U.S. patent application number 17/833002 was filed with the patent office on 2022-09-22 for mycoplasma bovis compositions.
The applicant listed for this patent is University of Melbourne, Zoetis Services LLC. Invention is credited to Glenn Francis Browning, Suman Mahan, Marc Serge Marenda, Philip Francis Markham, Fred H. Weber.
Application Number | 20220296694 17/833002 |
Document ID | / |
Family ID | 1000006381039 |
Filed Date | 2022-09-22 |
United States Patent
Application |
20220296694 |
Kind Code |
A1 |
Browning; Glenn Francis ; et
al. |
September 22, 2022 |
MYCOPLASMA BOVIS COMPOSITIONS
Abstract
Disclosed herein are Mycoplasma bovis immunogenic compositions
useful in raising an immune response in animals against M. bovis.
Also disclosed herein are methods for generating a protective
response against infections in mammals caused by M. bovis.
Inventors: |
Browning; Glenn Francis;
(Parkville, AU) ; Markham; Philip Francis;
(Parkville, AU) ; Marenda; Marc Serge; (Parkville,
AU) ; Weber; Fred H.; (Kalamazoo, MI) ; Mahan;
Suman; (Kalamazoo, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zoetis Services LLC
University of Melbourne |
Parsippany
Carlton |
NJ |
US
AU |
|
|
Family ID: |
1000006381039 |
Appl. No.: |
17/833002 |
Filed: |
June 6, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15751293 |
Feb 8, 2018 |
11389518 |
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PCT/US2016/046572 |
Aug 11, 2016 |
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17833002 |
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62205125 |
Aug 14, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/552 20130101;
C12N 1/20 20130101; A61K 2039/522 20130101; A61K 39/0241 20130101;
A61K 2039/521 20130101 |
International
Class: |
A61K 39/02 20060101
A61K039/02; C12N 1/20 20060101 C12N001/20 |
Claims
1-18. (canceled)
19. A Mycoplasma bovis strain, wherein said strain is deposited
with the American Type Culture Collection under accession number
PTA-122167, or wherein said strain is a progeny of M. bovis strain
PTA-122167 wherein said progeny of M. bovis strain PTA-122167 is
obtained by passaging strain PTA-122167.
20. An immunogenic composition comprising the Mycoplasma bovis
strain according to claim 19, wherein said strain is a modified
live strain.
21. The immunogenic composition according to claim 20, further
comprising at least one additional antigen.
22. The immunogenic composition of claim 21, wherein the additional
antigen is selected from the group consisting of a virus, a
bacterium, a fungus and a parasite.
23. The immunogenic composition of claim 20 formulated for
administration by a route selected from the group consisting of
intramuscular, subcutaneous, intranasal, intratracheal, oral and
aerosol.
24. A method for preventing disease caused by Mycoplasma bovis in a
bovine animal, comprising administering to the animal the
immunogenic composition of claim 20.
25. A method for reducing at least one clinical sign of disease
caused by Mycoplasma bovis in a bovine animal, comprising
administering to the animal the immunogenic composition of claim
19.
26. The method according to claim 25, wherein said at least one
clinical sign is selected form the group consisting of a lung
lesions, pyrexia, cough, lameness, and difficulty breathing.
27. A method for preventing diseases caused by Mycoplasma bovis and
another infectious agent of a bovine animal, comprising
administering to the animal the immunogenic composition of claim
22.
28. A method for preventing at least one clinical sign of diseases
caused by Mycoplasma bovis and said additional infectious agent,
comprising administering to the animal the immunogenic composition
of claim 22.
29. The method according to claim 28, wherein said at least one
clinical sign is selected form the group consisting of a lung
lesions, pyrexia, cough, lameness, and difficulty breathing.
30. A method of making a M. bovis vaccine, the method comprising:
a) obtaining a culture of M. bovis strain, wherein said strain is
deposited with the American Type Culture Collection under accession
number PTA-122167; b) further passaging said culture of M. bovis at
least once; c) obtaining a clone from said passaged culture; and d)
formulating said clone with a carrier or excipient and, optionally,
an adjuvant.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to Mycoplasma bovis
immunogenic compositions. The present invention further relates to
immunogenic compositions for use in raising an immune response in
animals against M. bovis, and methods for generating a protective
response against infections in mammals caused by M. bovis.
BACKGROUND OF THE INVENTION
[0002] Mycoplasma bovis causes multiple diseases in bovine herds
around the world, including, for example, mastitis, pneumonia,
arthritis, otitis, skin abscesses, infertility, abortion, or
combinations thereof. In each form of the disease, mortality can
occur. M. bovis is thus a significant concern to dairy and beef
producers, and veterinarians world-wide. A variety of antibiotics
have been developed and proposed as tools to mitigate the economic
losses and mortality produced by this pathogen. Preventing the
disease through the application of a safe and effective vaccine
offers significant advantages to antibiotic therapy through
enhancing animal welfare, improving producer economics, and
supporting the judicious use of antibiotics. Vaccines containing
inactivated (e.g., killed) M. bovis have been developed. However,
killed vaccines typically present long delays in the initiation of
a protective response, may require more than one dose, and in some
cases, may require adjuvants that are not suitable in certain
applications.
[0003] Live-attenuated, or modified-live, vaccines are advantageous
in that they can lead to the development of a more rapid immune
response, are often effective when administered in a single dose,
and are generally less expensive to produce. Thus, there is a need
for improved M. bovis vaccines, including live-attenuated ones.
SUMMARY OF THE INVENTION
[0004] Applicants have surprisingly identified immunogenic
compositions useful for generating a protective immune response
against Mycoplasma bovis infection in mammals. They can
additionally be combined with an adjuvant. Said compositions can
also be combined with additional antigens, yielding multivalent
compositions for combating multiple infections in mammals.
[0005] One embodiment of the invention provides an immunogenic
composition comprising an attenuated Mycoplasma bovis strain,
wherein said strain is deposited with the American Type Culture
Collection under accession number PTA-122167.
[0006] Another embodiment provides an immunogenic composition
comprising a chemically-attenuated Mycoplasma bovis strain which,
in comparison to a parent strain, has one or more mutations in one
or more of the proteins nitroreductase, histidine acid phosphatase,
excinuclease ABC subunit B, DNA polymerase Ill subunit beta, and
ATP synthase F1 subunit delta.
[0007] Another embodiment provides an immunogenic composition
comprising a Mycoplasma bovis strain, wherein said strain is
PTA-122167, further wherein said strain has been reverse engineered
to contain one or more of any of the original nucleotides found in
reference M. bovis strain, 3683 #22/3/12, at positions 115273,
194478, 194484, 194525, 194724, 204966, 207629, 262327, 271487,
271661, 271666, 272086, 272133, 309463, 310075, 310078, 310086,
310090, 311988, 334184, 358013, 365101, 365188, 397010, 397901,
399129, 410247, 410253, 410334, 410514, 410733, 414666, 453388,
468457, 502577, 502678, 502963, 543859, 560986, 632309, 632366,
632612, 633994, 686074, 687874, 687915, 696348, 729874, 730046,
761587, 761663, 761810, 761815, 765176, 765328, and 765403 thereof,
according to the nucleotide numbering of the reference genome of M.
bovis strain, Hubei-1.
[0008] Another embodiment provides an immunogenic composition
comprising the attenuated M. bovis strain of the previous
embodiment, wherein said M. bovis strain is inactivated.
[0009] Another embodiment provides the immunogenic composition of
any of the previous embodiments, and additionally an adjuvant.
[0010] Another embodiment provides the immunogenic composition of
any of the previous embodiments, further comprising at least one
additional antigen.
[0011] Another embodiment provides the immunogenic composition of
the previous embodiment, wherein the additional antigen is selected
from the group consisting of a virus, a bacterium, a fungus and a
parasite.
[0012] Another embodiment provides an immunogenic composition of
any of the previous embodiments, wherein said composition is
formulated for administration by a route selected from the group
consisting of intramuscular, subcutaneous, intranasal,
intratracheal, oral and aerosol.
[0013] Another embodiment provides a method for preventing disease
caused by M. bovis in a bovine animal, comprising administering to
the animal an immunizing amount of the immunogenic composition of
any of the previous embodiments.
[0014] Another embodiment provides a method for preventing diseases
caused by M. bovis and another infectious agent of a bovine animal,
comprising administering to the animal an immunizing amount of the
immunogenic composition of any of the previous embodiments, and an
additional antigen.
[0015] Another embodiment provides the use of an attenuated M.
bovis strain, or an inactivated attenuated M. bovis strain, in the
manufacture of a medicament for preventing disease caused by M.
bovis in a bovine animal.
[0016] Another embodiment provides a Mycoplasma bovis vaccine,
generated by the steps comprising: (A) obtaining a culture of M.
bovis deposited with the ATCC as PTA-122167; (B) further passaging
said culture of M. bovis at least once; (C) isolating a clone from
said passaged culture; and (D) formulating said clone with a
carrier or excipient, and optionally an adjuvant.
[0017] Another embodiment provides the vaccine of the previous
embodiment, wherein the clone obtained from the passaged culture is
more attenuated than PTA-122167.
[0018] Another embodiment provides a method of providing a
Mycoplasma bovis vaccine, wherein said vaccine is generated by the
steps comprising: (A) obtaining a culture of M. bovis deposited
with the ATCC as PTA-122167; (B) further passaging said culture of
M. bovis at least once; (C) isolating a clone from said passaged
culture; and (D) formulating said clone with a carrier or
excipient, and optionally an adjuvant.
[0019] Another embodiment provides a method of mutagenizing a
parental strain of M. bovis, wherein said method comprises
subjecting said strain to a chemical mutagen, followed by selection
of a progeny strain that remains viable after serial passaging in
vitro.
[0020] Another embodiment provides a method of mutagenizing a
parental strain of M. bovis, wherein the parental strain is
Mycoplasma bovis strain 3683.
[0021] Another embodiment provides a method of mutagenizing a
parental strain of M. bovis, wherein the progeny strain is
deposited with the American Type Culture Collection under accession
number PTA-122167.
[0022] Another embodiment provides a method of mutagenizing a
parental strain of M. bovis, wherein said method comprises
subjecting said strain to a chemical mutagen, followed by selection
of a progeny strain that remains viable after serial passaging in
vitro, wherein the progeny strain contains one or more of any of
the original nucleotides at positions 115273, 194478, 194484,
194525, 194724, 204966, 207629, 262327, 271487, 271661, 271666,
272086, 272133, 309463, 310075, 310078, 310086, 310090, 311988,
334184, 358013, 365101, 365188, 397010, 397901, 399129, 410247,
410253, 410334, 410514, 410733, 414666, 453388, 468457, 502577,
502678, 502963, 543859, 560986, 632309, 632366, 632612, 633994,
686074, 687874, 687915, 696348, 729874, 730046, 761587, 761663,
761810, 761815, 765176, 765328, and 765403, according to the
nucleotide numbering of the reference genome of M. bovis strain,
Hubei-1.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 provides a comparison of the growth rates at
33.degree. C. and 38.degree. C. of wild-type Mycoplasma bovis
strain 3683, M. bovis temperature-sensitive mutant #22/3/12, and M.
bovis mutant N2805-1.
DETAILED DESCRIPTION
[0024] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
[0025] Unless otherwise defined herein, scientific and technical
terms used in connection with the present embodiments shall have
the meanings that are commonly understood by those of ordinary
skill in the art. Further, unless otherwise required by context,
singular terms shall include pluralities, and plural terms shall
include the singular.
[0026] The term "adjuvant", as used herein, means a pharmacological
or immunological agent that modifies the effect of other agents,
such as a drug or immunogenic composition. Adjuvants are often
included in immunogenic compositions to enhance the recipient's
immune response to a supplied antigen. See below for a further
description of adjuvants.
[0027] The terms "antibody" or "antibodies", as used herein, mean
an immunoglobulin molecule able to bind to an antigen by means of
recognition of an epitope. Immunoglobulins are serum proteins
composed of "light" and "heavy" polypeptide chains, which have
"constant" and "variable" regions, and are divided into classes
(e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the
constant regions. An antibody that is "specific" for a given
antigen indicates that the variable regions of the antibody
recognize and bind a particular antigen exclusively. Antibodies can
be a polyclonal mixture, or monoclonal. They can be intact
immunoglobulins derived from natural or recombinant sources, or can
be immunoreactive portions of intact immunoglobulins. Antibodies
can exist in a variety of forms, including Fv, Fab', F(ab')2, Fc,
as well as single chain. An antibody can be converted to an
antigen-binding protein, which includes, but is not limited to,
antibody fragments. As used herein, the term "antigen binding
protein", "antibody" and the like, which may be used
interchangeably, refer to a polypeptide or polypeptides, or
fragment(s) thereof, comprising an antigen binding site. The term
"antigen binding protein" or "antibody" preferably refers to
monoclonal antibodies and fragments thereof, and
immunologic-binding equivalents thereof that can bind to a
particular protein and fragments thereof. As used herein, the term
encompasses not only intact polyclonal or monoclonal antibodies,
but also fragments thereof. For the purposes of the present
invention, "antibody" and "antigen binding protein" also includes
antibody fragments, unless otherwise stated. Exemplary antibody
fragments include Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, diabodies,
their antigen-recognizing fragments, small modular
immunopharmaceuticals (SMIPs) nanobodies and the like, all
recognized by one of skill in the art to be an antigen binding
protein or antibody fragment, and any of above-mentioned fragments
and their chemically or genetically manipulated counterparts, as
well as other antibody fragments and mutants thereof, fusion
proteins comprising an antibody portion, and any other modified
configuration of the immunoglobulin molecule that comprises an
antigen recognition site. Antibodies and antigen binding proteins
can be made, for example, via traditional hybridoma techniques
(Kohler et al., Nature 256:495-499 (1975)), recombinant DNA methods
(U.S. Pat. No. 4,816,567), or phage display techniques using
antibody libraries (Clackson et al., Nature 352:624-628 (1991);
Marks et al., J. Mol. Biol. 222:581-597 (1991)). For various other
antibody production techniques, see Antibodies: A Laboratory
Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988 as
well as other techniques that are well known to those skilled in
the art.
[0028] "Antigen", as used herein, means a molecule that contains
one or more epitopes (linear, conformational or both), that upon
exposure to a subject, will induce an immune response that is
specific for that antigen. An epitope is the specific site of the
antigen which binds to a T-cell receptor or specific B-cell
antibody, and typically comprises about 3 to about 20 amino acid
residues. The term "antigen" can also refer to subunit
antigens-antigens separate and discrete from a whole organism with
which the antigen is associated in nature--as well as killed,
attenuated or inactivated bacteria, viruses, fungi, parasites or
other microbes. The term "antigen" also refers to antibodies, such
as anti-idiotype antibodies or fragments thereof, and to synthetic
peptide mimotopes that can mimic an antigen or antigenic
determinant (epitope). The term "antigen" also refers to an
oligonucleotide or polynucleotide that expresses an antigen or
antigenic determinant in vivo, such as in DNA immunization
applications. An "antigen", as used herein, is a molecule or a
portion of a molecule capable of being specifically bound by an
antibody or antigen binding protein. In particular, an antibody, or
antigen binding protein, will bind to epitopes of the antigen. An
epitope, as used herein, refers to the antigenic determinant
recognized by the hypervariable region, or Complementarity
Determining Region (CDR), of the variable region of an antibody or
antigen binding protein.
[0029] The term "animal", as used herein, means any animal that is
susceptible to infection by Mycoplasma bovis, both domesticated and
wild. Preferably, "animal", as used herein, refers to a bovine.
[0030] The term "attenuated", as used herein, refers to a strain of
a microorganism whose pathogenicity has been reduced so that it
will generally initiate an immune response but without producing
disease. An attenuated strain is less virulent than the parental
strain from which it was derived. Attenuated microorganisms can be
screened in vitro or in vivo to confirm that they are less
pathogenic than its parental strain. Conventional means are used to
introduce attenuating mutations, such as in vitro passaging, as
well as chemical mutagenesis. An alternative means of attenuating
comprises making pre-determined mutations using site-directed
mutagenesis, where one or more mutations may be introduced. The
term "more attenuated", as used herein, refers to a strain which
has been further modified beyond the attenuated strain from which
it was derived. This further attenuation can be achieved through
additional in vitro passaging, or additional rounds of chemical or
site-directed mutagenesis. To be useful as a live vaccine, any
attenuated organism must nonetheless cause the host immune system
to initiate an effective immune response, which may require some
growth of the organism.
[0031] The terms "bacteria", "bacterial species", "bacterium", and
the like, as used herein, mean a large domain of prokaryotic
microorganisms.
[0032] The term "bovine", as used herein, means a diverse group of
medium- to large-sized ungulates, generally having cloven hoofs,
and at least one of the sexes having true horns. Bovines include,
but are not limited to, domestic cattle, bison, African buffalo,
water buffalo, yak, and four-horned or spiral-horned antelope.
[0033] The term "chemical mutagenesis", as used herein, means the
use of a compound that increases the frequency of some types of
mutation(s) occurring above the natural background level. The
compounds used can vary in their potency, since they can differ in
their ability to enter a cell, in the extent of their reactivity
with nucleic acids, in their general toxicity, and in the
likelihood that the type of chemical change they introduce into the
nucleic acid will be corrected by a endogenous repair system.
[0034] The term "immunogenic composition" or "immunizing amount",
as used herein, means a composition that generates an effective
immune response (i.e., has effective and/or at least partially
protecting immunogenic activity) when administered alone, or with a
pharmaceutically-acceptable carrier, to an animal. The immune
response can be a cellular immune response mediated primarily by
cytotoxic T-cells, or a humoral immune response mediated primarily
by helper T-cells, which in turn activate B-cells, leading to
antibody production. In addition, specific T-lymphocytes or
antibodies can be generated to allow for the future protection of
an immunized host.
[0035] The term "isolated", as used herein, means that the
referenced material is removed from some of the components of the
environment in which it is normally found. Thus, an isolated
biological material can be free of some cellular components, i.e.,
components of the cells in which the material is found or produced.
In the case of nucleic acid molecules, an isolated nucleic acid
includes, for example, a PCR product, an isolated mRNA, a cDNA, or
a restriction fragment. In another embodiment, an isolated nucleic
acid is preferably excised from the chromosome in which it may be
found, and more preferably is no longer joined to non-regulatory,
non-coding regions, or to other genes located upstream or
downstream of the nucleic acid molecule when found in the
chromosome. In yet another embodiment, the isolated nucleic acid
lacks one or more introns. Isolated nucleic acid molecules include
sequences inserted into plasmids, cosmids, artificial chromosomes,
and the like. Thus, in a specific embodiment, a recombinant nucleic
acid is an isolated nucleic acid. An isolated protein may be
associated with other proteins or nucleic acids, or both, with
which it associates in the cell, or with cellular membranes if it
is a membrane-associated protein. An isolated organelle, cell, or
tissue is removed from the anatomical site in which it is found in
an organism. An isolated material may be, but need not be,
purified. An "isolated" or "purified" polypeptide or
polynucleotide, e.g., an "isolated polypeptide," or an "isolated
polynucleotide", is purified to a state beyond that in which it
exists in nature. For example, the "isolated" or "purified"
polypeptide or polynucleotide, can be substantially free of
cellular material or other contaminating proteins from the cell or
tissue source from which the protein or polynucleotide is derived,
or substantially free from chemical precursors or other chemicals
when chemically synthesized. The preparation of antigen binding
protein having less than about 50% of non-antigen binding protein
(also referred to herein as a "contaminating protein"), or of
chemical precursors, is considered to be "substantially free." 40%,
30%, 20%, 10% and more preferably 5% (by dry weight), of
non-antigen binding protein, or of chemical precursors, is
considered to be substantially free.
[0036] The term "medicament", as used herein, means an agent that
promotes recovery from an infection, injury or ailment; a
medicine.
[0037] The term "mutant", as used herein, means an individual or
organism arising or resulting from an instance of mutation, which
is a base-pair sequence change within the nucleic acid or
chromosome of an organism, and results in the creation of a new
character or trait not found in the wild-type individual or
organism.
[0038] The term "parent" or "parental strain", as used herein,
means the entity from which offspring, or progeny, are derived. The
term "progeny", as used herein, means that produced by, or derived
from, one or more parents or parental strains.
[0039] The terms "prevent", "preventing" or "prevention", and the
like, as used herein, mean to inhibit the replication of a
microorganism, to inhibit transmission of a microorganism, or to
inhibit a microorganism from establishing itself in its host. These
terms, and the like, can also mean to inhibit or block one or more
signs or symptoms of infection.
[0040] The terms "reverse engineer" or "reverse mutagenize", as
used herein, mean to reintroduce by genetic means (e.g. polymerase
chain reaction, or PCR) the original nucleotide sequence occurring
at a particular position(s) within a microorganism's genome,
wherein that sequence had previously been changed.
[0041] The terms "serial passage" or "serial passaging", as used
herein, mean a method for purifying an organism, preferably a
microorganism, to obtain a clonally pure population. The terms can
also refer to a technique for attenuating, or weakening, the
virulence of an organism, preferably a microorganism.
[0042] The term "therapeutically effective amount" (or "effective
amount"), as used herein, means an amount of an active ingredient,
e.g., an agent according to the invention, with or without an
adjuvant, as appropriate under the circumstances, provided in a
single or multiple doses as appropriate, sufficient to effect
beneficial or desired results when administered to a subject or
patient. An effective amount can be administered in one or more
administrations, applications or dosages. A therapeutically
effective amount of a composition according to the invention may be
readily determined by one of ordinary skill in the art, and
provides a measureable benefit to a patient, such as protecting the
animal from subsequent challenge with a similar pathogen.
[0043] As used herein, the terms "therapeutic" or "treatment"
encompass the full spectrum of treatments for a disease or
disorder. By way of example, a "therapeutic" agent of the invention
may act in a manner, or a treatment may result in an effect, that
is prophylactic or preventive, including those that incorporate
procedures designed to target animals that can be identified as
being at risk (pharmacogenetics); or in a manner that is
ameliorative or curative in nature; or may act to slow the rate or
extent of the progression of at least one symptom of a disease or
disorder being treated.
[0044] The term "veterinarily-acceptable carrier", as used herein,
refers to substances which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of animals,
without undue toxicity, irritation, allergic response, and the
like, commensurate with a reasonable benefit-to-risk ratio, and
effective for their intended use.
[0045] The following description is provided to aid those skilled
in the art in practicing the present invention. Even so, this
description should not be construed to unduly limit the present
invention, as modifications and variations in the embodiments
discussed herein can be made by those of ordinary skill in the art,
without departing from the spirit or scope of the present inventive
discovery.
Bacteria; Immunogenic Compositions
[0046] In certain embodiments of the present invention, a
Mycoplasma bovis strain from a bovine has been attenuated, as well
as characterized. An isolate of said M. bovis strain was deposited
with the American Type Culture Collection ("ATCC"), Manassas, Va.,
USA, on May 12, 2015, and has been assigned accession number,
PTA-122167.
[0047] Immunogenic compositions of the present invention can be
attenuated or inactivated prior to use in an immunogenic
composition. Methods of attenuation and inactivation are well known
to those skilled in the art. Methods for attenuation include, but
are not limited to, serial passage in cell culture, ultraviolet
irradiation, and chemical mutagenesis. A large number of chemical
mutagens may interact directly with DNA. However, many chemicals,
such as aromatic amines and benzene are not necessarily mutagenic
by themselves, but through metabolic processes in cells, they
produce mutagenic compounds. Chemical mutagens can also include
reactive oxygen species (ROS). These may include superoxide,
hydroxyl radicals and hydrogen peroxide. A large number of these
highly reactive species are generated by normal cellular processes,
for example as by-products of mitochondrial electron transport, or
lipid peroxidation. A number of other chemical mutagens may also
generate these ROS. These ROS may result in the production of
various base adducts, as well as DNA strand breaks and crosslinks.
Chemical mutagens can also include deaminating agents, for example
nitrous acid, which can cause transition mutations by converting
cytosine to uracil. Polycyclic aromatic hydrocarbon (PAH), is also
a chemical mutagen. When activated, it can bind to DNA and form
adducts. Chemical mutagens can also include alkylating agents, such
as ethylnitrosourea. These compounds transfer methyl or ethyl group
to bases or the backbone phosphate groups. Guanine, when alkylated,
may be mispaired with thymine. Some of these alkylating agents may
cause DNA crosslinking and breakages. Nitrosamines are an important
group of mutagens, such as can be found in tobacco.
Methylnitronitrosoguanidine (MNNG, MNG, or NTG) is an alkylating
agent which acts by adding alkyl groups to the O.sup.6 of guanine
and O.sup.4 of thymine, which can lead to transition mutations
between GC and AT. These changes do not cause a heavy distortion in
the double helix of DNA, and thus are hard to detect by the DNA
mismatch repair system. Other alkylating agents can also include
mustard gas and vinyl chloride.
[0048] Methods for inactivation include, but are not limited to,
treatment with formalin, betapropriolactone (BPL) or binary
ethyleneimine (BEI), or other methods known to those skilled in the
art. Inactivation by formalin can be performed by mixing the
bacterial suspension with 37% formaldehyde, to a final formaldehyde
concentration of 0.05%. The bacteria-formaldehyde mixture is
stirred constantly for approximately 24 hours at room temperature.
The inactivated bacterial mixture is then tested for residual live
bacteria by assaying for growth in culture media. Inactivation by
BEI can be performed by mixing the bacterial suspension with 0.1 M
BEI (2-bromo-ethylamine in 0.175 N NaOH), to a final BEI
concentration of 1 mM. The bacteria-BEI mixture is stirred
constantly for approximately 48 hours at room temperature, followed
by the addition of 1.0 M sodium thiosulfate to a final
concentration of 0.1 mM. Mixing is continued for an additional two
hours. The inactivated bacterial mixture is tested for residual
live bacteria by assaying for growth in culture media.
[0049] Effective (i.e. protecting) Immunogenic compositions of the
present invention can include one or more veterinarily-acceptable
carriers, such as any and all solvents, dispersion media, coatings,
adjuvants, stabilizing agents, diluents, preservatives,
antibacterial and antifungal agents, isotonic agents, adsorption
delaying agents, and the like. Diluents can include water, saline,
dextrose, ethanol, glycerol, and the like. Isotonic agents can
include sodium chloride, dextrose, mannitol, sorbitol, and lactose,
among others known to those skilled in the art. Stabilizers include
albumin, among others known to the skilled artisan. Preservatives
include merthiolate, among others known to the skilled artisan.
[0050] Effective immunogenic compositions of the present invention
can include one or more adjuvants. Adjuvants include, but are not
limited to, the R1131 adjuvant system (Ribi Inc.; Hamilton, Mont.),
alum, aluminum hydroxide gel, oil-in water emulsions, water-in-oil
emulsions such as, e.g., Freund's complete and incomplete
adjuvants, Block co-n polymer (CytRx; Atlanta, Ga.), SAF-M (Chiron;
Emeryville, Calif.), AMPHIGEN.RTM. adjuvant, saponin, Quil A, QS-21
(Cambridge Biotech Inc.; Cambridge, Mass.), GPI-0100 (Galenica
Pharmaceuticals, Inc.; Birmingham, Ala.) or other saponin
fractions, monophosphoryl lipid A, Avridine lipid-amine adjuvant,
heat-labile enterotoxin from Escherichia coli (recombinant or
otherwise), cholera toxin, or muramyl dipeptide, among many others
known to those skilled in the art.
[0051] The amounts and concentrations of adjuvants and additives
useful in the context of the present invention can readily be
determined by the skilled artisan, and appropriate amounts of each
such substance are generally known or can be readily determined. In
one embodiment, the present invention contemplates immunogenic
compositions comprising from about 50 .mu.g to about 2000 .mu.g of
adjuvant. In another embodiment, adjuvant is included in an amount
from about 100 .mu.g to about 1500 .mu.g, or from about 250 .mu.g
to about 1000 .mu.g, or from about 350 .mu.g to about 750 .mu.g. In
another embodiment, adjuvant is included in an amount of about 500
.mu.g/2 ml dose of the immunogenic composition. Generally, vaccines
of the present invention are provided as 1-2 ML doses.
[0052] A number of cytokines or lymphokines have been shown to have
immune modulating activity, and thus may be used as adjuvants,
including, but not limited to, the interleukins 1-.alpha.,
1-.beta., 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Pat. No.
5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant forms), the
interferons-.alpha., .beta. and .gamma., granulocyte-macrophage
colony stimulating factor (see, e.g., U.S. Pat. No. 5,078,996 and
ATCC Accession Number 39900), macrophage colony stimulating factor,
granulocyte colony stimulating factor, GSF, and the tumor necrosis
factors .alpha. and .beta.. Still other adjuvants useful in this
invention include a chemokine, including without limitation, MCP-1,
MIP-1.alpha., MIP-1.beta., and RANTES. Adhesion molecules, such as
a selectin, e.g., L-selectin, P-selectin and E-selectin may also be
useful as adjuvants. Still other useful adjuvants include, without
limitation, a mucin-like molecule, e.g., CD34, GlyCAM-1 and
MadCAM-1, a member of the integrin family such as LFA-1, VLA-1,
Mac-1 and p150.95, a member of the immunoglobulin superfamily such
as PECAM, ICAMs, e.g., ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3,
co-stimulatory molecules such as CD40 and CD40L, growth factors
including vascular growth factor, nerve growth factor, fibroblast
growth factor, epidermal growth factor, B7.2, PDGF, BL-1, and
vascular endothelial growth factor, receptor molecules including
Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR,
LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6. Still
another adjuvant molecule includes Caspase (ICE). See, also
International Patent Publication Nos. WO98/17799 and WO99/43839,
incorporated herein by reference.
[0053] Suitable adjuvants used to enhance an immune response also
include, without limitation, MPL.TM. (3-O-deacylated monophosphoryl
lipid A; Corixa, Hamilton, Mont.), which is described in U.S. Pat.
No. 4,912,094, which is hereby incorporated by reference. Also
suitable for use as adjuvants are synthetic lipid A analogs or
aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or
analogs thereof, which are available from Corixa (Hamilton, Mont.),
and which are described in U.S. Pat. No. 6,113,918, which is hereby
incorporated by reference. One such AGP is
2-[(R)-3-Tetradecanoyloxytetradecanoylamino] ethyl
2-Deoxy-4-O-phosphono-3-O--[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3--
tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside, which is
also known as 529 (formerly known as RC529). This 529 adjuvant is
formulated as an aqueous form or as a stable emulsion.
[0054] Still other adjuvants include mineral oil and water
emulsions, aluminum salts (alum), such as aluminum hydroxide,
aluminum phosphate, etc., Amphigen, Avridine, L121/squalene,
D-lactide-polylactide/glycoside, pluronic polyols, muramyl
dipeptide, killed Bordetella, saponins, such as Stimulon.TM. QS-21
(Antigenics, Framingham, Mass.), described in U.S. Pat. No.
5,057,540, which is hereby incorporated by reference, and particles
generated therefrom such as ISCOMS (immunostimulating complexes),
Mycobacterium tuberculosis, bacterial lipopolysaccharides,
synthetic polynucleotides such as oligonucleotides containing a CpG
motif (U.S. Pat. No. 6,207,646, which is hereby incorporated by
reference), a pertussis toxin (PT), or an E. coli heat-labile toxin
(LT), particularly LT-K63, LT-R72, PT-K9/G129; see, e.g.,
International Patent Publication Nos. WO 93/13302 and WO 92/19265,
incorporated herein by reference.
[0055] Also useful as adjuvants are cholera toxins (CT) and mutants
thereof, including those described in published International
Patent Application number WO 00/18434 (wherein the glutamic acid at
amino acid position 29 is replaced by another amino acid, other
than aspartic acid, preferably a histidine). Similar CT toxins or
mutants are described in published International Patent Application
number WO 02/098368 (wherein the isoleucine at amino acid position
16 is replaced by another amino acid, either alone or in
combination with the replacement of the serine at amino acid
position 68 by another amino acid; and/or wherein the valine at
amino acid position 72 is replaced by another amino acid). Other CT
toxins are described in published International Patent Application
number WO 02/098369 (wherein the arginine at amino acid position 25
is replaced by another amino acid; and/or an amino acid is inserted
at amino acid position 49; and/or two amino acids are inserted at
amino acid positions 35 and 36). Said CT toxins or mutant can be
included in the immunogenic compositions either as separate
entities, or as fusion partners for the neurotoxic peptides of the
present invention.
[0056] The immunogenic compositions of the invention can also
include surface-active substances. Suitable surface-active
substances include, without limitation, quinone analogs,
hexadecylamine, octadecylamine, octadecyl amino acid esters,
lysolecithin, dimethyl-dioctadecylammonium bromide,
methoxyhexadecylgylcerol, and pluronic polyols; polyamines, e.g.,
pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl
peptide and dipeptide, dimethylglycine, tuftsin; oil emulsions; and
mineral gels, e.g., aluminum phosphate, etc., and
immune-stimulating complexes (ISCOMS).
[0057] All the aforementioned adjuvants and additional substances
can be used in amounts readily subject to determination by those
skilled in the art.
[0058] The immunogenic compositions of the invention can also
include antibiotics. Such antibiotics include, but are not limited
to, those from the classes of aminoglycosides, carbapenems,
cephalosporins, glycopeptides, macrolides, penicillins,
polypeptides, quinolones, sulfonamides, and tetracyclines. In one
embodiment, the present invention contemplates immunogenic
compositions comprising from about 1 .mu.g/ml to about 60 .mu.g/ml
of antibiotic. In another embodiment, the immunogenic compositions
comprise from about 5 .mu.g/ml to about 55 .mu.g/ml of antibiotic,
or from about 10 .mu.g/ml to about 50 .mu.g/ml of antibiotic, or
from about 15 .mu.g/ml to about 45 .mu.g/ml of antibiotic, or from
about 20 .mu.g/ml to about 40 .mu.g/ml of antibiotic, or from about
25 .mu.g/ml to about 35 .mu.g/ml of antibiotic. In yet another
embodiment, the immunogenic compositions comprise less than about
30 .mu.g/ml of antibiotic.
[0059] Other additives can also be included in the immunogenic
compositions of this invention, including preservatives,
stabilizing ingredients, and the like. Suitable exemplary
preservatives include chlorobutanol, potassium sorbate, sorbic
acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,
glycerin, phenol, and parachlorophenol. Suitable stabilizing
ingredients that may be used include, for example, casamino acids,
sucrose, gelatin, phenol red, N-Z amine, monopotassium diphosphate,
lactose, lactalbumin hydrolysate, and dried milk. The immunogenic
compositions may also be incorporated into liposomes for use as an
immunogenic composition, and may also contain other additives
suitable for the selected mode of administration of the
composition. The composition may include other
pharmaceutically-acceptable excipients for developing powder,
liquid or suspension dosage forms. See, e.g., Remington: The
Science and Practice of Pharmacy, Vol. 2, 19.sup.th edition (1995),
e.g., Chapter 95 Aerosols; and International Patent Publication No.
WO99/45966, the teachings of which are hereby incorporated by
reference.
[0060] Immunogenic compositions of the invention can include other
antigens. Antigens can be in the form of an inactivated whole or
partial preparation of the microorganism, or in the form of
antigenic molecules obtained by recombinant techniques or chemical
synthesis. Other antigens appropriate for use in accordance with
the present invention include, but are not limited to, those
derived from pathogenic bacteria, such as Haemophilus somnus,
Haemophilus parasuis, Bordetella bronchiseptica, Bacillus
anthracis, Actinobacillus pleuropneumonie, Pasteurella multocida,
Mannhemia haemolytica, Mycoplasma bovis, Mycobacterium bovis,
Mycobacterium paratuberculosis, Clostridial spp., Streptococcus
uberis, Staphylococcus aureus, Erysipelothrix rhusopathiae,
Chlamydia spp., Brucella spp., Vibrio spp., Salmonella enterica
serovars and Leptospira spp. Antigens can also be derived from
pathogenic fungi, such as Candida, protozoa such as Cryptosporidium
parvum, Neospora canium, Toxoplasma gondii, Eimeria spp., Babesia
spp., Giardia spp., or helminths such as Ostertagia, Cooperia,
Haemonchus, and Fasciola. Additional antigens can include
pathogenic viruses, such as bovine coronavirus, bovine rotavirus,
bovine herpesviruses, bovine parainfluenza virus, bovine viral
diarrhea virus, bovine adenovirus, bovine rhinovirus, bovine
reovirus, bovine respiratory syncytial virus, bovine leukosis
virus, rinderpest virus, foot and mouth disease virus, and rabies
virus.
Forms, Dosages, Routes and Timing of Administration
[0061] Immunogenic compositions of the present invention can be
administered to animals to induce an effective immune response
against M. bovis. Accordingly, the present invention provides
methods of stimulating an effective immune response against M.
bovis by administering to an animal a therapeutically effective
amount of an immunogenic composition of the present invention
described herein.
[0062] Immunogenic compositions of the present invention can be
made in various forms, depending upon the route of administration.
For example, the immunogenic compositions can be made in the form
of sterile aqueous solutions or dispersions suitable for injectable
use, or made in lyophilized forms using freeze-drying techniques.
Lyophilized immunogenic compositions are typically maintained at
about 4.degree. C., and can be reconstituted in a stabilizing
solution, e.g., saline or HEPES, with or without adjuvant.
Immunogenic compositions can also be made in the form of
suspensions or emulsions.
[0063] These immunogenic compositions can contain additives
suitable for administration via any conventional route of
administration. The immunogenic compositions of the invention can
be prepared for administration to subjects in the form of, for
example, liquids, powders, aerosols, tablets, capsules,
enteric-coated tablets or capsules, or suppositories. Thus, the
immunogenic compositions may also include, but are not limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles,
pastes, and implantable sustained-release or biodegradable
formulations. In one embodiment of a formulation for parenteral
administration, the active ingredient is provided in dry (i.e.,
powder or granular) form for reconstitution with a suitable vehicle
(e.g., sterile pyrogen-free water) prior to parenteral
administration of the reconstituted composition. Other useful
parenterally-administrable formulations include those which
comprise the active ingredient in microcrystalline form, in a
liposomal preparation, or as a component of a biodegradable polymer
system. Compositions for sustained release or implantation may
comprise pharmaceutically acceptable polymeric or hydrophobic
materials, such as an emulsion, an ion exchange resin, a sparingly
soluble polymer, or a sparingly soluble salt.
[0064] Immunogenic compositions of the present invention include a
therapeutically effective amount of M. bovis. Purified bacteria can
be used directly in an immunogenic composition, or can be further
attenuated, or inactivated. Typically, an effective immunogenic
composition contains between about 1.times.10.sup.2 and about
1.times.10.sup.12 bacterial particles, or between about
1.times.10.sup.3 and about 1.times.10.sup.11 bacterial particles,
or between about 1.times.10.sup.4 and about 1.times.10.sup.19
bacterial particles, or between about 1.times.10.sup.5 and about
1.times.10.sup.9 bacterial particles, or preferably between about
1.times.10.sup.6 and about 1.times.10.sup.8 bacterial particles.
The precise amount of a bacterium in an immunogenic composition
effective to provide a protective effect can be determined by a
skilled artisan.
[0065] Effective Immunogenic compositions generally comprise a
veterinarily-acceptable carrier in a volume of between about 0.5 ml
and about 5 ml. In another embodiment the volume of the carrier is
between about 1 ml and about 4 ml, or between about 2 ml and about
3 ml. In another embodiment, the volume of the carrier is about 1
ml, or is about 2 ml, or is about 5 ml. Veterinarily-acceptable
carriers suitable for use in immunogenic compositions can be any of
those described herein.
[0066] Such carriers include, without limitation, water, saline,
buffered saline, phosphate buffer, alcoholic/aqueous solutions,
emulsions or suspensions. Other conventionally employed diluents,
adjuvants and excipients, may be added in accordance with
conventional techniques. Such carriers can include ethanol,
polyols, and suitable mixtures thereof, vegetable oils, and
injectable organic esters. Buffers and pH adjusting agents may also
be employed. Buffers include, without limitation, salts prepared
from an organic acid or base. Representative buffers include,
without limitation, organic acid salts, such as salts of citric
acid, e.g., citrates, ascorbic acid, gluconic acid, histidine-Hel,
carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic
acid, Tris, trimethanmine hydrochloride, or phosphate buffers.
Parenteral carriers can include sodium chloride solution, Ringer's
dextrose, dextrose, trehalose, sucrose, lactated Ringer's, or fixed
oils. Intravenous carriers can include fluid and nutrient
replenishers, electrolyte replenishers, such as those based on
Ringer's dextrose and the like. Preservatives and other additives
such as, for example, antimicrobials, antioxidants, chelating
agents (e.g., EDTA), inert gases and the like may also be provided
in the pharmaceutical carriers. The present invention is not
limited by the selection of the carrier. The preparation of these
pharmaceutically acceptable compositions, from the above-described
components, having appropriate pH, isotonicity, stability and other
conventional characteristics, is within the skill of the art. See,
e.g., texts such as Remington: The Science and Practice of
Pharmacy, 20th ed., Lippincott Williams & Wilkins, pub., 2000;
and The Handbook of Pharmaceutical Excipients, 4.sup.th edit., eds.
R. C. Rowe et al, APhA Publications, 2003.
[0067] Those skilled in the art can readily determine whether a
bacterium needs to be attenuated or inactivated before
administration. In another embodiment of the present invention, M.
bovis can be administered directly to an animal without additional
attenuation. The amount of a bacterium that is therapeutically
effective can vary depending on the particular bacterium used, the
condition of the animal and/or the degree of infection, and can be
determined by a skilled artisan.
[0068] In accordance with the methods of the present invention, a
single dose can be administered to animals, or, alternatively, two
or more inoculations can take place with intervals of from about
two to about ten weeks. Boosting regimens can be required, and the
dosage regimen can be adjusted to provide optimal immunization.
Those skilled in the art can readily determine the optimal
administration regimen. When more than one dose is used, it is not
necessarily the case that the first and second doses are
administered to the same location in the animal.
[0069] Effective Immunogenic compositions can be administered
directly into the bloodstream, into muscle, or into an internal
organ. Suitable means for parenteral administration include
intravenous, intra-arterial, intraperitoneal, intrathecal,
intraventricular, intraurethral, intrasternal, intracranial,
intramuscular and subcutaneous. Suitable devices for parenteral
administration include needle (including microneedle) injectors,
needle-free injectors and infusion techniques.
[0070] Parenteral formulations are typically aqueous solutions
which can contain excipients such as salts, carbohydrates and
buffering agents (preferably to a pH of from about 3 to about 9, or
from about 4 to about 8, or from about 5 to about 7.5, or from
about 6 to about 7.5, or about 7 to about 7.5), but for some
applications, they can be more suitably formulated as a sterile
non-aqueous solution, or as a dried form to be used in conjunction
with a suitable vehicle such as sterile, pyrogen-free water. The
preparation of parenteral formulations under sterile conditions,
for example, by lyophilisation, can readily be accomplished using
standard pharmaceutical techniques well known to those skilled in
the art.
[0071] The solubility of compounds used in the preparation of
parenteral solutions can be increased by the use of appropriate
formulation techniques known to the skilled artisan, such as the
incorporation of solubility-enhancing agents including buffers,
salts, surfactants, liposomes, cyclodextrins, and the like.
[0072] Formulations for parenteral administration can be formulated
to be immediate and/or modified release. Modified release
formulations include delayed, sustained, pulsed, controlled,
targeted and programmed release. Thus compounds of the invention
can be formulated as a solid, semi-solid, or thixotropic liquid for
administration as an implanted depot, providing modified release of
the active compound. Examples of such formulations include
drug-coated stents and poly(dl-lactic-coglycolic)acid (PLGA)
microspheres.
[0073] Effective immunogenic compositions of the present invention
can also be administered topically to the skin or mucosa, that is,
dermally or transdermally. Typical formulations for this purpose
include gels, hydrogels, lotions, solutions, creams, ointments,
dusting powders, dressings, foams, films, skin patches, wafers,
implants, sponges, fibres, bandages and microemulsions; liposomes
can also be used. Typical carriers include alcohol, water, mineral
oil, liquid petrolatum, white petrolatum, glycerin, polyethylene
glycol and propylene glycol. Penetration enhancers can be
incorporated; see, for example, Finnin and Morgan, J. Pharm Sci, 88
(10):955-958 (1999). Other means of topical administration include
delivery by electroporation, iontophoresis, phonophoresis,
sonophoresis and microneedle or needle-free (e.g. Powderject.TM.
Bioject.TM., etc.) injection. Formulations for topical
administration can be designed to be immediate and/or modified
release. Modified release formulations include delayed, sustained,
pulsed, controlled, targeted and programmed release.
[0074] Effective immunogenic compositions can also be administered
intranasally or by inhalation, typically in the form of a dry
powder (either alone or as a mixture, for example, in a dry blend
with lactose, or as a mixed component particle, for example, mixed
with phospholipids, such as phosphatidylcholine), from a dry powder
inhaler, or as an aerosol spray from a pressurized container, pump,
spray, atomizer (preferably an atomizer using electrohydrodynamics)
to produce a fine mist. It can also be administered via a
nebulizer, with or without the use of a suitable propellant, such
as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane.
For intranasal use, the powder can comprise a bioadhesive agent,
for example, chitosan or cyclodextrin. The pressurized container,
pump, spray, atomizer, or nebulizer contains a solution or
suspension of the compound(s) of the invention comprising, for
example, ethanol, aqueous ethanol, or a suitable alternative agent
for dispersing, solubilizing, or extending release of the active, a
propellant(s) as solvent and an optional surfactant, such as
sorbitan trioleate, oleic acid, or an oligolactic acid.
[0075] Prior to use in a dry powder or suspension formulation, the
drug product is generally micronized to a size suitable for
delivery by inhalation (typically less than about 5 microns). This
can be achieved by any appropriate comminuting method, such as
spiral jet milling, fluid bed jet milling, supercritical fluid
processing (to form nanoparticles), high pressure homogenization,
or spray drying.
[0076] Capsules (made, for example, from gelatin or
hydroxypropylmethylcellulose), blisters, and cartridges for use in
an inhaler or insufflators, can be formulated to contain a powder
mix of the compound of the invention. A suitable powder base could
be lactose or starch, and a performance modifier could be
l-leucine, mannitol, or magnesium stearate. The lactose can be
anhydrous, or in the form of the monohydrate. Other suitable
excipients include dextran, glucose, maltose, sorbitol, xylitol,
fructose, sucrose and trehalose.
[0077] A suitable solution formulation for use in an atomizer,
using electrohydrodynamics to produce a fine mist, can contain from
about 1 .mu.g to about 20 mg of the compound of the invention per
actuation, and the actuation volume can vary from about 1 .mu.l to
about 100 .mu.l. In another embodiment, the amount of compound per
actuation can range from about 100 .mu.g to about 15 mg, or from
about 500 .mu.g to about 10 mg, or from about 1 mg to about 10 mg,
or from about 2.5 .mu.g to about 5 mg. In another embodiment, the
actuation volume can range from about 5 .mu.l to about 75 .mu.l, or
from about 10 .mu.l to about 50 .mu.l, or from about 15 .mu.l to
about 25 .mu.l. A typical formulation can comprise the compound of
the invention, propylene glycol, sterile water, ethanol and sodium
chloride. Alternative solvents which can be used instead of
propylene glycol include glycerol and polyethylene glycol.
[0078] Formulations for inhaled/intranasal administration can be
formulated to be immediate and/or modified release using, for
example, PLGA. Modified release formulations include delayed,
sustained, pulsed, controlled, targeted and programmed release.
[0079] In the case of dry powder inhalers and aerosols, the dosage
unit is generally determined by means of a valve which delivers a
metered amount. Units in accordance with the invention are
typically arranged to administer a metered dose or "puff"
containing from about 10 ng to about 100 .mu.g of the compound of
the invention. In another embodiment, the amount of compound
administered in a metered dose is from about 50 ng to about 75
.mu.g, or from about 100 ng to about 50 .mu.g, or from about 500 ng
to about 25 .mu.g, or from about 750 ng to about 10 .mu.g, or from
about 1 .mu.g to about 5 .mu.g. The overall daily dose will
typically be in the range from about 1 .mu.g to about 100 mg, which
can be administered in a single dose or, more usually, as divided
doses throughout the day. In another embodiment, the overall daily
dose can range from about 50 .mu.g to about 75 mg, or from about
100 .mu.g to about 50 mg, or from about 500 .mu.g to about 25 mg,
or from about 750 .mu.g to about 10 mg, or from about 1 mg to about
5 mg.
[0080] Effective immunogenic compositions of the present invention
can also be administered orally or perorally, that is, into a
subject's body through or by way of the mouth, and involves
swallowing or transport through the oral mucosa (e.g., sublingual
or buccal absorption, or both). Suitable flavors, such as menthol
and levomenthol, or sweeteners, such as saccharin or saccharin
sodium, can be added to those formulations of the invention
intended for oral or peroral administration.
[0081] Effective immunogenic compositions of the present invention
can be administered rectally or vaginally, for example, in the form
of a suppository, pessary, or enema. Cocoa butter is a traditional
suppository base, but various alternatives can be used as
appropriate. Formulations for rectal/vaginal administration can be
formulated to be immediate and/or modified release. Modified
release formulations include delayed, sustained, pulsed,
controlled, targeted and programmed release.
[0082] Effective immunogenic compositions of the present invention
can also be administered directly to the eye or ear, typically in
the form of drops of a micronized suspension, or solution in
isotonic, pH-adjusted, sterile saline. Other formulations suitable
for ocular and aural administration include ointments,
biodegradable (e.g. absorbable gel sponges, collagen) and
non-biodegradable (e.g. silicone) implants, wafers, lenses and
particulate or vesicular systems, such as niosomes or liposomes. A
polymer such as crossed-linked polyacrylic acid, polyvinylalcohol,
hyaluronic acid, a cellulosic polymer, for example,
hydroxypropylmethylcellulose, hydroxyethylcellulose, or methyl
cellulose, or a heteropolysaccharide polymer, for example, gelan
gum, can be incorporated together with a preservative, such as
benzalkonium chloride. Such formulations can also be delivered by
iontophoresis. Formulations for ocular/aural administration can be
formulated to be immediate and/or modified release. Modified
release formulations include delayed, sustained, pulsed,
controlled, targeted and programmed release.
[0083] The immunogenic compositions of the present invention, are
not limited by the selection of the conventional,
physiologically-acceptable carriers, adjuvants, or other
ingredients useful in pharmaceutical preparations of the types
described above. The preparation of these pharmaceutically
acceptable compositions, from the above-described components,
having appropriate pH isotonicity, stability and other conventional
characteristics, is within the skill of the art.
[0084] In general, selection of the appropriate "effective amount"
or dosage for the components of the immunogenic compositions of the
present invention will also be based upon the identity of the
antigen in the immunogenic composition(s) employed, as well as the
physical condition of the subject, most especially including the
general health, age and weight of the immunized subject. The method
and routes of administration, and the presence of additional
components in the immunogenic compositions, may also affect the
dosages and amounts of the compositions. Such selection, and upward
or downward adjustment of the effective dose, is within the skill
of the art. The amount of composition required to induce an immune
response, preferably a protective response, or produce an exogenous
effect in the subject without significant adverse side effects, can
vary, depending upon these factors. Suitable doses are readily
determined by persons skilled in the art.
[0085] Similarly, the order of immunogenic composition
administration and the time periods between individual
administrations, if more than one is necessary, may be selected by
one of skill in the art based upon the physical characteristics and
precise responses of the host to the application of the method.
Preferably, the effective immunogenic composition of the present
invention can be administered to a bovine animal that is 1 day to 3
weeks of age. More preferably, it can be administered to a bovine
animal that is 1-2 weeks of age. It should be noted, again, based
on the particular exposure risks present on any particular breeding
or ranching operation, that a further preferred aspect of the
invention includes vaccinating calves with the modified live
(attenuated) vaccines of the present invention at any age, such as
0 to 3 weeks of age, 0 to 2 months of age, and 0 to 6 months of
age. For an effective immunogenic composition comprising a
modified-live M. bovis, only a single administration is generally
necessary, and that administration can be carried out in a bovine
animal at an age as described above. However, it can be
administered more than once, for example, subsequent doses being
given at 2-4 week intervals following the first vaccination.
However, depending on various factors including the prevalence of
disease in a herd, and the presence of particularly pathogenic
circulating field strains, it may be appropriate to administer one
or multiple doses of the live vaccine of the present invention over
time to a given animal, irrespective of the age of the vaccinated
animal. Thus older animals may also be vaccinated, and repetitively
vaccined according to the practice of the present invention, which
is particularly important in respect of female bovines both before
the onset of, and during pregnancy. Thus in one further preferred
example, a female bovine is vaccinated between 2 and 4 months prior
to the start of pregnancy, even if the cow had been previously
vaccinated, and the calf is vaccinated soon after birth. Under some
circumstances it may be additionally appropriate to vaccinate the
mother cow during pregnancy.
[0086] For other types of M. bovis vaccines (e.g. killed or
subunit), the initial administration of the composition can be at
an age as outlined above, with subsequent administrations 2-4 weeks
following the previous dose.
Kits
[0087] Inasmuch as it may be desirable to administer an immunogenic
composition individually or in combination with additional
compounds, for example, for the purpose of treating a particular
disease or condition, it is within the scope of the present
invention that an immunogenic composition can conveniently be
included in, or combined in, the form of a kit suitable for
administration or co-administration of the compositions. Kits of
the present invention can comprise one or more separate
pharmaceutical compositions, at least one of which is an
immunogenic composition in accordance with the present invention,
and a means for separately retaining said compositions, such as
containers, a divided bottle, or a divided foil packet. An example
of such a kit is a syringe and needle, and the like. A kit of the
present invention is particularly suitable for administering
different dosage forms, for example, oral or parenteral, for
administering the separate compositions at different dosage
intervals, or for titrating the separate compositions against one
another. To assist one administering a composition of the present
invention, the kit typically comprises directions for
administration.
[0088] Another kit of the present invention can comprise one or
more reagents useful for the detection of M. bovis. The kit can
include reagents for analyzing a sample for the presence of whole
M. bovis, or M. bovis polypeptides, epitopes or polynucleotide
sequences. In certain embodiments, the kits can include a set of
printed instructions or a label indicating that the kit is useful
for the detection of animals infected with M. bovis.
Antibody, Antibodies
[0089] Antibodies can either be monoclonal, polyclonal, or
recombinant. The antibodies can be prepared against the immunogen
or a portion thereof. For example, a synthetic peptide based on the
amino acid sequence of the immunogen, or prepared recombinantly by
cloning techniques, or the natural gene product and/or portions
thereof, can be isolated and used as the immunogen. Immunogens can
be used to produce antibodies by standard antibody production
technology well known to those skilled in the art, such as
described generally in Harlow and Lane, "Antibodies: A Laboratory
Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
(1988), and Borrebaeck, "Antibody Engineering--A Practical Guide",
W.H. Freeman and Co. (1992). Antibody fragments can also be
prepared from the antibodies, and include Fab, F(ab').sub.2, and
Fv, by methods known to those skilled in the art.
[0090] In one embodiment of the present invention the antibody of
the invention further provides an intact immunoglobulin capable of
specific binding to a target, such as a carbohydrate,
polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site located in the variable region of the
immunoglobulin molecule. An intact antibody has two light and two
heavy chains. Thus a single isolated intact antibody may be a
polyclonal antibody, a monoclonal antibody, a synthetic antibody, a
recombinant antibody, a chimeric antibody, or a heterochimeric
antibody.
[0091] In the production of antibodies, screening for the desired
antibody can be accomplished by standard methods in immunology
known in the art. Techniques not specifically described are
generally followed as in Stites, et al. (eds), "Basic and Clinical
Immunology" (8th Edition), Appleton and Lange, Norwalk, Conn.
(1994), and Mishell and Shiigi (eds), "Selected Methods in Cellular
Immunology", W.H. Freeman and Co., New York (1980). In general,
ELISAs and Western blotting are the preferred types of
immunoassays; both assays are well known to those skilled in the
art. Both polyclonal and monoclonal antibodies can be used in the
assays. The antibody can be bound to a solid support substrate, or
conjugated with a detectable moiety, or be both bound and
conjugated as is well known in the art. (For a general discussion
of conjugation of fluorescent or enzymatic moieties, see Johnstone
and Thorpe, "Immunochemistry in Practice", Blackwell Scientific
Publications, Oxford (1982).) The binding of antibodies to a solid
support substrate is also well known in the art. (For a general
discussion, see Harlow and Lane (1988), as well as Borrebaeck
(1992).) The detectable moieties contemplated for use in the
present invention can include, but are not limited to, fluorescent,
metallic, enzymatic and radioactive markers such as biotin, gold,
ferritin, alkaline phosphatase, b-galactosidase, peroxidase,
urease, fluorescein, rhodamine, tritium, .sup.14C and
iodination.
Recombinant Techniques
[0092] In yet other embodiments of the invention, the immunogenic
composition may comprise a recombinant vaccine. Such recombinant
vaccines would generally comprise a vector and a heterologous
insert comprising a M. bovis antigen. Suitable M. bovis antigens
may include membrane and membrane-associated proteins, including
but not limited to the variable surface proteins (Vsps);
cytadhesins; lipoproteins, for example P48 and P68; as well as
potentially other hypothetical proteins that remain to be
investigated and characterized.
[0093] The insert may optionally comprise a heterologous promoter,
such as, for example, synthetic promoters known in the art.
Alternatively, the promoters of the host vector may exert
transcriptional control over the expression of the inserts.
Suitable non-limiting examples of promoters (which may be native or
heterologous, depending on the choice of the vector) are H6
vaccinia promoter, I3L vaccinia promoter, 42K poxviral promoter,
7.5K vaccinia promoter, and Pi vaccinia promoter.
[0094] Suitable vectors have been described elsewhere in this
application. In some embodiments, the vectors may be viral vectors,
including, without limitations, vaccinia and pox viral vectors,
such as parapox, racoonpox, swinepox, and different avipox vectors
(e.g., canarypox and fowlpox strains). Currently contemplated viral
strains include D1701, ALVAC, TROVAC, NYVAC strains. Generally,
sequences that are non-essential for the viral host are suitable
insertions sites for the inserts of the instant invention. The
strains recited above are well-characterized in the art and some
insertions sites in these vectors are well known. See, e.g., U.S.
Pat. Nos. 5,174,993; 5,505,941 5,766,599 5,756,103, 7,638,134,
6,365,393.
[0095] There are several known methods or techniques that can be
used to clone and express the nucleotide sequences of the present
invention. For example, the sequences can be isolated as
restriction fragments and cloned into cloning and/or expression
vectors. The sequences can also be PCR amplified and cloned into
cloning and/or expression vectors, or they can be cloned by a
combination of these two methods. Standard molecular biology
techniques known in the art, and not specifically described, can be
generally followed as described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
New York (1989); Ausubel et al., Current Protocols in Molecular
Biology, John Wiley and Sons, Baltimore, Md. (1989); Perbal, A
Practical Guide to Molecular Cloning, John Wiley & Sons, New
York (1988); Watson et al., Recombinant DNA, Scientific American
Books, New York; Birren et al (eds) Genome Analysis: A Laboratory
Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New
York (1998); and methodology set forth in U.S. Pat. Nos. 4,666,828;
4,683,202; 4,801,531; 5,192,659 and 5,272,057. Polymerase chain
reaction (PCR) is carried out generally as described in PCR
Protocols: A Guide to Methods and Applications, Academic Press, San
Diego, Calif. (1990).
[0096] The present invention encompasses the use of prokaryotic and
eukaryotic expression systems, including vectors and host cells,
which may be used to express both truncated and full-length forms
of the recombinant polypeptides expressed by the nucleotide
sequences of the present invention. A variety of host-expression
vector systems may be utilized to express the polypeptides of the
present invention. Such host-expression systems also represent
vehicles by which the coding sequences of interest may be cloned
and subsequently purified. The present invention further provides
for host cells which may, when transformed or transfected with the
appropriate vector or nucleotide sequence, express the encoded
polypeptide gene product of the invention. Such host cells include,
but are not limited to, microorganisms such as bacteria (e.g., E.
coli, B. subtilis) transformed with recombinant bacteriophage DNA,
plasmid DNA or cosmid DNA expression vectors containing coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing the gene product
coding sequences; insect cell systems infected with recombinant
virus expression vectors (e.g., baculovirus) containing the coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing coding sequences;
or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g., metallothionein promoter) or
from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter).
[0097] Generally, the vectors of the invention can be derived from,
but not limited to, bacterial plasmids, from bacteriophage, from
yeast episomes, from yeast chromosomal elements, from viruses
(e.g., as described above), from mammalian chromosomes, and from
combinations thereof, such as those derived from plasmid and
bacteriophage genetic elements including, but not limited to,
cosmids and phagemids.
[0098] Vectors of the present invention can be used for the
expression of M. bovis polypeptides. Generally, the vectors of the
invention include cis-acting regulatory regions operably linked to
the polynucleotide that encodes the polypeptides to be expressed.
The regulatory regions may be constitutive or inducible.
Appropriate trans-acting factors are supplied by the host by an in
vitro translation system, by a complementing vector, or by the
vector itself upon introduction into the host.
[0099] The vectors of the invention can include any elements
typically included in an expression or display vector, including,
but not limited to, origin of replication sequences, one or more
promoters, antibiotic resistance genes, leader or signal peptide
sequences, various tag sequences, stuffer sequences that may encode
a gene whose polypeptide confers antibiotic resistance, restriction
sites, ribosome binding sites, translational enhancers (sequences
capable of forming stem loop structures for mRNA stability
post-transcription), sequences that encode amino acids lacking a
stop codon, and sequences that encode a bacterial coat protein.
[0100] The present invention is further illustrated by, but by no
means limited to, the following examples.
Example 1. Generation and Isolation of a Mycoplasma bovis
Mutant
[0101] Mycoplasma bovis, isolated from the joint of a bovine
(Millmerran, Queensland; 1999), was passaged twice in Mycoplasma
bovis ("Bovis") broth (PPLO; yeast extract; phenol red;
heat-inactivated, .gamma.-irradiated swine serum), filter-cloned
4.times. on Bovis agar, and then further passsaged in broth. A
culture of the strain (10 ml) was prepared, and incubated at the
appropriate temperature for 24 hrs. After that time, the culture
was harvested by centrifugation at 13,000 g for 10 minutes at
4.degree. C. The resultant pellet was washed 3.times. with 1 ml of
phosphate-buffered saline (PBS). The final pellet was resuspended
in 1 ml PBS, and split between two 1.5 ml eppendorf tubes. The
strain (designated 3683) was mutagenized using the chemical,
methylnitronitrosoguanidine (NTG). Temperature-sensitive mutants of
M. bovis were generated and selected according to the protocol
described originally in Nonomura and Imada (Avian Dis.
26(4):763-775; 1982). Following mutagenesis and phenotypic
selection, filter cloning and passaging in Bovis broth was carried
out.
[0102] A calf was intranasally inoculated with a purified clone of
a temperature-sensitive mutant designated 3683 #22/3/12. M. bovis
isolate N2805-1 was subsequently obtained from a swab collected
from the nasopharynx of that specific calf, according to the
following procedure: Mycoplasma broth containing nasal swab
material collected 28 days after infection was plated on PPLO agar
plates, which were then incubated at 38.3.degree. C. in a 5%
CO.sub.2 atmosphere. An individual colony was picked and
transferred to PPLO broth, and incubated at 38.3.degree. C. in a 5%
CO.sub.2 atmosphere for 48 hours. The broth culture was then passed
through a sterile 0.45 micron filter, diluted in saline, and plated
on PPLO agar, in order to obtain individual colonies. A total of 8
passages were performed, alternating between broth and plates, with
the broth phase being filtered prior to plating, to obtain
individual colonies. This resulted in a total of 3 filter/cloning
steps. The final broth passage was divided into 2 aliquots, which
were then stored at -80.degree. C. A frozen aliquot was
subsequently thawed, and used to inoculate 60 ml of PPLO broth.
After 48 hours of incubation at 38.3.degree. C. in 5% CO.sub.2, the
culture was dispensed into 1.25 ml aliquots, and frozen at
-80.degree. C., to serve as working stock. One vial of stock was
thawed and used to inoculate 200 ml of PPLO broth, which was then
incubated for 24 hours at 37.degree. C. in 5% CO.sub.2. The culture
was then dispensed into 1.25 ml aliquots and frozen at -80.degree.
C. All subsequent work was performed using this material ("passage
10").
[0103] Because the strain used to vaccinate the calf from which
N2805-1 was eventually recovered was temperature-sensitive, the
phenotypic characteristics of N2805-1 at both the restrictive
(38.3.degree. C.) and permissive (33.degree. C.) temperatures were
evaluated. Growth of N2805-1 at the restrictive temperature was at
least as good as, if not better than, growth at the permissive
temperature (Table 1), indicating that N2805-1 no longer exhibited
a temperature-sensitive phenotype.
TABLE-US-00001 TABLE 1 Elapsed time (hrs) 33.degree. C.
38.3.degree. C. 0 7.67E+05 5.67E+05 15.5 2.33E+06 8.67E+06 18.5
2.00E+07 1.77E+07 21.5 4.80E+07 3.67E+07 24 4.33E+07 5.43E+07 39.5
1.83E+08 1.20E+08
[0104] In a separate study, the growth rate of N2805-1 was compared
to that of the temperature-sensitive mutant, 3683 #22/3/12, and the
parent strain (3683) from which it was derived. PPLO broth
containing 10% porcine serum was inoculated with 1% (v/v) of thawed
frozen stock culture of each strain. Duplicate broth cultures were
prepared, and one was incubated at 33.degree. C., and the other at
38.degree. C., both with 5% CO.sub.2. At 42 hours post-incubation,
a 0.1 ml aliquot was removed from each culture, and CFU/ml was
determined by serial dilution and plate count on PPLO agar. The
plates were incubated at the same temperature as the corresponding
broth culture for 5-7 days with 5% CO.sub.2. The results in FIG. 1
demonstrate that at 42 hrs post-incubation, N2805-1 was no longer
as temperature-sensitive as 3683 #22/3/12.
[0105] To characterize N2805-1 genotypically, the entire genome was
sequenced, and compared to that of the parental wild-type (WT,
premutagenized) strain 3683, from which it was derived. The result
of that endeavor was that 12 different single nucleotide
differences (single nucleotide polymorphisms, or SNPs) were
identified between the 3683 M. bovis strains and derived N2805-1
strains. To date, 5 of those 12 SNPs have been confirmed by PCR.
Exemplary changes in both the nucleotide and amino acid sequence of
those 5 SNPs are shown in Table 2 (going from strain 3683 to
isolate N2805-1).
TABLE-US-00002 TABLE 2 Nucleotide Nucleotide Resulting Sequence
Sequence Amino acid SNP # Description in 3683 in N2805-1 Change 3 a
Nitroreductase CAG AAG Glu > Lys 6 a Histidine acid GAT AAT Asp
> Asn phosphatase 8 a Excinuclease ABC GCC GTC Ala > Val
subunit B 9 a DNA polymerase III AGC AAC Ser > Asn subunit beta
10 a ATP synthase F1 CTT TTT Leu > Phe subunit delta
Example 2. Efficacy of a Modified-Live Mycoplasma bovis Vaccine
Strain Against a Virulent Mycoplasma bovis Heterologous
Challenge
[0106] Clinically healthy, colostrum-deprived (CD) calves were
enrolled in the study. All calves were serologically negative
(1:1600 titer) for M. bovis antibody by ELISA, and were PCR
negative for M. bovis in nasal swabs collected before Day 0.
[0107] All calves also were BVDV persistent infection negative in
ear notch tests. Animals were housed in separate rooms by treatment
group, in individual pens, during the vaccination phase of this
study. Treatment groups were co-mingled in four challenge rooms,
and housed by block during the challenge phase. Any adverse events
which occurred in calves during the course of the study were
recorded.
[0108] On Study Day 0 (SD 0), 1-2 week old animals were
intranasally administered (one nostril) one of the following
vaccines (Table 3): T01, saline; T02, M. bovis vaccine strain
N2805-1; T03; a reference experimental M. bovis vaccine
(live-attenuated); T04, temperature-sensitive, earlier passage of
M. bovis vaccine strain N2805-1. On SD 29-SD 31, all animals were
challenged by aerosol with M. bovis Kansas strain 110LA-11-10.
Calves were challenged using a small face mask (canine anesthetic
mask) connected to a nebulizer, via a chamber which contained the
aerosolized M. bovis challenge. Four nebulizers were connected to
each chamber, which was in turn connected, by individual tubes, to
four calves. Animals were subsequently necropsied on SD 56;
samples/tissues collected included: lung tissue, for PCR; lung
swabs, 1 for isolation of M. bovis, and 1 for other bacteria;
tonsil tissue; tracheobronchial lymph nodes; and lung lesion
tissue.
TABLE-US-00003 TABLE 3 Target Vaccine Target Target Treatment Route
and Dose Challenge Dose Necropsy Group N (CFU/2 mL dose) and
Exposure Day T01 16 IN, Aerosol on days 56 1 .times. 10.sup.9/calf
29, 30, 31 T02 16 IN, approximately 1 .times. 10.sup.9/calf 1
.times. 10.sup.8 T03 16 IN, (1X culture) 1 .times. 10.sup.9/calf 30
min/calf T04 16 IN, 1 .times. 10.sup.9/calf
[0109] Results. The back transformed least square means (BTLSM)
lung lesion scores (LLS) results are listed in Table 4. The vaccine
administered to treatment groups T02, T03 and T04 induced a
significant reduction in lung lesions compared to T01, saline
controls (0%, 1%, and 1%, versus 17%, respectively).
TABLE-US-00004 TABLE 4 Lower Upper Standard 90% 90% Range Error
Confidence Confidence % Number Back.sup.1 % Lung Limit Limit Lung
Treatment of Transform with of of with Number Animals LS mean
Lesions Mean Mean Lesions T01 16 17.sup.c 5 10 26 0 to 46 T02 16
O.sup.a 0 0 1 0 to 7 T03 15 l.sup.b 1 0 4 0 to 18 T04 15 l.sup.ab 1
0 4 0 to 59 1. Treatment groups with the same letter are not
significantly different at alpha =.10
[0110] Mean duration of pyrexia (>103.1.degree. F.) is indicated
in Table 5. All three vaccinated treatment groups had significantly
fewer days of pyrexia when compared to controls.
TABLE-US-00005 TABLE 5 Treatment number Estimate.sup.1 lower 90% Cl
upper 90% Cl T01 3.6.sup.b 1.7 6.8 T02 0.9.sup.a 0.1 2.2 T03
1.4.sup.a 0.4 3.2 T04 1.1.sup.a 0.2 2.5 .sup.1Different
superscripts within a column represent significant differences
between groups
[0111] Following challenge, the T01 group had a duration of
clinical disease associated with M. bovis respiratory disease that
averaged 4.7 days. This is contrasted with 0.7, 1.2 and 2.5 days
respectively for the T02, T03 and T04 groups (Table 6). The T02 and
T03 calves had a significant reduction in the mean duration of at
least one clinical sign compared to T01 calves (P<0.0009 and
P<0.0265).
[0112] The T02, T03 and T04 groups were protected from M.
bovis-induced disease, as fewer animals exhibited clinical signs of
cough, lameness and respiratory effort, and for a shorter duration
of time compared with the T01 group. No animals in any group
exhibited nasal discharge. Vaccinates did have droopy ear/head tilt
slightly more often than the T01 group (Table 7).
TABLE-US-00006 TABLE 6 Treatment number Estimate.sup.1 lower 95% Cl
upper 95% Cl T01 4.7.sup.a 2.3 9.1 T02 0.7.sup.b 0.1 1.5 T03
1.2.sup.b 0.2 2.9 T04 2.5.sup.a 0.9 5.2 .sup.1Different
superscripts within a column represent significant differences
between groups
TABLE-US-00007 TABLE 7 Ever T01 T02 T03 T04 Present? (%) N(1) (%)
N(1) (%) N(1) (%) N(1) Cough 56.3 16 18.8 16 26.7 15 46.7 15
Lameness 6.3 16 0.0 16 0.0 15 0.0 15 Nasal 0.0 16 0.0 16 0.0 15 0.0
15 Discharge Respiratory 68.8 16 12.5 16 20.0 15 20.0 15 Effort
Droopy 6.3 16 12.5 16 13.3 15 20.0 15 Ear/Head Tilt
[0113] Nasal swabs were collected and tested by PCR for M. bovis
weekly throughout the study. Detection by PCR peaked from day 7 to
day 21 for the vaccinate groups. The T01 controls remained PCR
negative during the vaccination phase. 100% of the T02 and T04
calves, and 93.3% of T03 calves were PCR positive during the study
(Table 8).
TABLE-US-00008 TABLE 8 Ever Treatment Day Day Day Day Day Day Day
Day Positive for No. N -1 7 14 21 27 36 43 55 Mycoplasma T01 16
0.0% 0.0% 0.0% 0.0% 0.0% 50.0% 25.0% 0.0% 56.3% T02 16 0.0% 75.0%
56.3% 87.5% 43.8% 81.3% 0.0% 12.5% 100.0% T03 15 0.0% 86.7% 66.7%
86.7% 46.7% 60.0% 20.0% 0.0% 93.3% T04 15 0.0% 75.0% 13.3% 20.0%
73.3% 73.3% 0.0% 6.7 100.0%
[0114] All animals were sero-negative for M. bovis (ELISA titers of
.ltoreq.1:1600, or Geometric Mean 800) prior to the start of the
study. Sero-conversion was detected at day 7 for T02 and T04
calves, and day 14 for the T03 group. Titer means were low across
all groups until after challenge (Table 9).
[0115] The mucosal IgA antibody response to vaccination and
challenge was monitored in nasal secretions using a M.
bovis-specific IgA ELISA. A cut-off value of >1:50 was used to
define a positive response. IgA antibody titers were induced by
vaccination, and generally increased with study progression. IgA
titers were higher for T02, T03 and T04 calves than T01 animals
from day 7 through day 55. The peak response was day 43 for T01 and
T02 groups, and day 55 for T03 and T04 groups (Table 10).
TABLE-US-00009 Day-1 Day 7 Day 14 Day 21 Geometric Geometric
Geometric Geometric Trt. Mean Range Mean Range Mean Range Mean
Range T01 800 800-800 800 800-800 800 800-800 800 800-800 T02 800
800-800 911 800-3200 872 800-3200 911 800-3200 T03 800 800-800 800
800-800 1038 800-12800 1346 800-12800 T04 800 800-800 872 800-1809
838 800-1600 962 800-3200 Day 27 Day 36 Day 43 Day 55 Geometric
Geometric Geometric Geometric Trt. Mean Range Mean Range Mean Range
Mean Range T01 911 800-1600 911 800-1600 2810 800-12800 17689
3200-51200 T02 3200 800-6400 3200 800-6400 9871 1600-25600 8668
3200-51200 T03 4223 800-12800 4223 800-12800 14018 1600-102400
11145 800-102400 T04 2788 800-6400 2788 800-6400 6704 800-102400
8445 1600-102400
TABLE-US-00010 TABLE 10 Day-1 Day 7 Day 14 Day 21 Geometric
Geometric Geometric Geometric Trt. Mean Range Mean Range Mean Range
Mean Range T01 1 1-2 3 1-50 5 1-10 9 2-50 T02 1 1-2 4 1-10 113
10-6250 152 50-1250 T03 2 1-10 6 2-50 92 10-250 278 50-1250 T04 1
1-2 6 1-50 69 10-250 132 50-6250 Day 27 Day 36 Day 43 Day 55
Geometric Geometric Geometric Geometric Trt. Mean Range Mean Range
Mean Range Mean Range T01 13 1-50 28 10-250 186 50-1250 234 50-1250
T02 185 50-1250 251 50-1250 458 50-6250 506 250-6250 T03 205
50-1250 227 50-1250 1018 250-3150 914 250-6260 T04 226 50-6250 249
250-250 729 50-6250 588 250-6250
[0116] Lung, tonsil, and tracheobronchial lymph node samples were
tested by PCR for M. bovis*. (* Mycoplasma agalactiae can also be
amplified in this PCR assay.) M. bovis recovery was less frequent
in all three vaccinated treatment groups in lung and lymph node
tissue samples, compared to controls. Recovery from tonsils was
high for all treatment groups (Table 11).
TABLE-US-00011 TABLE 11 Treatment Lymph Node Tonsil Lung Tissue
number N % N % N % T01 12/16 75.0 16/16 100.0 16/16 100.0 T02 7/16
43.8 16/16 100.0 9/16 56.3 T03 9/15 60.0 15/15 100.0 8/15 53.3 T04
6/15 40.0 12/15 80.0 9/15 60.0 * PCR data is from tissues collected
at necropsy.
[0117] The rate of isolation of M. bovis from the lung swabs (day
of necropsy) was lower in the T02, T03 and T04 calves, at 31.3%,
40.0% and 33.3%, compared to 87.5% for T01 controls. Lung swabs
were negative for all other bacterial pathogens tested (Table
12).
TABLE-US-00012 TABLE 12 Mycoplasma Histophilus Mannheimia
Pasteurella Beberstenia Treatment bovis* somni haemolytica
multocida trehalosi number % N % N % N % N % N TO1 87.5 14 of 16 0
0 of 16 0 0 of 16 0 0 of 16 0 0 of 16 T02 31.3 5 of 16 0 0 of 16 0
0 of 16 0 0 of 16 0 0 of 16 T03 40.0 6 of 15 0 0 of 15 0 0 of 15 0
0 of 15 0 0 of 15 T04 33.3 5 of 15 0 0 of 15 0 0 of 15 0 0 of 15 0
0 of 15 *Mycoplasma agalactiae can also be amplified in this PCR
assay.
[0118] The IFN gamma ELISPOT assay measured the induction of
cell-mediated immunity after vaccination and challenge. IFN gamma
spot-forming cells (SFCs) per million BTLSM (day -1 and day 42) and
geometric means (day 7 and day 14) are listed in Table 13.
TABLE-US-00013 TABLE 13 Day -1 Day 7 Day 14 Day 42 Treatment BTLSM
Geometric Mean Geometric Mean BTLSM No T02 T03 T04 T02 T03 T04 T02
T03 T04 T02 T03 T04 T01 491.8 300.9 456.6 556.8 231.5 243.1 219.8
258.8 246.5 1244 976.7 1101 T02 273.8 160.4 210.4 797.6 350.8 599.3
195.8 214.7 163.4 1040.2 598.6 978.5 T03 481.5 351.0 462.4 637.9
415.9 414.7 343.8 274.6 214.7 763.9 662.0 372.0 T04 303.5 224.5
370.5 808.3 600.8 706.6 431.8 448.6 352.3 695.3 831.4 860.5
[0119] In conclusion, the N2805-1 vaccine candidate (T02) provided
a significant reduction in lung lesion scores. Rectal temperatures
and clinical signs of disease post-challenge were reduced in
severity and duration by N2805-1. Serum antibody titers, IgA
titers, and PCR and culture results also support the efficacy of
this strain. Thus, the N2805-1 vaccine is a good candidate to
evaluate for safety, and in further efficacy studies.
Example 3. Genomic Characterization of Temperature-Sensitive
Mutant, 3863 22/3/12, and Non-Temperature Sensitive Mutant,
N2805-1
[0120] In an effort to characterize the genomic differences between
the M. bovis temperature-sensitive mutant, 3683 #22/3/12, and the
non-temperature sensitive mutant, N2805-1, isolated from a calf
which had been inoculated with 3683 #22/3/12, the genomes of both
were sequenced using Illumina technology. DNA libraries were
prepared, and barcoded samples were sequenced to obtain an average
of 60.times. coverage of paired-end sequences per sample. Quality
control of all reads was carried out, and failed and low quality
reads were removed prior to mapping and single nucleotide
polymorphism (SNP) analysis. Sequence reads were mapped to a
reference genome (Hubei-1; Genbank accession number CP002513) using
CLC Genomics workbench software (Qiagen; Redwood City, Calif.). The
variant detection module from CLC Genomics workbench was used for
SNP discovery. The identity and location of the SNPs are shown in
Table 14. Between the two mutant strains, 56 SNPs were identified,
17 of which resulted in amino acid changes (including 2 which
resulted in the introduction of a stop codon), 6 resulted in
frameshift mutations, and 33 occurred in non-coding regions.
TABLE-US-00014 TABLE 14 SNP Nucleotide Amino Acid Location*
Change.sup.# Change.sup.# 115273 T > C (non-coding region)
194478 C > T (non-coding region) 194484 T > C (non-coding
region) 194525 T > C Thr > Ala 194724 G > A (non-coding
region) 204966 T > A (non-coding region) 207629 A > G
(non-coding region) 262327 A > T (non-coding region) 271487 C
> T Gly > Ser 271661 G > A Gln > Stop 271666 G > --
Thr > frameshift 272086 T > A Lys > Ile 272133 A > G
(non-coding region) 309463 C > -- Pro > frameshift 310075 A
> T Lys > Met 310078 T > A Ile > Lys 310086 A > C
Asn > His 310090 A > C Lys > Thr 311988 C > T
(non-coding region) 334184 A > -- (non-coding region) 358013 G
> A (non-coding region) 365101 T > C (non-coding region)
365188 C > T (non-coding region) 397010 T > -- Lys >
frameshift 397901 T > -- (non-coding region) 399129 A > T
(non-coding region) 410247 A > T (non-coding region) 410253 T
> C (non-coding region) 410334 G > A (non-coding region)
410514 T > A Arg > Ser 410733 T > C (non-coding region)
414666 G > A Gln > Stop 453388 A > G (non-coding region)
468457 A > -- (non-coding region) 502577 A > G Ile > Val
502678 C > T (non-coding region) 502963 T > C (non-coding
region) 543859 G > T (non-coding region) 560986 G > -- Gly
> frameshift 632309 C > T (non-coding region) 632366 A > G
(non-coding region) 632612 A > G (non-coding region) 633994 T
> -- Lys > frameshift 686074 T > C (non-coding region)
687874 G > A (non-coding region) 687915 C > A Ala > Ser
696348 C > -- Gly > frameshift 729874 C > T (non-coding
region) 730046 C > T Arg > Cys 761587 T > C Thr > Ala
761663 G > A (non-coding region) 761810 A > G (non-coding
region) 761815 T > C Thr > Ala 765176 G > A Ala > Thr
765328 T > C (non-coding region) 765403 G > A Met > Ile
*Numbering of SNP locations is based on Mycoplasma bovis reference
strain Hubei-1 (GenBank accession number CP002513).
.sup.#Nucleotide and amino acid changes that occurred going from
strain, 3863 #22/3/12, to strain N2805-1.
[0121] A Mycoplasma bovis strain from a bovine has been attenuated,
as well as characterized. An isolate of said M. bovis strain was
deposited with the American Type Culture Collection ("ATCC"),
Manassas, Va., USA, on May 12, 2015, and has been assigned
accession number, PTA-122167.
* * * * *