CA2288433A1 - Compositions and methods for conferring tick immunity and preventing tick borne diseases - Google Patents
Compositions and methods for conferring tick immunity and preventing tick borne diseases Download PDFInfo
- Publication number
- CA2288433A1 CA2288433A1 CA002288433A CA2288433A CA2288433A1 CA 2288433 A1 CA2288433 A1 CA 2288433A1 CA 002288433 A CA002288433 A CA 002288433A CA 2288433 A CA2288433 A CA 2288433A CA 2288433 A1 CA2288433 A1 CA 2288433A1
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- Prior art keywords
- scapularis
- polypeptide
- tick
- polypeptides
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43527—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from ticks
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/14—Ectoparasiticides, e.g. scabicides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Tropical Medicine & Parasitology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Insects & Arthropods (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Methods and compositions for conferring tick immunity and preventing or reducing the transmission of tick-borne pathogens. I. scapularis polypeptides and fragments, fusion and multimeric proteins, DNA molecules encoding them, antibodies directed against the polypeptides, fusion proteins or multimeric proteins. Vaccines comprising I. scapularis polypeptides alone or in addition to other protective polypeptides. Methods comprising the polypeptides, antibodies and vaccines.
Description
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.
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.
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 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 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.
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 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.
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 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 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.
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 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.
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.
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.
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, 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 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 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 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 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 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).
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 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.
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, 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 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 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 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 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 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).
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 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.
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 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.
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 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.
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 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.
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.
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 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.
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.
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 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 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 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 WO 98!49303 PCT/US98/08371 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 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-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 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.
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 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.
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.
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 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.
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 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 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 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 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.
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.
WO 98!49303 PCT/US98/08371 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, 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 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 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 WO 98/49303 PCT/US98/083?1 and 10 caused in increase in the CBH response. The protein bands of Fractions 9 and 10 are 28 kD an 40 kD respectively.
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 Numbrr ojDtposir'J
Accession Number of Deposit ~ 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) 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:
MetPheLys LeuLysPhe PheIleLeuPhe AlaLeuAla GlyLeuCys PheGlyAsp ThrSerPro SerGluThrGly AlaSerSer SerAspGly GluAlaGly SerGluPro AlaGlySerGlu ThrValAsp GlnThrSer GluGlyLys AspGlySer GlyAspIleGln LysSerLys SerIleGly SUBSTITUTE SHEET (RULE 26) GACCATTTG CCAGACT'TCATCGGTACT AACCAGGAC AAA TCCTAT 290 GTA
AspHisLeu ProAspPhe IleGlyThr AsnGlnAsp LysValSerTyr CTGAACAGG CTACTGT~T GTCTGCAAT AAAAAGCAC AACCTTCGCAAG 288 LeuAsnArg LeuLeuSer ValCysAsn LysLysHis AsnLeuArgLys ATAAACAAA GTAAATA'TTACGTTCGAA CTCTGCACT TTCGTCTGTCTG 336 IleAsnLys ValAsnI1e ThrPheGlu LeuCysThr PheValCysLeu SerGluSer IleThrGly ThrAsnGln GluGluArg IleProThrAsp CTGGTTTGC AACAGCA,~1CAAAGACAAA TGCCCCAAA GAAGGATCCTGC 932 LeuValCys AsnSerA.snLysAspLys CysProLys GluGlySerCys ProThrPro ProLeuPro SerCys (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 Phe Gly Asp Thr Ser Pro Ser Glu Thr Gly Ala Ser Ser Ser Asp Gly Glu Ala Gly Ser Glu Pro Ala Gly Ser Glu Thr Val Asp Gln Thr Ser Glu Gly Lys Asp Gly Ser Gly Asp Ile Gln Lys Ser Lys Ser Ile Gly Asp His Leu Pro Asp Phe Ile Gly Thr Asn Gln Asp Lys Val Ser Tyr Leu Asn Arg Leu Leu Ser Val Cys Asn Lys Lys His Asn Leu Arg Lys Ile Asn Lys Val Asn Ile Thr Phe Glu Leu Cys Thr Phe Val Cys Leu Ser Glu Ser Ile Thr Gly Thr Asn Gln Glu Glu Arg Ile Pro Thr Asp SUBSTITUTE SHEET (RULE 26) Leu Val Cys Asn Ser Asn Lys Asp Lys Cys Pro Lys Glu Gly Ser Cys Pro Thr Pro Pro Leu Pro Ser Cys (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:
(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:
SUBSTITUTE SHEET (RULE 26)
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 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.
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 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.
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 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.
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.
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 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.
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.
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 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 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 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 WO 98!49303 PCT/US98/08371 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 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-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 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.
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 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.
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.
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 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.
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 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 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 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 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.
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.
WO 98!49303 PCT/US98/08371 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, 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 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 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 WO 98/49303 PCT/US98/083?1 and 10 caused in increase in the CBH response. The protein bands of Fractions 9 and 10 are 28 kD an 40 kD respectively.
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 Numbrr ojDtposir'J
Accession Number of Deposit ~ 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) 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:
MetPheLys LeuLysPhe PheIleLeuPhe AlaLeuAla GlyLeuCys PheGlyAsp ThrSerPro SerGluThrGly AlaSerSer SerAspGly GluAlaGly SerGluPro AlaGlySerGlu ThrValAsp GlnThrSer GluGlyLys AspGlySer GlyAspIleGln LysSerLys SerIleGly SUBSTITUTE SHEET (RULE 26) GACCATTTG CCAGACT'TCATCGGTACT AACCAGGAC AAA TCCTAT 290 GTA
AspHisLeu ProAspPhe IleGlyThr AsnGlnAsp LysValSerTyr CTGAACAGG CTACTGT~T GTCTGCAAT AAAAAGCAC AACCTTCGCAAG 288 LeuAsnArg LeuLeuSer ValCysAsn LysLysHis AsnLeuArgLys ATAAACAAA GTAAATA'TTACGTTCGAA CTCTGCACT TTCGTCTGTCTG 336 IleAsnLys ValAsnI1e ThrPheGlu LeuCysThr PheValCysLeu SerGluSer IleThrGly ThrAsnGln GluGluArg IleProThrAsp CTGGTTTGC AACAGCA,~1CAAAGACAAA TGCCCCAAA GAAGGATCCTGC 932 LeuValCys AsnSerA.snLysAspLys CysProLys GluGlySerCys ProThrPro ProLeuPro SerCys (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 Phe Gly Asp Thr Ser Pro Ser Glu Thr Gly Ala Ser Ser Ser Asp Gly Glu Ala Gly Ser Glu Pro Ala Gly Ser Glu Thr Val Asp Gln Thr Ser Glu Gly Lys Asp Gly Ser Gly Asp Ile Gln Lys Ser Lys Ser Ile Gly Asp His Leu Pro Asp Phe Ile Gly Thr Asn Gln Asp Lys Val Ser Tyr Leu Asn Arg Leu Leu Ser Val Cys Asn Lys Lys His Asn Leu Arg Lys Ile Asn Lys Val Asn Ile Thr Phe Glu Leu Cys Thr Phe Val Cys Leu Ser Glu Ser Ile Thr Gly Thr Asn Gln Glu Glu Arg Ile Pro Thr Asp SUBSTITUTE SHEET (RULE 26) Leu Val Cys Asn Ser Asn Lys Asp Lys Cys Pro Lys Glu Gly Ser Cys Pro Thr Pro Pro Leu Pro Ser Cys (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:
(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:
SUBSTITUTE SHEET (RULE 26)
Claims (44)
1. An isolated, recombinant or synthetic DNA molecule comprising a DNA sequence which encodes an I. scapularis polypeptide, wherein said polypeptide is selected from the group consisting of:
(a) the SP16 polypeptide of SEQ ID NO: 2;
(b) fragments comprising at least 8 amino acids taken as a block from the polypeptide of (a);
(c) a derivative of any one of the polypeptides of (a), said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a).
(a) the SP16 polypeptide of SEQ ID NO: 2;
(b) fragments comprising at least 8 amino acids taken as a block from the polypeptide of (a);
(c) a derivative of any one of the polypeptides of (a), said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a).
2. The DNA molecule according to claim 1, wherein the DNA sequence is the sequence set forth in SEQ ID NO: 1.
3. An isolated, recombinant or synthetic DNA molecule comprising a DNA sequence which encodes a 32 kD I.
scapularis polypeptide expressed by clone 1 (ATCC accession No _____), and fragments and derivatives thereof.
scapularis polypeptide expressed by clone 1 (ATCC accession No _____), and fragments and derivatives thereof.
4. An isolated, recombinant or synthetic DNA molecule comprising a DNA sequence which encodes a 28 kD I.
scapularis polypeptide which appears as a single band on SDS-PAGE of Fraction 9 of I. scapularis salivary gland extract
scapularis polypeptide which appears as a single band on SDS-PAGE of Fraction 9 of I. scapularis salivary gland extract
5. An isolated, recombinant or synthetic DNA molecule comprising a DNA sequence which encodes a 40 kD I.
scapularis polypeptide which appears as a single band on SDS-PAGE of Fraction 10 of I. scapularis salivary gland extract.
scapularis polypeptide which appears as a single band on SDS-PAGE of Fraction 10 of I. scapularis salivary gland extract.
6. An isolated, recombinant or synthetic DNA molecule comprising a DNA sequence which encodes a 65 kD I.
scapularis polypeptide which appears as a single band on SDS-PAGE of I. scapularis saliva.
scapularis polypeptide which appears as a single band on SDS-PAGE of I. scapularis saliva.
7. The DNA molecule according to any one of claims 1-6, wherein said polypeptide comprises a protective epitope.
8. A DNA molecule comprising a DNA sequence encoding a fusion protein, wherein the fusion protein comprises an I.
scapularis polypeptide encoded by a DNA molecule according to any one of claims 1 to 7.
scapularis polypeptide encoded by a DNA molecule according to any one of claims 1 to 7.
9. A DNA molecule comprising a DNA sequence encoding a multimeric protein, which multimeric protein comprises an I. scapularis polypeptide encoded by a DNA molecule according to any one of claims 1 to 7.
10. An expression vector comprising a DNA molecule according to any one of claims 1 to 9.
11. A host cell transformed with a DNA molecule according to any one of claims 1 to 10 or the expression vector according to claim 12.
12. The host cell according to claim 11, wherein said host cell is selected from the group consisting of: strains of E. coli; Pseudomonas, Bacillus; Streptomyces; yeast, fungi; animal cells, including human cells in tissue culture; plant cells; and insect cells.
13. A polypeptide encoded by a DNA molecule according to any one of claims 1 to 7.
14. A method for producing a polypeptide according to claim 13, comprising the step of culturing a host cell according to claim 11 or claim 12.
15. An I scapularis polypeptide selected from the group consisting of:
(a) the SP16 polypeptide of SEQ ID NO: 2;
(b) fragments comprising at least 8 amino acids taken as a block from the polypeptide of (a);
(c) a derivative of the polypeptide of (a), said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a).
(a) the SP16 polypeptide of SEQ ID NO: 2;
(b) fragments comprising at least 8 amino acids taken as a block from the polypeptide of (a);
(c) a derivative of the polypeptide of (a), said derivative being at least 80% identical in amino acid sequence to the corresponding polypeptide of (a).
16. A 32 kD I. scapularis polypeptide expressed by clone 1 (ATCC accession No. ), and fragments and derivatives thereof.
17. A 28 kD I, scapularis polypeptide which appears as a single band on SDS-PAGE of Fraction 9 of I. scapularis salivary gland extract.
18. A 40 kD I. scapularis polypeptide which appears as a single band on SDS-PAGE of Fraction 10 of I. scapularis salivary gland extract.
19. A 65 kD I. scapularis polypeptide which appears as a single band on SDS-PAGE of I. scapularis saliva.
20. A fusion protein comprising an I. scapularis polypeptide according to any one of claims 15 to 19.
21. The fusion protein according to claim 20, wherein said fusion protein comprises two or more I. scapularis polypeptides.
22. A multimeric protein comprising an I. scapularis polypeptide according to any one of claims 15 to 19.
23. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a component selected from the group consisting of: a polypeptide according to any one of claims 15-19; a fusion protein according to claim 20 or 21 ; and a multimeric protein according to claim 22.
24. The pharmaceutical composition according to claim 23, wherein the component is crosslinked to an immunogenic carrier.
25. The pharmaceutical composition according to claim 23 or 24, further comprising at least one additional non-I. scapularis polypeptide.
26. The pharmaceutical composition according to claim 25, wherein the non-I. scapularis polypeptide is a protective polypeptide from a tick-borne pathogen.
27. The pharmaceutical composition according to claim 26, wherein the tick-borne pathogen is selected from the group consisting of: Borrelia burgdorferi, aoHGE, Babesia microti and arboviruses.
28. The pharmaceutical composition according to claim 27, wherein the non-I, scapularis polypeptide is a B.
burgdorferi polypeptide.
burgdorferi polypeptide.
29. A method for conferring tick immunity, comprising the step of administering to a subject a pharmaceutical composition according to any one of claims 23 to 28.
30. A method for preventing infection by a tick-borne pathogen or a tick-borne disease, wherein the method comprises the step of administering to a subject a pharmaceutical composition according to any one of claims 23-28.
31. A diagnostic kit comprising a component selected from the group consisting of: a polypeptide according to any one of claims 15-19; a fusion protein according to claim 20 or 21; and a multimeric protein according to claim 22, and also comprising a means for detecting binding of said component to an antibody.
32. An antibody that binds to a polypeptide according to any one of claims 15-19.
33. The antibody according to claim 32 which is polyclonal.
34. The antibody according to claim 32 which is monoclonal.
35. A diagnostic kit comprising an antibody according to any one of claims 32-34.
36. A method for detecting tick immunity comprising the step of contacting a body fluid of a subject with a polypeptide according to any one of claims 15-19; a fusion protein according to claim 20 or 21 ; and a multimeric protein according to claim 22.
37. A pharmaceutical composition comprising an antibody according to any one of claims 32-34.
38. A vaccine comprising an anti-I. scapularis polyclonal antibody.
39. A vaccine comprising a monoclonal anti-I.
scapularis antibody.
scapularis antibody.
40. A method for conferring tick immunity comprising administering to a subject an antibody according to any one of claims 32-34, a pharmaceutical composition according to claim 37 or 44 or a vaccine according to claim 38 or 39.
41. Peak 1 of I. scapularis salivary gland extract, obtained by ion exchange chromatography with a MonoQ column in a Pharmacia FPLC apparatus.
42. Fraction 9 of I. scapularis salivary gland extract, obtained by electroelution with a Bio Rad mini whole gel eluter.
43. Fraction 10 of I. scapularis salivary gland extract, obtained by electroelution with a Bio Rad mini whole gel eluter.
44. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a component selected from the group consisting of: Peak 1 of claim 41, Fraction 9 of claim 42 and Fraction 10 of claim 43.
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US60/043,154 | 1997-04-29 | ||
PCT/US1998/008371 WO1998049303A2 (en) | 1997-04-29 | 1998-04-29 | Compositions and methods for conferring tick immunity and preventing tick borne diseases |
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WO (1) | WO1998049303A2 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000027873A2 (en) * | 1998-11-06 | 2000-05-18 | Research Corporation Technologies, Inc. | Interleukin-2 binding protein from arthropods |
US20010046499A1 (en) * | 1999-12-03 | 2001-11-29 | Kantor Fred S. | Tick antigens and compositions and methods comprising them |
WO2001058941A1 (en) * | 2000-02-11 | 2001-08-16 | Evolutec Limited | Cytokine activity regulator molecules from tick salivary glands |
WO2022241312A1 (en) * | 2021-05-14 | 2022-11-17 | University Of Maryland, College Park | Tick mouthpart antigens as effective anti-tick vaccines |
-
1998
- 1998-04-29 WO PCT/US1998/008371 patent/WO1998049303A2/en not_active Application Discontinuation
- 1998-04-29 CA CA002288433A patent/CA2288433A1/en not_active Abandoned
- 1998-04-29 AU AU72584/98A patent/AU7258498A/en not_active Abandoned
- 1998-04-29 JP JP54721298A patent/JP2001523964A/en active Pending
- 1998-04-29 EP EP98919901A patent/EP1017806A2/en not_active Withdrawn
Also Published As
Publication number | Publication date |
---|---|
WO1998049303A3 (en) | 1999-01-28 |
WO1998049303A2 (en) | 1998-11-05 |
JP2001523964A (en) | 2001-11-27 |
EP1017806A2 (en) | 2000-07-12 |
AU7258498A (en) | 1998-11-24 |
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Legal Events
Date | Code | Title | Description |
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FZDE | Discontinued |