 2.2 graphically depicts average daily weight gain data
according to dose of immunomodulator administered as described in
Example 2.
[0013] FIG. 2.3 graphically depicts the model-adjusted lung lesion
scores with respect to dose of immunomodulator administered as
described in Example 2.
[0014] FIG. 3.1 graphically depicts the model-adjusted lung lesion
scores with respect to dose of immunomodulator administered as
described in Example 3
[0015] FIG. 3.2 graphically depicts the model-adjusted lung lesion
scores with respect to day of immunomodulator administration as
described in Example 3.
[0016] FIG. 4.1 graphically depicts % of protected animals by
treatment group as described in Example 4.
[0017] FIG. 4.2 graphically depicts percent of animals protected by
treatment group (<1% lung lesions and no lung lesions) as
described in Example 4.
[0018] FIG. 5.1 graphically depicts measurements of the CD 25 EI
expression index (y-axis) in cells infected with BHV-1 across all
five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0019] FIG. 5.2 graphically depicts measurements of the CD 25 EI
expression index (y-axis) in cells infected with BRSV across all
five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0020] FIG. 5.3 graphically depicts measurements of the CD 25 EI
expression index (y-axis) in cells infected with BVDV type 1 across
all five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0021] FIG. 5.4 graphically depicts measurements of the CD 25 EI
expression index (y-axis) in cells infected with BVDV type 2 across
all five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0022] FIG. 5.5 graphically depicts measurements of the IFN.gamma.
expression index (y-axis) in cells infected with BHV-1 across all
five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0023] FIG. 5.6 graphically depicts measurements of the IFN.gamma.
expression index (y-axis) in cells infected with BRSV across all
five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0024] FIG. 5.7 graphically depicts measurements of the IFN.gamma.
expression index (y-axis) in cells infected with BVDV type 1 across
all five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0025] FIG. 5.8 graphically depicts measurements of the IFN.gamma.
expression index (y-axis) in cells infected with BVDV type 2 across
all five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0026] FIG. 5.9 graphically depicts measurements of the IL-4
expression index (y-axis) in cells infected with BHV-1 across all
five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0027] FIG. 5.10 graphically depicts measurements of the IL-4
expression index (y-axis) in cells infected with BRSV across all
five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0028] FIG. 5.11 graphically depicts measurements of the IL-4
expression index (y-axis) in cells infected with BVDV type 1 across
all five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0029] FIG. 5.12 graphically depicts measurements of the IL-4
expression index (y-axis) in cells infected with BVDV type 2 across
all five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0030] FIG. 5.13 graphically depicts Model adjusted serum antibody
titer estimates (y-axis) in cells infected with BVDV type 1 across
all five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0031] FIG. 5.14 graphically depicts Model adjusted serum antibody
titer estimates (y-axis) in cells infected with BVDV type 2 across
all five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0032] FIG. 5.15 graphically depicts Model adjusted serum antibody
titer estimates (y-axis) in cells infected with BHV-1 across all
five cell types for each of the 6 treatment groups (x-axis) as
described in Example 5.
[0033] FIG. 5.16 graphically depicts model-adjusted average daily
gain outcomes as described in Example 5.
[0034] FIG. 6.1 graphically depicts the BHV1 SNT titers for the
treatment groups as described in Example 6.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The method of eliciting an immune response in a member of
the bovine species of the present invention includes administering
to the member of the bovine species an effective amount of an
immunomodulator composition to elicit an immune response. The
immunomodulator composition includes a liposome delivery vehicle
and at least one nucleic acid molecule. In addition, the
immunomodulator elicits a non-antigen-specific immune response that
is effective alone or enhances the operation of at least one
biological agent such as a vaccine, when administered prior to such
a vaccine, co-administered with such a vaccine, administered post
vaccination, or mixed with the vaccine.
[0036] The methods provide new treatment strategies for protecting
the bovine species from infectious diseases and treating
populations having infectious disease. Finally, the method of the
present invention provides a more rapid, a longer and better
protection against a disease when the immunomodulator is used in
combination with a vaccine.
[0037] 1. Composition
[0038] a. Immunomodulator
[0039] In one embodiment of the invention, the immunomodulator
composition includes a liposome delivery vehicle and at least one
nucleic acid molecule, as described in U.S. Pat. No. 6,693,086, and
incorporated herein by reference.
[0040] A suitable liposome delivery vehicle comprises a lipid
composition that is capable of delivering nucleic acid molecules to
the tissues of the treated subject. A liposome delivery vehicle is
preferably capable of remaining stable in a subject for a
sufficient amount of time to deliver a nucleic acid molecule and/or
a biological agent. In one embodiment, the liposome delivery
vehicle is stable in the recipient subject for at least about 5
minutes. In another embodiment, the liposome delivery vehicle is
stable in the recipient subject for at least about 1 hour. In yet
another embodiment, the liposome delivery vehicle is stable in the
recipient subject for at least about 24 hours.
[0041] A liposome delivery vehicle of the present invention
comprises a lipid composition that is capable of fusing with the
plasma membrane of a cell to deliver a nucleic acid molecule into a
cell. In one embodiment, when delivered a nucleic acid: liposome
complex of the present invention is at least about 1 picogram (pg)
of protein expressed per milligram (mg) of total tissue protein per
microgram (pg) of nucleic acid delivered. In another embodiment,
the transfection efficiency of a nucleic acid: liposome complex is
at least about 10 pg of protein expressed per mg of total tissue
protein per pg of nucleic acid delivered; and in yet another
embodiment, at least about 50 pg of protein expressed per mg of
total tissue protein per pg of nucleic acid delivered. The
transfection efficiency of the complex may be as low as 1 femtogram
(fg) of protein expressed per mg of total tissue protein per pg of
nucleic acid delivered, with the above amounts being more
preferred.
[0042] A preferred liposome delivery vehicle of the present
invention is between about 100 and 500 nanometers (nm), in another
embodiment, between about 150 and 450 nm and in yet another
embodiment, between about 200 and 400 nm in diameter.
[0043] Suitable liposomes include any liposome, such as those
commonly used in, for example, gene delivery methods known to those
of skill in the art. Preferred liposome delivery vehicles comprise
multilamellar vesicle (MLV) lipids and extruded lipids. Methods for
preparation of MLV's are well known in the art. More preferred
liposome delivery vehicles comprise liposomes having a polycationic
lipid composition (i.e., cationic liposomes) and/or liposomes
having a cholesterol backbone conjugated to polyethylene glycol.
Exemplary cationic liposome compositions include, but are not
limited to, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium
chloride (DOTMA) and cholesterol,
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTAP) and cholesterol,
1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium
chloride (DOTIM) and cholesterol, dimethyldioctadecylammonium
bromide (DDAB) and cholesterol, and combinations thereof. A most
preferred liposome composition for use as a delivery vehicle
includes DOTIM and cholesterol.
[0044] A suitable nucleic acid molecule includes any nucleic acid
sequence such as coding or non-coding sequence, and DNA or RNA.
Coding nucleic acid sequences encode at least a portion of a
protein or peptide, while non-coding sequence does not encode any
portion of a protein or peptide. According to the present
invention, "non-coding" nucleic acids can include regulatory
regions of a transcription unit, such as a promoter region. The
term, "empty vector" can be used interchangeably with the term
"non-coding", and particularly refers to a nucleic acid sequence in
the absence of a protein coding portion, such as a plasmid vector
without a gene insert. Expression of a protein encoded by the
nucleic acid molecule is not required for elicitation of a
non-antigen-specific immune response; therefore the nucleic acid
molecule does not necessarily need to be operatively linked to a
transcription control sequence. However, further advantages may be
obtained (i.e., antigen-specific and enhanced immunity) by
including in the composition nucleic acid sequence (DNA or RNA)
which encodes an immunogen and/or a cytokine.
[0045] Complexing a liposome with a nucleic acid molecule may be
achieved using methods standard in the art or as described in U.S.
Pat. No. 6,693,086, and incorporated herein by reference. A
suitable concentration of a nucleic acid molecule to add to a
liposome includes a concentration effective for delivering a
sufficient amount of nucleic acid molecule into a subject such that
a systemic immune response is elicited. In one embodiment, from
about 0.1 .mu.g to about 10 .mu.g of nucleic acid molecule is
combined with about 8 nmol liposomes, in another embodiment, from
about 0.5 .mu.g to about 5 .mu.g of nucleic acid molecule is
combined with about 8 nmol liposomes, and in yet another
embodiment, about 1.0 .mu.g of nucleic acid molecule is combined
with about 8 nmol liposomes. In one embodiment, the ratio of
nucleic acids to lipids (.mu.g nucleic acid: nmol lipids) in a
composition is at least about 1:1 nucleic acid: lipid by weight
(i.e., 1 .mu.g nucleic acid: 1 nmol lipid), and in another
embodiment, at least about 1:5, and in yet another embodiment, at
least about 1:10, and in a further embodiment at least about 1:20.
Ratios expressed herein are based on the amount of cationic lipid
in the composition, and not on the total amount of lipid in the
composition. In another embodiment, the ratio of nucleic acids to
lipids in a composition of the invention is from about 1:1 to about
1:80 nucleic acid: lipid by weight; and in another embodiment, from
about 1:2 to about 1:40 nucleic acid: lipid by weight; and a
further embodiment, from about 1:3 to about 1:30 nucleic acid:
lipid by weight; and in yet another embodiment, from about 1:6 to
about 1:15 nucleic acid: lipid by weight.
[0046] b. Biological Agent
[0047] In another embodiment of the invention, the immunomodulator
includes a liposome delivery vehicle, a nucleic acid molecule, and
at least one biological agent.
[0048] Suitable biological agents are agents that are effective in
preventing or treating bovine disease. Such biological agents
include immune enhancer proteins, immunogens, vaccines,
antimicrobials or any combination thereof. Suitable immune enhancer
proteins are those proteins known to enhance immunity. By way of a
non-limiting example, a cytokine, which includes a family of
proteins, is a known immunity enhancing protein family. Suitable
immunogens are proteins which elicit a humoral and/or cellular
immune response such that administration of the immunogen to a
subject mounts an immunogen-specific immune response against the
same or similar proteins that are encountered within the tissues of
the subject. An immunogen may include a pathogenic antigen
expressed by a bacterium, a virus, a parasite or a fungus.
Preferred antigens include antigens which cause an infectious
disease in a subject. According to the present invention, an
immunogen may be any portion of a protein, naturally occurring or
synthetically derived, which elicits a humoral and/or cellular
immune response. As such, the size of an antigen or immunogen may
be as small as about 5-12 amino acids and as large as a full length
protein, including sizes in between. The antigen may be a multimer
protein or fusion protein. The antigen may be purified peptide
antigens derived from native or recombinant cells. The nucleic acid
sequences of immune enhancer proteins and immunogens are
operatively linked to a transcription control sequence, such that
the immunogen is expressed in a tissue of a subject, thereby
eliciting an immunogen-specific immune response in the subject, in
addition to the non-specific immune response.
[0049] In another embodiment of the invention, the biological agent
is a vaccine. The vaccine may include a live, infectious, viral,
bacterial, or parasite vaccine or a killed, inactivated, viral,
bacterial, or parasite vaccine. In one embodiment, one or more
vaccines, live or killed viral vaccines, may be used in combination
with the immunomodulator composition of the present invention.
Suitable vaccines include those known in the art for the cattle
species. Exemplary vaccines, without limitation, include those used
in the art for protection against infectious bovine rhinotracheitis
(IBR) (Type 1 bovine herpes virus (BHV1)), parainfluenza virus type
3 (PI3), bovine respiratory syncytial virus (BRSV), bovine viral
diarrhea virus (BVDV Type 1 and 2), Histophilus somni, Mycoplasma
bovis, and other diseases known in the art. In an exemplary
embodiment, a vaccine for the protection against Mannheimia
haemolytica may be used in combination with the immunomodulator
composition of the present invention.
[0050] In yet another embodiment of the invention, the biological
agent is an antimicrobial. Suitable antimicrobials include:
quinolones, preferably fluoroquinolones, .beta.-lactams, and
macrolide-streptogramin-lincosamide (MLS) antibiotics.
[0051] Suitable quinolones include benofloxacin, binfloxacin,
cinoxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin,
enoxacin, enrofloxacin, fleroxacin, gemifloxacin, ibafloxacin,
levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin,
norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pradofloxacin,
perfloxacin, temafloxacin, tosufloxacin, sarafloxacin,
gemifloxacin, and sparfloxacin. Preferred fluoroquinolones include
ciprofloxacin, enrofloxacin, moxifloxacin, danofloxacin, and
pradofloxacin. Suitable naphthyridones include nalidixic acid.
[0052] Suitable .beta.-lactams include penicillins, such as
benzathine penicillin, benzylpenicillin (penicillin G),
phenoxymethylpenicillin (penicillin V), procaine penicillin,
methicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin,
flucloxacillin, temocillin, amoxicillin, ampicillin, co-amoxiclav
(amoxicillin and clavulanic acid), azlocillin, carbenicillin,
ticarcillin, mezlocillin, piperacillin; cephalosporins, such as
cefalonium, cephalexin, cefazolin, cefapririn, cefquinome,
ceftiofur, cephalothin, cefaclor, cefuroxime, cefamandole,
defotetan, cefoxitin, ceftriaxone, cefotaxime, cefpodoxime,
cefixime, ceftazidime, cefepime, cefpirome; carbapenems and penems
such as imipenem, meropenem, ertapenem, faropenem, doripenem,
monobactams such as aztreonam (Azactam), tigemonam, nocardicin A,
tabtoxinine-B-lactam; and .beta.-lactamase inhibitors such as
clavulanic acid, tazobactam, and sulbactam. Preferred
.beta.-lactams include cephalosporins, in particular,
cefazolin.
[0053] Suitable MLS antibiotics include any macrolide, lincomycin,
clindamycin, pirlimycin. A preferred lincosamide is pirlimycin.
[0054] Other antimicrobials include 2-pyridones, tetracyclines,
sulfonamides, aminoglycosids, trimethoprim, dimetridazoles,
erythromycin, framycetin, furazolidone, various pleuromutilins such
as tiamulin, valnemulin, various, streptomycin, clopidol,
salinomycin, monensin, halofuginone, narasin, robenidine, etc.
[0055] 2. Methods
[0056] a. Methods of Immune Stimulation
[0057] In one embodiment of the invention, an immune response is
elicited in a member of the bovine species by administering an
effective amount of an immunomodulator composition to the member of
the bovine species. The effective amount is sufficient to elicit an
immune response in the member of the bovine species. The
immunomodulator includes a liposome delivery vehicle and a nucleic
acid molecule.
[0058] In one embodiment, the effective amount of the
immunomodulator is from about 1 micrograms to about 1000 micrograms
per animal. In another embodiment, the effective amount of the
immunomodulator is from about 5 micrograms to about 500 micrograms
per animal. In yet another embodiment, the effective amount of the
immunomodulator is from about 10 micrograms to about 100 micrograms
per animal. In a further embodiment, the effective amount of the
immunomodulator is from about 10 micrograms to about 50 micrograms
per animal.
[0059] In another embodiment of the invention, an immune response
is elicited in a member of the bovine species by administering an
effective amount of an immunomodulator, which includes a liposome
delivery vehicle, an isolated nucleic acid molecule, and a
biological agent. It is contemplated that the biological agent may
be mixed with or co-administered with the immunomodulator or
independently thereof. Independent administration may be prior to
or after administration of the immunomodulator. It is also
contemplated that more than one administration of the
immunomodulator or biological agent may be used to extend enhanced
immunity. Furthermore, more than one biological agent may be
co-administered with the immunomodulator, administered prior to the
immunomodulator, administered after administration of the
immunomodulator, or concurrently.
[0060] b. Diseases
[0061] The methods of the invention elicit an immune response in a
subject such that the subject is protected from a disease that is
amenable to elicitation of an immune response. As used herein, the
phrase "protected from a disease" refers to reducing the symptoms
of the disease; reducing the occurrence of the disease, and
reducing the clinical or pathologic severity of the disease or
reducing shedding of a pathogen causing a disease. Protecting a
subject can refer to the ability of a therapeutic composition of
the present invention, when administered to a subject, to prevent a
disease from occurring, cure, and/or alleviate or reduce disease
symptoms, clinical signs, pathology, or causes. Examples of
clinical signs of BRD include lung lesions, increased temperature,
depression (e.g. anorexia, reduced responsiveness to external
stimuli, droopy ears), nasal discharge, and respiratory character
(e.g. respiratory rate, respiratory effort). As such, to protect a
member of the bovine species from a disease includes both
preventing disease occurrence (prophylactic treatment) and treating
a member of the bovine species that has a disease (therapeutic
treatment). In particular, protecting a subject from a disease is
accomplished by eliciting an immune response in the member of the
bovine species by inducing a beneficial or protective immune
response which may, in some instances, additionally suppress,
reduce, inhibit, or block an overactive or harmful immune response.
The term "disease" refers to any deviation from the normal health
of a member of the bovine species and includes a state when disease
symptoms are present, as well as conditions in which a deviation
(e.g., infection, gene mutation, genetic defect, etc.) has
occurred, but symptoms are not yet manifested.
[0062] Methods of the invention may be used for the prevention of
disease, stimulation of effector cell immunity against disease,
elimination of disease, alleviation of disease, and prevention of a
secondary disease resulting from the occurrence of a primary
disease.
[0063] The present invention may also improve the acquired immune
response of the animal when co-administered with a vaccine versus
administration of the vaccine by itself. Generally a vaccine once
administered does not immediately protect the animal as it takes
time to stimulate acquired immunity. The term "improve" refers, in
the present invention, to elicitation of an innate immune response
in the animal until the vaccine starts to protect the animal and/or
to prolong the period of protection, via acquired immunity, given
by the vaccine.
[0064] Methods of the invention include administering the
composition to protect against infection of a wide variety of
pathogens. The composition administered may or may not include a
specific antigen to elicit a specific response. It is contemplated
that the methods of the invention will protect the recipient
subject from disease resulting from infectious microbial agents
including, without limitation, viruses, bacteria, fungi, and
parasites. Exemplary viral infectious diseases, without limitation,
include those resulting from infection with infectious bovine
rhinotracheitis (IBR) (Type 1 bovine herpes virus (BHV1)),
parainfluenza virus type 3 (PI3), bovine respiratory syncytial
virus (BRSV), bovine viral diarrhea virus (BVDV Type 1 and 2),
bovine adenovirus, bovine coronavirus (BCV), bovine calicivirus,
bovine parvovirus, BHV4, bovine reovirus, bovine enterovirus,
bovine rhinovirus, malignant catarrhal fever virus, bovine leukemia
virus, rabies virus, Vesicular stomatitis virus (VSV), bluetongue
(Orbivirus), recombinants thereof, and other viruses known in the
art. Exemplary bacterial infections, without limitation, include
those resulting from infection with gram positive or negative
bacteria and Mycobacteria such as Escherichia coli, Pasteurella
multocida, Clostridium perfringens, Clostridium colinum,
Campylobacter jejuni, Clostridium botulinum, Clostridium novyi,
Clostridium chauveoi, Clostridium septicum, Clostridium
hemolyticum, Clostridium tetani, Mannheimia haemolytica, Ureaplasma
diversum, Mycoplasma dispar, Mycoplasma bovis, Mycoplasma
bovirhinis, Histophilus somni, Campylobacter fetus, Leptospira
spp., Arcanobacterium pyogenes, Bacillus anthrax, Fusobacterium
necrophorum, Fusobacterium spp., Treponema spp., Corynebacterium,
Brucella abortus, Mycobacterium paratuberculosis, Mycobacterium
spp., Histophilus spp., Moraxella spp., Muellerius spp., Mycoplasma
spp., Salmonella spp., Bacillus anthracis, and other bacteria known
in the art. Exemplary fungi or mold infection, without limitation,
include those resulting from infection with Actinobacterim spp.,
Aspergillus spp., and Histomonas spp., and other infectious fungi
or mold known in the art. Exemplary parasites include, without
limitation, Neospora spp., Trichostrongylus, Cooperia, Anaplasma
spp, Babesia spp, Chorioptes spp, Cysticercus spp, Dermatophilus
spp, Damalinia bovis, Dictylocaulus spp, Eimeria spp, Eperythrozoon
spp, Haemonchus spp., Melophagus spp, Muellerius spp, Nematodirus
spp, Oestrus spp, Ostertagia spp, Psoroptes spp, Sarcoptes spp,
Serpens spp, Strongyloides spp, Toxoplasma spp, Trichuris spp,
Trichophyton spp, and Tritrichomas spp, Fascioloides spp, Anaplasma
marginale, and other parasites known in the art.
[0065] c. Subjects
[0066] The methods of the invention may be administered to any
subject or member of the bovine species, whether domestic or wild.
In particular, it may be administered to those subjects that are
commercially reared for breeding, meat or milk production. Suitable
bovine subjects, without limitation, include antelopes, buffalos,
yaks, cattle, and bison. In one embodiment, the member of the
bovine species is cattle. Species of cattle include, without
limitation, cows, bulls, steers, heifer, ox, beef cattle, or dairy
cattle. A skilled artisan will appreciate that the methods of the
invention will be largely beneficial to cattle reared for breeding,
meat or milk production, since they are especially vulnerable to
environmental exposure to infectious agents.
[0067] d. Administration
[0068] A variety of administration routes are available. The
particular mode selected will depend, of course, upon the
particular biological agents selected, the age and general health
status of the subject, the particular condition being treated and
the dosage required for therapeutic efficacy. The methods of this
invention may be practiced using any mode of administration that
produces effective levels of an immune response without causing
clinically unacceptable adverse effects. The compositions may
conveniently be presented in unit dosage form and may be prepared
by any of the methods well known in the art.
[0069] Vaccination of the bovine species can be performed at any
age. The vaccine may be administered intravenously,
intramuscularly, intradermal, intraperitoneal, subcutaneously, by
spray/aerosol, orally, intraocularly, intratracheally, intranasal,
or by other methods known in the art. Further, it is contemplated
that the methods of the invention may be used based on routine
vaccination schedules. The immunomodulator may also be administered
intravenously, intramuscularly, subcutaneously, by spray, orally,
intraocularly, intratracheally, nasally, or by other methods known
in the art. In one embodiment, the immunomodulator is administered
subcutaneously. In another embodiment, the immunomodulator is
administered intramuscularly. In yet another embodiment, the
immunomodulator is administered as a spray. In a further
embodiment, the immunomodulator is administered orally.
[0070] In one embodiment, the immunomodulator is administered by
itself to the animal prior to challenge (or infection). In another
embodiment, the immunomodulator is administered by itself to the
animal post challenge (or infection). In yet another embodiment,
the immunomodulator is administered by itself to the animal at the
same time as challenge (or infection). In a further embodiment, the
immunomodulator composition is co-administered at the same time as
the vaccination prior to challenge. In yet a further embodiment,
the immunomodulator composition is co-administered at the same time
as the vaccination at the same time as challenge (or infection).
The co-administration may include administering the vaccine and
immunomodulator in the same general location on the animal at two
different sites next to each other (i.e., injections next to each
other at the neck of the animal), on opposing sides of the animal
at the same general location (i.e., one on each side of the neck),
or on different locations of the same animal. In another
embodiment, the immunomodulator composition is administered prior
to vaccination and challenge. In a further embodiment, the
immunomodulator composition is administered after vaccination but
prior to challenge. In a further embodiment, the immunomodulator
composition is administered after challenge to an animal that has
been vaccinated prior to challenge (or infection).
[0071] In one embodiment, the immunomodulator is administered from
about 1 to about 14 days prior to challenge or from about 1 to
about 14 days post challenge. In another embodiment, the
immunomodulator is administered from about 1 to about 7 days prior
to challenge or from about 1 to about 7 days post challenge. In yet
another embodiment, the immunomodulator is administered 1, 2, 3, 4,
5, 6, 7days prior to challenge or 1, 2, 3, 4, 5, 6, 7 days post
challenge.
[0072] Other delivery systems may include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the compositions therefore
increasing convenience. Many types of release delivery systems are
available and known to those of ordinary skill in the art. They
include polymer based systems such as poly(lactide-glycolide),
copolyoxalates, polycaprolactones, polyesteramides,
polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.
Microcapsules of the foregoing polymers containing drugs are
described in, for example, U.S. Pat. No. 5,075,109. Delivery
systems also include non-polymer systems that are lipids including
sterols such as cholesterol, cholesterol esters and fatty acids or
neutral fats such as mono-di and tri-glycerides; hydrogel release
systems; sylastic systems; peptide based systems; wax coatings;
compressed tablets using convention binders and excipients;
partially fused implants; and the like. Specific examples include,
but are not limited to erosional systems in which an agent of the
invention is contained in a form within a matrix such as those
described in U.S. Pat. Nos. 4,452,775, 4,675,189 and 5,736,152, and
diffusional systems in which an active component permeates at a
controlled rate from a polymer such as described in U.S. Pat. Nos.
3, 854,480, 5,133,974 and 5,407,686. In addition, pump-based
hardware delivery systems can be used, some of which are adapted
for implantation.
[0073] As various changes could be made in the above composition,
products and methods without departing from the scope of the
invention, it is intended that all matter contained in the above
description and in the examples given below, shall be interpreted
as illustrative and not in a limiting sense.
Definitions
[0074] The term "effective amount" refers to the amount necessary
or sufficient to realize a desired biologic effect. For example, an
effective amount of immunomodulator for treating or preventing an
infectious disease is that amount necessary to cause the
development of an immune response upon exposure to the microbe,
thus causing a reduction in the amount of microbe within the
subject and preferably to the eradication of the microbe. The
effective amount for any particular application can vary depending
on such factors as the disease or condition being treated, the size
of the subject, or the severity of the disease or condition. One of
ordinary skill in the art can empirically determine the effective
amount of immunomodulator without necessitating undue
experimentation.
[0075] The term "cytokine" refers to an immune enhancing protein
family. The cytokine family includes hematopoietic growth factor,
interleukins, interferons, immunoglobulin superfamily molecules,
tumor necrosis factor family molecules and chemokines (i.e.
proteins that regulate the migration and activation of cells,
particularly phagocytic cells). Exemplary cytokines include,
without limitation, interleukin-2 (IL-2), interleukin-12 (IL12),
interleukin-15 (IL-15), interleukin-18 (IL-18), interferon-.alpha.
(IFN.alpha.), and interferon-.gamma. (IFN.gamma.).
[0076] The term "elicit" can be used interchangeably with the terms
activate, stimulate, generate or upregulate.
[0077] The term "eliciting an immune response" in a subject refers
to specifically controlling or influencing the activity of the
immune response, and can include activating an immune response,
upregulating an immune response, enhancing an immune response
and/or altering an immune response (such as by eliciting a type of
immune response which in turn changes the prevalent type of immune
response in a subject from one which is harmful or ineffective to
one which is beneficial or protective).
[0078] The term "operatively linked" refers to linking a nucleic
acid molecule to a transcription control sequence in a manner such
that the molecule is able to be expressed when transfected (i.e.,
transformed, transduced or transfected) into a host cell.
Transcriptional control sequences are sequences which control the
initiation, elongation, and termination of transcription.
Particularly important transcription control sequences are those
which control transcription initiation, such as promoter, enhancer,
operator and repressor sequences. A variety of such transcription
control sequences are known to those skilled in the art. Preferred
transcription control sequences include those which function in
avian, fish, mammalian, bacteria, plant, and insect cells. While
any transcriptional control sequences may be used with the
invention, the sequences may include naturally occurring
transcription control sequences naturally associated with a
sequence encoding an immunogen or immune stimulating protein.
[0079] The terms "nucleic acid molecule" and "nucleic acid
sequence" can be used interchangeably and include DNA, RNA, or
derivatives of either DNA or RNA. The terms also include
oligonucleotides and larger sequences, including both nucleic acid
molecules that encode a protein or a fragment thereof, and nucleic
acid molecules that comprise regulatory regions, introns, or other
non-coding DNA or RNA. Typically, an oligonucleotide has a nucleic
acid sequence from about 1 to about 500 nucleotides, and more
typically, is at least about 5 nucleotides in length. The nucleic
acid molecule can be derived from any source, including mammalian,
fish, bacterial, insect, viral, plant, or synthetic sources. A
nucleic acid molecule can be produced by methods commonly known in
the art such as recombinant DNA technology (e.g., polymerase chain
reaction (PCR), amplification, cloning) or chemical synthesis.
Nucleic acid molecules include natural nucleic acid molecules and
homologues thereof, including, but not limited to, natural allelic
variants and modified nucleic acid molecules in which nucleotides
have been inserted, deleted, substituted, or inverted in such a
manner that such modifications do not substantially interfere with
the nucleic acid molecule's ability to encode an immunogen or
immune stimulating protein useful in the methods of the present
invention. A nucleic acid homologue may be produced using a number
of methods known to those skilled in the art (see, for example,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Labs Press, 1989), which is incorporated herein by
reference. Techniques to screen for immunogenicity, such as
pathogen antigen immunogenicity or cytokine activity are known to
those of skill in the art and include a variety of in vitro and in
vivo assays.
EXAMPLES
[0080] The following examples illustrate various embodiments of the
invention.
Example 1
Evaluation of Cattle Receiving a DNA Immunomodulator Before or
After Developing Natural Bovine Respiratory Disease
[0081] The purpose of this study was to determine the efficacy of
the DNA immunomodulator administered to calves prior to and after
developing natural cases of BRD.
Immunomodulator
[0082] The immunomodulator used in this study was a composition
comprising a cationic lipid and non-coding DNA. The synthetic
immunomodulator lipid components
[1-[2-[9-(Z)-octadecenoyloxy]]-2-[8](Z)-heptadecenyl]-3-[hydroxyethyl]imi-
dazolinium chloride (DOTIM) and a synthetic neutral lipid
cholesterol were formulated to produce liposomes approximately 200
nm in diameter (See, U.S. Pat. No. 6,693,086). The DNA component
was a 4242 base-pair non-coding DNA plasmid produced in E. coli,
which, being negatively charged, associates with the
positively-charged (cationic) liposomes (See, U.S. Pat. No.
6,693,086).
Study Animals
[0083] 84 Holstein steer calves of weaning age were selected from a
herd without a current history of respiratory disease. Each
individual calf was initially evaluated and determined to be in
good health. The 84 calves were divided into seven treatment groups
of 12 calves each. Only animals not vaccinated for Mannheimia
haemolytica were included in the study. None of the animals had
received an antimicrobial agent within 30 days prior to
administration of DNA immunomodulator.
[0084] The treatment groups were administered varying doses of the
DNA immunomodulator describe above on the day of treatment as
indicated in Table 1.1 below. The dilution scheme of the DNA
immunomodulator is provided in Table 1.2. The DNA immunomodulator
was administered intramuscularly and cranial to the left shoulder,
ventral to the nuchal ligament, and caudo-dorsal to the jugular
groove of the calves.
[0085] As referred to below, Treatment Day -1 refers to the start
date of the study after initial selection in which the calves were
evaluated and determined to be suitable for the study. Treatment
Day 0 is one day subsequent to Day -1, and so on.
TABLE-US-00001 TABLE 1.1 Administration Schedule of Immunomodulator
Animals DNA Day of per Treatment Immunomodulator Immunomodulator
Treatment number Dose (.mu.g) Administration group 1 500 -1 12 2
200 -1 12 3 50 -1 12 4 500 0 12 5 200 0 12 6 50 0 12 7 0 NA 12
(Control)
[0086] A large proportion of the calves were observed to be
experiencing variable levels of BRD on the morning of Day 0. By Day
5 all of the calves remaining in the study population were observed
to have met the case definition for BRD morbidity. Cattle were only
removed from the study population if euthanasia was indicated due
to severe BRD. No other infectious/non-infectious diseases were
observed and thereby required removal in this study.
Evaluation
[0087] On Days 1-5 of the study the calves were evaluated for
various health indicators. For example, rectal temperature and
average daily weight were determined for each of the calves per day
through the length of the study. Animals were evaluated at
approximately the same time each day (+/-3 hours) from Day 1 to Day
5. FIGS. 1.1 and 1.2 present the averages of rectal temperatures
and average daily weight gain according to dose of immunomodulator
administered.
[0088] On Day 5, all calves were euthanized and necropsied. Lung
lesion scores were determined (based upon the degree lung
consolidation estimated by visual inspection and manual palpation)
for each individual calf at the time of necropsy.
[0089] FIG. 1.3 presents the lung lesion scores with respect to
dose of immunomodulator administered. The overall lung lesion
scores for each day of administration were approximately 11% and
14% for Day -1 and Day 0, respectively. Lung lesion scores of
11.2%, 9.0%, 10.8% and 19.9% were exhibited for 500, 200, 50 and
negative control groups, respectively. The largest difference
between the control group and a treated group (200 .mu.g) was about
an 11% reduction.
[0090] Model-adjusted estimates on FIG. 1.3 reflect the raw
averages that are adjusted for all statistical model covariates
(i.e. dose, day, and dose x day) as well as for the pen in which
the calves were housed throughout the study. Therefore,
model-adjusted estimates may display differences compared to the
raw averages.
[0091] Subsequent bacteriology (lung cultures) and virology (nasal
swabs) were also performed. Of the remaining calves (69) that were
euthanized on Day 5, 11.6% were found to be shedding bovine herpes
virus type 1 (BHV-1) in nasal secretions. With regard to lung
cultures from all of the study animals, 41% were positive for Mh,
31.3% were culture positive for Pasteurella multocida (Pm), 10.8%
were culture positive for both Mh and Pm, and no Histophilus somni
was isolated throughout the study population. Cultures for
Mycoplasma bovis were not performed in this study
Results
[0092] In this study, the dose of the DNA immunomodulator (i.e. 500
.mu.g, 200 .mu.g, and 50 .mu.g) approached a significant reduction
in lung lesion scores compared to the negative control (P=0.1284;
See FIG. 1.3). However, the day of DNA immunomodulator
administration (i.e., Day -1 or 0) was not significantly associated
with lung lesion scores. No statistical differences in lung lesion
scores were observed among the DNA immunomodulator dose groups.
Rectal temperature tended to be significantly associated with the
dose of DNA immunomodulator (P=0.1190) but was not associated with
the day of administration. No obvious differences were observed
between the dose of the DNA immunomodulator and the negative
control with regard to average daily weight gain.
[0093] There was a strong tendency for the DNA immunomodulator to
reduce lung lesions compared to negative control, thereby,
providing evidence that this product has the potential to protect
lung tissue during a BRD outbreak. In this study, the day of
treatment administration was not associated with lung lesions
thereby indicating that it does not matter if cattle received the
DNA immunomodulator one day prior or the same day as the onset of
clinical signs associated with BRD. This outcome is important as
the timing of exposure to BRD pathogens is generally unknown among
typical production systems and is further complicated by the impact
of various stressors experienced by cattle throughout the chain of
production. Therefore, providing producers with a product that
offers flexibility in the timing of administration, in relation to
the onset of BRD, is of extreme value in the beef and dairy
industries.
Example 2
Evaluation of Cattle Receiving a DNA Immunomodulator Concurrently
With or One Day After an Experimental Challenge With Mannheimia
haemolytica
[0094] The purpose of this study was to determine the efficacy of
the DNA immunomodulator administered to calves concurrently with or
one day after an experimental challenge with Mannheimia
haemolytica.
Immunomodulator
[0095] The immunomodulator used in this study was the composition
described above in Example 1.
Study Animals
[0096] 84 Holstein steer calves of weaning age and weighing on
average about 300 lbs (136 kg) were selected from a herd without a
current history of respiratory disease. Each individual calf was
initially evaluated and determined to be in good health. The 84
calves were divided into seven treatment groups of 12 calves each.
Only animals not vaccinated for Mannheimia haemolytica were
included in the study. None of the animals had received an
antimicrobial agent within 30 days prior to administration of DNA
immunomodulator. The treatment groups were administered varying
doses of the DNA immunomodulator on the day of treatment as
indicated in Table 2.1 below. The dilution scheme of the DNA
immunomodulator is provided in Table 2.2. The DNA immunomodulator
was administered intramuscularly and cranial to the left shoulder,
ventral to the nuchal ligament, and caudo-dorsal to the jugular
groove of the calves.
[0097] As referred to below, Treatment Day 0 refers to the start
date of the study after initial selection in which the calves were
evaluated and determined to be in good health. Treatment Day 1 is
one day subsequent to Day 0, and so on.
TABLE-US-00002 TABLE 2.1 Administration Schedule of Immunomodulator
and Mh Challenge Animals DNA Day of Day of Mh per Treatment
Immunomodulator Immunomodulator Challenge Treatment number Dose
(.mu.g) Administration Administration group 1 500 0 0 12 2 200 0 0
12 3 50 0 0 12 4 500 1 0 12 5 200 1 0 12 6 50 1 0 12 7 0 NA 0 12
(Control)
Experimental Challenge
[0098] On Day 0, the calves were challenged a total of
3.12.times.10.sup.7 colony forming units (CFU) of Mannheimia
haemolytica. The inoculum was administered via the respiratory
tract. By Day 3, all of the calves in the study population were
observed to have met the case definition for BRD morbidity. The
median day of onset was one day.
Evaluation
[0099] As in the previous example, on Days 1-5 of the study the
calves were evaluated for various health indicators. Rectal
temperature and average daily weight were determined for each of
the calves per day through the length of the study. Animals were
evaluated at approximately the same time each day. FIGS. 2.1 and
2.2 present the averages of rectal temperatures and average daily
weight gains with respect to dose of immunomodulator
administered.
[0100] On Day 5, all calves were euthanized and necropsied. Lung
lesion scores were determined for each individual calf at the time
of necropsy according to the formula described in Example 1.
[0101] FIG. 2.3 presents the model-adjusted lung lesion scores with
respect to dose of immunomodulator administered.
Results
[0102] In this study, the dose of the DNA immunomodulator (i.e. 500
.mu.g, 200 .mu.g, and 50 .mu.g) significantly reduced lung lesion
scores compared to the negative control. However, the lower doses
(200 .mu.g, and 50 .mu.g) outperformed the 500 .mu.g dose in
reducing lung lesions. The day of DNA immunomodulator
administration (i.e., Day 0 or 1) was not significantly associated
with lung lesion scores. No statistical differences in lung lesion
scores were observed among the DNA immunomodulator dose groups.
Rectal temperature was significantly reduced in calves administered
the DNA immunomodulator compared to the negative control, but was
not associated with dose. No obvious differences were observed
between the dose of the DNA immunomodulator and the negative
control with regard to average daily weight gain.
[0103] There was a strong tendency for the DNA immunomodulator to
reduce lung lesions compared to negative control, thereby,
providing evidence that this product has the potential to protect
lung tissue during a BRD outbreak. In this study, the day of
treatment administration was not associated with lung lesions
thereby indicating that it did not matter if cattle received the
DNA immunomodulator one day prior, or the same day as, the onset of
clinical signs associated with BRD. This outcome is important as
the timing of exposure to BRD pathogens is generally unknown among
typical production systems and is further complicated by the impact
of various stressors experienced by cattle throughout the chain of
production. Therefore, providing producers with a product that
offers flexibility in the timing of administration, in relation to
the onset of BRD, is of extreme value in the beef and dairy
industries.
Example 3
Evaluation of Cattle Receiving a DNA Immunomodulator Two Days
Before or Concurrently With an Experimental Challenge with
Mannheimia haemolytica
[0104] The purpose of this study was to determine the efficacy of
the DNA immunomodulator administered to calves two days before or
concurrently with an experimental challenge with Mannheimia
haemolytica.
Immunomodulator
[0105] The immunomodulator used in this study was the composition
described above in Example 1.
Study Animals
[0106] 96 Holstein steer calves weighing on average about 800-1000
lbs (363-454 kg) were selected from a herd without a current
history of respiratory disease. Each individual calf was initially
evaluated and determined to be in good health. The 96 calves were
divided into eight treatment groups of 12 calves each. Only animals
not vaccinated for Mannheimia haemolytica were included in the
study. None of the animals had received an antimicrobial agent
within 30 days prior to administration of DNA immunomodulator. The
treatment groups were administered varying doses of the DNA
immunomodulator on the day of treatment as indicated in Table 3.1
below. The dilution scheme of the DNA immunomodulator is provided
in Table 3.2. The DNA immunomodulator was administered
intramuscularly and cranial to the left shoulder, ventral to the
nuchal ligament, and caudo-dorsal to the jugular groove of the
calves.
[0107] As referred to below, Treatment Day -2 refers to the start
date of the study when Treatment Groups 1-3 were administered the
immunomodulator. Treatment Day 0 is two days subsequent to Day -2,
and so on.
TABLE-US-00003 TABLE 3.1 Administration Schedule of Immunomodulator
and Mh Challenge Animals DNA Day of Day of Mh per Treatment
Immunomodulator Immunomodulator Challenge Treatment number Dose
(.mu.g) Administration Administration group 1 200 -2 0 12 2 50 -2 0
12 3 25 -2 0 12 4 200 0 0 12 5 50 0 0 12 6 25 0 0 12 7 0 -2 0 12
(Control) 8 0 0 0 12 (Control)
Experimental Challenge
[0108] On Day 0, the calves were challenged with a total of
1.9.times.10.sup.10 CFUs. The inoculum was administered via the
respiratory tract.
Evaluation
[0109] As in the previous examples, on Days 1-5 of the study the
calves were evaluated for various health indicators. On Day 5, all
calves were euthanized and necropsied. Lung lesion scores were
determined for each individual calf at the time of necropsy.
[0110] FIG. 3.1 presents the model-adjusted lung lesion scores with
respect to dose of immunomodulator administered. FIG. 3.2 presents
the model-adjusted lung lesion scores with respect to day of
immunomodulator administration.
Results
[0111] In this study, the dose of the DNA immunomodulator (i.e. 200
.mu.g, 50 .mu.g, and 25 .mu.g) significantly reduced lung lesion
scores compared to the negative controls. However, no statistical
differences in lung lesion scores were observed among the DNA
immunomodulator dose groups. The day of DNA immunomodulator
administration (i.e. Days -2 and 0) was significantly associated
with lung lesion scores. Significant reduction in lung lesions was
observed when the immunomodulator was administered on Day 0 when
compared to Day -2.
Example 4
Mh Challenge Co-Administration of Immunomodulator and Killed Mh
Vaccine
[0112] The purpose of this study was to determine the efficacy of
the DNA immunomodulator co-administered with killed Mh vaccine to
calves subjected to an experimental challenge with Mannheimia
haemolytica.
Immunomodulator
[0113] The immunomodulator used in this study was the composition
described above in Example 1.
Study Animals
[0114] 81 Holstein bull calves, 12 weeks old, were selected from a
herd without a current history of respiratory disease. Each
individual calf was evaluated and determined to be in good health.
Only animals not vaccinated for Mannheimia haemolytica were
included in the study. None of the animals had received an
antimicrobial agent within 30 days prior to administration of
inoculum.
Experimental Infection and Challenge
[0115] The challenge, or experimental infection, included exposure
to an inoculum of Mannheimia haemolytica. The organisms were used
at a concentration of 1.7.times.10.sup.8 per animal for the first
inoculum and 2.4.times.10.sup.10 animal for the second inoculum.
The animals were also challenged with a spray by another
respiratory route. The concentration of the organisms in the spray
inoculum was 1.9.times.10.sup.10 per animal.
[0116] The efficacy of the immunomodulator, as described above,
administered to calves followed by exposure to Mannheimia
haemolytica was determined by the twelve treatment groups as
detailed on Table 3.
TABLE-US-00004 TABLE 4.3 Study Treatment Groups. Treatment Days
Number of Group Targeted Dose Day Contact Animals T1 Killed MH
(oil) vaccine (SC) 0 X 7 T2 Killed MH (oil) vaccine + 0 X 7
Immunomodulator 500 .mu.g (SC) T3 Killed MH (oil) vaccine (SC) 7 X
6 T4 Killed MH (oil) vaccine + 7 X 7 Immunomodulator 500 .mu.g (SC)
T5 Immunomodulator 500 .mu.g (SC) 7 X 7 T6 Immunomodulator 500
.mu.g (SC) 13 X 7 T7 Immunomodulator 500 .mu.g (IM) 13 X 7 T8
Immunomodulator 500 .mu.g (SC) 15* X 7 T9 Control NC NA NA 7 T10
Control CC NA X 5 T11 Control SE NA X 7 T12 Killed MH (aqueous)
vaccine + 0 X 7 Immunomodulator 500 .mu.g (SC) Oil MH = Mannheimia
haemolytica vaccine (Pulmo-Guard .RTM. PHM) Aqueous MH = Mannheimia
haemolytica vaccine (One Shot .RTM.) NC = Not commingled and not
spray challenged (for background gross pathology) CC = Contact and
spray challenged SE = Used as Seeder challenge (Challenged
intratracheal) All animals, except SE and NC were spray challenged
SC = Subcutaneous route of injection IM = Intramuscular route of
injection NA = Not Applicable *Animals in group T8 will be treated
after intranasal challenge
[0117] On day 0 of the study, all animals in groups T1, T2 and T12
were administered the immunomodulator subcutaneously. The
immunomodulator was administered subcutaneously on Day 7 to Groups
T3, T4, and T5. The immunomodulator was administered subcutaneously
on Day 13 to Group T6 and intramuscularly to T7. The
immunomodulator was administered subcutaneously on Day 15 to Group
T8.
[0118] All animals receiving the vaccine were vaccinated according
to label instructions. Immunomodulator and the vaccine were
administered as close together near a lymph node (neck)--two
injections (one for vaccine and the other for the immunomodulator).
All animals receiving the subcutaneous route of injection were
injected near a lymph node in the sub scapular region.
[0119] On study day 10, all T11 calves were transported off site in
a stock trailer for approximately 24 hours to stress the calves. On
Study day 11, 20 mL of an inoculum containing Mannheimia
haemolytica was administered transtracheally to all the T11
animals, followed 4 hours later with 25 mL of inoculum. On study
day 14, all groups, except T9 were commingled and transported off
site in a stock trailer for approximately 24 hours to stress the
calves. All animals except in group NC were commingled in a large
pen for 12 to 16 hours on Study day 14 and then returned to their
separate pens (each animal had a separate pen). On Study day 15, 20
mL of Mannheimia haemolytica was administered by another
respiratory route to all groups except T9 and T11. The animals were
observed daily throughout the study for clinical abnormalities and
mortality. All animals were negative or had low titers at screening
prior to purchase of animals. The animals had high titers prior to
treatment, which indicates that the animals serologically converted
to Mannheimia haemolytica prior to receiving treatment.
Results
[0120] The animals of group T8 had significantly lower lung
lesions.
[0121] The study suggests that there is an onset of early
protection (day 7) with or without vaccine (groups T4 and T5
compared to T3). See FIGS. 4.1 and 4.2.
Example 5
Evaluation of Acquired Immunity in Cattle Vaccinated With a
Commmercial-Live Vaccine When Co-Administered With a DNA
Immunomodulator
[0122] The purpose of this study was to determine if
co-administration of the DNA immunomodulator augmented the acquired
immunity afforded by modified-live viral (MLV) vaccines.
Immunomodulator
[0123] The immunomodulator used in this study was the composition
described above in Example 1.
Study Animals
[0124] 72 Holstein steers calves of weaning age were selected from
a herd without a current history of respiratory disease. The 72
calves were divided into six treatment groups of 12 calves each.
Each individual calf was evaluated and determined to be in good
health. All calves were free of serum antibodies to BHV-1, BVDV
types 1 and 2, and BRSV. In addition, all calves were found to be
serum antibody negative to PI-3. The calves were subsequently
determined to be negative for bovine viral diarrhea virus
persistent infection by immunohistochemistry.
[0125] The treatment groups were administered the vaccine and
varying doses of the DNA immunomodulator intramuscularly on the day
of treatment as indicated in Table 5.1 below. The dilution scheme
of the DNA immunomodulator is provided in Table 5.2. On day 0 of
the study, all animals in groups T1-T4 were administered the
immunomodulator. All animals receiving the vaccine were vaccinated
according to label instructions. Immunomodulator and the vaccine
were administered as close together cranial to the front of the
shoulder--two injections (one for vaccine and the other for the
immunomodulator).
TABLE-US-00005 TABLE 5.1 Administration Schedule of Immunomodulator
and Vaccine Day of Vaccine and/or Immunomodulator Number of Group
Targeted Dose Administration Animals T1 MLV + Immunomodulator 0 12
(500 .mu.g) IM T2 MLV + Immunomodulator 0 12 (200 .mu.g) IM T3 MLV
+ Immunomodulator 0 12 (100 .mu.g) IM T4 MLV + Immunomodulator 0 12
(50 .mu.g) IM T5 MLV 0 12 T6 No treatment NA 12 MLV = Mannheimia
haemolytica vaccine (Bovi-shield .RTM.) - modified-live 4-way viral
respiratory vaccine IM = Intramuscular route of injection
Evaluation
[0126] Immunological testing was performed on samples from
appropriate hematological specimens collected from the calves on
Days 0, 13, 28, 27, 34 and 41. Cell mediated immunity (CMI)
measurements were conducted for each specimen. The target pathogens
for this study were BHV-1, BVDV 1 and 2, and BRSV. Laboratories
used standardized procedures and methods as appropriate for the
previously specified target pathogens.
Results
[0127] Model-adjusted data for CMI outcomes on each Day of sample
collection among all treatment groups were determined. Across all
treatment groups, cell types, and antigens no statistical
differences (P>0.10) were detected when comparing DNA
immunomodulator treatment groups--MLV vaccine combinations to
cattle receiving only the MLV vaccine (See FIGS. 5.1-5.12). In
particular, FIGS. 5.1-5.4 present the measurements of the CD 25 EI
expression index (y-axis) across all five cell types for each of
the 6 treatment groups (x-axis). FIGS. 5.5-5.8 present the
measurements of the IFN.gamma. expression index (y-axis) across all
five cell types for each of the 6 treatment groups (x-axis). FIGS.
5.9-5.12 present the measurements of the IL-4 expression index
(y-axis) across all five cell types for each of the 6 treatment
groups (x-axis). Estimates were produced for each of the 4 BRD
viral pathogens represented in their respective graph. For these
statistical evaluations, all comparisons were made to the "MLV
only" treatment group.
[0128] Statistically significant (P<0.10) treatment x Day
interactions were detected for BVDV 1 (Days 28 and 35) and BVDV 2
(Day 42). No significant findings (P>0.10) were detected for
BHV-1 at any of the listed time points. A graphical representation
of these findings is displayed on FIGS. 5.13-5.15. The BRSV data
was removed from analysis due to observance of antibody
seroconversion within the negative control treatment group. Note
that, for all statistical evaluations, all comparisons were made to
the "MLV only" treatment group.
[0129] Individual animal weights were also collected during the
study. A graphical representation of model-adjusted average daily
gain outcomes is displayed in FIG. 5.16. No significant findings
(P>0.10) were detected across treatment groups when compared to
the MLV only group.
[0130] In summary, the DNA immunomodulator did not enhance CMI when
co-administered with a MLV vaccine compared to the sole
administration of MLV vaccine. However, 500 .mu.g of the DNA
immunomodulator may augment humoral immunity when co-administered
with a MLV vaccine (specifically BVDV). Nonetheless, it should be
noted that despite a lack of consistent improvement in acquired
immunity, co-administration of the DNA immunomodulator, at doses of
500 .mu.g, 200 .mu.g, 100 .mu.g, and 50 .mu.g, did not impair the
positive immunologic effects induced by the MLV vaccine. In
addition, performance (e.g. ADG) was not negatively impacted by
administration of the DNA immunomodulator.
Example 6
Evaluation of Acquired Immunity in Cattle Vaccinated With a
Commmercial-Vaccine When Co-Administered With a DNA
Immunomodulator
[0131] The purpose of this study was to determine if
co-administration of the DNA immunomodulator augmented the acquired
immunity afforded by vaccines containing inactivated antigens.
Immunomodulator
[0132] The immunomodulator used in this study was the composition
described above in Example 1.
Study Animals
[0133] 48 Holstein female cattle of 3-5 month age were selected
from a herd without a current history of respiratory disease. The
48 cattle were divided into six treatment groups of 8 animals each.
Each individual animal was evaluated and determined to be in good
health. All animals were free of serum antibodies to BHV-1, BVDV
types 1 and 2. The animals were also determined to be negative for
bovine viral diarrhea virus persistent infection by PCR. The
animals were not selected on SNT titers against BRS virus and PI3
virus.
[0134] The treatment groups were administered the vaccine and
varying doses of the DNA immunomodulator intramuscularly on the day
of treatment as indicated in Table 5.1 below. The vaccine contained
BHV1 and BVDV type1 and 2 as inactivated antigens, and modified
live PI3 virus and BRS virus. The Immunomodulator and the vaccine
were either given separately on the same side of the animal cranial
to the front of the shoulder, or separately on the opposite side of
the animal in the same region, or mixed in one syringe. The
dilution scheme of the DNA immunomodulator is provided in Table
5.2.
TABLE-US-00006 TABLE 6.1 Administration Schedule of Immunomodulator
and Vaccine Day of Vaccine and/or Immunomodulator Number of Group
Targeted Dose Administration Animals T1 Placebo (Dextrose 5%) 0 8
T2 Vaccine + Dextrose 0 8 IM, separately T3 Vaccine +
Immunomodulator 0 8 (20 .mu.g) IM, mixed T4 Vaccine +
Immunomodulator 0 8 (200 .mu.g) IM, mixed T5 Vaccine +
Immunomodulator 0 8 (200 .mu.g) IM, separately same side T6 Vaccine
+ Immunomodulator 0 8 (200 .mu.g) IM, separately opposite side
Vaccine = combined (inactivated and modified live)4-way viral
respiratory vaccine (Rispoval .RTM.) IM = Intramuscular route of
injection
Evaluation
[0135] Immunological testing was performed on samples from
appropriate hematological specimens collected from the cattle on
Days 0, 3, 5, 7, 9, 11, 14, 17, 20, 23 and 27. The target pathogens
for this study were BHV-1, BVDV 1 and 2. For information also the
antibody titers against BRS virus and P13 virus were determined.
Laboratories used standardized Serum Neutralization Tests (SNT) as
procedures for the previously specified target pathogens.
Results
[0136] Statistically significant (P<0.010) treatment x Day
interactions were detected for BHV1 (Day 27). No significant
findings (P>0.10) were detected for all other time points for
BHV1 and for BVDV type 1 at 2 at any of the listed time points. The
results of the BRSV and PI3 titers were not further evaluated
because the animals were not serologically negative at the
beginning of the study. An effect of treatment could therefore not
be verified. A graphical representation of these findings is
displayed on FIGS. 6.1. Note that, for all statistical evaluations,
all comparisons were made to the "Vaccine and Dextrose 5%"
treatment group.
* * * * *