Note: Descriptions are shown in the official language in which they were submitted.
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VACCINES AND METHODS TO TREAT LYME DISEASE IN DOGS
Field of the invention
This invention is in the field of veterinary medicine. More particularly, this
invention is in the field of vaccines treating or preventing Lyme disease in
dogs.
Background
Lyme disease is a bacterial infection caused by pathogenic spirochetes of
the genus Borrelia. The infection can occur in humans, dogs, deer, mice and
other animals, and is transmitted by arthropod vectors, most notably ticks of
the
genus Ixodes. Borrelia burgdorferi, the most common cause of Lyme disease in
North America, was first cultured in 1982. Borrelia are introduced into the
host at
the site of the tick bite and this is also the location of the initial
characteristic skin
lesion, erythema chronicum migrans (ECM). In dogs, Lyme disease manifests
with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy,
and
in some cases, fatal glomerulonephritis. A recent study revealed that the
percentage of actively infected dogs in endemic areas can be as high as 1 1%.
The infection may be treated at any time with antibiotics such as penicillin,
erythromycin, tetracycline, and ceftriaxone. Once infection has occurred,
however, the drugs may not purge the host of the spirochete, but may only act
to
control the chronic forms of the disease. Complications such as arthritis and
fatigue may continue for several years after diagnosis and treatment.
The canine Lyme disease vaccines were developed to provide protection
by primarily inducing OspA borreliacidal antibodies. B. burgdorferi OspC is
another potential target for borreliacidal antibody-mediated immunity. This
protein appears to have an epitope that is responsible for inducing
borreliacidal
antibodies, and is not conserved among the pathogenic Borrelia spp. Although
the specific function of the OspC protein remains unknown, it has been
suggested that OspC expression is required for infection of mammals, but not
for
infection of ticks. Borrelia express OspC shortly after the tick begins
feeding, and
must continue to express OspC in order to establish an infection in mammals.
Therefore, the "window of effectiveness" of the OspC borreliacidal antibodies
is
increased significantly, compared to OspA borreliacidal antibodies.
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Ca!lister et al., (U.S. Pat. Nos. 6,210,676 and 6,464,985)
have suggested employing an immunogenic polypeptide
fragment of OspC, alone or in combination with an OspA polypeptide, to prepare
a vaccine to protect humans and other mammals against Lyme disease. Livey et
al. (U.S. Pat. No. 6,872,550) also proposed a
vaccine for immunizing against Lyme disease prepared from a combination of
recombinant OspA, OspB, and OspC proteins.
However, at least two obstacles need to be overcome before a successful
vaccine can be created. First, there are over twenty OspC phylotypes, and it
is
1.0 unclear which ones should be included into a vaccine. Second, suitable
epitopes for development of borreliacidal anti-OspC antibodies need to be
determined.
Therefore, there remains a longstanding need in the art for an improved
vaccine to protect mammals, and especially canines, from Lyme disease.
Summary of Invention
The instant invention addresses these and other needs by providing, in
one aspect, an immunogenic composition comprising: a first protein comprising
an amino acid sequence at least 95% identical to SEQ ID NO: 1
(MDPNTVSSFQVDSFLWHVRKRVADQELG DAPFLDRLRRDQKSLRG RGSTLG
LDI ETATRAGKQI VERI LKEESDEALKMTMG KQNVSSLDEKNSVSVDLPGEMNV
LVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDL
GQTTLEVFKEDG KTLVSKKVTSKDKSSTEEKFN EKG EVSEKI ITRADGTRLEYT
EIKSDGSGKAKEVLKSYVLEGTLTAEKTTLVVKEGTVTISKNISKSGEVSVELND
TDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEG
SAVEITKLDEIKNALK); and a second protein, comprising immunodominant
epitopes of OspC phylotypes F and N.
In a set of embodiments, the second protein comprises a plurality of
peptides at least 95% identical to immunodominant epitopes from loop 5 (loop
peptide) and alpha helix 5 (helix peptide) of one or more OspC phylotypes I,
H,
C, M, and D, wherein further the loop peptides and the helix peptide from each
phylotype are adjacent to each other and wherein the loop peptides and the
helix
peptides are arranged sequentially; and at least one of: a loop peptide and a
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helix peptide of OspC phylotype F adjacent to each other, or an amino acid
sequence 95% identical to SEQ ID NO 32. In one set of embodiments, if said
amino acid sequence 95% identical to SEQ ID NO: 32 is present, it is at the
carboxy terminus of said second protein.
In one set of embodiments, the first protein is SEQ ID NO: 1. In another
set of embodiments, the loop and helix peptides of phylotypes I, H, N, C, M, D
and F are at least 95% identical to SEQ ID NOs: 4-17, respectively.
In another set of embodiments, the immunogenic composition may also
comprise additional loop and helix peptides from one or more OspC phylotypes
F, T, U, E, A, B, and K, which are, in some embodiments, identical to SEQ ID
NOs 16-29, respectively.
In yet another set of embodiments, the immunogenic composition may
further comprise at least one additional antigen protective against a
microorganism that can cause disease in dogs. The microorganism may be
selected from the group comprising canine distemper (CD) virus, canine
adenovirus type 2 (CAV-2), canine parainfluenza (CPI) virus, canine parvovirus
(CPV), canine coronavirus (CCV), canine herpesvirus, and rabies virus.
Antigens
from these pathogens for use in the vaccine compositions of the present
invention can be in the form of a modified live viral preparation or an
inactivated
viral preparation. Other pathogens also include Leptospira bratislava,
Leptospira
canicola, Leptospira grip potyphosa, Leptospira icterohaemorrhagiae,
Leptospira
pomona, Leptospira hardjobovis, Porphyromonas spp., Bacteriodes spp.,
Leishmania spp., Ehrlichia spp., Mycoplasma ssp. and Microsporum canis.
In particular embodiments, the immunogenic composition comprises SEQ
ID NO: 1 and either SEQ ID NO: 30 or SEQ ID NO: 31.
In another aspect, the instant invention provides a vaccine composition
comprising the immunogenic composition as described above. The vaccine can
also comprise an adjuvant and a pharmaceutically acceptable carrier. In
different embodiments, adjuvants include, without limitations mineral salts,
surface-active agents and microparticles, bacterial products, cytokines and
hormones, carriers, oil-in-water emulsions and water-in-oil emulsions.
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In yet another aspect, the invention also provides a method of preventing
Lyme disease in a canine comprising administering to the canine in need
thereof
an immunologically effective dose of the vaccine composition.
Brief Description of the Figures
Figure 1 illustrates neighbor joining tree of OspC types identified by
cloning from skin biopsy samples taken from dogs.
Figure 2 illustrates neighbor joining tree of OspC types identified by
sequencing Borrelia burgdorferi clones isolated from skin biopsies taken from
dogs.
Figures 3A and 3B illustrate chimeric sequences Al2CF and A10CF
(SEQ ID NO: 31 and 30, respectively), suitable as the second protein of the
immunogenic composition described herein.
Figure 4 illustrates protein sequence for OspC phylotype A strain B31 and
other OspC phylotypes.
Detailed Description of Preferred Embodiments
For a better understanding of the instant application, the following non-
limiting definitions are provided:
The term "at least 95% identical" includes all percentages of identity
including and between 95% and 100%, for example, 96%, 97%, 98%, 99%, etc.
The term "alpha helix 5 region" or "helix 5 region" refers to amino acid
sequence located between residues 160 and 200 of OspC phylotype A strain
B31, and contains secondary structural elements including a portion of loop 6,
alpha helix 5, and the unstructured C-terminal domain (Kumaran et al., 2001).
The term "conservative substitution" denotes the replacement of an amino
acid residue by another biologically similar residue, or the replacement of a
nucleotide in a nucleic acid sequence such that the encoded amino acid residue
does not change or is another biologically similar residue. Examples of
conservative substitution include the substitution of one hydrophobic residue
such as isoleucine, valine, leucine or methionine for another hydrophobic
residue, or the substitution of one polar residue for another polar residue,
such
as the substitution of arginine for lysine, glutamic for aspartic acid, or
glutamine
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for asparagine, and the like. The term "conservative substitution " also
includes
the use of a substituted amino acid in place of an unsubstituted parent amino
acid, provided that antibodies raised to the substituted polypeptide also
immunoreact with the unsubstituted polypeptide.
The term "conservative variation" of a reference protein or a reference
nucleic acid refers to a protein or a nucleic acid, respectively, which
differs from
the reference molecule by only conservative substitution(s).
The term "construct" preceded by a phylotype name (e.g., N-construct or
l-construct) refers to an amino acid sequence comprising the loop peptide and
the helix peptide.
The term "helix peptide" or "alpha helix peptide" of a certain phylotype of
OspC refers to a peptide which is at least 95% identical to an immunodominant
epitope from alpha helix 5 region of OspC protein of that phylotype. Thus, for
example, helix peptide N refers to a peptide which is at least 95% identical
to an
immunodominant epitope from alpha helix 5 region of OspC phylotype N.
The term "immunodominant epitope" refers to an epitope on a molecule
that induces a dominant, or intense, immune response when compared to other
epitopes, including one or both B- and T-cell responses.
The term "linear epitope" refers to an epitope comprising a single, non-
interrupted, contiguous chain of amino acids joined together by peptide bonds
to
form a peptide or polypeptide. Such an epitope can be described by its primary
structure, i.e. the linear sequence of amino acids in the chain. Such an
epitope,
when expressed in a recombinant protein subunit of OspC, retains the ability
to
bind infection-induced antibodies in a manner similar to the binding of wild-
type
protein.
The term "loop peptide" of a certain phylotype of OspC refers to a peptide
which is at least 95% identical to an immunodominant epitope from loop 5
region
of OspC protein of that phylotype. Thus, for example, loop peptide N refers to
a
peptide which is at least 95% identical to an immunodominant epitope from loop
5 region of OspC phylotype N.
The term "loop 5 region" refers to amino acid sequence generally located
between residues 131 and 159 of OspC phylotype A strain B31 and contains
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secondary structural elements, including a portion of alpha helix 3, loop 5
and
alpha helix 4. See Kumaran et al., 2001. The sequence for OspC phylotype A
strain B31 is provided in SEQ ID NO: 35 and in Fig. 4.
The term "therapeutically effective amount" as used herein means an
amount of a microorganism, or a subunit antigen, or polypeptides, or
polynucleotide molecules, and combinations thereof, sufficient to elicit an
immune response in the subject to which it is administered. The immune
response can comprise, without limitation, induction of cellular and/or
humoral
immunity.
The terms "vaccine" and "vaccine composition," as used herein, mean a
composition which prevents or reduces an infection, or which prevents or
reduces one or more signs or symptoms of infection. The protective effects of
a
vaccine composition against a pathogen are normally achieved by inducing in
the subject an immune response, either a cell-mediated or a humoral immune
response or a combination of both. Generally speaking, abolished or reduced
incidences of infection, amelioration of the signs or symptoms, or accelerated
elimination of the microorganism from the infected subjects are indicative of
the
protective effects of a vaccine composition.
In a broad aspect, the instant invention provides an immunogenic
composition capable of inducing antibodies against OspA and OspC proteins of
Borrelia burgdorferi. Thus, the composition will include two proteins: the
first
protein comprising an OspA or a fragment thereof, and a second protein,
comprising an OspC protein or a fragment thereof. In some embodiments, the
second protein is a chimeric protein comprising multiple fragments of OspC
proteins of different phylotypes.
In some embodiments, the first protein comprises a fragment of OspA
protein (SEQ ID NO: 2)
(MGKQNVSSLDEKNSVSVDLPG EMNVLVSKEKN KDG KYDLIATVDKLELKGTS
DKN NGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDG KTLVSKKVTSKDKS
STEEKFN EKG EVSEKI ITRADGTRLEYTEI KSDGSGKAKEVLKSYVLEGTLTAET
TLVVKEGTVTLS KN I SKSG EVSVE LN DTDSSAATKKTAAW NSGTSTLTITVNS K
KTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEI KNALK), which is
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immediately downstream of a viral protein, such as, for example, a fragment of
the influenza virus NS1 protein, which is SEQ ID NO: 3
(MDPNTVSSFQVDSFLW HVRKRVADQELG DAPFLDRLRRDQKSLRG RGSTLG
LDIETATRAGKQIVERILKEESDEALKMT). An important requirement for the first
protein is its ability to generate anti-OspA antibodies in a vaccinated
animal.
Thus, the full length sequence of the OspA fragment is not necessary, and
neither is the 100% identity to SEQ ID NO: 2.
As noted elsewhere in the application, 95% sequence identity is likely to
be sufficient to provide suitable level of antibody production. The differing
amino
acids can be conservative substitutions, and/or are located outside of
immunodominant epitope(s) of the OspA fragment.
In other embodiments, shorter OspA fragments can be used. A person of
ordinary skill in the art would know how to determine which OspA fragments
contain immunodominant epitopes capable of generating borreliacidal
antibodies.
The inventors have surprisingly found that the first protein comprising,
from N- to C- terminus, a fragment of the influenza virus NS-1 protein,
followed
by OspA protein with its signal sequence removed, is particularly suitable for
the
immunogenic compositions of the instant invention.
Prior art studies are silent as to what phylotypes of OspC are prevalent in
invasive Lyme disease in dogs. Most studies have been performed on human
samples. Jones et al reports that the most prevalent phylotypes found in joint
fluid of the human patients with arthritis are K and A, and typically, and
phylotypes A, B, C, D, H, K, N were discovered. Arthritis Rheum 2009 60(7)
2174. Earnhart et al have discovered phylotypes A, B, I, K, C, D, N in blood
and/or CSF samples Infect Immun. 2005 73(12): 7869. Other studies typically
associated phylotypes A, B, I and K with invasive forms of Lyme disease in
humans.
However, it was surprisingly discovered that in dogs, the most prevalent
phylotype is OspC F, which was not previously associated with invasive form of
Lyme disease, whether in humans or in dogs. Phylotype N, which is associated
with invasive Lyme disease in humans, was also associated with invasive Lyme
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disease in dogs. Additionally, the inventors have discovered that phylotypes T
and U, previously not associated with invasive Lyme disease in humans, may
cause invasive Lyme disease in dogs.
According to some embodiments, the second protein contains
immunodominant epitopes capable of generating immune response against
different OspC protein phylotypes. More specifically, the second protein of
the
immunogenic composition claimed in the instant invention is a chimeric protein
that comprises immunodominant epitopes of OspC phylotypes F and N. The
immunodominant epitopes may be in the form of loop and/or helix peptides as
discussed below, or they may be present within larger fragments of the target
OspC protein. A suitable non-limiting example of such fragments is SEQ ID NO:
32
(NNSGKDGNTSANSADESVKGPNLTEISKKITESNAVVLAVKEIETLLSSIDELAT
KAIGQKI DANG LGVQANQNGSLLAGAYAISTLITQKLSALNSEDLKEKVAKVKKC
SEDFTNKLKNGNAQLGLAAATDDNAKAAI LKTNGTNDKGAKELKDLSDSVESLV
KAAQVMLTNSVKELTSPVVAESPKKP), which is a fragment of OspC phylotype
F protein.
Previous studies demonstrate that the inclusion of conserved region of
OspC protein (i.e., conserved among different phylotypes) is important for
generation of anti-borrelicidal antibody in mice and humans but not in dogs.
See
Lovrich et al, Clin .and Vaccine Immunol. May 2007, p. 635-637. Nevertheless,
the inventors have surprisingly discovered that the addition of the longer
fragment of one of the OspC phylotypes (e.g., phylotype F) is beneficial for
the
expression level and thus makes the manufacturing of the second protein more
efficient.
Buckles et al demonstrated that loop 5 of OspC protein is surface
exposed and may be a suitable target for generating borreliacidal antibodies.
Clin Vaccine Immunol. 2006 Oct;13(10):1162-5. See also W009135118.
However, considering that at least 21 phylotypes of OspC have been described
(Seinost et al., Infect Immun. 1999 Jul;67(7):3518-24 1999), it remains to be
determined what combination provides suitable protection against Lyme disease.
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Thus, in some embodiments, the second protein comprises linear
epitopes from loop 5 region (loop peptides) and helix 5 regions (helix 5
peptides)
of OspC proteins of different phylotypes. Currently considered phylotypes are
T,
U, E, A, B, K, I, H, N, C, M, D and F. The second protein may thus comprise
loop and helix peptides from 2- 13 phylotypes of OspC, e.g., 2, 3, 4, 5, 6, 7,
8, 9,
10, 11, 12, 13 phylotypes. The order of the peptides is not crucial. In some
embodiments, the loop peptides are interspaced with the helix peptides, and
vice
versa. In other words, the loop and helix peptides are arranged sequentially:
in
such embodiments, no two loop peptides should be present in the second
1.0 protein without a helix peptide between them, and no two helix peptides
should
be present without a loop peptide between them.
A person of ordinary skill in the art would be aware how to determine
immunodominant epitopes from the loop regions and helix regions of various
OspC phylotypes. For example, sera from subjects infected with Borrelia
burgdorferi of different phylotypes may be reacted with specific peptides from
the
loop regions and helix regions of the corresponding phylotypes, and the
binding
of the antibodies present in the sera to the loop peptides and/or helix
peptides
can be quantified (e.g., by ELISA, immunoblot, etc), thus providing clues as
to
which peptides contain immunodominant linear epitopes from a given OspC
phylotype.
Similarly, the borreliacidal activity of the antibodies may be determined by
methods well known in the art, e.g., generally, by co-incubating cultured
Borrelia
burgdorferi with the sera from subjects challenged with the immunodominant
linear epitopes as described above, and quantification of living and dead
Borrelia.
In some embodiments, the sequences for the loop peptides and helix
peptides are as follows:
Loop peptide I is at least 95% identical to SEQ ID NO: 4
(AKLKGEHTDLGKEGVT);
Helix peptide I is at least 95% identical to SEQ ID NO: 5
(KGADELEKLFESVKNLSKAAKEMLTNSVKE);
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Loop peptide H is at least 95% identical to SEQ ID NO: 6
(SEKFAGKLKNEHASLGKKDAT);
Helix peptide H is at least 95% identical to SEQ ID NO: 7
(KGAKELKDLSDSVESLVKA);
Loop peptide N is at least 95% identical to SEQ ID NO: 8
(SDDFTKKLQSSHAQLGVAGGATT);
Helix peptide N is at least 95% identical to SEQ ID NO: 9
(ADELEKLFKSVESLAKAAQDALANSVNELTS);
Loop peptide C is at least 95% identical to SEQ ID NO: 10
(KKLKEKHTDLGKKDAT);
Helix peptide C is at least 95% identical to SEQ ID NO: 11
(AAELEKLFESVENLAKAAKEMLSNS);
Loop peptide M is at least 95% identical to SEQ ID NO: 12
(NKAFTDKLKSSHAELGIANGAAT);
Helix peptide M is at least 95% identical to SEQ ID NO: 13
(KGAQELEKLFESVKNLSKAAQETLNNSVKE);
Loop peptide D is at least 95% identical to SEQ ID NO: 14
(SESFTKKLSDNQAELGIENAT);
Helix peptide D is at least 95% identical to SEQ ID NO: 15
(KGAEELVKLSESVAGLLKAAQAILANSVKELTSPVVAESPKKP);
Loop peptide F is at least 95% identical to SEQ ID NO: 16
(SEDFTNKLKNGNAQLGLAAAT);
Helix peptide F is at least 95% identical to SEQ ID NO: 17
(KGAKELKDLSDSVESLVKAAQVMLTNS);
Loop peptide T is at least 95% identical to SEQ ID NO: 18
(STGFTNKLKSGHAELGPVGGNAT);
Helix peptide T is at least 95% identical to SEQ ID NO: 19
(KGAKELKDLSESVEALAKAAQAMLTNS);
Loop peptide U is at least 95% identical to SEQ ID NO: 20
(SEKFTKKLSESHADIGIQAAT);
Helix peptide U is at least 95% identical to SEQ ID NO: 21
(KGAEELDKLFKAVENLSK);
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Loop peptide E is at least 95% identical to SEQ ID NO: 22
(STEFTN KLKSEHAVLG LDN LT);
Helix peptide E is at least 95% identical to SEQ ID NO: 23
(KGAAE LEKLKAVEN LSKAAQDTLKNAVKE LTS PIVAES PKKP);
Loop peptide A is at least 95% identical to SEQ ID NO: 24
(SETFTNKLKEKHTDLGKEGVT);
Helix peptide A is at least 95% identical to SEQ ID NO: 25
(KGAEELGKLFESVEVLSKAAKEMLANSVKELTS);
Loop peptide B is at least 95% identical to SEQ ID NO: 26
(SEEFSTKLKDNHAQLGIQGVT);
Helix peptide B is at least 95% identical to SEQ ID NO: 27
(KGVEELEKLSGSLESLS);
Loop peptide K is at least 95% identical to SEQ ID NO: 28
(SEDFTKKLEGEHAQLGIENVT); and
Helix peptide K is at least 95% identical to SEQ ID NO: 29
(AAELEKLFKAVENLAKAAKEM).
In some embodiments, loop and helix peptides from the same phylotype
are positioned together, i.e., adjacent to each other. For example, loop
peptide
from OspC phylotype A and a helix peptide from OspC phylotype A should not
be separated by either the loop or the helix peptide from any other OspC
phylotype.
Further, while in some embodiments, the loop and helix peptides from the
same OspC phylotype are immediately adjacent to each other, in other
embodiments, the loop peptide and the helix peptide may be separated by a
linker sequence which does not affect the structure of the final protein. The
properties of amino acids and their effects on protein structure are well
known in
the art and persons of ordinary skill in the art would be able to recognize
which
amino acids are suitable for the linkers.
As will be demonstrated in the Examples, the inventors have surprisingly
found that F and N are the most prevalent OspC phylotypes associated with
Lyme disease in dogs. The inventors have also found that the presence of loop
and helix peptides from phylotypes I, H, N, C, M, D and F provides a very good
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level of protection against Lyme disease in dogs. While the order of the loop
and
helix peptides from different phylotypes is not crucial, in some embodiments,
the
second protein comprises, in N- to C-orientation, an l-construct, a H-
construct, a
N-construct, a C-construct, a M-construct, a D-construct, followed by an amino
acid sequence which is at least 95% identical to a fragment of OspC phylotype
F
protein (e.g., SEQ ID NO: 32). Thus, in some embodiments, the second protein
will comprise an amino acid sequence at least 95% (e.g., 96%, 97%, 98%, 99%,
and preferably, 100%) identical to SEQ ID NO: 31 (Al2CF).
In other embodiments, the loop and the helix peptides from phylotypes F,
T, U, E, A, B, K are included within the second protein. In some embodiments,
the second protein, thus, would comprise the following, in N- to C-
orientation: a
T-construct, a U-construct, a E-construct, an A-construct, a B-construct, a K-
construct, the l-construct, the H-construct, the N-construct, the C-construct,
the
M-construct, and the D-construct. Optionally, the second protein can also
comprise an F-construct, which is, in some embodiments, is upstream of the T-
construct. Alternatively, or additionally, the second protein can contain the
amino acid sequence which is at least 95% (e.g., 96%, 97%, 98%, 99%)
identical to the fragment of OspC phylotype F protein (SEQ ID NO: 32).
Other suitable examples of the second protein, as well as methods of
making and using same are provided in Application PCT/U52011/056854 (filed
on Oct 19, 2011, inventors R. Marconi and C. Earnhart).
In certain embodiments, the immunogenic composition would comprise
SEQ ID NO: 1; and either one of SEQ ID NO: 30 or SEQ ID NO: 31.
The sequences described herein may be manufactured by methods well
known in the art. The polypeptides may be produced by direct peptide synthesis
using solid-phase techniques (see, e.g., Stewart et al. (1969) Solid-Phase
Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J. (1963) J Am
Chem Soc 85:2149-2154). Peptide synthesis may be performed using manual
techniques or by automation. Automated synthesis may be achieved, for
example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer,
Foster City, Calif.), in accordance with the instructions provided by the
manufacturer. For example, subsequences may be chemically synthesized
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separately and combined using chemical methods to provide full-length
polypeptides or fragments thereof. Alternatively, such sequences may be
ordered from any number of companies which specialize in production of
polypeptides. Most commonly, polypeptides may be produced by expressing
coding nucleic acids and recovering polypeptides, as described below.
For example, in embodiments where loop peptides and the helix peptides
are 100% identical to the fragments of OspC proteins of the target phylotypes,
the nucleic acid sequences of such loop and helix peptides are also known or
easily accessible from publicly available databases, e.g., Genbank. If the
selected loop/helix peptides are somewhat different from the naturally
occurring
fragments of OspC proteins, the encoding nucleic acid sequences can be easily
designed using well known genetic code.
Many organisms display bias for use of particular codons to code for
insertion of a particular amino acid in a growing peptide chain. Codon
preference
or codon bias, differences in codon usage between organisms, is well
documented among many organisms. Codon bias often correlates with the
efficiency of translation of messenger RNA (mRNA), which is in turn believed
to
be dependent on, inter alia, the properties of the codons being translated and
the
availability of particular transfer RNA (tRNA) molecules. The predominance of
selected tRNAs in a cell is generally a reflection of the codons used most
frequently in peptide synthesis. Accordingly, since the majority of amino
acids
are encoded by multiple codons (methionine is the exception), the nucleic acid
sequences can be tailored for optimal gene expression in a given organism
based on codon optimization.
Methods for producing recombinant polypeptides are also included. One
such method comprises introducing into a population of cells any nucleic acid
as
described above, which is operatively linked to a regulatory sequence
effective
to produce the encoded polypeptide, culturing the host cells (e.g., yeast,
insect,
mammalian cells, plant cells, etc) in a culture medium to express the
polypeptide, and isolating the polypeptide from the cells or from the culture
medium. The nucleic acid is introduced into such cells by any delivery method
as
is known in the art, including, e.g., transformation, transfection, injection,
gene
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gun, passive uptake, etc. As one skilled in the art will recognize, the
nucleic acid
may be part of a vector, such as a recombinant expression vector, including a
DNA plasmid vector, or any vector as known in the art.
Alternatively, cell-free prokaryotic or eukaryotic-based expression
systems may be used.
In some embodiments, the nucleic acid sequence encoding the first
and/or second protein, may further comprise a sequence encoding a polypeptide
(the "fusion partner") that is fused to the first and/or second protein,
thereby
facilitating purification of the fusion protein. In certain embodiments of
this aspect
of the invention, the fusion partner is a hexa-histidine peptide (SEQ ID NO:
47,
HHHHHH), as provided in the pQE vector (Qiagen, Inc.), and described in Gentz
et al., Proc Natl Acad Sci USA 86:821-824 (1989), or it may be the HA tag,
which
corresponds to an epitope derived from the influenza hemagglutinin protein
(Wilson, I., et al., Cell 37:767, 1984). The polynucleotide may also contain
non-
coding 5' and 3' sequences, such as transcribed, non-translated sequences,
splicing and polyadenylation signals, ribosome binding sites and sequences
that
stabilize mRNA.
The immunogenic compositions described herein are particularly suitable
for preventing or diminishing the severity of symptoms of Lyme disease in
dogs.
Thus, in another aspect, the instant invention provides a vaccine, comprising
the
immunogenic composition according to any of the embodiments described
above, and a suitable adjuvant.
The first and the second proteins of the immunogenic composition of the
instant invention should be present in immunologically effective amount, i.e.,
in
an amount sufficient to trigger the immune response in the dog. In some
embodiments, the concentration of the first protein is between 1 and 100 ug/ml
(e.g., 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 ug/ml), and the concentration of
the
second protein is between 1 and 200 ug/ml (e.g., 5, 10, 20, 30, 40, 50, 60,
70,
80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 ug/ml). In some
embodiments, the amount of the first protein is between about 10 and 50 ug/ml
and the amount of the second protein is between 20 and 100 ug/ml.
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Adjuvants suitable for use in accordance with the present invention
include, but are not limited to several adjuvant classes such as; mineral
salts,
e.g., Alum, aluminum hydroxide, aluminum hydroxide gels (e.g., Rehydrage10),
aluminum phosphate and calcium phosphate; surface-active agents and
microparticles, e.g., nonionic block polymer surfactants, cholesterol,
virosomes,
saponins (e.g., Quil A, QS-21 and GPI-0100), proteosomes, immune stimulating
complexes, cochleates, quarterinary amines (dimethyl diocatadecyl ammonium
bromide (DDA)), pyridine, vitamin A, vitamin E; bacterial products such as the
RIB! adjuvant system (Ribi Inc.), cell wall skeleton of Mycobacterum ph/el
(Detox0), muramyl dipeptides (MDP) and tripeptides (MTP), monophosphoryl
lipid A, Bacillus Ca!mete-Guerin, heat labile E. coli enterotoxins, cholera
toxin,
trehalose dimycolate, CpG oligodeoxnucleotides; cytokines and hormones, e.g.,
interleukins (IL-1, IL-2, IL-6, IL-12, IL-15, IL-18), granulocyte-macrophage
colony
stimulating factor, dehydroepiandrosterone, 1,25-dihydroxy vitamin D3;
polyanions, e.g., dextran; polyacrylics (e.g., polymethylmethacrylate,
Carbopol
934P); carriers e.g., tetanus toxid, diptheria toxoid, cholera toxin B
subnuit,
mutant heat labile enterotoxin of enterotoxigenic E. coli (rmLT), heat shock
proteins; oil-in-water emulsions e.g., AMPHIGENO (Hydronics, USA); and water-
in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants. In
other embodiments, SP oil may also be used. As used herein, the term "SP oil"
designates an oil emulsion comprising a polyoxyethylene-polyoxypropylene
block copolymer, squalane, polyoxyethylene sorbitan monooleate and a buffered
salt solution. In general, the SP oil emulsion will comprise about 1 to 3%
vol/vol
of block copolymer, about 2 to 6% vol/vol of squalane, more particularly about
3
to 6% of squalane, and about 0.1 to 0.5% vol/vol of polyoxyethylene sorbitan
monooleate, with the remainder being a buffered salt solution.
The vaccines described herein may be combination vaccines which
include the immunogenic composition described above, in combination with at
least one antigen from other canine pathogens, capable of inducing a
protective
immune response in dogs against disease caused by such other pathogens.
Such other pathogens include, but are not limited to, canine distemper
(CD) virus, canine adenovirus type 2 (CAV-2), canine parainfluenza (CPI)
virus,
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canine parvovirus (CPV), canine coronavirus (CCV), canine herpesvirus, and
rabies virus. Antigens from these pathogens for use in the vaccine
compositions
of the present invention can be in the form of a modified live viral
preparation, an
inactivated viral preparation, or a subunit protein preparation. In other
embodiments, a recombinant CDV (Canine Distemper Virus) may also be used.
Methods of attenuating virulent strains of these viruses, and methods of
making
an inactivated viral preparation, are known in the art, and are described in,
e.g.,
U.S. Pat. Nos. 4,567,042 and 4,567,043.
Other pathogens also include Leptospira bratislava, Leptospira canicola,
Leptospira grippotyphosa, Leptospira icterohaemorrhagiae, Leptospira pomona,
Leptospira hardjobovis, Porphyromonas spp., Bacteriodes spp., Leishmania
spp., Ehrlichia spp., Mycoplasma ssp., Anaplasma spp. and Microsporum canis.
Antigens from these pathogens for use in the vaccine compositions of the
present invention can be in the form of an inactivated whole or partial cell
preparation, using methods well-known in the art. For example, methods of
making an inactivated whole or partial Leptospira cell preparation are known
in
the art and are described in, e.g., Yan, K-T, "Aspects of Immunity to
Leptospira
borgpetersenii serovar hardjo", PhD Thesis, Appendix I, 1996. Faculty of
Agriculture and Food Science, The Queen's University of Belfast; Mackintosh et
al., "The use of a hardjo-pomona vaccine to prevent leptospiruria in cattle
exposed to natural challenge with Leptospia interrogans serovarhardjo", New
Zealand Vet. J. 28:174-177, 1980; Bolin et. al., "Effect of vaccination with a
pentavalent leptopsiral vaccine on Leptospira interrogans serovar hardjo type
hardjo-boivs infection of pregnant cattle", Am. J. Vet. Res. 50:161-165, 1989.
In accordance with the present invention, vaccines can be administered to
a dog of at least 6 weeks old, or at least 7 weeks old, or at least 8 or 9
weeks
old. The administration can be done by any known routes, including the oral,
intranasal, mucosal topical, transdermal, and parenteral (e.g., intravenous,
intraperitoneal, intradermal, subcutaneous or intramuscular). Administration
can
also be achieved using needle-free delivery devices. Administration can also
be
achieved using a combination of routes, e.g., first administration using a
parental
route, and subsequent administration using a mucosa! route. In some
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embodiments, routes of administration include subcutaneous and intramuscular
administrations.
All publications cited in the specification, both patent publications and
non-patent publications, are indicative of the level of skill of those skilled
in the
art to which this invention pertains.
For a clearer understanding of the invention, the following examples are
set forth below. These examples are merely illustrative, and are not
understood
to limit the scope or underlying principles of the invention in any way.
Indeed,
various modifications of the invention, in addition to those shown and
described
herein, will become apparent to those skilled in the art from the examples set
forth hereinbelow and the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims.
EXAMPLES
Example 1. Determination of B. burgdorferi OspC phylotypes
associated with Lyme disease in dogs.
Adult &odes scapularis ticks were collected in southern Rhode Island by
flagging. The percentage of ticks infected with B. burgdorferi was determined
through direct fluorescent microscopy using standard methods and labeled anti-
B. burgdorferi antibody.
All procedures were conducted in compliance with regulations of the
Animal Welfare Act and the dogs were maintained in accordance with Farm
Canine Husbandry Standard Operating Procedures. Fifteen purpose-bred dogs
of both sexes (7 males, 8 females; 9 to 10 weeks of age; Marshall
Bioresources)
were assigned identification numbers and divided into four study groups
designated as 101 (n=4), T02 (n=4), 103 (n=4) and T04 (n=3). The dogs were
fitted with Elizabethan collars and housed in one-over-one condo style cages.
One day prior to tick infestation serum was collected from each dog. Dogs in
study groups T01, T02, 103 and 104 were infested with 0, 25, 50 or 75 adult
lxodes scapularis ticks, respectively, using secured infestation chambers
placed
on each side of the midthorax. The ticks were fed to repletion, removed and
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serum samples and skin biopsies were collected at 49 and 90 days (relative to
the start of infestation). Seroconversion was assessed with the SNAP 4DX test
(IDEXX). To cultivate spirochetes, a portion of each skin biopsy was placed in
BSK-H media (6% rabbit serum; 37EC, 5% CO2). Clonal populations were
obtained from the cultures by sub-surface plating as previously described.
Colonies were excised from the plates and placed in BSK-H media for
cultivation.
DNA was extracted from skin biopsies using the Qiagen DNeasy Kit as
instructed by the supplier. In addition, DNA was extracted from cultures of
the
clonal populations of B. burgdorferi as previously described. The ospC gene
was
PCR amplified using DNA extracted from tissues (100 ng) and from DNA
obtained from boiled B. burgdorferi cell lysates (1:1 supernatant; GoTaq
polymerase). All PCR was performed using standard conditions. A portion of
each reaction was assessed by agarose gel electrophoresis and ethidium
bromide staining. The remaining PCR products were excised from the gels
(Qiagen Gel Extraction Kit; QIAGEN) and annealed with the pET46Kk/LIC vector
(Novagen). The plasmids were propagated in E. coli NovaBlue cells (Novagen).
Colonies were screened for the ospC gene by PCR. The templates for PCR
were generated by boiling a portion of each ospC positive, E. coli colony.
Portions of the colonies were also inoculated into LB media (2 ml), grown
overnight, harvested by centrifugation and plasmid extracted using the Qiagen
MiniPrep kit (QIAGEN). The primers used for PCR are as follows (5' to 3'):
OspC-F1
GACGACGACAAGATTGAATACATTAAGTGCAATATTAATGAC (SEQ
ID NO: 33)
OspC-R1
GAG GAG AAG CCCGG TTTACAAATTAATCTTATAATATTGATCTTAAT
TAAGG (SEQ ID NO: 34)
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DNA sequencing was performed by Eurofins MWG Operon. Neighbor
joining trees were generated using ClustaIX 2Ø10 software in the multiple
alignment mode with the default settings and a Gonnet matrix and visualized
using N-J Plot version 2.2.
Results
Analysis of the prevalence of B. burgdorferi in ticks collected from
Rhode Island. Using direct fluorescent microscopy it was determined that ¨50%
of the Ixodes scapularis ticks field-collected in southern Rhode Island were
infected with B. burgdorferi. This is consistent with previously reported tick
infection rates in this area.
Infection of dogs with Borrelia burgdorferi through tick infestation.
At the start of this study, all dogs were confirmed to be sero-negative for
through
immunoblot analyses and through the use of the B. burgdorferi using the SNAP
4DX assay (IDEXX). To infect dogs with B. burgdorferi via the natural
transmission route, field collected ticks were fed on dogs. Since the
infection
rate in the ticks was ¨50% increasing numbers of ticks (0, 25, 50 or 75) were
placed on the dogs. Serum samples were collected 49 days after tick
infestation and immunological status evaluated. Of the dogs infested with
ticks,
10 of 11 were sero-positive for B. burgdorferi. All negative controls dogs
(not
infested with ticks) were seronegative. Total DNA was extracted from skin
biopsies collected from each dog and tested for B. burgdorferi by PCR with
ospC
and flaB primer sets. All seropositive dogs yielded ospC and flaB amplicons of
the predicted size. All seronegative dogs were PCR negative for both genes.
Analysis of OspC diversity in strains found in infected dog tissues.
To determine the ospC genotype of strains that persisted in the skin of dogs
exposed to ticks, ospC was PCR amplified from DNA extracted from skin
biopsies. The resulting amplicons were cloned into pET46 Ek/LIC and the
plasmids propagated in E. co/i. Plasmid was then isolated from no less than 5
separate E. coli colonies and the ospC sequences determined. Sequence
alignment and dendogram construction (Figure 1) demonstrated the persistence
of strains producing several different ospC types in 6 of the 10 dogs (Table
1).
Since multiple ticks were used to infect each dog this observation is not
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surprising. OspC types A, B, F, 1 and N were identified with types F and N
being
the most prevalent (5/10 and 7/10 dogs, respectively).
To further define the range of ospC genotypes present in the infected
dogs, cultures from the skin biopsies were plated to yield clonal populations.
By
this approach, strains expressing ospC types that were not detected by PCR of
biospy samples can be identified. Individual B. burgdorferi colonies were then
tested for ospC by PCR (Figure 2). Additional ospC types were identified in 3
of
6 dogs. Two of the identified OspC types, both of which originated from the
same
dog, had not been previously identified. These phyletic types were designated
as
DRI85a and DRI85e. Other ospC types identified by this approach included
types E, F, H, 1, N, U and T (Table 1). Collectively, in these analyses a
total of 11
different OspC types were detected.
Table 1. OspC types from sequencing biopsies and clonal isolates per
group and per individual dog.
Study group OspC Types
Group # Dog ID Biopsy Culture
TO1 controls negative negative
T02 DRI85 A, N U, DRI85a, DRI85e
DRI63 N not analyzed
DRI16 A, N E
DR103 B, F, N H, N
T03 DR109 1, N not analyzed
DR105 F E, U
DRI41 B, 1, N not analyzed
DRI83 F E
T04 DRI40 F, N E, 1, F, T
DRI73 F not analyzed
In this study the inventors determined the ospC genotype of Lyme
disease spirochete strains that successfully established infection and
persisted
in dogs. Field collected Ixodes scapularis ticks from Rhode Island were fed on
laboratory dogs and the ospC genotype of strains present in skin after 49 days
was determined. A total of 11 different OspC types were identified. OspC type
F,
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which has not been previously detected in humans, was the most frequently
detected OspC type (50% of infected dogs). Types B, N and U, which occur with
very low frequency in humans were also detected. Two ospC types that have not
been previously defined (DRI85a and DRI85e) were also recovered. The
diversity observed in this study is consistent with earlier studies that
demonstrated the maintenance of several ospC phyletic types within a local B.
burgdorferi population. In that the ticks used in this study were collected
from a
single geographic region, it is possible that strains expressing other OspC
types
that are not well represented in Rhode Island are also competent to infect
dogs.
In spite of this caveat, this study is the first to demonstrate that OspC
types not
previously associated with human infection can efficiently infect dogs, thus
facilitating the rational design of a new generation canine Lyme disease
vaccine.
Example 2. Efficacy of recombinant chimeric Borrelia burgdoferi
OspC/OspA vaccines in dogs.
Thirty dogs, all in good general health, were chosen for the study. Blood
samples were collected prior to the initial vaccination. Dogs received one of
the
following vaccines, as described in Table 2: T01: PBS (control product); T02:
2Oug/m1 OspA + 30 ug/ml Al 2CF (SEQ ID NO: 31); T03: 2Oug/m1 OspA + 30
ug/ml Al OCF (SEQ ID NO: 30). (Al2CF consists of epitopes from multiple OspC
phylotypes, linked together to form a single polypeptide. Al OCF also consists
of
epitopes from multiple OspC phylotypes; its design is similar to that of Al
2CF.)
Dogs were vaccinated twice, at 8 and 11 weeks of age, and then challenged at
14 weeks of age. Following vaccination, dogs were observed for 20 minutes for
reactions or abnormalities. Injection sites were observed on Days 1, 2, 3 and
22,
23, 24 for swelling, pain, heat, abscess, drainage, etc. Each dog was fitted
with
an Elizabethan [E] collar one day prior to placing the ticks, and the dogs
were
monitored for their ability to move, eat and drink with E-collars in place.
Twenty
to forty pairs (male, female) of Ixodes scapularis adult ticks, collected from
the
northeast USA, were placed along the dorsal midline of each dog, and allowed
to
feed until repletion for a period of 7 to 10 days. Serum samples and skin
biopsies were collected at prescribed intervals, and assayed to monitor
infection.
Replete or unattached, non-viable ticks were collected and stored at 4 C. At
the
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end of the challenge, remaining ticks were removed and stored, and dogs were
treated with a topical acaricide according to label, followed by a second
application 30 days later. Dogs were observed daily for overall physical
appearance and behavior. Clinical observations were performed (lameness and
ataxia); if either was observed, that dog's body temperature (tympanic) was
measured / recorded daily, until the clinical signs subsided. Blood
collections
and skin biopsies were performed per protocol, other than deviations in
scheduling / timing, based on receipt of ticks and subsequent infestation.
Punch
skin biopsies were taken near the general site of tick attachments on the
dorsal
cervical region, and timed to coincide with blood collections. Final biopsies
were
taken immediately after euthanasia, and prior to necropsy.
Table 2. Study Design
Study Days ( 7 after Day 21)
Tick Blood
Treatment N IVP I CP Vaccination Infestation Collection Biopsy Necropsy
TO1 10 PBS
Al2CF +
OspA -1,20, 100,
TO2 10
Rehydragel 0 and 21 106-117 100,145,
146,177, 197,198
LV 176, 196 197, 198
A10CF +
T03 10 OspA
Rehydragel
LV
Results
No reactions or abnormalities were observed in any dogs following
vaccination, nor were any abnormalities at the injection site (swelling, pain,
heat,
abscess, drainage, etc.). Body temperatures, measured using a tympanic probe,
did not show an appreciable or sustained elevation. Intermittent lameness
occurred in two dogs vaccinated with Al2CF + OspA (T02), and one dog in the
non-vaccinated group (T01) was lame on Days 192-193. Ataxia was not
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observed in any dogs during the study. Abnormal health events, including
pyoderma, bite wounds, abrasions, loose stools, otitis externa, etc., were
observed in some dogs during the study, but none were attributed to the
vaccines or vaccinations.
A serological response, indicative of active Borrelia burgdoferi infection,
was observed on Day 146 in 8 of 10 control dogs (T01), and in one dog in T02.
Nine control dogs (T01) were serologically positive on Day 177, and all dogs
in
T01 were positive at the study conclusion. In contrast, there was only one dog
in
each vaccinated group that was positive from Day 177 to the conclusion of the
study.
The ticks used in the study were dual infected with B. burgdoferi and
Anaplasma. The results of the serological assay indicate that ticks
successfully
transmitted Anaplasma to the dogs. This supports the specificity of the
vaccine
constructs (T02, T03) against B. burgdoferi only.
ELISA values, expressed as geometric mean titers to each of OspA and
OspC, were significantly different when comparing TO1 versus T02, and TO1
versus T03, on all days, with the exception of the comparison of TO1 vs. T02
for
OspC on Day 146 (Table 3).
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Table 3. ELISA Geometric Means OspA, OspC by Group, and by Phase
Pre-challenge Post-challenge
Antigen Treatment Day 20 Day 100 Day
146 Day 177 Day 198
TO1 89.82a
81.23a 162.45a 151.57a 263.90a
TO2
Al 2CF+ 1299.60 b
5571.52 b 3200.00 b 2599.21 b 1600.00 b
OspA OspA
TO3
Al OCF+ 1437.16 b
5198.41 b 3939.66 b 2262.74 b 1600.00 b
OspA
TO1 100.34 a 93.30
a 1392.88 a 1969.83 a 1714.84 a
TO2
Al 2CF+ 373.21 b 696.44 b 800.00
a'b 696.44 b 527.80 b
OspC OspA
TO3
Al OCF+ 335.91 b 606.29 b 565.69
b 565.69 b 492.46 b
OspA
a, b: values with different superscripts are significantly different P 0.10
Serum samples collected during the post-challenge phase from control
dogs (T01) and dogs in T02 (Al2CF+OspA) were assayed in an ELISA which is
specific for live B. burgdoferi organisms. The geometric mean titers for T01
vs
T02 were: at Day 146, 90 vs 6; Day 177, 116 vs 7; and Day 198, 87 vs 7. Thus,
these results support the vaccine's protective effect against B. burgdoferi.
Skin punch biopsy samples were cultured for viable spirochetes. In group
T01, 4 dogs on Day 146, and 5 dogs on Day 177, were culture positive. One
dog in each of T02 and T03 had a spirochete-positive skin culture on Day 177.
No positive culture was obtained from any group at the conclusion of the
study.
Skin punch biopsies were also assessed by PCR, using tiaB- and ospC- specific
primers, for the presence of B. burgdoferi on Day 146. Five dogs in TO1 were
positive for flaB, while 3 were positive for ospC. No dogs in either T02 or
T03
were positive for either PCR reaction.
Examinations of joints and skin sections microscopically demonstrated
that vaccination with either the T02 or T03 vaccine protected against
infection
(data not shown). Vaccinated dogs had fewer changes in their joints and skin
as
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is characteristic of Lyme disease. If such changes were present, they were
less
severe in vaccinated dogs when compared to tissues from non-vaccinated
control dogs. There was a slight difference between the two vaccines (T02;
T03), based on the number of dogs with lesions in their joints (6 for T02; 7
for
T03). However, a definitive conclusion cannot be drawn as to which construct
provided better protection.
In conclusion, both Al 2CF+OspA (T02) and Al OCF+OspA (T03) were
efficacious in protecting dogs against Borrelia burgdoferi infection as
transmitted
by Ixodes scapularis ticks.
1.0
