WO1998049303A2 - Compositions and methods for conferring tick immunity and preventing tick borne diseases - Google Patents

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 IMMUNITY AND PREVENTING TICK BORNE DISEASES

This application claims priority under 35 U.S.C. § 120 from pending United States provisional application Serial Number 60/043,154, filed April 29, 1997.

This invention was made with government support under Grant numbers Al 30548, Al 37993, Al 41440 and Al 39002 awarded by the National Institutes of Health. The government may have certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION This invention relates to compositions and methods for conferring immunity to tick bites and for the prevention of tick-borne diseases.

More particularly, this invention relates to polypeptides, and DNA sequences which encode them, from the Ixodes scapulari s tick. Such polypeptides and DNA sequences are useful to detect tick 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 infection of a host with tick-borne pathogens. Also within the scope of this invention are antibodies directed against J. scapularis polypeptides, compositions including vaccines comprising the antibodies.

This invention also relates to vaccines comprising one or more of the J. scapulari s polypeptides or antibodies of this invention. Also within the scope of this invention are diagnostic kits comprising I. scapularis polypeptides or antibodies of this invention.

This invention also relates to methods for using the aforementioned polypeptides, 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. MMWR 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," Parasi tology, 84, pp. 195-204 (1982); M. Brossard et al . , "Jxodes ricinus L: Mast Cell, Basophils and Eosinophils In the Sequence of Cellular Events In the Skin of Infested or RE-infested Rabbits," Parasi tology, 85, pp. 583-592 (1982); Fivaz et al . , "Cross- resistance Between Instars of the Brown Ear-tick Rhipicephal us appendicula tus (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, transmission of some tick-borne pathogens might be reduced.

Ixodid ticks are the most important arthropod vectors of infectious agents. Ixodes scapulari s 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. scapulari s for use in inducing tick immunity.

DISCLOSURE OF THE INVENTION

The present 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. scapulari s polypeptides, DNA sequences that encode the polypeptides, antibodies directed against the polypeptides and compositions and methods comprising the polypeptides, DNA sequences and antibodies.

This invention further provides a single or multicomponent vaccine comprising one or more I. scapulari s polypeptides or antibodies of this invention.

This invention relates to DNA sequences that code for I. scapularis antigens, recombinant DNA molecules that are characterized 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 J. 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 J. scapulari s 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. scapulari s 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 depicts the duration of attachment of nymphal ticks on guinea pigs sensitized to J. scapularis larvae.

Figure 4 show the results of individual 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 B31 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).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to J. 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 . 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. scapulari s 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 J. scapularis polypeptide isolated as a single band on a 12% 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. scapulari s polypeptide isolated as a single band on a 12% SDS-PAGE gel from tick saliva, and compositions and methods comprising the polypeptide.

In another embodiment, this invention provides a Peak 1 fraction of J. 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 12% PAGE gel and gel elution of the extract, and compositions and methods comprising the polypeptide. In another embodiment, this invention provides Fraction 10 of I. scapularis salivary gland extract obtained by separation on a 12% 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 embodiments are characterized by immunogenic polypeptides. As used herein, an "immunogenic J. scapulari s polypeptide" is any I . scapularis polypeptide that, when administered to an animal, is capable of eliciting a corresponding antibody. In particular, immunogenic I . scapularis polypeptides are intended to include additional polypeptides 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 polypeptides 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 preferred embodiment, this invention provides a vaccine comprising one or more I. scapulari s 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 J. scapularis polypeptides of this invention. Such antibodies may be used in a variety of applications, including to detect expression of J. 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 J. 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 J. scapularis salivary gland extract, as described in Example XIII; or a 65 kD 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 JB. burgdorferi , and preventing or lessening the severity of infection includes erythema migrans, arthritis, carditis, neurological disorders, and other Lyme disease related disorders. It may be evidenced by a decrease in or absence of spirochetes in the treated animal. And, it may be evidenced by a decrease in the level of spirochetes in infected ticks which have fed on treated animals.

One of skill in the art will understand that probes and oligonucleotide primers derived from the DNA encoding an J. scapulari s polypeptide may be used to isolate and clone further variants of I. scapulari s proteins from other Ixodes isolates and perhaps from other hard bodied ticks as well, which are useful in the methods and compositions of this invention.

As used herein, a "derivative" an J. 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 (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 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-J. scapularis polypeptide antibody of this invention includes antibodies directed against polypeptides expressed by J. scapularis, or fragments or derivatives thereof, that are immunologically cross-reactive with any one of the aforementioned polypeptides. Finally, an anti-I. scapularis polypeptide antibody of this invention includes antibodies directed against other I. scapularis polypeptides identified according to methods taught herein.

As used herein, an "anti-I. scapulari s polypeptide antibody" is an immunoglobulin molecule, or portion thereof, that is immunologically reactive with an I. scapularis polypeptide of the present invention and that was either elicited by immunization with J. scapularis or an J. scapulari s polypeptide of this invention or was isolated or identified by its reactivity with an J. 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 immunologically 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 Laboratory 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 polypeptides disclosed herein in order to alter the lipidation state is also a method which may be used to increase their immunogenicity or alter their biochemical properties. For example, the polypeptides or fragments thereof may be expressed with or without the signal and other sequences that may 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 express the polypeptides as fusion proteins comprising other I. scapularis or non-I. scapularis sequences .

In accordance with this invention, derivatives of the I. scapularis polypeptides may be prepared by a variety of methods, including by in vi tro 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. Those of skill in the art will understand that conservative substitution is preferred, e.g., 3-methylhistidine may be substituted for histidine, 4-hydroxyproline may be substituted for proline,

5-hydroxylysine may be 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 5%, more typically less than 1%) 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) . Causing amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties. Such substitutions would include for example, substitution of a hydrophilie residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, 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 residue having a net negative charge.

When the result of a given substitution cannot be predicted with certainty, the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired 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 polypeptides 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 I. scapularis polypeptide.

In a preferred embodiment of this invention, fusion proteins comprising I. scapularis polypeptides are constructed comprising B cell and/or T cell epitopes from multiple serotypic 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 of 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. scapulari s polypeptides are fused to moieties, such as immunoglobulin domains, which may increase the stability and prolong the in vi vo 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 vaccine. 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, human monocytic ehrlichiosis, babesiosis, diphtheria, polio, hepatitis, and measles. Such a vaccine, by virtue of its ability to elicit antibodies to a variety of protective I. scapulari s 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 multicomponent vaccine may contain the I. scapulari s polypeptides as part of a multimeric molecule in which the various components are covalently associated. Alternatively, it may contain multiple individual components. For example, a multicomponent vaccine may be prepared comprising two 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.

Alternatively, 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 SP16 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 cell 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. scapulari s 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 the I. scapulari s 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 well 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. scapulari s 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 epitopes may be B cell epitopes, T cell epitopes, or combinations thereof.

According to another embodiment of this invention, we describe a method which 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, of infection by a tick-borne pathogen. Again, the polypeptides, fusion proteins and multimeric proteins that are preferred for use in such methods are those that contain protective epitopes, which may be B cell epitopes, T cell epitopes, or combinations thereof. The most 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 theory, we believe that this is the best way to 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 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 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 polypeptides of this invention in a form that will favor the production of T-helper cells type 1 (T_H1), which help activate macrophages, and/or T-helper cells type 2 (T_H2), which help B cells to generate antibody responses. Aside from administering epitopes which are strong T cell or B cell epitopes, the induction of T_H1 or T_H2 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 (T_H1 response) or interleukin-4 or interleukin-10 (T_H2 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. Alternatively, the polypeptides may be used to produce monoclonal 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 vi tro by testing them for their ability to stimulate proliferation and/or cytokine production by T cell clones generated 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 immunogenic I. scapulari s polypeptides containing strong T cell epitopes. The fusion protein that carries both strong T cell and B cell epitopes is able to participate in elicitation of a high titer antibody response effective to confer tick immunity.

Strong T cell epitopes may also be provided by non-I. scapularis molecules. For example, strong T cell epitopes have been observed 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, [D.R. Milich et al . , "Antibody Production To The Nucleocapsid And Envelope Of The Hepatitis B Virus Primed By A Single Synthetic T Cell Site", Nature, 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. scapularis 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. scapularis 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 transcriptional and translational expression control sequences that are functional in the chosen host. Preferably, the expression control sequences, and the gene of interest, will be contained in an expression vector that further comprises a selection marker.

The DNA sequences 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 sequence 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 expressed 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, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus and retroviruses including lentiviruses . Useful expression vectors for bacterial 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. λGTIO and λGTll, and other phages. Useful expression vectors for yeast cells include the 2μ plasmid and derivatives thereof. Useful vectors for insect 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 trp 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 -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, monolayers or the like. Host cells may also be derived directly or indirectly from tissues.

A wide variety of unicellular host cells are useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli , Pseudomonas, Bacillus, Stre tomyces, 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, 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 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 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. unit (e.g., which RNA polymerase 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 promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase 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 hosts 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 proteins 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 purification 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 encoded 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 , Synthesi s, 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 protecting groups known in the field of peptide synthesis and recognized by conventional abbreviations therein, may be found in T. Greene, Protecti ve Groups In Organic Synthesis, Academic Press (1981) .

According to another embodiment of this invention, antibodies directed against the I. scapulari s polypeptides are generated. Such antibodies are immunoglobulin molecules or portions thereof that are immunologically reactive with an I. scapulari s polypeptide of the present invention. It should be understood that the antibodies of this invention include antibodies immunologically reactive with fusion proteins and multimeric proteins comprising an I. scapularis polypeptide . Antibodies directed against an I. scapulari s polypeptide may be generated by a variety of means including immunizing a mammalian host with I. scapularis extract or tick infestation, or by immunization of a mammalian host with an I. scapularis polypeptide of the present invention. Such antibodies may be 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 Biochem.. 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 immunoglobulin sequences from different species (e.g., mouse and human ) or from portions of immunoglobulin light and heavy chain sequences from the same species. It may 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 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 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 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 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 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 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 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. scapulari s polypeptides, either in libraries constructed from I. scapulari s DNA 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 infection 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 only a classical model for tick immunity but also displays skin reactivity that mimic hypersensitivity reactions in humans. Thus, by administering a particular I. scapularis polypeptide or anti-I. scapularis polypeptide antibody to guinea pigs, one of skill in the art may determine without undue experimentation whether that polypeptide or antibody would be useful in the methods and compositions claimed herein.

The administration of the I. scapulari s polypeptide or antibody 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 such techniques, see Antibodies , A Laboratory Manual , 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-polyoxypropylene-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 similar 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 compositions similar to those employed for other pharmaceutically important polypeptides (e.g., the vaccine against hepatitis) .

Any pharmaceutically acceptable dosage route, including parenteral, intravenous, intramuscular, intralesional or subcutaneous injection, may be used to administer the polypeptide or antibody composition. For example, the composition may be administered to the patient in any pharmaceutically acceptable dosage form including those which may be administered to a patient intravenously as bolus or by continued infusion over a period of hours, days, weeks or months, 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 μg to 100 mg of the purified polypeptide, and preferably, the dosage will consist of 10-1000 μg. Generally, the dosage for an antibody will be 0.5 mg-3.0 g.

In a preferred embodiment of this invention, the I. scapulari s 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 Typhimuri um Vaccine Expressing Circumsporozoite Protein 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 Antigen Induces Partial Protection Against Coccidiosis", Inf. Immun.. 57, pp. 2434-40 (1989); M. Dunne 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. Pis . , 164:1224 (1991) .

Moreover, the level of pathogens in ticks feeding on such animals may be lessened or eliminated, thus inhibiting transmission to the next animal.

According to yet another embodiment, the I. scapulari s 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. scapularis antigens as a result of a tick bite. The detection of I. scapularis antigens is evidence of tick attachment and at least some feeding. Such information is an important aid in the early diagnosis of I. scapularis- borne diseases. Such diagnostic agents may be included in a kit which may also comprise 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 125I or 51Cr 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 C,

15 0, 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. scapulari s 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. scapularis polypeptides and antibodies directed against those polypeptides provide much more specific diagnostic reagents than whole ticks and thus may alleviate such pitfalls as false positive and false negative results. One skilled in the art will realize that it may also be advantageous in the preparation of detection reagents to utilize epitopes 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 microti , aoHGE (the agent of human granulocytic ehrlichiosis) as well as some arboviruses, such as the Eastern equine encephalitis virus, and instructions for their use.

The polypeptides and antibodies of the present invention, and compositions and methods comprising them, may also be useful for prevention of tick bites by other species of ticks which may express proteins sharing amino acid sequence or conformational similarities with the I. scapularis polypeptides of the present invention.

In order that 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 - Guinea Piσ Model of I. scapulari s Immunity

We chose the guinea pig for our model even though it is not a natural host for Ixodes ticks because guinea pigs are the classical model for tick immunity and because their immune skin reactions closely mimic those in humans. We infested naive guinea pigs with 100 larval I. scapulari s 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-mercaptoethanol until all dissections were complete to prevent degradation by RNases.

We isolated RNA using Stratagene 's RNA Micro Isolation Kit®. Briefly, we added 30 μl of 2M Na acetate, 300 μl if water-saturated phenol and 60 μl if chloroform: isoamyl alcohol to a 300 μl aliquot of salivary gland in GTIC/mercaptoethanol . We capped the tube, vortexed and microfuged for 5 min. 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 75% 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 μg total RNA per tick. We sent the isolated RNA to Clonetech where a Lambda ZAPII expression library was made after initial amplification of the message.

We also prepared a whole-tick cDNA library using a substantially similar method.

B. Screening Ixodes 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 μg 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 μM 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 the 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. 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 15 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 -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 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 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. 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 R408 helper phage and digested the vectors with the inserts with EcoRl endonuclease . Clone 1 had a 700 bp insert; Clone 2, an 800 bp insert, Clone 3, a 600 bp 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 individual inserts in XL1 blue cells with IPTG. We lysed the cells and separated the lysate on SDS-PAGE, transferred to nitrocellulose membrane and probed with tick-immune or salivary gland immune sera. Tick immune sera bound to a 32 kD band from Clones 1 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 band was recognized in both uninduced and IPTG induced cells. Thus, the proteins are not expressed from the lac promoter.

To identify additional I. scapularis antigens capable of conferring tick immunity, we rescreen the expression libraries with immune sera from mice, rabbits and humans according to the methods described herein. C. Sequencing the Inserts

The inserts of Clones 1-3 were sequenced by the Sanger method in the W. Keck DNA sequencing Laboratory at Yale. All 3 of the clones were found to have the same open reading frame. The gene, which we designated spl 6, encodes a 16 kD protein. The DNA sequence and deduced amino acid sequence of spl β axe 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 spl β 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 spl β 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, spl β is expressed in the salivary gland.

EXAMPLE III - Recombinant Expression of SP16 To obtain enough DNA for expression, we amplified the spl β gene sequence from the BLUESCRIPT plasmid and added Xhol an Hindlll sites to a fragment of spl β 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 spl β into E. coli DH5α 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 with SP16

To test SP16 for the ability to confer tick immunity, we immunize naive guinea pigs with 10 μg 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 with Anti-SP16 Antiserum We prepared anti-SP16 antiserum by immunizing C3H/HeN mice with 10 μg of recombinant SP16 fusion protein an boosted twice with the 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 challenged with 5 nymphal ticks.

EXAMPLE VI - Isolation of Proteins From I. scapularis Saliva We collected saliva from I. scapularis according to the methods of Ewing et al . [C. Ewing et al . , "Isolation of Borrelia burgdorferi From Saliva of The Tick Vector, Ixodes scapulari s . " 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' backs with the cap glued on. We allowed ticks to feed for approximately 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 sterile glass micropipette around the hypostome to collect saliva.

We induced salivation by applying 2 μl of pilocarpine (50 mg/ml in 95% ethanol) to the scutum of the tick. We added additional 1 μl aliquots of pilocarpine at 20 min. intervals for 2.5 hours at 35°C in a humid chamber. We collected saliva from the micropipettes into a 0.5 ml sterile tube and frozen at -20°C. We added 3 μl of saliva to 2 μl of sample buffer and 5 μl running buffer, boiled and ran the sample on 12% 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 Novex 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 μg 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 DEAE 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-Tick Antiserum

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 50% reduction in the number of attached ticks compared to naive animals (p<0.05) . Ticks fed on passively immunized animals weighed 24% 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-Protection At Different Tick Stages

We were interested in determining if immunity to

I. scapulari s is stage-specific. This is of interest because the nymph and adult ticks transmit B. burgdorferi 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. scapulari s and passively immunized 2 guinea pigs with 5 ml i.v. of anti-larval immune serum. We used naive animals as controls. We challenged the animals with 50 I. scapularis nymphs each. We counted and weighed ticks recovered from the water pans daily.

We observed that actively and passively immunized animals had reduced duration of attachment (Figure 3) . Passively immunized animals had a 40% reduction in the number of ticks attached compared to controls at 96 hours.

The weight of ticks recovered from actively and passively immunized animal was also significantly reduced compared to controls . Thus, different stages of tick development share at least some protective antigens.

EXAMPLE X - Prevention of B. burgdorferi Transmission Before testing the effect of tick immunity on the transmission of B. burgdorferi , the agent of Lyme Disease, we determined whether guinea pigs could be infected by challenge with B . burgdorferi infected ticks. We challenged naive guinea pigs with 5 B31 or N40 strain infected I. scapularis nymphs. Skin punches at the site of tick attachment and elsewhere 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 . 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. scapulari s larvae or nymphs and 5 weeks later, challenged the sensitized animals with 5 ticks from a pool with an 80% 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-S. burgdorferi antibody confirmed that 70-100% 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 preliminary 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, aoHGE 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 transmission 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 Appl . 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 with 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 μg of the salivary gland extract to the column using a salt gradient. The starting buffer consisted of 0.02 M Tris-HCl pH 7.5 and the elution buffer was 0.02 M Tris-HCl 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

56% 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 Salivary 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 μg of total protein. We electrophoresed 500 μg of the on a 12% 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 CBH response in the skin regions injected with Fractions 9 and 10 as well as whole .

Fractions 9 and 10 have a protein band at 28 kD and 40 kD respectively. Thus, we have identified specific proteins from I. scapularis which appear to have a role in inducing tick immunity in guinea pigs.

EXAMPLE XIII - Separation of I. scapularis Salivary Gland Extract

We thawed 800 salivary glands from I. scapularis obtained as described above and pooled them into a 1.5 ml low adhesion microcentrifuge tube. We removed as much supernatant from the pellet as possible, checking that there were no salivary glands in the supernatant. We added 259 μl of distilled water and 0.002% TWEEN 80® to the pellet and vortexed carefully. We 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 pellet the debris, added 30 μl if 10X PBS and removed 55 μl for another use. We added 50 μl of 5X sample buffer to the remaining extract, boiled for 5 min. and froze at -20°C.

We electrophoresed 500 μg (approximately 300 μl) of extract on a 12% SDS-PAGE at 100 V. We then put the gel into Tris-Boric acid, 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 18 min. at 90 mv constant current reversing for 10 sec. We obtained 14 fractions which we concentrated using Ultrafree MC concentrators. We then ran 3 μl 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 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 reference number YU-105 PCT International app cationNo. PCT/US98/08371

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3bis)

A. The indications made below relate to the microorganism referred to in the description on _DageP6, L9; P40, L27; P55, LIB; P 57, LI 1 _|jne

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | |

Name of depositary institution

American Type Culture Collection

Address of depositary institution (including postal code and country)

12301 Parklawn Drive Rockville, Maryland 20852 United States of America

Identification Reference by Depositor: Ixodes scapularis salplό-pBLUESCRIPT plasmid

Date of deposit Accession Number

28 April 1998 (28.04.98)

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet [~~j

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 WHICH INDICATIONS ARE NfADE (if the indications are not for all designated States)

EPO

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The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications tg, 'Accession Number of Deposit")

Accession Number of Deposit

For receiving Office use only For International Bureau use only

I I This sheet was received with the international application j I This sheet was received by the International Bureau on:

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Form PCI7RO/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 REABABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln 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 NO:l:

(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 NO:l:

ATG TTC AAA CTG AAG TTC TTT ATT CTC TTC GCA CTC GCT GGA TTA TGT 48 Met Phe Lys Leu Lys Phe Phe He Leu Phe Ala Leu Ala Gly Leu Cys 1 5 10 15

TTC GGG GAT ACA AGT CCC AGT GAG ACA GGA GCA TCA TCT TCT GAT GGT 96 Phe Gly Asp Thr Ser Pro Ser Glu Thr Gly Ala Ser Ser Ser Asp Gly 20 25 30

GAA GCT GGC AGC GAA CCA GCG GGA TCA GAA ACT GTT GAC CAA ACG TCG 144 Glu Ala Gly Ser Glu Pro Ala Gly Ser Glu Thr Val Asp Gin Thr Ser 35 40 45

GAG GGT AAG GAT GGT TCC GGT GAC ATC CAA AAA AGC AAA TCA ATA GGC 192 Glu Gly Lys Asp Gly Ser Gly Asp He Gin Lys Ser Lys Ser He Gly 50 55 60 GAC CAT TTG CCA GAC TTC ATC GGT ACT AAC CAG GAC AAA GTA TCC TAT 240 Asp His Leu Pro Asp Phe He Gly Thr Asn Gin Asp Lys Val Ser Tyr 65 70 75 80

CTG AAC AGG CTA CTG TCT GTC TGC AAT AAA AAG CAC AAC CTT CGC AAG 288 Leu Asn Arg Leu Leu Ser Val Cys Asn Lys Lys His Asn Leu Arg Lys 85 90 95

ATA AAC AAA GTA AAT ATT ACG TTC GAA CTC TGC ACT TTC GTC TGT CTG 336 He Asn Lys Val Asn He Thr Phe Glu Leu Cys Thr Phe Val Cys Leu 100 105 110

AGC GAA AGT ATA ACC GGA ACA AAT CAA GAA GAA CGA ATT CCA ACA GAC 384 Ser Glu Ser He Thr Gly Thr Asn Gin Glu Glu Arg He Pro Thr Asp 115 120 125

CTG GTT TGC AAC AGC AAC AAA GAC AAA TGC CCC AAA GAA GGA TCC TGC 432 Leu Val Cys Asn Ser Asn Lys Asp Lys Cys Pro Lys Glu Gly Ser Cys 130 135 140

CCA ACA CCC CCC TTG CCA AGC TGC TAA 459

Pro Thr Pro Pro Leu Pro Ser Cys 145 150

(2) INFORMATION FOR SEQ ID NO: 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 NO: 2:

Met Phe Lys Leu Lys Phe Phe He Leu Phe Ala Leu Ala Gly Leu Cys

1 5 10 15

Phe Gly Asp Thr Ser Pro Ser Glu Thr Gly Ala Ser Ser Ser Asp Gly 20 25 30

Glu Ala Gly Ser Glu Pro Ala Gly Ser Glu Thr Val Asp Gin Thr Ser 35 40 45

Glu Gly Lys Asp Gly Ser Gly Asp He Gin Lys Ser Lys Ser He Gly 50 55 60

Asp His Leu Pro Asp Phe He Gly Thr Asn Gin Asp Lys Val Ser Tyr 65 70 75 80

Leu Asn Arg Leu Leu Ser Val Cys Asn Lys Lys His Asn Leu Arg Lys 85 90 95

He Asn Lys Val Asn He Thr Phe Glu Leu Cys Thr Phe Val Cys Leu 100 105 HO

Ser Glu Ser He Thr Gly Thr Asn Gin Glu Glu Arg He Pro Thr Asp 115 120 125 Leu Val Cys Asn Ser Asn Lys Asp Lys Cys Pro Lys Glu Gly Ser Cys 130 135 140

Pro Thr Pro Pro Leu Pro Ser Cys 145 150

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 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 NO: 3: TGTAGGCGGT TCGGTAAGTT AAAG 24

(2) INFORMATION FOR SEQ ID NO: 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 NO: 4: GCACTCATCG TTTACAGCGT G 21

Claims

Claims :

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) .

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 J. 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 J. scapularis polypeptide which appears as a single band on SDS-PAGE of Fraction 9 of J. scapulari s salivary gland extract

5. An isolated, recombinant or synthetic DNA molecule comprising a DNA sequence which encodes a 40 kD J. scapularis polypeptide which appears as a single band on SDS-PAGE of Fraction 10 of I. scapulari s 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 J. 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 J. scapulari s 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, Bacill us; 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 scapulari s 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) .

16. A 32 kD I. scapul ari s polypeptide expressed by clone 1 (ATCC accession No. ), and fragments and derivatives thereof.

17. A 28 kD J. scapularis polypeptide which appears as a single band on SDS-PAGE of Fraction 9 of J. scapularis salivary gland extract.

18. A 40 kD I. scapularis polypeptide which appears as a single band on SDS-PAGE of Fraction 10 of J. scapularis salivary gland extract.

19. A 65 kD J. scapularis polypeptide which appears as a single band on SDS-PAGE of J. scapulari s 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 J. scapularis polypeptides.

22. A multimeric protein comprising an I. scapulari s 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-J. 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-J. scapularis polypeptide is a B . 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-J. scapulari s polyclonal antibody.

39. A vaccine comprising a monoclonal anti-J. scapulari s 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 J. scapularis salivary gland extract, obtained by electroelution with a Bio Rad mini whole gel eluter.

43. Fraction 10 of J. 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.