Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS .AND METHODS FOR CONFERRING
TICK IMMUPJITY AID PREVENTING TICK BORNE DISEASES
This application claims priority under 35 U.S.C.
~ 120 from pending United States provisional application
Serial Number 60/043,:L54, filed April 29, 1997.
This invention was made with government support
under Grant numk~ers A=f 30548, AI 37993, AI 41440 and AI
39002 awarded by the National Institutes of Health. The
government may have cf~rtain rights in the invention.
TE HNICi~L FIELD OF THE INVENTION
This invention relates to compositions and methods
for conferring ~_mmunii~y to tick bites and for the prevention
of tick-borne d~_sease:~ .
More particularly, this invention relates to
polypeptides, and DNA sequences which encode them, from the
Ixodes scapular_is ticlt. Such polypeptides and DNA sequences
are useful to detect i=ick immunity in a subject, to elicit
an immune response which is effective to prevent or lessen
the duration of tick attachment and feeding and to prevent
or lessen infect=ion o:E a host with tick-borne pathogens.
Also within the scope of this invention are antibodies
directed against. I. suapularis polypeptides, compositions
including vaccines comprising the antibodies.
This inven'~ion also relates to vaccines
comprising one or more of the I. scapularis polypeptides or
antibodies of this invention. Also within the scope of this
invention are d:Lagnosvic kits comprising I. scapularis
polypeptides or antibodies of this invention.
This invention also relates to methods for using
the aforementioned po:lypeptides, DNA sequences and
antibodies are also within the scope of this invention.
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BACKGROUND OF THE INVENTION
Ticks are the most common vector transmitting
diseases to humans in the United States [CDC, 1989. Lyme
Disease - United States, 1987 and 1988. NINIWR Morb. Mortal.
Wkly Rep., 38, 668-672]. They transmit the agents of
important human diseases, such as Lyme disease, babesiosis,
Rocky Mountain spotted fever, ehrlichiosis, and tick-borne
encephalitis. The incidence of tick-borne disease is rising
to the point that such diseases are a major public health
problem. Early treatment, which requires early diagnosis,
is ideal. However, some tick-borne diseases, particularly
Lyme disease and ehrlichiosis, are difficult to diagnose.
As a result, the diseases are often missed and and treatment
early in the disease is not possible. There is an urgent
need, thus, for new methods for the early diagnosis of tick-
borne disease.
Another approach to the problem of tick-borne
diseases is controlling the ticks. However, chemical
control using acaricides poses significant problems for the
environment and public health. In addition, ticks are
developing resistance to the chemicals, making this approach
also not effective. Accordingly, there is an urgent need
for alternative methods for controlling tick infestation.
One method utilizes host immunity to ticks. Tick
immunity is the capacity of previously exposed hosts to
interfere with tick feeding and development. A reduction in
tick weight, duration of attachment, number of ticks
feeding, size of egg mass an molting success are parameters
to measure immunity. Tick immunity, induced by repeated
tick exposure, has been shown in rabbits, cattle, dogs and
guinea pigs [J.R. Allen, "Observation on the Behavior of
Dermacentor andersoni Larvae Infesting Normal and Tick
Resistant Guinea Pigs," Parasitology, 84, pp. 195-204
(1982); M. Brossard et al., "Ixodes ricinus L: Mast Cell,
Basophils and Eosinophils In the Sequence of Cellular Events
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In the Skin of :Cnfested or RE-infested Rabbits,"
Parasitology, 8.'~, pp. 583-592 (1982); Fivaz et al., "Cross-
resistance BetwE~en Instars of the Brown Ear-tick
Rhipicephalus a~~pendiculatus (Acarina:Ixodidae)," Exp. Appl.
Acarol., 11, pp. 323-326 (1991)].
The transmission of tick-borne pathogens, such as
B. burgdorferi requires a prolonged period of feeding. If
the feeding timE~ can :be shortened as a result of tick
immunity, transrnissio:n of some tick-borne pathogens might be
reduced.
Ixodid ticks are the most important arthropod
vectors of infectious agents. Ixodes scapularis is the
vector for Lyme disease, human granulocytic ehrlichiosis
(HGE), babesia and tick-borne encephalitis. Accordingly,
there is an urgent need to identify antigens of I.
scapularis for use in inducing tick immunity.
DISChOSURE OF THE INVENTION
The p~°esent invention solves the problems referred
to above by providing compositions and methods for
conferring and detecting tick immunity and for preventing or
lessening the transmission of tick-borne pathogens. More
particularly, this invention provides I. scapularis
polypeptides, DZJA sequences that encode the polypeptides,
antibodies directed against the polypeptides and
compositions anc~ methods comprising the polypeptides, DNA
sequences and antibodies.
This =invention further provides a single or
multicomponent vaccine comprising one or more I. scapularis
polypeptides or antibodies of this invention.
This invention relates to DNA sequences that code
for I. scapular_is antigens, recombinant DNA molecules that
are characterizE~d by the DNA sequences, unicellular hosts
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transformed with those DNA sequences and molecules, and
methods of using those sequences, molecules and hosts to
produce the I. scapularis polypeptides and vaccines
comprising them. The DNA sequences of the invention are
advantageously used to make oligonucleotides probes and
polymerase chain reaction primers for use in isolating
additional I. scapularis genes.
Also within the scope of this invention are
diagnostic means and methods characterized by I. scapularis
polypeptides or antibodies directed against the
polypeptides. These means and methods are useful for the
detection of tick immunity. They are also useful in
following the course of immunization against tick bites. In
patients previously inoculated with the vaccines of this
invention, the detection means and methods disclosed herein
are also useful for determining if booster inoculations are
appropriate.
This invention further provides an I. scapularis
salivary gland extract and fractions thereof,including
fractions containing protective I. scapularis antigens.
Finally, this invention also provides methods for
the identification and isolation of additional I. scapularis
polypeptides, as well as compositions and methods comprising
such polypeptides.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the duration of attachment of I.
scapularis nymphal ticks to tick immune or naive guinea
pigs. Each point represents the mean of 5 animals ~ SE.
Figure 2 depicts the average weight of ticks
recovered after attachment to the same tick-immune or naive
guinea pigs shown in Figure 1.
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Figure 3 de~~icts the duration of attachment of
nymphal ticks on guinea pigs sensitized to I, scapularis
larvae.
Figure 4 show the results of individual
5 experiments comparing the rate of B. burgdorferi infection
in tick-immune guinea pigs with that of naive guinea pigs
challenged with B. burgdorferi infected nymphal ticks. In
Experiment 1, strain H31 was used. In all subsequent
experiments, strain N40 was used. The infection rate was
determined by the number of guinea pigs with positive
cultures and development of serological conversion.
Figure 5 depicts the separation into 4 peaks of
salivary gland extract from partially fed nymphs on an anion
exchange column.
Figure 6 is a representation of the results of a
cutaneous anaphylaxis assay showing dye extravasation from
the reaction of salivary gland extract or fractions thereof
resolved by anion exchange chromatography to antibodies
present in a salivary-gland immune guinea pig.
Figure 7 sets forth the results of a cutaneous
anaphylaxis assay with 14 fractions of salivary gland
extract in a salivary gland immune guinea pig. Rare: scarce
presence of mononuclear leukocytes, heterophils and
eosinophils in papillary dermis; +: slight but real
increase; ++: definite increase; +++: relatively marked
increase.
Figure 8 depicts the DNA and amino acid sequences
of the SP16 polypeptide (SEQ ID NOS: 1 and 2).
DETA7:LED DhSCRIPTION OF THE INVENTION
This i:avention relates to I. scapularis
polypeptides and DNA sequences encoding them, antibodies
directed against those polypeptides, compositions comprising
the polypeptides, DNA sequences or antibodies. This
invention further relates to methods for identifying
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additional I. scapularis polypeptides and antibodies and
methods for conferring and detecting tick immunity and for
preventing or lessening the transmission of tick-borne
pathogens.
S More specifically, in one embodiment, this
invention provides a 16 kD I. scapularis polypeptide and
compositions and methods comprising the polypeptide.
In another embodiment, this invention provides a
32 kD polypeptide expressed by Clones 1 and 2 (ATCC
accession No. ), and compositions and methods comprising
the polypeptides.
In another embodiment, this invention provides a
28 kD I, scapularis polypeptide isolated as a single band on
a 12'~ SDS-PAGE gel from Fraction 9 of I. scapularis salivary
gland extract, and compositions and methods comprising the
polypeptide.
In another embodiment, this invention provides a
40 kD I. scapularis polypeptide isolated as a single band on
a 12o SDS-PAGE gel from Fraction 10 of I. scapularis
salivary gland extract, and compositions and methods
comprising the polypeptide.
In another embodiment, this invention provides a
65 kD I. scapularis polypeptide isolated as a single band on
a 12o SDS-PAGE gel from tick saliva, and compositions and
methods comprising the polypeptide.
In another embodiment, this invention provides a
Peak 1 fraction of I. scapularis salivary gland extract
obtained by partial separation of the extract by ion
exchange chromatography and compositions and methods
comprising the polypeptide.
In another embodiment, this invention provides
Fraction 9 of I. scapularis salivary gland extract obtained
by separation on a 12o PAGE gel and gel elution of the
extract, and compositions and methods comprising the
polypeptide.
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S
In another Embodiment, this invention provides
Fraction 10 of .1~. scapularis salivary gland extract obtained
by separation on a 12<'s PAGE gel and gel elution of the
extract, and compositions and methods comprising the
polypeptide.
The preferred compositions and methods of each of
the aforementioned emx~odiments are characterized by
immunogenic polypeptides. As used herein, an "immunogenic
I. scapularis polypept:ide" is any I. scapularis polypeptide
that, when admin.istere:d to an animal, is capable of
eliciting a corresponding antibody. In particular,
immunogenic I. ~~capularis polypeptides are intended to
include additional pol.ypeptides which may be identified
according to the methods disclosed herein.
The most preferred compositions and methods of
each of the aforementioned embodiments are characterized by
I. scapularis polypept.ides which elicit in treated animals,
the formation of a tick immune response. As used herein, a
"tick immune response"' or "tick immunity" is manifested by a
reduction in the duration of tick attachment to a host or a
reduction in the weight of ticks recovered after detaching
from the host compared to those values in ticks that attach
to non-immune hosts, failure of the ticks to complete their
development or failure to lay the normal number of viable
eggs.
In another F~referred embodiment, this invention
provides a vaccine comprising one or more I. scapularis
polypeptides or fractions of this invention or one or more
antibodies directed against the polypeptides or fractions of
this invention.
As used herein, a substantially pure polypeptide
is a polypeptide that is detectable as a single band on an
immunoblot probed with. polyclonal anti-I. scapularis anti-
serum.
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In yet another embodiment, this invention provides
antibodies directed against the I. scapularis polypeptides
of this invention, and pharmaceutically effective
compositions and methods comprising those antibodies. The
antibodies of this embodiment are those that are reactive
with the I. scapularis polypeptides of this invention. Such
antibodies may be used in a variety of applications,
including to detect expression of I_ scapularis antigens, to
screen for expression of novel I. scapularis polypeptides,
to purify novel I. scapularis polypeptides and to confer
tick immunity.
In still another embodiment, this invention
relates to diagnostic means and methods characterized by the
I. scapularis polypeptides, DNA sequences or antibodies of
the invention.
A further embodiment of this invention provides
methods for inducing tick immunity in a host by
administering an I, scapularis polypeptide or antibody of
the invention.
A preferred embodiment of this invention is a
method for preventing or reducing the transmission of tick-
borne pathogens by administering polypeptides or antibodies
of this invention that are effective to induce tick
immunity. A particularly preferred embodiment is a method
for preventing or reducing the severity for some period of
time of B. burgdorferi infection.
In order to further define this invention, the
following terms and definitions are herein provided.
As used herein, an "I. scapularis polypeptide" is
a polypeptide encoded by a DNA sequence of I. scapularis.
For example, I. scapularis polypeptides include the SP16
polypeptide, the 32 kD polypeptides expressed by clones 1
and 2 and appearing as a single band on a Western blot after
reacting with sera from tick immune animals, as described in
Example II; a 28 kD or 40 kD polypeptide detectable as a
single band on SDS-PAGE of Fractions 9 and 10, respectively,
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of I. scapularis salivary gland extract, as described in
Example XIII; or a 65 1tD polypeptide detectable as a single
band on SDS-PAGE of I. scapularis saliva, and fragments or
derivatives thereof .
As used herein, a "protective I. scapularis
polypeptide" is any I. scapularis polypeptide that, when
administered to an animal, elicits an immune response that
is effective to confer tick immunity or to prevent or lessen
the severity, for some period of time, of infection by a
tick-borne pathogen. Preventing or lessening the severity
of infection may be evidenced by a change in the
physiological manifestations of infection with that
pathogen. In a preferred embodiment, the tick-borne
pathogen is B. b~.~rgdorferi, and preventing or lessening the
severity of infection includes erythema migrans, arthritis,
carditis, neurological disorders, and other Lyme disease
related disorder;. It may be evidenced by a decrease in or
absence of spirochetes in the treated animal. And, it may
be evidenced by ;~ decrease in the level of spirochetes in
infected ticks w;nich have fed on treated animals.
One of skill in the art will understand that
probes and oligo:nucleotide primers derived from the DNA
encoding an I. scapularis polypeptide may be used to isolate
and clone further variants of I. scapularis proteins from
other Ixodes isolates and perhaps from other hard bodied
ticks as well, which a.re useful in the methods and
compositions of this invention.
As used herein, a "derivative" an I. scapularis
polypeptide is a polypeptide in which one or more physical,
chemical, or biological properties has been altered. Such
modifications include, but are not limited to: amino acid
substitutions, modifications, additions or deletions;
alterations in the pattern of lipidation, glycosylation or
phosphorylation; reactions of free amino, carboxyl, or
hydroxyl side groups of the amino acid residues present in
the polypeptide with other organic and non-organic
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molecules; and other modifications, any of which may result
in changes in primary, secondary or tertiary structure.
As used herein, a "protective epitope" is (1) an
epitope which is recognized by a protective antibody, and/or
5 (2) an epitope which, when used to immunize an animal,
elicits an immune response sufficient to confer tick
immunity or to prevent or lessen the severity for some
period of time, of infection with a tick-borne pathogen. A
protective epitope may comprise a T cell epitope, a B cell
10 epitope, or combinations thereof.
As used herein, a "protective antibody" is an
antibody that confers tick immunity or protection for some
period of time, against infection by a tick-borne pathogen
or any one of the physiological disorders associated with
such infection. In a preferred embodiment, the antibody
confers protection against B. burgdorferi infection.
As used herein, a "T cell epitope" is an epitope
which, when presented to T cells by antigen presenting
cells, results in a T cell response such as clonal expansion
or expression of lymphokines or other immunostimulatory
molecules. A strong T cell epitope is a T cell epitope
which elicits a strong T cell response.
As used herein, a "B cell epitope" is the simplest
spatial conformation of an antigen which reacts with a
specific antibody.
As used herein, a "therapeutically effective
amount" of a polypeptide or of an antibody is the amount
that, when administered to an animal, elicits an immune
response that is effective to confer tick immunity or to
prevent or lessen the severity, for some period of time, of
infection by a tick borne pathogen.
As used herein, an "an anti-I. scapularis
polypeptide antibody," also referred to as "an antibody of
this invention," is an antibody directed against an I.
scapularis polypeptide of this invention. An anti-I.
scapularis polypeptide antibody of this invention includes
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antibodies directed against polypeptides expressed by I.
scapularis, or fragments or derivatives thereof, that are
immunologically cross--reactive with any one of the
aforementioned ~~olypeptides. Finally, an anti-I. scapularis
polypeptide antibody of this invention includes antibodies
directed against. other I. scapularis polypeptides identified
according to methods t=aught herein.
As used herein, an "anti-I. scapularis polypeptide
antibody" is an immunoglobulin molecule, or portion thereof,
that is immunological:Ly reactive with an I. scapularis
polypeptide of t:he present invention and that was either
elicited by immunization with I. scapularis or an I.
scapularis polypeptide of this invention or was isolated or
identified by it:s rea~~tivity with an I. scapularis
polypeptide of this invention.
An anti-I. scapularis polypeptide antibody may be
an intact immunoglobulin molecule or a portion of an
immunoglobulin molecule that contains an intact antigen
binding site, including those portions known in the art as
F(v), Fab, Fab' and F(ab')2. It should be understood that
an anti-I. scapularis polypeptide antibody may also be a
protective antibody.
The I. scapularis polypeptides disclosed herein
are immunologic~311y reactive with antisera generated by
immunization with I. scapularis extracts or by tick bite.
Accordingly, they are useful in methods and compositions to
detect tick immunity.
In addition, because at least some, if not all of
the I. scapularis polypeptides disclosed herein are
protective proteins, they are particularly useful in single
and multicomponent vaccines against tick bites and infection
by tick-borne pathogens. In this regard, multicomponent
vaccines are preferred because such vaccines may be
formulated to more closely resemble the immunogens presented
by tick bite, and because such vaccines are more likely to
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confer broad-spectrum protection than a vaccine comprising
only a single I. scapularis polypeptide.
Multicomponent vaccines according to this
invention may also contain polypeptides which characterize
other vaccines useful for immunization against diseases such
as, for example, Lyme disease, human monocytic ehrlichiosis,
babesiosis, diphtheria, polio, hepatitis, and measles. Such
multicomponent vaccines are typically incorporated into a
single composition.
The preferred compositions and methods of this
invention comprise I. scapularis polypeptides having
enhanced immunogenicity. Such polypeptides may result when
the native forms of the polypeptides or fragments thereof
are modified or subjected to treatments to enhance their
immunogenic character in the intended recipient.
Numerous techniques are available and well known
to those of skill in the art which may be used, without
undue experimentation, to substantially increase the
immunogenicity of the I. scapularis polypeptides herein
disclosed. For example, I. scapularis polypeptides of this
invention may be modified by coupling to dinitrophenol
groups or arsanilic acid, or by denaturation with heat
and/or SDS. Particularly if the polypeptides are small,
chemically synthesized polypeptides, it may be desirable to
couple them to an immunogenic carrier. The coupling, of
course, must not interfere with the ability of either the
polypeptide or the carrier to function appropriately. For a
review of some general considerations in coupling
strategies, see Antibodies, A Laborator~r Manual, Cold Spring
Harbor Laboratory, ed. E. Harlow and D. Lane (1988).
Useful immunogenic carriers are well known in the
art. Examples of such carriers are keyhole limpet
hemocyanin (KLH); albumins such as bovine serum albumin
(BSA) and ovalbumin, PPD (purified protein derivative of
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tuberculin); red blood cells; tetanus toxoid; cholera
toxoid; agarose beads; activated carbon; or bentonite.
Modification of the amino acid sequence of the I.
scapularis polypeptidE:s disclosed herein in order to alter
the lipidation state is also a method which may be used to
increase their immunoc~enicity or alter their biochemical
properties. For example, the polypeptides or fragments
thereof may be expressed with or without the signal and
other sequences that raay direct addition of lipid moieties.
As will be apparent from the disclosure to follow,
the polypeptides may also be prepared with the objective of
increasing stability or rendering the molecules more
amenable to purification and preparation. One such
technique is to expre:~s the polypeptides as fusion proteins
comprising other I. s~~apularis or non-I. scapularis
sequences.
In accordance with this invention, derivatives of
the I. scapular~:s pol;ypeptides may be prepared by a variety
of methods, inc7_uding by in Vitro manipulation of the DNA
encoding the native polypeptides and subsequent expression
of the modified DNA, by chemical synthesis of derivatized
DNA sequences, or by chemical or biological manipulation of
expressed amino acid sequences.
For example, derivatives may be produced by
substitution of one or more amino acids with a different
natural amino acid, an amino acid derivative or non-native
amino acid. These of skill in the art will understand that
conservative substitution is preferred, e.g.,
3-methylhistidine may be substituted for histidine,
4-hydroxyprolin~~ may be substituted for proline,
5-hydroxylysine may b~e substituted for lysine, and the like.
Furthermore., one of skill will recognize that
individual substitutions, deletions or additions which
alter, add or delete a single amino acid or a small
percentage of amino acids (typically less than 50, more
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typically less than lo) in an encoded sequence are
"conservatively modified variations" where the alterations
result in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution
tables providing functionally similar amino acids are well
known in the art. The following six groups each contain
amino acids that are conservative substitutions for one
another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V) ; and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
See also, Creighton (1984) Proteins W.H. Freeman and
Company.
Conservative substitutions typically include the
substitution of one amino acid for another with similar
characteristics such as substitutions within the following
groups: valine, glycine; glycine, alanine; valine,
isoleucine; aspartic acid, glutamic acid; asparagine,
glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. The non-polar (hydrophobic) amino
acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. The polar neutral
amino acids include glycine, serine, threonine, cysteine,
tyrosine, asparagine and glutamine. The positively charged
(basic) amino acids include arginine, lysine and histidine.
The negatively charged (acidic) amino acids include aspartic
acid and glutamic acid.
Other conservative substitutions can be taken from
Table 1, and yet others are described by Dayhoff in the
Atlas of Protein Sequence and Structure (1988).
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Causincl amino acid substitutions which are less
conservative may also :result in desired derivatives, e.g.,
by causing changes in ~~harge, conformation and other
biological properties. Such substitutions would include for
5 example, substitution of a hydrophilic residue for a
hydrophobic residue, substitution of a cysteine or proline
for another resi<~ue, substitution of a residue having a
small side chain for a residue having a bulky side chain or
substitution of a residue having a net positive charge for a
10 residue having a net negative charge.
When the result of a given substitution cannot be
predicted with certainty, the derivatives may be readily
assayed accordin~~ to the methods disclosed herein to
determine the presence or absence of the desired
15 characteristics. In particular, the immunogenicity,
immunodominance and/or protectiveness of a derivative of
this invention can be readily determined using methods
disclosed in the Examples.
In a preferred embodiment of this invention, the
I. scapularis.polypept.ides disclosed herein are prepared as
part of a larger fusion protein. For example, an I.
scapularis polypeptide of this invention may be fused at its
N-terminus or C-terminus to a different immunogenic I.
scapularis polypeptide, to a non-I. scapularis polypeptide
or to combinations thereof, to produce fusion proteins
comprising the T. scapularis polypeptide.
In a preferred embodiment of this invention,
fusion proteins compr'.sing I. scapularis polypeptides are
constructed comF~risin<1 B cell and/or T cell epitopes from
multiple seroty~>ic variants of I. scapularis, each variant
differing from another with respect to the locations or
sequences of the epitopes within the polypeptide. In a more
preferred embodiment, fusion proteins are constructed which
comprise one or more c~f the I. scapularis polypeptides fused
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to other I. scapularis polypeptides. Such fusion proteins
are particularly effective in the induction of tick immunity
against a wide spectrum of isolates.
In another preferred embodiment of this invention,
the I. scapularis polypeptides are fused to moieties, such
as immunoglobulin domains, which may increase the stability
and prolong the in vivo plasma half-life of the polypeptide.
Such fusions may be prepared without undue experimentation
according to methods well known to those of skill in the
art, for example, in accordance with the teachings of United
States patent 4,946,778, or United States patent 5,116,964.
The exact site of the fusion is not critical as long as the
polypeptide retains the desired biological activity. Such
determinations may be made according to the teachings herein
or by other methods known to those of skill in the art.
It is preferred that the fusion proteins
comprising the I. scapularis polypeptides be produced at the
DNA level, e.g., by constructing a nucleic acid molecule
encoding the fusion protein, transforming host cells with
the molecule, inducing the cells to express the fusion
protein, and recovering the fusion protein from the cell
culture. Alternatively, the fusion proteins may be produced
after gene expression according to known methods.
The I. scapularis polypeptides may also be part of
larger multimeric molecules which may be produced
recombinantly or may be synthesized chemically. Such
multimers may also include the polypeptides fused or coupled
to moieties other than amino acids, including lipids and
carbohydrates.
Preferably, the multimeric proteins will consist
of multiple T or B cell epitopes or combinations thereof
repeated within the same molecule, either randomly, or with
spacers (amino acid or otherwise) between them.
In a preferred embodiment of this invention, I.
scapularis antigens are incorporated into a vaccine.
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In another preferred embodiment of this invention,
an I. scapularis polypeptide of this invention which is also
a protective I. scapularis polypeptide is incorporated into
a single component vac~~ine. In a more preferred embodiment
of this invention, I. scapularis polypeptides of this
invention which are also protective polypeptides are
incorporated into a multicomponent vaccine comprising other
protective polypeptides. In addition, a multicomponent
vaccine may also contain protective polypeptides useful for
immunization against other diseases such as, for example,
Lyme disease, hwnan monocytic ehrlichiosis, babesiosis,
diphtheria, poli«, hepatitis, and measles. Such a vaccine,
by virtue of its ability to elicit antibodies to a variety
of protective I. scapularis polypeptides, will be effective
to protect against tick bite by a broad spectrum of ticks,
even those that may not express one or more of the I.
scapularis proteins.
The multicom.ponent vaccine may contain the I.
scapvlaris polypeptides as part of a multimeric molecule in
which the various components are covalently associated.
Alternatively, it may contain multiple individual
components. For exam~~le, a multicomponent vaccine may be
prepared comprising tyro or more of the I. scapularis
polypeptides, wherein each polypeptide is expressed and
purified from independent cell cultures and the polypeptides
are combined prior to or during formulation.
Alternative7.y, a multicomponent vaccine may be
prepared from heterodimers or tetramers wherein the
polypeptides have been fused to immunoglobulin chains or
portions thereof. Such a vaccine could comprise, for
example, an SP1E~ polypeptide fused to an immunoglobulin
heavy chain and polypeptide from Fraction 9, fused to an
immunoglobulin light chain, and could be produced by
transforming a host cE:ll with DNA encoding the heavy chain
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18
fusion and DNA encoding the light chain fusion. One of
skill in the art will understand that the host cell selected
should be capable of assembling the two chains
appropriately. Alternatively, the heavy and light chain
fusions could be produced from separate cell lines and
allowed to associate after purification.
The desirability of including a particular
component and the relative proportions of each component may
be determined by using the assay systems disclosed herein,
or by using other systems known to those in the art. Most
preferably, the multicomponent vaccine will comprise
numerous T cell and B cell epitopes of protective I.
scapularis polypeptides.
This invention also contemplates that the I.
scapularis polypeptides of this invention, either alone or
combined, may be administered to an animal via a liposome
delivery system in order to enhance their stability and/or
immunogenicity. Delivery of the I. scapularis polypeptides
via liposomes may be particularly advantageous because the
liposome may be internalized by phagocytic cells in the
treated animal. Such cells, upon ingesting the liposome,
would digest the liposomal membrane and subsequently present
the polypeptides to the immune system in conjunction with
other molecules required to elicit a strong immune response.
The liposome system may be any variety of
unilamellar vesicles, multilamellar vesicles, or stable
plurilamellar vesicles, and may be prepared and administered
according to methods well known to those of skill in the
art, for example in accordance with the teachings of United
States patents 5, 169, 637, 4, 762, 915, 5, 000, 958 or 5, 185, 154 .
In addition, it may be desirable to express the I.
scapularis polypeptides of this invention, as well as other
selected I. scapularis polypeptides, as lipoproteins, in
order to enhance their binding to liposomes.
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I9
Any of i:he I. scapularis polypeptides of this
invention may be used in the form of a pharmaceutically
acceptable salt. Suitable acids and bases which are capable
of forming salts with the polypeptides of the present
invention are wel:L known to those of skill in the art, and
include inorganic and organic acids and bases.
According to this invention, we describe a method
which comprises the steps of treating an animal with a
therapeutically effective amount of an I. scapularis
polypeptide, or a fusion protein or a multimeric protein
comprising an I. scapularis polypeptide, in a manner
sufficient to confer tick immunity or prevent or lessen the
severity, for some period of time, of infection by a tick-
borne pathogen. The polypeptides that are preferred for use
in such methods are those that contain protective epitopes.
Such protective epitope~s may be B cell ep.itopes, T cell
epitopes, or combinations thereof.
According to another embodiment of this invention,
we describe a method wr~ich comprises the steps of treating
an animal with a multicomponent vaccine comprising a
therapeutically effective amount of an I. scapularis
polypeptide, or a fusion protein or multimeric protein
comprising such polypeptide in a manner sufficient to confer
tick immunity or prevent or lessen the severity, for some
period of time, cf infE~ction by a tick-borne pathogen.
Again, the polype:ptides, fusion proteins and multimeric
proteins that are: prefE~rred for use in such methods are
those that contain protective epitopes, which may be B cell
epitopes, T cell epitohes, or combinations thereof.
The mo:>t preferred polypeptides, fusion proteins
and multimeric proteins for use in these compositions and
methods are those containing both strong T cell and B cell
epitopes. Without being bound by theary, we believe that
this is the best way t~o stimulate high titer antibodies that
are effective to confer tick immunity. Such preferred
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polypeptides will be internalized by B cells expressing
surface immunoglobulin that recognizes the B cell
epitope(s). The B cells will then process the antigen and
present it to T cells. The T cells will recognize the T
5 cell epitope(s) and respond by proliferating and producing
lymphokines which in turn cause B cells to differentiate
into antibody producing plasma cells. Thus, in this system,
a closed autocatalytic circuit exists which will result in
the amplification of both B and T cell responses, leading
10 ultimately to production of a strong immune response which
includes high titer antibodies against the I. scapularis
polypeptide.
One of skill in the art will also understand that
it may be advantageous to administer the I. scapularis
15 polypeptides of this invention in a form that will favor the
production of T-helper cells type 1 (THl), which help
activate macrophages, and/or T-helper cells type 2 (T"2),
which help B cells to generate antibody responses. Aside
from administering epitopes which are strong T cell or B
20 cell epitopes, the induction of THl or TH2 cells may also be
favored by the mode of administration of the polypeptide.
For example, I. scapularis polypeptides may be administered
in certain doses or with particular adjuvants and
immunomodulators, for example with interferon-gamma or
interleukin-12 (THl response) or interleukin-4 or
interleukin-10 (T"2 response).
To prepare the preferred polypeptides of this
invention, in one embodiment, overlapping fragments of the
I. scapularis polypeptides of this invention are constructed
as described herein. The polypeptides that contain B cell
epitopes may be identified in a variety of ways for example
by their ability to (1) remove protective antibodies from
polyclonal antiserum directed against the polypeptide or
(2) elicit an immune response which is effective to confer
tick immunity.
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21
Alternai;ively, the polypeptides may be used to
produce monoclona=L antibodies which are screened for their
ability to confer tick immunity when used to immunize naive
animals. Once a given monoclonal antibody is found to
confer protection,, the particular epitope that is recognized
by that antibody may then be identified.
As recognition of T cell epitopes is MHC
restricted, the polypeptides that contain T cell epitopes
may be identified in vitro by testing them for their ability
to stimulate pro liferation and/or cytokine production by T
cell clones gener,~ted from humans of various HLA types, from
the lymph nodes, spleens, or peripheral blood lymphocytes of
C3H or other laboratory mice, or from domestic animals.
Compositions comprising multiple T cell epitopes recognized
by individuals with different Class II antigens are useful
for prevention and treatment of human granulocytic
ehrlichiosis in a broad spectrum of patients.
In a preferred embodiment of the present
invention, an I. scapularis polypeptide containing a B cell
epitope is fused to one or more other irr~~unogenic I.
scapularis polypeptides containing strong T cell epitopes.
The fusion protein that. carries both strong T cell and B
cell epitopes is able t:o participate in elicitation of a
high titer antibody re~~ponse effective to confer tick
immunity.
Strong T cell. epitopes may also be provided by
non-I. scapulari~; molecules. For example, strong T cell
epitopes have been obsE:rved in hepatitis B virus core
antigen (HBcAg). Furthermore, it has been shown that
linkage of one of these segments to segments of the surface
antigen of Hepatitis B virus, which are poorly recognized by
T cells, results in a major amplification of the anti-HBV
surface antigen response, jD.R. Milich et al., "Antibody
Production To The NuclE~ocapsid And Envelope Of The Hepatitis
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22
B Virus Primed By A Single Synthetic T Cell Site", N re,
329, pp. 547-49 (1987) ] .
Therefore, in yet another preferred embodiment, B
cell epitopes of the I. scapularis polypeptides are fused to
segments of HBcAG or to other antigens which contain strong
T cell epitopes, to produce a fusion protein that can elicit
a high titer antibody response against I. scapularis
antigens. In addition, it may be particularly advantageous
to link an I. scapularis polypeptide of this invention to a
strong immunogen that is also widely recognized, for example
tetanus toxoid.
It will be readily appreciated by one of ordinary
skill in the art that the I. scapularis polypeptides of this
invention, as well as fusion proteins and multimeric
proteins containing them, may be prepared by recombinant
means, chemical means, or combinations thereof.
For example, the polypeptides may be generated by
recombinant means using the DNA sequence as set forth in the
sequence listing contained herein. DNA encoding serotypic
variants of the polypeptides may likewise be cloned, e.g.,
using PCR and oligonucleotide primers derived from the
sequence herein disclosed.
In this regard, it may be particularly desirable
to isolate the genes encoding I. scapvlaris polypeptides
from isolates that differ antigenically, i.e., Ixodes
isolates against which I. scapularis polypeptides are
ineffective to protect, in order to obtain a broad spectrum
of different epitopes which would be useful in the methods
and compositions of this invention.
Oligonucleotide primers and other nucleic acid
probes derived from the genes encoding the I, scapvlaris
polypeptides of this invention may also be used to isolate
and clone other related proteins from I. scapularis and
related ticks which may contain regions of DNA sequence
homologous to the DNA sequences of this invention.
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23
If the I. scapularis polypeptides of this
invention are produced recombinantly, they may be expressed
in unicellular hosts. As is well known to one of skill in
the art, in order to obtain high expression levels of
foreign DNA sequences in a host, the sequences are generally
operatively linked to t:ranscriptional and translational
expression control sequences that are functional in the
chosen host. Preferab=Ly, the expression control sequences,
and the gene of interest, will be contained in an expression
vector that further comprises a selection marker.
The DNA sequE~nces encoding the polypeptides of
this invention may or may not encode a signal sequence. If
the expression host is eukaryotic, it generally is preferred
that a signal seduence be encoded so that the mature protein
is secreted from the eukaryotic host.
An amino terminal methionine may or may not be
present on the e~~press~~d polypeptides of this invention. If
the terminal methionine is not cleaved by the expression
host, it may, if desired, be chemically removed by standard
techniques.
A wide variety of expression host/vector
combinations may be employed in expressing the DNA sequences
of this invention. Useful expression vectors for eukaryotic
hosts, include, :Eor example, vectors comprising expression
control sequences from SV40, bovine papilloma virus,
adenovirus, aden«-associated virus, cytomegalovirus and
retroviruses including lentiviruses. Useful expression
vectors for bact~'rial hosts include bacterial plasmids, such
as those from E. coli, including pBluescript, pGEX-2T, pUC
vectors, col El, pCRl, pBR322, pMB9 and their derivatives,
pET-15, wider host range plasmids, such as RP4, phage DNAs,
e.g., the numerous derivatives of phage lambda, e.g. 1~GT10
and AGT11, and other phages. Useful expression vectors for
yeast cells include the 2u plasmid and derivatives thereof.
Useful vectors for in:~ect cells include pVL 941.
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24
In addition, any of a wide variety of expression
control sequences -- sequences that control the expression
of a DNA sequence when operatively linked to it -- may be
used in these vectors to express the DNA sequences of this
invention. Such useful expression control sequences include
the expression control sequences associated with structural
genes of the foregoing expression vectors. Examples of
useful expression control sequences include, for example,
the early and late promoters of SV40 or adenovirus, the lac
system, the ,~r~ system, the TAC or TRC system, the T3 and T7
promoters, the major operator and promoter regions of phage
lambda, the control regions of fd coat protein, the promoter
for 3-phosphoglycerate kinase or other glycolytic enzymes,
the promoters of acid phosphatase, e.g., Pho5, the promoters
of the yeast a-mating system and other constitutive and
inducible promoter sequences known to control the expression
of genes of prokaryotic or eukaryotic cells or their
viruses, and various combinations thereof.
In a preferred embodiment, DNA sequences encoding
the I. scapularis polypeptides of this invention are cloned
in the expression vector lambda ZAP II (Stratagene, La
Jolla, CA), in which expression from the lac promoter may be
induced by IPTG.
In another preferred embodiment, DNA encoding the
I. scapularis polypeptides of this invention is inserted in
frame into an expression vector that allows high level
expression of the polypeptide as a glutathione S-transferase
fusion protein. Such a fusion protein thus contains amino
acids encoded by the vector sequences as well as amino acids
of the I. scapularis polypeptide.
The term "host cell" refers to one or more cells
into which a recombinant DNA molecule is introduced. Host
cells of the invention include, but need not be limited to,
bacterial, yeast, animal and plant cells. Host cells can be
unicellular, or can be grown in tissue culture as liquid
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cultures, monola~~ers o:r the like. Host cells may also be
derived directly or in~3irectly from tissues.
A wide variety of unicellular host cells are
useful in expres:~ing t:he DNA sequences of this invention.
S These hosts may :include well known eukaryotic and
prokaryotic host:, such as strains of E. coli, Pseudomonas,
Bacillus, Strept~~myces, fungi, yeast, insect cells such as
Spodoptera frugiperda (SF9), animal cells such as CHO and
mouse cells, African green monkey cells such as COS 1,
10 COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as well
as plant cells.
A host cell is "transformed" by a nucleic acid
when the nucleic acid is translocated into the cell from the
extracellular environment. Any method of transferring a
IS nucleic acid into the cell may be used; the term, unless
otherwise indicated herein, do not imply any particular
method of delivering a nucleic acid into a cell, nor that
any particular cell type is the subject of transfer.
An "expression control sequence" is a nucleic acid
20 sequence which regulates gene expression (i.e.,
transcription, RNA formation and/or translation).
Expression control sequences may vary depending, for
example, on the chosen host cell or organism (e. g., between
prokaryotic and eukaryotic hosts), the type of transcription
25 unit (e.g., which RNA polymerise must recognize the
sequences), the cell type in which the gene is normally
expressed (and, in turn, the biological factors normally
present in that cell type) .
A "promoter" is one such expression control
sequence, and, as used herein, refers to an array of nucleic
acid sequences which control, regulate and/or direct
transcription of downstream (3') nucleic acid sequences. As
used herein, a ~~romotE~r includes necessary nucleic acid
sequences near t:he st<~rt site of transcription, such as, in
the case of a polymerise II type promoter, a TATA element.
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A "constitutive" promoter is a promoter which is
active under most environmental and developmental
conditions. An "inducible" promoter is a promoter which is
inactive under at least one environmental or developmental
condition and which can be switched "on" by altering that
condition. A "tissue specific" promoter is active in
certain tissue types of an organism, but not in other tissue
types from the same organism. Similarly, a developmentally-
regulated promoter is active during some but not all
developmental stages of a host organism.
Expression control sequences also include distal
enhancer or repressor elements which can be located as much
as several thousand base pairs from the start site of
transcription. They also include sequences required for RNA
formation (e.g., capping, splicing, 3' end formation and
poly-adenylation, where appropriate); translation (e. g.,
ribosome binding site); and post-translational modifications
(e. g., glycosylation, phosphorylation, methylation,
prenylation, and the like).
The term "operatively linked" refers to functional
linkage between a nucleic acid expression control sequence
(such as a promoter, or array of transcription factor
binding sites) and a second nucleic acid sequence, wherein
the expression control sequence directs transcription of the
nucleic acid corresponding to the second sequence.
The term "polypeptide" refers to any polymer
consisting essentially of amino acids regardless of its
size. Although "protein" is often used in reference to
relatively large polypeptides, and "peptide" is often used
in reference to small polypeptides, usage of these terms in
the art overlaps and varies. The term "polypeptide" as used
herein thus refers interchangeably to peptides, polypeptides
and proteins, unless otherwise noted.
The term "amino acid" refers to a monomeric unit
of a peptide, polypeptide or protein.
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It should of course be understood that not all
vectors and expression control sequences will function
equally well to express the DNA sequences of this invention.
Neither will all hosts function equally well with the same
expression system. However, one of skill in the art may
make a selection among these vectors, expression control
sequences and ho:>ts without undue experimentation and
without departing from the scope of this invention. For
example, in selecting a vector, the host must be considered
because the vector must be replicated in it. The vector's
copy number, the ability to control that copy number, the
ability to control integration, if any, and the expression
of any other prot=eins .encoded by the vector, such as
antibiotic or other selection markers, should also be
considered.
In selecting an expression control sequence, a
variety of factors should also be considered. These
include, for example, the relative strength of the promoter
sequence, its controllability, and its compatibility with
the DNA sequence of this invention, particularly with regard
to potential secondary structures. Unicellular hosts should
be selected by consideration of their compatibility with the
chosen vector, the toxicity of the product coded for by the
DNA sequences of this invention, their secretion
characteristics, their ability to fold the polypeptide
correctly, their fermentation or culture requirements, and
the ease of puri:Eication from them of the products coded for
by the DNA sequences of this invention.
Within these parameters, one of skill in the art
may select various vector/expression control sequence/host
combinations that will express the DNA sequences of this
invention on fermentation or in other large scale cultures.
The molecules comprising the I. scapularis
polypeptides enc~~ded by the DNA sequences of this invention
may be isolated from the fermentation or cell culture and
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purified using any of a variety of conventional methods
including: liquid chromatography such as normal or reversed
phase, using HPLC, FPLC and the like; affinity
chromatography (such as with inorganic ligands or monoclonal
antibodies); size exclusion chromatography; immobilized
metal chelate chromatography; gel electrophoresis; and the
like. One of skill in the art may select the most
appropriate isolation and purification techniques without
departing from the scope of this invention. If the
polypeptide is membrane bound or suspected of being a
lipoprotein, it may be isolated using methods known in the
art for such proteins, e.g., using any of a variety of
suitable detergents.
In addition, the I. scapularis polypeptides may be
generated by any of several chemical techniques. For
example, they may be prepared using the solid-phase
synthetic technique originally described by R. B.
Merrifield, "Solid Phase Peptide Synthesis. I. The
Synthesis Of A Tetrapeptide", J. Am. Chem. Soc., 83,
pp. 2149-54 (1963), or they may be prepared by synthesis in
solution. A summary of peptide synthesis techniques may be
found in E. Gross & H. J. Meinhofer, 4 The Peptides:
Analysis, Synthesis, Biology; Modern Techniques Of Peptide
And Amino Acid Analysis, John Wiley & Sons, ( 1981 ) and
M. Bodanszky, Principles Of Peptide Synthesis, Springer-
Verlag (1984).
Typically, these synthetic methods comprise the
sequential addition of one or more amino acid residues to a
growing peptide chain. Often peptide coupling agents are
used to facilitate this reaction. For a recitation of
peptide coupling agents suitable for the uses described
herein see M. Bodansky, supra. Normally, either the amino
or carboxyl group of the first amino acid residue is
protected by a suitable, selectively removable protecting
group. A different protecting group is utilized for amino
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acids containing a reactive side group, e.g., lysine. A
variety of prote~~ting groups known in the field of peptide
synthesis and recognized by conventional abbreviations
therein, may be found in T. Greene, Protective Groups In
Organic Synthesis, Academic Press (1981).
According to another embodiment of this invention,
antibodies directed against the I. scapularis polypeptides
are generated. ;such antibodies are immunoglobulin molecules
or portions thereof that are immunologically reactive with
an I. scapularis polypeptide of the present invention. It
should be understood that the antibodies of this invention
include antibodies imm.unologically reactive with fusion
proteins and multimeric proteins comprising an I. scapularis
polypeptide.
Antibodies directed against an I. scapularis
polypeptide may be generated by a variety of means including
immunizing a mammalian. host with I. scapularis extract or
tick infestation, or ~~y immunization of a mammalian host
with an I. scapularis polypeptide of the present invention.
Such antibodies may beg polyclonal or monoclonal; it is
preferred that they are monoclonal. Methods to produce
polyclonal and monoclonal antibodies are well known to those
of skill in the art. For a review of such methods, see
Antibodies, A Laboratory Manual, supra, and D.E. Yelton,
et al., Ann. Rev. of E3iochem., 50, pp. 657-80 (1981).
Determination of immunoreactivity with an I. scapularis
polypeptide of this invention may be made by any of several
methods well known in the art, including by immunoblot assay
and ELISA.
An antibody of this invention may also be a hybrid
molecule formed from ummunoglobulin sequences from different
species (e.g., mouse and human ) or from portions of
immunoglobulin light and heavy chain sequences from the same
species. It ma~~ be a molecule that has multiple binding
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specificities, such as a bifunctional antibody prepared by
any one of a number of techniques known to those of skill in
the art including: the production of hybrid hybridomas;
disulfide exchange; chemical cross-linking; addition of
5 peptide linkers between two monoclonal antibodies; the
introduction of two sets of immunoglobulin heavy and light
chains into a particular cell line; and so forth.
The antibodies of this invention may also be human
monoclonal antibodies produced by any of the several methods
10 known in the art. For example, human monoclonal antibodies
may be produced by immortalized human cells, by SCID-hu mice
or other non-human animals capable of producing "human"
antibodies, by the expression of cloned human immunoglobulin
genes, by phage-display, or by any other method known in the
15 art .
In addition, it may be advantageous to couple the
antibodies of this invention to toxins such as diphtheria,
pseudomonas exotoxin, ricin A chain, gelonin, etc., or
antibiotics such as penicillins, tetracyclines and
20 chloramphenicol.
In sum, one of skill in the art, provided with the
teachings of this invention, has available a variety of
methods which may be used to alter the biological properties
of the antibodies of this invention including methods which
25 would increase or decrease the stability or half-life,
immunogenicity, toxicity, affinity or yield of a given
antibody molecule, or to alter it in any other way that may
render it more suitable for a particular application.
One of skill in the art will understand that
30 antibodies directed against an I. scapularis polypeptide may
have utility in prophylactic compositions and methods
directed against tick bite and infection with a tick-borne
pathogen. For example, the level of pathogens in infected
ticks may be decreased by allowing them to feed on the blood
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31
of animals immunized with the I. scapularis polypeptides of
this invention.
The antibodies of this invention also have a
variety of other uses. For example, they are useful as
reagents to screen for expression of the I. scapularis
polypeptides, either in libraries constructed from I.
scapularis DNR or from other samples in which the proteins
may be present. Moreover, by virtue of their specific
binding affinities, the antibodies of this invention are
also useful to purify or remove polypeptides from a given
sample, to block or bind to specific epitopes on the
polypeptides and to direct various molecules, such as
toxins, to ticks.
To screen the I. scapularis polypeptides and
antibodies of this invention for their ability to confer
protection against tick bite or their ability to lessen the
severity of infecaion with tick-borne pathogens, guinea pigs
are preferred as an animal model. Of course, while any
animal that is susceptible to tick immunity may be useful,
guinea pigs are not on:Ly a classical model for tick immunity
but also displays skin reactivity that mimic
hypersensitivity react:LOns in humans. Thus, by
administering a particular I. scapularis polypeptide or
anti-I. scapular_is polypeptide antibody to guinea pigs, one
of skill in the <irt ma:y determine without undue
experimentation whether that polypeptide or antibody would
be useful in the methods and compositions claimed herein.
The administration of the I. scapularis
polypeptide or a~atibody of this invention to the animal may
be accomplished by any of the methods disclosed herein or by
a variety of other standard procedures. For a detailed
discussion of su~~h techniques, see Antibodies, A Laboratory
Manval, supra. Preferably, if a polypeptide is used, it
will be administered with a pharmaceutically acceptable
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adjuvant, such as complete or incomplete Freund's adjuvant,
RIBI (muramyl dipeptides) or ISCOM (immunostimulating
complexes). Such adjuvants may protect the polypeptide from
rapid dispersal by sequestering it in a local deposit, or
they may contain substances that stimulate the host to
secrete factors that are chemotactic for macrophages and
other components of the immune system. Preferably, if a
polypeptide is being administered, the immunization schedule
will involve two or more administrations of the polypeptide,
spread out over several weeks.
Once the I. scapularis polypeptides or antibodies
of this invention have been determined to be effective in
the screening process, they may then be used in a
therapeutically effective amount in pharmaceutical
compositions and methods to confer tick immunity and to
prevent or reduce the transmission of tick-borne pathogens.
The pharmaceutical compositions of this invention
may be in a variety of conventional depot forms. These
include, for example, solid, semi-solid and liquid dosage
forms, such as tablets, pills, powders, liquid solutions or
suspensions, liposomes, capsules, suppositories, injectable
and infusible solutions. The preferred form depends upon
the intended mode of administration and prophylactic
application.
Such dosage forms may include pharmaceutically
acceptable carriers and adjuvants which are known to those
of skill in the art. These carriers and adjuvants include,
for example, RIBI, ISCOM, ion exchangers, alumina, aluminum
stearate, lecithin, serum proteins, such as human serum
albumin, buffer substances, such as phosphates, glycine,
sorbic acid, potassium sorbate, partial glyceride mixtures
of saturated vegetable fatty acids, water, salts or
electrolytes such as protamine sulfate, disodium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-
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based substances, and polyethylene glycol. Adjuvants for
topical or gel base forms may be selected from the group
consisting of sodium carboxymethylcellulose, polyacrylates,
polyoxyethylene-~>olyox~,rpropylene-block polymers,
polyethylene glycol, and wood wax alcohols.
The vaccines and compositions of this invention
may also include other components or be subject to other
treatments during preparation to enhance their immunogenic
character or to improve their tolerance in patients.
Compositions comprising an antibody of this
invention may be administered by a variety of dosage forms
and regimens sim~_lar to those used for other passive
immunotherapies and well known to those of skill in the art.
Generally, the I" scapularis polypeptides may be formulated
and administered to the patient using methods and composi-
tions similar to those employed for other pharmaceutically
important polypeptides (e. g., the vaccine against
hepatitis).
Any pharmaceutically acceptable dosage route,
including parentc~ral, intravenous, intramuscular,
intralesional or subcutaneous injection, may be used to
administer the pc~lypeptide or antibody composition. For
example, the com~~osition may be administered to the patient
in any pharmaceutically acceptable dosage form including
those which may he administered to a patient intravenously
as bolus or by continued infusion over a period of hours,
days, weeks or m~~nths, intramuscularly -- including
paravertebrally and periarticularly -- subcutaneously,
intracutaneously, intra-articularly, intrasynovially,
intrathecally, intralesionally, periostally or by oral or
topical routes. Preferably, the compositions of the
invention are in the form of a unit dose and will usually be
administered to the patient intramuscularly.
The I. scapularis polypeptides or antibodies of
this invention may be administered to the patient at one
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34
time or over a series of treatments. The most effective
mode of administration and dosage regimen will depend upon
the level of immunogenicity, the particular composition
and/or adjuvant used for treatment, the severity and course
of the expected infection, previous therapy, the patient's
health status and response to immunization, and the judgment
of the treating physician.
For example, in an immunocompetent patient, the
more highly immunogenic the polypeptide, the lower the
dosage and necessary number of immunizations. Similarly,
the dosage and necessary treatment time will be lowered if
the polypeptide is administered with an adjuvant.
Generally, the dosage will consist of 10 ~g to 100 mg of the
purified polypeptide, and preferably, the dosage will
IS consist of 10-1000 ug. Generally, the dosage for an
antibody will be 0.5 mg-3.0 g.
In a preferred embodiment of this invention, the
I. scapularis polypeptide is administered with an adjuvant,
in order to increase its immunogenicity. Useful adjuvants
include RIBI, and ISCOM, simple metal salts such as aluminum
hydroxide, and oil based adjuvants such as complete and
incomplete Freund's adjuvant. When an oil based adjuvant is
used, the polypeptide usually is administered in an emulsion
with the adjuvant.
In yet another preferred embodiment, E.coli
expressing proteins comprising an I, scapularis polypeptide
are administered orally to non-human animals according to
methods known in the art, to confer tick immunity and to
prevent or reduce the transmission of tick-borne pathogens.
For example, a palatable regimen of bacteria expressing an
I. scapularis polypeptide, alone or in the form of a fusion
protein or multimeric protein, may be administered with
animal food to be consumed by wild mice or other animals
that act as alternative hosts for I. scapularis ticks.
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Ingestion of such bacteria may induce an immune
response comprising both humoral and cell-mediated
components. See J.C. Sadoff et al., "Oral Salmonella
Typhimvrivm Vaccine Expressing Circumsporozoite Protein
5 Protects Against Malaria", Science, 240, pp. 336-38 (1988)
and K.S. Kim et al., "Immunization Of Chickens With Live
Escherichia coli Expressing Eimeria acervulina Merozoite
Recombinant Anti~3en Induces Partial Protection Against
Coccidiosis", Inf. Immin., 57, pp. 2434-40 (1989); M. Dunne
10 et al., "Oral Vaccination Against Human granulocytic
ehrlichiosis Using Salmonella Expressing OspA," Inf. and
Immun., 63:1611 (1995); E. Fikrig et al., "Protection of
Mice From Lyme Borreliosis By Oral Vaccination With
Escherichia coli Expressing OspA," J. Infec. Dis., 164:1224
15 ( 1991 ) .
Moreover, th.e level of pathogens in ticks feeding
on such animals ::nay be lessened or eliminated, thus
inhibiting transmission to the next animal.
According to yet another embodiment, the I.
20 scapularis polypeptides of this invention, and the DNA
sequences encoding them are useful as diagnostic agents for
detecting tick immunity and tick bite. The polypeptides are
capable of binding to antibody molecules produced in
animals, including humans, that have been exposed to I.
25 scapularis antigens a~~ a result of a tick bite. The
detection of I. scapularis antigens is evidence of tick
attachment and at lea:>t some feeding. Such information is
an important aid in the early diagnosis of I. scapularis-
borne diseases.
30 Such diagno:>tic agents may be included in a kit
which may also compri:>e instructions for use and other
appropriate reagents, preferably a means for detecting when
the polypeptide or antibody is bound. For example, the
polypeptide or antibody may be labeled with a detection
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means that allows for the detection of the polypeptide when
it is bound to an antibody, or for the detection of the
antibody when it is bound to I. scapularis or an antigen
thereof.
The detection means may be a fluorescent labeling
agent such as fluorescein isocyanate (FIC),
fluorescein isothiocyanate (FITC), and the like, an enzyme,
such as horseradish peroxidase (HRP), glucose oxidase or the
like, a radioactive element such as 1251 or 5lCr that
produces gamma ray emissions, or a radioactive element that
emits positrons which produce gamma rays upon encounters
with electrons present in the test solution, such as 11C,
15~~ or 13N. Binding may also be detected by other methods,
for example via avidin-biotin complexes.
The linking of the detection means is well known
in the art. For instance, monoclonal antibody molecules
produced by a hybridoma can be metabolically labeled by
incorporation of radioisotope-containing amino acids in the
culture medium, or polypeptides may be conjugated or coupled
to a detection means through activated functional groups.
The diagnostic kits of the present invention may
be used to detect the presence of anti-I. scapularis
antibodies in a body fluid sample such as serum, plasma or
urine. Thus, in preferred embodiments, an I. scapularis
polypeptide or an antibody of the present invention is bound
to a solid support typically by adsorption from an aqueous
medium. Useful solid matrices are well known in the art,
and include crosslinked dextran; agarose; polystyrene;
polyvinylchloride; cross-linked polyacrylamide;
nitrocellulose or nylon-based materials; tubes, plates or
the wells of microtiter plates. The polypeptides or
antibodies of the present invention may be used as
diagnostic agents in solution form or as a substantially dry
powder, e.g., in lyophilized form.
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I. sca_pulari.s polypeptides and antibodies directed
against those polypept.ides provide much more specific
diagnostic reagents than whole ticks and thus may alleviate
such pitfalls as falser positive and false negative results.
S One skilled in the art will realize that it may
also be advantageous i.n the preparation of detection
reagents to utilize e~>itopes from more than one I.
scapularis protein and antibodies directed against such
epitopes.
The skilled artisan also will realize that it may
be advantageous to prepare a diagnostic kit comprising
diagnostic reagents to detect I. scapularis as well as
pathogens found in the same tick vector, for example,
Borrelia burgdorferi, Babesia micro n, aoHGE (the agent of
human granulocytic ehrlichiosis) as well as some
arboviruses, such as t:he Eastern equine encephalitis virus,
and instructions for t:heir use.
The polypept:ides and antibodies of the present
invention, and compositions and methods comprising them, may
also be useful for prevention of tick bit:es by other species
of ticks which m.ay express proteins sharing amino acid
sequence or conformat~_onal similarities with the I.
scapular.is polypeptides of the present invention.
In order th<it this invention may be better
understood, the following examples are set forth. These
examples are for purposes of illustration only, and are not
to be construed as limiting the scope of the invention in
any manner.
EXAMPLE I - Guir~ Pick Model of I. scapularis Immunity
We chase thf~ guinea pig for our model even though
it is not a natural host for Ixodes ticks because guinea
pigs are the classica:L model for tick immunity and because
their immune skin rea~~tions closely mimic those in humans.
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We infested naive guinea pigs with 100 larval I.
scapularis ticks. We placed the guinea pigs in wire-bottom
cages over a water pan to allow recovery of ticks that fall
off after feeding to repletion. We examined the guinea pigs
daily and counted the ticks remaining on them. We followed
the duration of attachment and the weight of recovered ticks
as parameters of immunity.
After 14 days, we rechallenged the guinea pigs in
a similar fashion. After the second exposure, sites of tick
attachment became grossly reddened. We biopsied the sites
and notes infiltrates of basophils in a characteristic
cutaneous basophil hypersensitivity. We found a marked
decrease in the duration of attachment (Figure 1) and weight
of ticks recovered (Figure 2) from guinea pigs actively
immunized by prior infestations compared to naive controls.
These results indicate that the guinea pigs developed tick
immunity.
EXAMPLE II - Cloning I. scapularis Salivary
Gland Protein Genes
A. Preparing cDNA Libraries
To obtain I. scapularis salivary glands for
preparation of a cDNA expression library, over a 4 week
period, we fed 1000 I. scapularis nymphs on naive 5-6 week
old C3H/HeJ mice. After 72 hours, we pulled off the ticks
and kept them under humidified conditions until dissection,
which was within 24 hours of being pulled.
For dissection, we placed the ticks over a drop of
PBS on a cover slip and cut them in half using a spear and
sharp-pointed tweezers. We transferred the upper half of
the body to a second drop of PBS within the cover slip and
cut lengthwise. We scooped the interior content of the
upper segment from the shell and recovered the pair of
salivary glands. We kept the salivary glands under
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guanidium/B-merca.ptoethanol until all dissections were
complete to prevent decFradation by RNases.
We isolated FZNA using Stratagene's RNA Micro
Isolation Kit~. Brief=Ly, we added 30 ul of 2M Na acetate,
300 ul if water-saturat=ed phenol and 60 ul if
chloroform:isoam~;~l alcohol to a 300 ul aliquot of salivary
gland in GTIC/mer~captoE~thanol. We capped the tube, vortexed
and microfuged for 5 m=Ln. at maximum speed. We transferred
the upper phase containing the RNA to a new tube.
We added gycogen carrier and isopropanol an
microfuged for 30 min. in the cold to precipitate RNA. We
washed the pellet: in 7',~o ethanol and dried in a vacuum for 5
min. We resuspended the RNA in water and read an aliquot in
a spectrophotometer at 260 nm. Our yield was 0.1-0.27 ug
total RNA per tick. WEB sent the isolated RNA to Clonetech
where a Lambda Z~~PII e:~pression library was made after
initial amplific~~tion of the message.
We also prepared a whole-tick cDNA library using a
substantially similar method.
B. Screening I3sodes :Libraries With
Hyperimmune and Immune Sera
To identify antigens recognized by tick-immune
sera, we screened the cDNA libraries as follows.
We prepared salivary gland-immune sera by
immunizing 3 guinea pigs with 10 ug of salivary gland
extract prepared as described above with some modifications.
We collected the salivary glands in 10 mM PBS, 20 mM EGTA
and 100 ~M PMSF at pH 7.2 and kept on ice to prevent
degradation. We then freeze-thawed the pooled salivary
gland preparation 3 times and sonicated for 3 pulses of one
minute until the mixture clarified. We determined protein
content using thEs microtiter method of the Bradford assay.
The average yield from fed ticks was 2-3 micrograms of
protein per tick.
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We immunized first with extract in complete
Freund's adjuvant and boosted twice with the same amount of
antigen in incomplete Freund's. A control group of 3 guinea
pigs received DNFB as he antigen and were treated similarly.
5 To prepare whole tick immune sera, we infested 3 guinea pigs
with 20-25 nymphs 3 times with at 15-20 day intervals.
We sacrificed the animals I5 days after the final
tick feeding and collected blood by heart puncture. We
isolated the immune sera and anti-DNFB sera and stored it at
10 -20°C until further use.
We grew approximately 1,000 Lambda phage on E.
coli XL Blue cell lawns in 90 mm culture plates. We then
induced expression of the cDNA with 10 mM IPTG in a soaked
nitrocellulose membrane for 3 hours and probed the membranes
15 with salivary gland-immune or whole tick-immune sera in 2-10
fold dilutions. As controls, we probed replica plates with
anti-DNFB or normal guinea pig sera.
After washing, we incubated the filters with
alkaline phosphate conjugated goat anti-guinea pig antibody
20 to detect clones.
The tick-immune sera recognized 3 clones (Clones
1-3) from the salivary gland library and 1 clone (Clone 4)
from the whole-tick library. The salivary gland immune sera
recognized 1 clone (Clone 5) from the whole-tick library.
25 We deposited Clone 1 on April 28, 1998 with the American
Type Culture Collection, 12301 Parklawn Drive, Rockville,
Maryland 20852 under ATCC accession number
We excised the inserts from the clones using 8408
helper phage and digested the vectors with the inserts with
30 EcoRl endonuclease. Clone 1 had a 700 by insert; Clone 2,
an 800 by insert, Clone 3, a 600 by insert; Clone 4, a 4-5
kb insert and Clone 5, a 5-6 kb insert.
We confirmed binding to the immune sera, we
induced expression of the pBluescript vectors containing
35 individual inserts in XLl blue cells with IPTG. We lysed
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the cells and separate<~ the lysate on SDS-PAGE, transferred
to nitrocellulose membrane and probed with tick-immune or
salivary gland ir:unune sera. Tick immune sera bound to a 32
kD band from Clones I and 2 and to an 85 kD band from Clone
4. Salivary gland sera bound to a 90 kD band from Clone 5.
The same sized bind wa:; recognized in both uninduced and
IPTG induced cel=Ls. Thus, the proteins are not expressed
from the 1ac promoter.
To identify additional I. scapularis antigens
capable of conferring tick immunity, we rescreen the
expression libra~°ies with immune sera from mice, rabbits and
humans according to th~~ methods described herein.
C. Sequencing l~he In;~ r s
The in:>erts ~~f Clones 1-3 were sequenced by the
Sanger method in the W. Keck DNA sequencing Laboratory at
Yale. All 3 of ~~he clones were found to have the same open
reading frame. 'rhe gene, which we designated spl6, encodes
a 16 kD protein. The DNA sequence and deduced amino acid
sequence of spl6 are set forth in SEQ ID NOS: 1 and 2. The
sequence had a ribosome binding site in the proper position,
start and stop codons and a poly A tail, indicating active
expression of this gene in the salivary gland.
To confirm that the spl6 gene is expressed in the
salivary gland, we isolated total RNA from 20 salivary
glands of partially fed ticks and prepared cDNA from the RNA
using reverse transcriptase and oligo dT primer. We
amplified the spl6 from the salivary gland cDNA and
separated on an agarose gel. We excised the amplified band
from the gel and resequenced it. The sequence of the
amplified band matched the sequence of Clone 1-3. Thus,
spl6 is expressed in the salivary gland.
EXAMPLE III - R_ecombin~nt Expression of SP16
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To obtain enough DNA for expression, we amplified
the spl6 gene sequence from the BLUESCRIPT plasmid and added
XhoI an HindIII sites to a fragment of spl6 lacking the
signal sequence. We cloned the amplified gene fragments
into the pGEX-2T vector system, in frame with glutathione-S-
transferase to generate a GST-fusion protein. We
electroporated the vector containing the spl6 into E. coli
DHSa and induced expression with IPTG. We purified the
fusion protein on a glutathione column.
Those of skill in the art will recognize that
additional I, scapularis antigens can be isolated using the
methods described herein. Recombinant antigen can be
purified in a number of ways. For example, recombinant
antigen without the fusion protein can be purified using
thrombin to cleave at a thrombin cleavage site located
between the GST and the recombinant I. scapularis antigen.
Alternatively, the antigens can be cloned into the PET 15b
vector which produces recombinant antigens with a histidine
leader sequence. The recombinant histidine fusion protein
can then be purified using a nickel column and eluting with
EDTA. Finally, recombinant antigens can be recovered by
equilibrium dialysis after purification of the antigen from
SDS-PAGE gels.
Purified SP16 is tested for the ability to confer
tick immunity by active immunization assay or the CBH assay.
EXAMPLE IV - Active Immunization wi h SP16
To test SP16 for the ability to confer tick
immunity, we immunize naive guinea pigs with 10 ug of the
GST-SP16 fusion protein an boost twice. Fourteen days after
the last boost, we challenge the actively immunized animals
with 5 nymphs to detect immunity.
EXAMPLE V - Passive Immunization wi h Anti-SP16 An isPr m
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We prey>ared anti-SP16 antiserum by immunizing
C3H/HeN mice with 10 ug of recombinant SP16 fusion protein
an boosted twice with i~he same amount. Fourteen days after
the last boost, we sacrificed the immunized animals and
collected the antiserum.
We immunized guinea pigs with the anti-SP16
antiserum and ch~~llengE~d with 5 nymphal ticks.
EXAMPLE VI - Isolation of Proteins From I. scax~ularis Saliva
We col7.ected saliva from I. scapularis according
to the methods of: Ewing et al. [C. Ewing et al., "Isolation
of Borrelia burgc~orfer.i From Saliva of The Tick Vector,
Ixodes scapulari:~." J. Clin. Microbiol., 32, pp. 755-758
(1994)]. Briefly, we affixed ticks onto the backs of naive
guinea pigs in the tops of a plastic bottle taped to the
guinea pigs' bac}cs wit:n the cap glued on. We allowed ticks
to feed for approximat~sly 13 days. We pulled off the ticks
with forceps, rinsed them with distilled water and
immediately fixed to glass slides with double-sided tape.
We place a steri:Le glass micropipette around the hypostome
to collect saliva.
We ind2~ced salivation by applying 2 ul of
pilocarpine (50 mg/ml in 95o ethanol) to the scutum of the
tick. We added additional 1 ul aliquots of pilocarpine at
20 min. interval: for 2.5 hours at 35°C in a humid chamber.
We col:Lected saliva from the micropipettes into a
0.5 ml sterile tube and frozen at -20°C. We added 3 ul of
saliva to 2 ul o:f sample buffer and 5 ul running buffer,
boiled and ran the sample on 12o SDS-PAGE gels at 125 volts
for 1.25 hours. We stained the gels with Coomassie Blue for
30 min. and destained until the background cleared and dried
the gel with Nov~ex Gel-Dry~ drying solution. The gels
showed one protein band at 65 kD.
EXAMPLE VII - Preparation of Fab Fragments of Immune Serum
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To obtain Fab fragments of immune serum for use in
screening the salivary gland expression library, we first
made rabbit and guinea pig anti-tick antiserum. We
repeatedly infested rabbits and guinea pigs with larval or
nymphal I, scapularis ticks. We determined that the animals
were tick immune if the site of tick attachment became red
of if tick feeding was less than 48 hours. We bled tick
immune animals to collect tick immune serum.
We also prepared guinea pig anti-tick salivary
gland antiserum by immunizing guinea pigs subcutaneously
with 20 ug of salivary gland extract prepared as described
above, in incomplete Freund's adjuvant. We boosted twice
with the same amount of crude extract.
To prepare the Fab fragment, we precipitated the
antiserum with ammonium sulfate and isolated the IgG
fraction using DEAF chromatography. We digested the IgG
preparation using a solid phase papain column. We purified
Fab fragments from the papain digestion using a protein A
affinity column to remove Fc and intact IgG molecules.
EXAMPLE VIII - Passive Immunization with Anti-TickAntiserum
We bled tick immune guinea pigs an passively
immunized naive animals i.v. with 5 ml of the immune
antiserum. We then challenged the passively immunized
animals with 100 larval I. scapularis ticks. We used naive
guinea pigs as negative controls and actively immunized
animals as positive controls.
At 72 hours, passively immunized animals had a 500
reduction in the number of attached ticks compared to naive
animals (p<0.05). Ticks fed on passively immunized animals
weighed 240 less than ticks fed on naive animals at 120
hours after tick challenge (p<0.04).
Thus, we were able to transfer partial tick
immunity with sera.
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EXAMPLE IX - Cross-Pro:ection At Different Tick Stages
We werE: interested in determining if immunity to
I. scapularis is stage--specific. This is of interest
because the nymph and ~idult ticks transmit B. burgdorferi
5 while larvae are more readily available and thus easier to
obtain in sufficient numbers for testing.
We actively immunized 2 guinea pigs with larval I.
scapu.Iaris and passive7_y immunized 2 guinea pigs with 5 ml
i.v. of anti-larval immune serum. We used naive animals as
10 controls. We challenged the animals with 50 I. scapularis
nymphs each. We counted and weighed ticks recovered from
the water pans daily.
We observed i~hat actively and passively immunized
animals had reduced duration of attachment (Figure 3).
15 Passively immunia:ed animals had a 40o reduction in the
number of ticks ~ittachf~d compared to controls at 96 hours.
The weight of ticks recovered from actively and passively
immunized animal was a:Lso significantly reduced compared to
controls.
20 Thus, different stages of tick development share
at least some protective antigens.
EXAMPLE X - PrevE:ntion ~f B. buradorferi Transmission
Before test_Lng the effect of tick immunity on the
25 transmission of B. burgdorferi, the agent of Lyme Disease,
we determined whE~ther guinea pigs could be infected by
challenge with B. burgdorferi infected ticks. We challenged
naive guinea pig:~ with 5 B31 or N40 strain infected I.
scapularis nymph,. Skin punches at the site of tick
30 attachment and e:Lsewhere 2, 4 and 7 weeks after tick
challenge were consistently positive for spirochetes by
culture.
To confirm infection, we determined that guinea
pigs develop an .immune response against B. burgdorferi.
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Western blots of s of cloned N40 spirochetes probed with
serum from the challenged animals showed antibodies to
flagellin, P39 and OspC antigens. Sera from animal exposed
to uninfected ticks and those exposed to infected ticks but
that were not culture positive failed to develop such
antibodies.
We have therefore demonstrated B. burgdorferi
infection of guinea pigs by tick challenge.
We then determined if tick immunity affected the
transmission of B. burgdorferi. We sensitized guinea pigs
with I. scapularis larvae or nymphs and 5 weeks later,
challenged the sensitized animals with 5 ticks from a pool
with an 80o infection rate of N40 spirochetes. We obtained
3mm skin punch biopsies at the tick attachment site and
serum samples at 2, 4 and 7 weeks after tick challenge. At
8 weeks after challenge we sacrificed the animals and
collected blood, bladder and spleen for culture.
As shown in Figure 3, only 1 out of 18 tick immune
animals had a positive skin culture while 10 out of 18 naive
animals had positive cultures. Cultures of blood, bladder
and spleen were negative for both groups.
As determined by Western blot, tick immune animals
failed to develop anti-B. burgdorferi antibodies while naive
animals developed antibodies to flagellin and P39. Staining
of ticks recovered from both groups of animals with FITC-
conjugated polyclonal anti-B, burgdorferi antibody confirmed
that 70-1000 of the ticks were infected.
Our results demonstrate that tick immunity
prevents or markedly reduces B. burgdorferi transmission.
We conducted a similar experiment to test the
effect of tick immunity on aoHGE transmission. We first
determined that guinea pigs could be infected with aoHGE.
We confirmed infection of the guinea pigs by PCR
amplification of an aoHGE 16S rDNA target from blood,
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seroconversion to the aoHGE-specific 44-kDa antigen and
infectivity of the guinea pig blood in mice.
Our prE~liminary results did not indicate that
transmission of aoHGE was prevented in tick immune animals.
There are a number of :possible explanations for these
results. First, unlike B. burgdorferi which resides in the
tick mid-gut, aol3GE resides in the salivary glands.
Accordingly, the time frame for tranmsmission to a host may
be quite fast. In a more immune host (either a host which
mounts a stronger immune response and/or a host with an
increased immunizing dose), ticks may drop off sooner and
aoHGE transmissi«n would be prevented. Further, we
challenged the immune animals with 5 ticks. Natural
infection occurs with 1 tick. Accordingly, the challenge
dose may have been so high that any reduction in
transmission was masked.
EXAMPLE XI - Isolation of I. scapularis
Antigens from Salivary Gland Extract
We used a cutaneous basophil hypersensitivity (CBH)
assay to screen for I. scapularis antigens for their ability
to induce tick immunity Z. Ovary et al., "Passive Cutaneous
Anaphylaxis With Antibody Fragments," Science, 140, pp. 193-
195 (1963); Z. Ovary et al., "PCA and rPCA in Guinea Pigs
With Rabbit and Guinea Pig Antibodies And Different
Antigens," J. Immunol., 97, pp. 559-563 (1966); Z. Ovary,
"Passive Cutaneous Anaphylaxis in the Guinea Pig," Int.
Arch. of Allergy and ~.ppl. Immunol., 14, pp. 18-26 (1959)].
In this assay, an actively or passively immunized
animal is injected with Evan's blue dye intravenously.
Immediately afterward, injections of test substances are
placed intradermally on the back at about 10-15 minute
intervals allowing 20-30 substances to be tested in a single
animal. If protective antigen is present in the test
substance, it reacts ~~ith homocytotropic antibody to cause
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release of vasomediators. The dye that is bound to serum
albumin extravasates into the tissues producing a blue spot.
We prepared I. scapularis salivary gland extract
as described above. To better characterize the preparation,
we purified it with a MonoQ column on a Pharmacia FPLC
apparatus. We applied 20 ug of the salivary gland extract
to the column using a salt gradient. The starting buffer
consisted of 0.02 M Tris-HC1 pH 7.5 and the elution buffer
was 0.02 M Tris-HC1 with 50 mM NaCl pH 7.5. Figure 4
depicts the absorption curve for protein at 280 nm an the
gradient profile. Four peaks can be seen in the eluate at
560 of the elution buffer.
We tested a guinea pig immunized with whole
salivary gland extract and previously shown to be tick
immune, with dilutions of the unseparated extract in PBS and
with the peaks shown above, incompletely separated by FPLC,
diluted in Tris Hcl buffer.
After injecting dye intravenously, we made
intradermal injections of 0.1 ml of antigen. At about 10
minutes, blue-spots began to appear. As shown in Figure 5,
the Peak 1 showed strong activity, indicating the presence
of a protective antigen.
EXAMPLE XII - Identification of Protective
I. scapularis Antigens in
Fractionated Salivar5r Gland Extract
To identify protective I. scapularis salivary gland
antigens, we prepared salivary gland extract as described
above. We used 800 fed salivary glands to prepare an that
yielded 600 ug of total protein. We electrophoresed 500 ug
of the on a 12o SDS-PAGE gel and separated with a BioRad
gel eluter. The elution yielded 14 fractions ranging in
size from 14-100 kD.
We conducted a CBH assay as described above,
injecting an immunized guinea pig with 0.1 ml of each
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fraction. As shown in Figure 6, we observed a definite
increase in the C:BH response in the skin regions injected
with Fractions 9 and 1() as well as whole .
Fractions 9 and 10 have a protein band at 28 kD
and 40 kD respect:ively. Thus, we have identified specific
proteins from I. scapu_laris which appear to have a role in
inducing tick immunity in guinea pigs.
EXAMPLE XIII - Separat:ion of I. scapularis
S~~livar~~ Gland Extract
We thawed 800 salivary glands from I. scapularis
obtained as described above and pooled them into a 1.5 rnl
low adhesion microcentrifuge tube. We removed as much
supernatant from the pallet as possible, checking that there
were no salivary glands in the supernatant. We added 259 ul
of distilled water and 0.0020 TWEEN 800 to the pellet and
vortexed careful~.y. WEB then sonicated the salivary glands
for 5 min. in an ice water bath and vortexed again. We
repeated the sonication twice, each time for 5 min. We spun
at 14,000 rpm to pellei~ the debris, added 30 ul if lOX PBS
and removed 55 u7. for another use . We added 50 ul of 5X
sample buffer to the rs~maining extract, boiled for 5 min.
and froze at -20°C.
We eleca rophoresed 500 ug (approximately 300 ul)
of extract on a 7.2 o SD:3-PAGE at 100 V. We then put the gel
into Tris-Boric ~icid, pH 8.3 and 0.5% SDS for 10 min. to
equilibrate. We cut the gel to fit into the BioRad gel
eluter and eluted for :L8 min. at 90 my constant current
reversing for 10 sec.
We obt~iined :14 fractions which we concentrated
using Ultrafree MC concentrators. We then ran 3 ul of each
fraction on a 12« gel. We used one fourth of each fraction
in a cutaneous anaphylaxis assay to determine which fraction
had protective antigens. As seen in Figure 7, Fractions 9
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and 10 caused in increase in the CBH response. The protein
bands of Fractions 9 and 10 are 28 kD an 40 kD respectively.
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Applicant's or agent's file x_105 PCT ~ Intetnat:onal appiicationNo.
pCTNS98/08371
reference number
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis)
A. 'Ihe indications made below
relate to the microorganism
referred to in the description
on page P6, L9; P40, L27; P55,
L18; P 57, L11 line
B. IDENTIFICATION OF DEPOSItT
Further deposits are identified
on an additional sheet Q
Name of depository institution
American Type Culture Collection
Addross of depository institution
(including postal code and country)
12301 Parklawn Drive
Rockville, Maryland 2085:2
United States of America
Identification Reference by Depositor:
Ixodes scapularis salpl6-pBLUESCRIPT
plasmid
Date of deposit Accession Number
28 April 1998 (28.04.98)
C. ADDITIONAL INDICATIONS (lrava
blank ij'nd applicoblr) 'llis
information is continued on
an additional sheet Q
In respect of the designation
of the EPO, samples of the deposited
microorganisms will
be made available until the publication
of the mention of the grant
of the European patent
or until the date on which the
application is refused or withdrawn
or is deemed to be
withdrawn, as provided in Rule
28(3) of the Implementing Regulations
under the EPC
only by the issue of a sample
to an expert nominated by requester
(Rule 28(4) EPC).
D. DESIGNATED STATES FOR VVHICH
INDICATIONS ARE MADE (i/tleindicotionrarend~orall
daignotodState)
EPO
E. SEPARATE FURNISHING OF INDICATIONS
(lraveblankif not applicabk)
The indications listed below
will be submitted to
thelnternationalBureaulater(specifytltegawolnatareo/tlmindicatiomaa.,
Acceviae
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~ For receiving Office use only For lnternationa! Bureau use only
'ibis sheet was received with the international application ~ 'I3is sheet was
received by the international Bureau on:
Autb~ized officer / / Authorized offices
Form PCT/R0/134 (July 1992)
CA 02288433 1999-10-28
WO 98/49303 PCT/US98/0837I
52
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Yale University
(B) STREET: 451 College Street
(C) CITY: New Haven
(D) STATE: CT
(E) COUNTRY: USA
(F) ZIP: 06520
(ii) TITLE OF INVENTION: TICK IMMUNITY
(iii) NUMBER OF SEQUENCES: 4
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(v) CURRENT APPLICATION DATA:-
(A) APPLICATION NUMBER: PCT Unassigned
(B) FILING DATE: 29-APR-1998
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 459 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..456
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:1:
ATGTTCAAA CTGAAGTTC TTTATTCTCTTC GCACTCGCT GGATTATGT 48
MetPheLys LeuLysPhe PheIleLeuPhe AlaLeuAla GlyLeuCys
5 10 15
TTCGGGGAT ACAAGTCCC AGTGAGACAGGA GCATCATCT TCTGATGGT 96
PheGlyAsp ThrSerPro SerGluThrGly AlaSerSer SerAspGly
20 25 30
GAAGCTGGC AGCGAACCA GCGGGATCAGAA ACTGTTGAC CAAACGTCG 144
GluAlaGly SerGluPro AlaGlySerGlu ThrValAsp GlnThrSer
35 40 45
GAGGGTAAG GATGGTTCC GGTGACATCCAA AAAAGCAAA TCAATAGGC 192
GluGlyLys AspGlySer GlyAspIleGln LysSerLys SerIleGly
50 55 60
SUBSTITUTE SHEET (RULE 26)
CA 02288433 1999-10-28
WO 98149303 PCT/US98/08371
53
GACCATTTG CCAGACT'TCATCGGTACT AACCAGGAC AAA TCCTAT 290
GTA
AspHisLeu ProAspPhe IleGlyThr AsnGlnAsp LysValSerTyr
65 70 75 80
CTGAACAGG CTACTGT~T GTCTGCAAT AAAAAGCAC AACCTTCGCAAG 288
LeuAsnArg LeuLeuSer ValCysAsn LysLysHis AsnLeuArgLys
85 90 95
ATAAACAAA GTAAATA'TTACGTTCGAA CTCTGCACT TTCGTCTGTCTG 336
IleAsnLys ValAsnI1e ThrPheGlu LeuCysThr PheValCysLeu
100 105 110
AGCGAAAGT ATAACCGGA ACAAATCAA GAAGAACGA ATTCCAACAGAC 384
SerGluSer IleThrGly ThrAsnGln GluGluArg IleProThrAsp
115 120 125
CTGGTTTGC AACAGCA,~1CAAAGACAAA TGCCCCAAA GAAGGATCCTGC 932
LeuValCys AsnSerA.snLysAspLys CysProLys GluGlySerCys
130 135 140
CCAACACCC CCCTTGCCA AGCTGCTAA 459
ProThrPro ProLeuPro SerCys
145 150
(2) INFORMATION FOR S:EQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 152 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Phe Lys Leu Lys P:he Phe Ile Leu Phe Ala Leu Ala Gly Leu Cys
1 5 10 15
Phe Gly Asp Thr Ser Pro Ser Glu Thr Gly Ala Ser Ser Ser Asp Gly
20 25 30
Glu Ala Gly Ser Glu Pro Ala Gly Ser Glu Thr Val Asp Gln Thr Ser
35 40 45
Glu Gly Lys Asp Gly Ser Gly Asp Ile Gln Lys Ser Lys Ser Ile Gly
50 55 60
Asp His Leu Pro Asp Phe Ile Gly Thr Asn Gln Asp Lys Val Ser Tyr
65 70 75 80
Leu Asn Arg Leu Leu Ser Val Cys Asn Lys Lys His Asn Leu Arg Lys
85 90 95
Ile Asn Lys Val Asn Ile Thr Phe Glu Leu Cys Thr Phe Val Cys Leu
100 105 110
Ser Glu Ser Ile Thr Gly Thr Asn Gln Glu Glu Arg Ile Pro Thr Asp
115 120 125
SUBSTITUTE SHEET (RULE 26)
CA 02288433 1999-10-28
WO 98/49303 PCT/US98/08371
54
Leu Val Cys Asn Ser Asn Lys Asp Lys Cys Pro Lys Glu Gly Ser Cys
130 135 140
Pro Thr Pro Pro Leu Pro Ser Cys
145 150
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
TGTAGGCGGT TCGGTAAGTT AAAG 29
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GCACTCATCG TTTACAGCGT G 21
SUBSTITUTE SHEET (RULE 26)
