CA2144651A1 - Immortalized cells and uses therefor - Google Patents

Abstract

The present invention provides immortalized tick cell lines, an immortalized bovine T cell line and uses therefor, including diagnostic reagents and vaccine compositions.

Description

:1, , " ~r ~VO 94/06463 ~ g 6 S 1 PCl/US93/08606 IMMORTP~T ~7Fn CELLS AND USES TH~REFOR

Field of the Invention This invention relates generally to il~ ~ ~lized pnmary cells, and, more specific~11y, to i~llnlul Lalized cell lines useful for the production of antigens and biologically active cv~ oullds.

P~aek~round of the Invention Continnous or immortalized cell lines which are chara~ten7ed by the ability to grow indto.finitely are valuable as a souree of immunogens, a source of new biologically active compounds and for the production and replication of relevantpathogens. The ability to establish a eulture that will grow indçfinitely variesdepending on the animal speeies from whieh the eells originate. For example, rodent eells routinely generate eontin~lous cell lines, but ehieken eells almost never beeome immortal. Human eells, with the exeeption of tumor eells, are not good sourees of immortalized eell lines.
Tick cell cultures are of interest for the research and study of tick-borne pathogens and microor~nism~ Presently available }n vitro primary or continuous tick cell cultures are not capable of long term, eonsistent maintenance of tick-borne

2 0 pathogens.
There exists a need in the art for imrnortalized vitro eell eultures of cells capable of supporting the growth of pathogenie org~nicmc, for use in developing vaccines to seleeted pathogens and for research purposes.

2 5 Summary of the Invention The invention provides imrnortalized cultures of primary eells. These cell lines are useful, for example, as ~ntigçns in vaecine compositions, as vessels for growth and production of antigens, as diagnostic reagents, and in therapeutic compositions.

3 0 Other aspects and advantages of the present invention are described further in the following 3et~i1e~1 description of the preferred embotiimentc thereof.

Detailed Descl;u~ion of the Invention The present invention provides i~n~ alized tick cell lines and an 3 5 immortalized bovine T cell line. Such immortalized cell lines according to this invention are useful as, or in the production of, vaccinal agents, e.g., against ticks, and may provide protection against ~ise~es caused by pathogens, e.g., tick-bornepathogens.

WO 94/06463 2 1 ~ ~ 6 5 ~ PCr/US93/08~

As used herein, the term "i~ Ol L~lized cell line" refers to the cells depositedwith the ~m~ ~c~n Type Culture Collection (ATCC) as stated below. Immortalized cells of this invention may be clonally expanded by conventional techniques to produce a homogeneous population of progeny cell lines which can be m~int~ined 5 continuously in an appropriate culture m~ m Illlmol L~lized cultures of Dermacentor andersoni and Amblyomma amçricanum tick gut are provided. One example is the tick cell line called DGEC~This cell line, is describeA in detail and char~cteri7~d in Example 1. The line has remained stable for over 26 months and was deposited with the American Type Culture Collection (ATCC) on July 8, 1992 [ATCC Accession No. CRL 11084].
Also deposited with with ATCC on July 8, 1992 [ATCC Accession No. CRL 11083~, was the AGEC-1 imrnortalized tick cell line of the invention, which is discussedbelow in more detail.
Further, the invention provides an immortalized bovine T cell line, designated Bpbl-T1 (bovine peripheral blood lymphocyte-T cell). This immortalized bovine T
cell line is useful, inter alia, for T-cell dependent antigen targeting assays, T-cell receptor and antigen recognition studies, adoptive transfer experiments, and in vitro screening of cytokines, antigens and/or other bioresponse mo lifiers. This line has remained stable for over 8 months and was deposited with the ATCC on September 2 o l l , 1992 [ATCC Accessi~n No. CRL11120].
This invention further includes progeny and derivatives of these cell lines, e.g., cells which have been derived from the specific identified cell line by passaging or clonal expansion. All st~tements made herein relating to the il~ Ol L~lized cell lines of the invention are equally applicable to their progeny and derivatives.
2 5 Immortalized tick cell cultures of this invention are useful for, among other things, growing, in vitro, tick-borne pathogens, such as Borrelia burgdorferi. the causative agent of Lyme ~iice~ce~ and for producing antigens to these pathogens.These cell lines have also proven useful in m~int~ining vitro cultures of ~A.
marginale~ Fhrlichia species, and Borrelia bur~dorferi.
3 0 The immortalized T cells of the invention are also useful for growing a selected pathogen, as well as for producing antigens to these pathogens.
Additionally, the immortalized T cells of the invention are useful for the study of pathogens capable of infecting T cells, e.g. lentiviruses, and the study of T-cell signal transduction.
The i~ llulLalized cells of this invention provide a system for mass production of antigens, e.g., preferably cellular components such as proteins or non-proteinaceous material capable of inducing an immune response directed against a W O 94/06463 2 l ~ ~ G 5 1 PC~r/US93/08606 parasite borne pathogen. According to one embodiment, an immortalized cell of the invention may itself produce a biolog*al or therapeutic agent, e.g., a tick cellpolypeptide or protein, or another component or fraction of a tick cell which enh~nres the ability of a vaccine composition cnnt~ining a selected pathogenic 5 antigen to stim~ te a protective immune response in the vaccine. For exarnple, the o-lalized tick cell lines, AGEC-1 or DGEC-1, when cultured may naturally produce peptides, polypeptides, proteins, or other cellular fractions which are useful as anti-co~ nt~, anti-infl~mm~tnry agents and diuretics, for pharmaceutical and veterinary purposes. These exemplary and other agents are among the biological 10 products naturally produced by an imrnortalized cell of this invention upon culture.
Appropriate culture conditions to obtain maximum production of such natural products can be determined by one of skill in the art. These biological materials may be produced intr~cellnl~rly and obtained from the cultured tick cell by conventional cell disruption, e.g., lysis, and purification of the material or a cellular fraction 15 containing it from the lysate, based on its chPmiral identity or biological activity.
~ltrm~tively, the immortalized cell of the invention may secrete the agent into the media. Methods of isolating and purifying such biological materials or cell fractions are known in the art and may be utilized as desired.
According to another embodiment, the present invention provides a method 2 0 for producing antigens directed against a selected pathogen by infecting an imlllol Lalized cell culture of the invention with a selected pathogenic microorganism, e.g., ~3orrelia burgdorferi. Ehrlichia canis, Ana~ ma marginalis. Babesia bovis,Theileria parva. The infected iml.~ alized cell may, upon culturing permit the natural replication of the pathogen, and the production of pathogenic proteins in 2 5 culture. Either the pathogen itself, whether it be a virus or microorgani~m, or desirable proteinaceous m~trri~lc including subunits, polypeptides, cell fractions, fr~gmP,nt~ thereof, or other macromolecules such as carbohydrates, lipids, and lipoproteins may be produced in, and isolated from, the immortalized cell culture by conventional biological and genetic en ineering techniques. Such proteinaceous and 3 0 non-protein~reous materials, including the i~ lol ~lized cell or pathogen itself, are defined herein as antigens. Alternatively, the pathogens may be co-harvested from the cell culture infected by the pathogen.
It is understood that proteinaceous and non-proteinaceous materials produced by the pathogen-infected hlllllol ~lized cell lines may be include materials produced 35 from the immortalized cell biosynthetic activity. Pathogen-produced materialsisolated from the cell culture are expected to have use in vaccine compositions. For example, association of such pathogen-produced material with tick cell derived WO 94/06463 - `2 1 4 4 6 ~; 1 PCr/US93/0869 m~teri~l may provide vaccinal compositions with enhanced ability to stimnl~te immllnity in the vaccinee due to the influence of the tick cell environment upon the development and growth of the pathogen in the tick cell culture. In a similar manner, the illlll~Ul ~alized bovine cell line and products produced therein may be used to enhance vaccine compositions cont~ining bovine pathogens or antigenic materials t thereof. These cells and m~ten~l~ may further be of use in aiding identification of bovine antigens and in assaying for bovine cytokine production and activity.
Still an alternative embodiment for producing desirable antigens or polypeptides using an immortalized cell line of this invention involves transfecting the cell line with a recombinant molecule containing a heterologous polypeptide or protein having desirable antigenic properties under the control of a suitable regulatory sequence capable of directing the replication and expression thereof in the ihlllllolL~lized cell line. The transfected immortalized cell line containing the recombinant molecule is cultured to enable ~ ssion of the heterologous protein or polypeptide in the cell line. The methods employed in the design of the recombinant molecule, selection of the heterologous protein and regulatory sequences, and incorporation thereof into the cell line are within the skill of the art. [See, e.g., Maniatis et al., Molecular Cloning (A Laboratory M~mual), Cold Spring Harbor Press, Cold Spring Harbor New York (1989)].
2 0 Exemplary suitable vectors or plasmids for tr.msfecting the imlllol ~lized tick cells include those with an operational promotor, including insect viruses, insect recomhin~nt pl~mi(ls, Entomopox virus, and arboviruses. Currently, it is expected that insect promoters, such as polyhedron of baculovirus, spheroidin of entomopox virus, drosophila promoters or arbovirus (Semliki Forest Virus) would produce the 2 5 best results in the illllllOl Lalized tick cells. Similarly, vector components for m~mm~ n cells and known vectors may be readily selected by one of skill in the art for use in transfecting the bovine cell line Bpbl-Tl. See, for example, Maniatis et al, cited above.
The production of a desirable antigen from an immortalized tick cell line of 3 0 this invention is e~emrli~ l below (Example 3). Analogous procedures may beemployed to produce ~ntigens in the immortalized bovine cell line of the invention.
Media and cells from pathogen-infected illllllOl L~lized cells are generally collected at 24-108 hours post-infection and are used as the source of antigen for imm~lni7~tions.
If desirable, antigen-cont~ining media can be clarified from cell associated m~te~i~l by 3 5 centrifugation, aliquoted and stored at -20C until used. Alternatively, the pathogen ~ntigeniC m~teri~l may remain associated with any cell material from the imrnortalized cell and be incorporated into a vaccine. Immortalized tick cell produced material in ~094/06463 21~t651 PCI/US93/08606 association with the seleGte~l pathogen antigen may enhance the immunostim~ tQryeffect of the pathogen antigen. All antigen preparations can be quantified for parasite-specific protein (PSP) with ELISA. Cell-associated antigen may be prepared by sonication on ice in serum-free media followed by the centrifugation step 5 described above. Protein concentrations are determined by the method of Bradford, Anal. Biochem.~ ~:248 (1976). Sonicated parasite suspensions are adjusted to a final concentration of 10-100 ~lg/mL in serum-free media, aliquoted, and stored at -20C
until use.
The pathogens may express receptors on the cell surface of the illllllOl L~lized10 cell of the invention or intracellularly. Alternatively, the pathogens are contained intr~cell~ rly and released when the immortalized cell is disrupted in vitro or is processed in vivo by the animal vaccinated with the pathogen-containing immortalized cell. Disruption may be accomplished using known means, e.g. by freeze-thawing or other biochemical or mechanical disruption. In still another 15 alternative, antigenic portions of the pathogen are purified or left in combin~tion~ i.e.
the antigens may comprise a mixture of unpurified cellular material, viral subunits or fr~gmentc, media, and, optionally an adjuvant, from the imlllol~lized cell in which they are produced and used in a vaccine formulation.
The antigens produced as described above can be employed in the preparation 2 0 of a vaccine. The vaccine comprises an immunogenic amount of one or more antigen produced by the invention in a form suitable for internal ~(iministration.
Such a vaccine, directed against the selected pathogen or parasite, comprises an immunogenic amount of at least one pathogenic antigen which is produced by growing an immortalized cell culture of this invention infected with the selected 2 5 pathogen, including viruses and microorganicmc. The pathogenic antigen, as defined above, includes the entire pathogen, desirable subunits, polypeptides, cell fractions or fr~gmentc thereof, or desirable non-proteinaceous material.
Alternatively, the vaccine contains a whole in mortalized cell and the pathogen antigen. The pathogenic a~ntigen may be expressed in the i~ alized cell3 0 intracellularly, on the cell surface, or secreted during the natural processing of the cell or a vaccine fc)rmlll~tion containing a pathogen-infected hlllllollalized cell by the host. ~ltern~tively, such a vaccine composition may contain some cellular component of the immortalized cell which is not the whole cell, e.g., an irnmunogenic protein or polypeptide fragment of the immortalized cell, a subunit non-proteinaceous 3 5 material, or mixtures thereof.
In another embodiment, a vaccine is designed to protect against a tick. Such a vaccine composition contains an immunogenic amount of an immortalized tick cell _, 7, -- 6 5 1 of the invention, a cellular fraction, an antigenic protein or fragment of the cell, or other suitable immllnogenic fragments of an i~ o~lized cell of the invention. Inanother embodiment, the anti-parasite vaccine of the invention may contain both of the immortalized tick cell lines of the invention, or combinations thereof, whether as 5 whole cells, cellular fractions, or only proteinaceous or desirable non-proteinaceous materials from the cells. Further, these vaccine compositions may include other conventional anti-parasitic agents suitable for internal ~lmini~tration and/or be ~-lmini~tered in connection with other, known tick vaccinal compositions. Example 2 illustrates cross-species reactivity with an anti-tick vaccine of the invention.The AGEC-1 and DGEC-l immortalized tick cell lines of the present invention and the Bpbl-T1 bovine cell line are used to prepare vaccines with or without incorporated and replicating pathogens (See Example 3) and a pharmaceutically acceptable carrier. For example, as a tick vaccine, the uninfected or non-pathogen associated tick cells are replicated to the desired volume and cell15 density using large scale cell culture procedures known to those in the art such as (e.g., roller bottles, microcarrier, suspension, hollow fiber, etc.). The cells are harvested by standard procedures and concentrated by ultrafiltration or centrifugation. The cells are inactivated by 1-3 cycles of freeze-thaw, or heat or suitable chemical inactivation. The inactivated cells are then adjuvanted under 2 0 optimal conditions to provide a suspension of cells and adjuvant. The bovine cells may be analogously manipulated for the preparation of a vaccine composition.
In another formulation, the membrane proteins of the immortalized cells harvested can be fractionated by standard methods to form a vaccine containing only a part of the immortalized cell of the invention alone or in combination with other 2 5 antigens. This vaccine would not need an inactivation step, but may optionally be adjuvanted and arlminictçred in the manner described above. Similarly, one of skill in the art can identify desirable immunogenic proteins, peptides, or polypeptides derived from an i~ lized cell of the invention for inclusion in a vaccine composition ofthe invention. Such proteins, peptides, or polypeptides once iclentifie A can be3 0 isolated and purified, produced recombin~ntly, or synthesized by known means.
Vaccines of this invention may be employed in a method of immnni7ing hum~ns or anim~ls against a selected pathogen by injecting a vaccine of this invention into the animal.
As one example, a vaccine composition may contain as one of its active 3 5 ingredients a selected Borrelia antigen produced in cell line AGEC- 1 or DGEC-1, and either purified from the cell line or used in association with cellular material from the tick cell line. Such a vaccine is desirably ~mini~tered to humans and animals to ~O 94/06463 2 1 4 4 6 5 1 PCI/US93/08606 stim~ te ill " "llllily against Lyme disease or ticks. Where the vaccine comprises Borrelia antigens only, the vaccinee may develop ;"",~,.;ly against the spirochete, the causative agent of Lyme ~lice~e Where the vaccine also contains tick cell antigenic m~t~ri~l, the vaccinee may develop an immune response which destroys the tick - 5 before it can transfer the spirochete to the vaccinee. In the same manner, a vaccine of this invention can be used to protect against Rocky Mountain spotted fever, Ehrlichiosis, arboviruses, anaplasmosis, theileriosis, babesiosis, cutaneous irritations, and allergic dermatitis, where the antigen produced in the cell line is derived from one of the pathogens causing that condition.
~ltern~tively, where a vaccine of the invention is form~ te-l to be protective against Lyme tli~e~e, the whole immortalized tick cell, or a cellular fraction or subunit, a tick cell protein or peptide fragm~nt, or combin~tion thereof, from the immortalized cells may provide one vaccine component to stim--l~te immllnity to the tick cell. A tick cell vaccine component may be separately ~rlmini~tered with a vaccine containing a whole, inactivated pathogenic cell or subunit vaccine prepared from Borrelia. In other words, the Borrelia antigen does not have to be produced in the immortalized cells. It may be produced in another conventional way and co-~rlminictered with an immortalized tick cell component-containing composition.
Alternatively, in the present invention, Borrelia are grown in the immortalized cells 2 0 and then the entire il~ ol ~lized cells and spirochete material are harvested, inactivated, and preferably adjuvanted as described above. Another embodiment involves se~ting the spirochete or spirochete-produced proteins or antigenic fragments from the immortalized cells after growth and preparing the spirochetes or proteinaceous fr~gm~nts separately from the cells as a vaccine. In this case, the 2 5 spirochete is inactivated, or a protein isolated and adjuvanted in a similar manner.
Thus, the present invention provides a vaccine useful in preventing infection with any tick-borne pathogen. Such a vaccine composition contains an immortalized tick cell of the invention, or an immunogenic or antigenic fragment thereof, and an antigen directed to the selected pathogen. This antigen may be a whole inactivated 3 0 viral or cellular pathogen, or an antigenic protein, macromolecule or polypeptide thereof. For example, in a preferred embodiment, an anti-Lyme disease vaccine contains both the immortalized cells and any Borrelia bur~doferi antigen. An example of such an antigen is the spirochete, described in Example 9. However, this invention is not limited to an antigen produced in association with the illllllOI lalized 3 5 tick cells of the invention, but the Borrelia antigen may be obtained from other sources. It is anticipated that such a vaccine would offer superior protection over a vaccine containing solely the spirochete or subunits thereof.

WO 94/06463 2 1 ~ 4 6 5 1 PCI/US93/0861~
~. .
An immortalized cell of the invention can also be used as a recombinant vector for expression of a gene from a selected pathogen, e.g., Borrelia. Thus the need for growing and m~int~ining the spirochete would be elimin~t~rl Such a recombinant veetor expressing the gene for Borrelia is introduced into the immortalized tick cells. The tick cells e~lt;s~.ing the gene are harvested as described above, inaetivated, adjuvanted, and optimiæd for stability and efficaey.
For example, certain Borrelia express outer surface proteins (OSPs) which are capable of dir~elllially distinguishing between Borrelia grown in standard laboratory media and Borrelia passaged through live ticks. Using a recombinant DNA
approach, genes representing the OSPs from Borrelia vectored by live ticks could be isolated and expressed in an ap~ iate vector. Particularly useful vectors include the Semliki forest virus vector, the baeulovirus vector and the entomopox virus vector. The ill~lllulL~lized tick cells of the invention, infected with these viruses to produce the desired antigen are processed as whole cell or subunit extracts as described above.
Typically, antigenic proteins produced in association with the imrnortalized cells of this invention are desirably employed in vaccine compositions. For example, an immunogenic amount of an antigenic protein or desirable non-proteinaceous material are mixed with a ph~Tm~eutically acceptable carrier. An immunogenic 2 0 amount of a selected pathogenic antigen is generally between about 0.01 llg to 10.0 mg antigen, more preferably 0.05 ~Lg - 1 mg, and may be determined by one of skill in the art depending on the identify of the antigen, pathogen, and host animal.
An imm~lnogenic amount of the illlmollalized tick cells is about 101 to 108, and preferably about 106 cells/dose. Alternatively, the total immortalized cell 2 5 proteinaceous material, cellular fractions, or desirable non-proteinaceous macromolecules is in the range of 0.05 llg to 1 mg, as described above for the pathogenic antigens above. However, one of skill in the art ean make appl.,pliate adj~lstm~t~ depending upon the vaecine.
In adrlitiQn to the aetive ingredients discussed in the preeeding paragraphs, 3 0 other optional ingredients inelll-ling, for example, stabilizers, e~riers, and adjuvants may be added to the vaccine compositions of the invention. Stabilizers are addedoptionally to provide longer shelf life or enhance the poteney of the forrn~ tedvaecines of the invention. Typieally, stabiliærs, adjuvants, and inaetivation agents are Optil~ ed to determine the best form~ tion for efficacy in the target animal.
3 5 Suitable stabilizers, as with the other op~ional ingredients, are well known to those of skill in the art. Examples of such st~bili7ers inelude e~c~mino acids, sucrose, gelatin, phenol red, N-Z amine AS, monopotassium glllt~m~te, potassium ~0 94/06463 PCI/US93/08606 ~1~4651 monophosphate, potassium diphosphate, bovine albumin fraction V, lactose, lactalburnin hydrolysate, dried milk, and heat inactivated serum.
Suitable ph~rm~ceutically acceptable carriers facilitate ~tlmini~tration of the proteins and antigens but are E3hysiologically inert and/or nonharmful. Carriers may 5 be selected by one of skill in the art. Exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, and water. Additionally, the carrier or diluent includes a time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax. In addition, slow release polymer formulations can be used.
The vaccine compositions of this invention may be incorporated into convention~l sustained-release matrices, implant formulations, tablet formulations or injectable sustained release formulations and matrices. Components of such formulations are well known to the art.
One or more of the above described vaccine components may be admixed or adsorbed with a conventional adjuvant. The adjuvant is used as a non-specific irritant to attract leukocytes or enhance an immllne response. Such adjuvants include, among others, mineral oil and water, aluminum hydroxide, Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronic polyols, muramyl dipeptide, killed Bordetella. saponins, saponin-derivatives such as Quil A, and Immune 2 0 Stimul~tory Complexes (ISCOMs).
Inactivation agents may also be included in the vaccine formulations of the invention. Suitable inactivation agents include formalin, glutaraldehyde, binaryethylçneimine (BEI), beta-propriolactone, and heat, and freeze/thaw techniques. Other suitable agents are well known in the art.
A desirable dosage of a vaccine of the invention involves the ~lministration of 1 to 3 doses of desired vaccine composition, where the desired antigenic content of each fraction is as stated herein. The vaccine is preferably ~tlmini.ctered in two injections intramuscularly or subcutaneously, approximately 2 weeks to 3 months apart to immllni7e and boost the immune system of the target animal. However, the 3 0 mode of ~ministration of the vaccines of the invention may be any suitable route, including intradermally, intravenously, intraperitoneally, transdermally, topologically (e.g. by patch ointment) and by implant.
The specific dose level, mode and timing of aflmini~tration for any particular animal depends upon a variety of factors including the age, general health and diet of 3 5 . the animal; the species of the animal; and the degree of protection being sought. Of course, the ~lmini~tration can be repeated at suitable intervals, such as annually, if necessary or desirable.

21~6~:~
W O 94/06463 `~ PC~r/US93/086 For example, in cattle, the volume of the dose may vary from 0.1 to ~ mL of a sterile preparation of an immunogenic amount of the active vaccine component or co~ onents to 0.1 to 1 mL in a small animal such as a dog or cat. The immnne measurements to detect active immnni7~tion of the t~rget animal can be determined 5 by testing titers of antibody to cell protein using vitro detection systems such as ELISA.
In another aspect, the immortalized cells of the invention may provide si~ni~lc~nt thel~peuLic products. The ap~ liate me~linm and other conditions under which to culture an illlll~l L~lized cell line of this invention to enable it to produce a 10 desired therapeutic agent can be readily determined by one of skill in the art by conventional techniques. Such a therapeutic agent may be secreted directly into the culture medium and isolated by conventional techniques by one of skill in the art.
~ltern~tively, if expressed internally, the agent can be isolated from a culture lysate of such cells using known techniques.
Thus, the present invention also provides a method of immllni7ing animals against tick infest~tion, Lyme rlise~e, and other tick-borne diseases by ~lmini~tering to an animal a vaccine compostion of the invention. For the method of hl,mu"izing ~nim~l~ against tick infestation, the vaccine composition of the invention contains an immunogenic amount immortalizing tick cell of the invention, DGEC- l, AGEC- l, 2 0 progeny or derivatives thereof, or combinations of these cells. For the method of ir~ ing animals against Lyme disease, the vaccine composition of the invention may contain an immunogenic amount of one or more B. bur~dorferi antigen produced in, on, or by an illllllc,llalized tick cell line of the invention, an immortalized tick cell line of the invention alone or in combination with one or more ~3. burgdorferi antigens, or a combin~riQn of these.For the method of immnni7ing ~nim~ls against a selecte~l tick-bome ~lise~e, the vaccine composition of the invention may contain an immnnogeniC amount of one or more antigen from the pathogen which causes the tick-bome disease produced in, on, or by an illmlol~alized tick cell line of theinvention, an immortalized tick cell line of the invention alone or in combination with 3 0 one or more of the pathogen, or a combination of these.
The present invention further provides a method of immuni7ing animals against a selected pathogen, where said pathogen is capable of being grown in association with the immortalized bovine T cells of the invention. In par~cular,bovine lellk~mi~ virus, bovine immunQdeficiency virus or other pathogens which 3 5 infect bovine lymophocytes are anticipated to be suitable pathogens for growth in the c Bpbl-Tl i~llmol~lized bovine T cells:

~ro 94/06463 2 1 ~ 4 6 5 I PCr/US93/08606 The imrnortalized cells of the invention also provide a source of diagnostic reagents. For example, the DGEC-1 or AGEC-1 i~ ol~lized tick cell lines of the invention, or preferably protein sequences thelc;Lulll, may be used as diagnostic reagents in an in vitro assay to detect the presence of a pathogen, particularly a tick-borne pathogen, in a sample of body fluids from an animal suspected of infection. In addition, pathogens produced in the immortalized cells of the invention, and their protein sequences may also provide diagnostic agents for the pathogen. Further, the specificity of the illllllOl ~alized cell receptors can be used to determine typing of org~nicmc isolated from patients suspected to have been exposed to such org~nicms.
1 0 Once the ,cce~ is identified, receptor specific typing can be performed.
Such a diagnostic assay preferably involves the association of the cell line of the invention with a detectable label and the incubation of this diagnostic reagent with the sample fluids. For example, the specificity of the receptors on the immortalized tick cell sof the invention, can be used to type org~nicmc isolated from patients suspected of being bitten by a tick. Because the DGEC- 1 cell specifically agglutinates strain 297 of Borrelia burgdorferi~ agglutination could be used to type other B. burgdorferi strains. Binding of a pathogen to the cells can be detected by a variety of conventional assays, including agglutination, amplification, stim-llRtion of a pathogen antigen, e.g., OSP on Borrelia, and competition binding lltili7ing a MAb to 2 0 the pathogen, e.g., Borrelia, or a receptor on an immortalized cell line of the invention.
In one embodiment, B. bur~dorferi specifically agglutinates after attachment to DGEC-1 cells in vitro, intlic~ting a specific receptor on tne B. burgdorferi and DGEC-1 cells. After several days of attachment, the B. burgdorferi are inducecl to 2 5 replicate. Normally, B. burgdorferi will not replicate in DGEC- 1 media. The binding, ag~lutin~tion and det~chm~nt denotes an activation (synergy) step between B. bur~dorferi and DGEC- 1. Thus, the immortalized cells and/or B. burgdorferi may elaborate growth factors in the media.
Antibodies to a receptor(s) on the il~l,lol~lized tick cell lines of the invention, 3 0 DGEC- 1 or AGEC- 1, may be used in diagnostic assays. Conventional techniques for making suitable polyclonal, recombinant, or more desirably, monoclonal antibodies are well known to those of skill in the art [see, e.g., Kohler and Milstein; W. D. Huse et al., Science. ~ 1275-1281 (1988); PCT Patent Publication No.
PCT/W086/01533, published March 13, 1986; British Patent Application No.
GB2188638A, published October 7, 1987; Amit et ~1-, Science~ ~:747-753 (1986);
Queen et al., Proc. Natl. Acad. Sci. USA~ ~:10029-10033 (1989); PCT Patent Publication No~ PCT/WO90/07861, pubiished July 26, 1990; and Riechmann et al., 21~551 WO 94/06463 ; ~ ; PCr/US93/08 ~ature, ;~:323-327 (1988)]. For ~ gnostic purposes, the antibodies may be associated with individual labels, which are preferably interactive to produce adetectable signal. Most preferably, the signal is visually detectable. For colorimetric detection, a variety of enzyme systems have been described in the art which will5 operate ap~lop~;ately.
Antibodies specific for receptors on DGEC-l or AGEC-1 may also be used therapeutically as targeting agents to deliver therapeutic compounds. Rather than being associated with a label, such a therapeutic agent employs the antibody linked to an agent or ligand capable of disabling the replicating mechanism of the pathogen.
10 Alternatively, the antibody may block binding of the pathogen to the target cell.
Such antibodies may also be useful in therapeutic compositions for treating tickinfestations.
The following examples are merely illustrative of the different aspects of this invention and are not intended to limit the scope of the present invention.
F.xample 1 - Characterization of Transforrned Cell Line The im~lloll~lized cells DGEC-1 derived from the intestinal epithelial tract of Dermacentor andersoni show continuous growth and replication by a budding process, which can result in "chain-like" clusters of cells. Growth characteristics 2 0 change with increased duration in culture. However, replication rates among gut cells transformed according to this method vary, as does the ease of transformation.
At approximately nine months after initiation of cultures, the cell replication rate increased and slight morphological changes occurred. A distinct nucleus became visible for the first time. It is possible that cells required this time to accommodate to 2 5 culture conditions and to delete transfected DNA, which is not needed for cellular growth and development.
Three types of cells have been observed in the heterogeneous population:
small, round and clear; large, granular and brown; and large clear cells. Cells possess phagocytic activity throughout the culture period. ~ntimif~robials have been removed 3 0 from est~bliche~l cultures without any detectable changes in cellular growth characteristics.
The DGEC-l cell line was further characterized by (1) selective ~tt:~chment by Borrelia burgdorferi (receptor metli~tor), (2) differential phagocytosis of red blood cells in mixed blood cell populations and (3) induction of immnne response in guinea 3 5 pigs that reduced tick infestation and feeding. The line has remained stable for over 15 monthc.

~ro 94/06463 2 1 g 4 6 5 1 PCr/US93/08606 Further, this cell line, as well as illll~lUl ~lized intestinal epithelial cellsfrom ,~mblyomma ~rnericanum (AGEC-1), have proven useful in maintaining in vitrocultures of A. n~ ale. ~, bur~dorferi. and F~hrlichia species. It is expected that these cell lines will also be useful in maintaining in ViVO cultures of Arboviruses, 5 Babesia, and other tick-borne pathogens.
The growth of certain of these pathogens is demonstrated in Example 3 below.

Exarnple 2 - Immunization of Guinea Pi~s with Il--n~ol lalized Tick Gut cells Successful vaccination of guinea pigs with tick gut extracts is always accompanied with a specific antibody titer than can be enhanced upon repeat vaccinations. These antibody titers are normally measured by ELISA tests. To determine whether the immortalized gut cells have retained the ability to synthesize important antigens for immunological control of ticks, the cells were used to vaccinate guinea pigs. Three ~al~lllcters are used to ~letermin~ whether the guinea pig has been successfully immllni7eA against tick infestation: (a) death during or shortly after the blood meal is taken, (b) reduction or elimin~tion of ovipositing (egg laying), (c) reduction or lack of hatching from egg mass that is laid. These parameters are additive in the clinical measurements against tick infestation since any 2 0 can block the tick life cycle. This example describes the results of vaccination and challenge studies in the guinea pig model and demonstrates production of antibody against the immortalized tick cells that cross react with native tick gut tissue in an ELISA format.
The continuously growing tick gut cells of the invention were harvested from L-15B complete medium, centrifuged (at 150 x g; 10 minutes), and washed 3x in L-l5B incomplete ..,~li-.... The cells were then resuspended in L-15B incomplete medium and adjusted to 1.0 x 106 cells/mL. One million cells were a~lmini~t~red by subcutaneous injection to each guinea pig on days 0 and 14. Each injection was in a volume of 1 mL, and given in a dose divided between two sites. A total of 12 guinea 3 û pigs: 6 for Amblyomma americanum cells and 6 for Dennacentor andersoni cells were used. Two weeks after the last injection of cells, the ~nim~l~ were bled bycardiac puncture, 4 mL of blood from each animal was collected, serum separated and stored at -20C. Tmmllni7ed ~nim~l~ developed strong antibody responses as determined by enzyme linked immunosorbent assay (ELISA) as described below.
3 5 The antigen was prepared as follows. Adult, female, unfed DelTnacentorandersoni and Amblyomma americanum (200 each) were surface sterilized and their gut tissue was collected in sterile PBS (pH 7.2, 0.15 M). The gut preparation was W0 94/06463 2 t ~ Bsl PCl/US93/08 sonic~te~ and the protein content det~rmined by bicinchoninic (BCA) assay as described in P. K. Smith et al, Analytical Biochernistry. 150:76-85 (1985).
The coating for the wells was made up of 5 ~lg/mL of gut antigen suspended in carbonate-bicarbonate buffer, loaded 100 ~Vwell, and incubated at 4C overnight.
5 The wells were blocked with 1 % bovine serum albumin.
Serum dilutions (both control and immune) were made from 1:10 to 1:20480.
The second antibody used was rabbit anti-guinea pig (IgG, H&L) in HRPO, 1: 1000 in 1% BSA (PBS, 0.05% Tween-20). The substrate was o-phenylene dramine [OPD;
Eastman Kodak, Rochester, NY] and the optical density was read at 490 nm. The 1 0 results of these assays are set out in Tables 1-3 below.
Tables 1 and 2 set out the results of an assay performed on guinea pigs immllni7ed with Amblyomma americanum gut cells ill-l,lol~alized by the techniquedescribed in Example 5 above. The native gut antigen (5 ~Lg/mL) was 500 ng/well in 100 ~LL-as determine~ by enzyme-linked immunosorbent assay, ELISA, using known 1 5 techniques. Tick gut protein concentrations were deterrnined prior to testing as a standard. Each well was coated i~le~tir~lly, each using the same antibody dilution and detector antibody. Labelled antibody was rabbit anti-guinea pig-HRPO (IgG, H&L), 1:1000. The pre-imml-ni~tion titers (OD160) of A. americanum antigen were 0.037 and of D. andersoni antigen were 0.064.
2 0 Table 1 represents the detennin~hon of antibody response in anim~ls imml-ni7P~l with irnmortalized A. americanum cells (AGEC-1) and not challenged with ticks. The time periods indicate the time after inoculation at which the sera was tested. The antigen refers to the antigen used in the test.

2 5 Table 1 FIVE WEEK POST-INJ. S~X WEEK POST-INJ.
NATIVE GUT ANTIGEN NATI~E GUT ANTIGEN
Guinea Pi~ Amblyomma l:)ermacentor Amblyomma Dçrmaçentor 1009 1.313 1.187 1.267 1.306 1010 1.144 1.036 1.230 1.292 1011 0.687 0.785 1.064 1.047 The following data represents the deterrnination of antibody response by ELISA in animals immnni7ecl with immortaliæd A. americanum cells and which were 3 0 tick challenged.

~Vo 94/06463 2 1 4 4 S ~ii 1 PCr/US93/08606 Table 2 NATIVE GUT ANTIGEN
Guinea Pi~ Amblyomma Dermacentor 1018 0.326 0.340 1019 0.545 0.667 1020 0.459 0.711 1021 0.381 0.203 1022 0.303 0.429 1023 0.461 0.613 Single cell suspensions were prepared from D. andersoni freshly isolated gut.
5 The isolated cells were frozen and thawed. Guinea pigs were imm~ni7e~l with this preparation at 0.5 mg per mL per animal. A total of three booster injections were given with a 10 day interval between inoculations. As can be seen from the data in Table 3 below, ~nim~l~ immllni7ed with the immortalized ~ermacentor andersoni gut cells (DGEC-l) developed strong antibody responses. The antigen of Table 3 is that 1 0 used for screening.

Table 3 NATIVE GUT ANTIGEN
Guinea Pi~ Arnblyomma l:~ermacentor 1024 0.167 0.343 1025 0.208 0.334 1026 - 0.160 0.442 1027 0.617 0.947 1028 0.495 0.458 1029 0.485 0.468 1 5 The antibodies generated against freshly prepared tick gut tissue by imm-lni7~tion of guinea pigs were used in immllnofluorescence assays to detect reactive epitopes on immortalized tick gut cells m~int~ine~l in culture for 11 months.
These guinea pig antibodies were obtained from animals that were subsequently challenged with ticks. After collection of sera for immunoloc~l;7~tion, immnni7e~
2 0 anim~lc were challenged with larvae, nymphs or adults of the sen~iti7ing species.
This immunolocalization data correlates with the protective study results.

WO 94/06463 2 1 4 ~ ~'1 PCI/US93/086~

Table 4 below illustrates the effect on the life cycle patterns of Dermacentor andersoni ticks on control ~nim~l~ and may be con.~a,ed with Table S which illustrates similar data on animals immllni7~d with Dermacentor andersoni native gut antigen.

Table 4 ~infest/ Feeding Physical *replete davs Wei~ht Dead/live Appearance Larvae 280/275 4 5/275 Hatched Nymphs 60/60 6 0/60 Molted Adults 8/8 8-10 946 mgs* 0/8 1034 mgs*
1023 mgs*
943 mgs*
1045 mgs*
988 mgs*
1038 mgs*
1011 mgs*
Table 5 ~infestl Feeding Dead/ Physical ~replete davs Wei~ht live Appearance Larvae 230/216 4 14/216 Adults 8/4 18-19 392 mgs 4/4 435 mgs 643 mgs 572 mgs This data shows that animals ;,.",~ni,ed with freshly prepared D. andersoni gut cells were resistant to infestation with adult ticks. None of the four female ixodids allowed to infest ;.~ i7efl guinea pigs produced ova, inrlicating a clear anti-1 5 tick response.
Tables 6 and 7 below illustrate this effect on Amblyomma americanum and Dermacentor andersoni ticks in animals immllni7Pd with Amblyomma americanum gut cells immortalized by the technique described above.

~VO 94/06463 2 1 ~4 6 51~ PCl/US93/08606 Table 6 - ~. americanum ticks #infes~/ Feeding Dead/ Physical #replete davs ~k'E~ li~ A~pearance Larvae 220/253 4 228 mg/0.9 mg nl Molted Nymphs 60/4~ 6 572 mg/12.71 mg n' Molted 1019 (75.0%) Adults 1020 8/8 12 23 mg 12 434 mg Dead Reddish 13 690 mg Reddish 14 55 mg Dark red 19 35.6 mg Dead Dark red 19 34.8 mg Dead Dark red 19 15.6 mg Dead Dark red (100 % dead) n1 (100% fed larvae survived) n2 {45 Molted to adults (7S.0%), 15 dead (25.0%)}

The red color indicates that the tick guts apparently rlicintegrated. One tick burst in capsule. The adult ticks in this study either died or were incapable of laying eggs. None produced egg mass.

WO 94/06463 2 1 ~ 4 6 5 1 PCI/US93/089 rable 7 - D. andersoni ticks #infest/ Feeaing Dead/ Physical #re~lete davs Weight ~ A~earance Larvae Nymphs 60/52 8 1421 mg/27.33 mg 23/29 29 grey 1022 (86.7%) nl 23 black Adults ~Eick) (E~ Mass) 1023 8/8 12 485 mg 244.9 mg* Dark red 12 792 mg 523.0 mg Grey 12 579 mg 358.6 mg* Grey 13 786 mg 465.8 mg Grey 13 539 mg 235.9 mg Grey 13 405 mg 198.9 mg Grey 14 498 mg 323.6 mg Grey 250 mg Grey nl { 17 molted to adult (28.3%), 43 dead t81.8%)) 5 * For the adults, in~licates that the ova produced by females did not hatch.

The lack of data for larvae inclic~tes that they did not attach to the host.
However, while there was no feeding, a severe cutaneous reaction was seen at thebite site. No egg mass was produced for those feeding 15 days. The data further 10 in~1ic~tes that the i~ i7~tion induced cross-species protection which is indicated by longer times to replete, lower finished weight and reduced viable egg mass.
Tables 8 and 9 below illustrate this effect on Amblyomma americanum and Dermacentor andersoni ticks in animals il~ lul-i~;d with il....lol~lized Dermacentor andersoni gut cells obtained by the technique described above.

~0 94/06463 PCI/US93/08606 Table 8 - 1~). andersonl t~
#infest/ Feeding Dead/ Physical #rçplete davs Weight live Appearance Larvae:

Nymphs:
1025 60/38 8 1058 mg/27.84 mg nl 31 grey (63.3%) 7 black Adults:
1026 8/6 12 768 mg dead black 12 972 mg dead dark red 12 791 mg* grey 12 936 mg** grey 13 970 mg dead reddish 19 15.6 mg grey nl {25 molted to adults (41.7%), 35 dead (58.4%)}
5 * Tn-lic~tes the production of egg mass (324.6 mg), which did not hatch.
** Tn~ ates the production of 353.5 mg egg mass.

Two ticks died early during feeding period. The lack of data for larvae indicates that there was no attachment. However, while no feeding occurred for 1 0 larvae, a severe cutaneous reaction was seen at the bite site.

_ WO 94/06463 2 1 4 ~ 6 S 1 PCI/US93/08~

Table 9 - A. americanum ticks ~infest/ Feeding Dead/ Physical ~replete ~ Weight 1~ Ap~earance Larvae 200/210 4 174 mg/0.83 mg Molted Nymphs 1028 60/38 6 366 mg/9.63 mg all grey {37 molted to adults (61.7%), 23 dead (38.4%)}
Adults 1029 8+8 14 296 mg dark red 14 370 mg* grey 14 417 mg** grey 276 mg dead black 18 158 mg dk grey 18 315 mg dk grey 19 37.5 mg grey 19 23.4 mg dead black * Indicates production of 131.5 mg egg mass.
5 ~* Tnclic~tes the production of 167.4 mg egg mass.

Anirnals immnni7e~ with immortalized cells were challenged with larvae, nymphs or adults of the homologous species. Prelimin~ry observations indicated that resistance to adult and nymphal challenge was present. Adult ticks required longer to 0 feed and engol~,e~ t was altered. Many male, female and nymphal ticks died after engorgement. In addition, production of ova was reduced. Immortalized digestive tract cells of ~. americanum appear to srimlll~te a more solid resistance to infestation than cells of D. andersoni origin.
This data indicates that the immun;7~tion induced some cross-species 1 5 protection.
Exam~le 3 - Growing Patho~ens in Immortalized Tick Cells A. Ehrlichia canis and Granulocytic Canine Ehrlichia Both Ehrlichia canis and Granulocvtic canine Ehrlichia (GCE) have been successfully grown in the illlll~ol~alized gut cells of Derrnacentor andçrsoni 2 0 (DGEC- 1} and Arnblyomrna arnericanum (AGEC- 1). The Ehrlichia was obtained from peripheral blood collected from dogs which were exp~rimellt~lly infected with these or~ni~m~. ~ canis is associated with lymphocytes and monocytes. GCE is associated with granulocytes. The peripheral blood was collected in vacutainers .
cont~lnlng hepann.

~O 94/06463 2 1 4 4 6 5 1 PCr/US93/08606 Peripheral blood lymphocytes (PBL) were isolated by Ficoll gradient (Histopaque) and washed 3x in L-lSB incomplete me~ m The cell numbers were then adjusted to 1.0 x 106 cells/mL and added to a flask containing gut cells (6 mL) (a few erythrocytes also were present in the peripheral blood lymphocyte 5 preparations.
Granulocytes were isolated from blood by the method described by Sigma Chemic~l~ using Histopaque 1077 and 1119 in a double gradient method.
Isolated granulocytes were washed 2x in L-15B incomplete medium inoculated to gut cells (1.0 x 106 cells/mL).
1 0 Microscopic ex~min~tion of cultures 2-3 days post infection showed aggregation of PBL/granulocytes around the gut cells. This was very evident in Amblyornma americanum gut cells, since they are big, oval and larger than PBLs/granulocytes. About 2-3 weeks post-inoculation, the cont~min~tecl RBC
degenerated, PBL/granulocytes showed rough surfaces and numerous granular 1 5 m~t~ri~l was observed in cultures. In addition, Amblyomma cells showed phagocytosis of hemoglobin (appeared red in color).
Eight to ten weeks after infecting cells, the minute bodies increased in number and many of these bodies studded on to the surface of the gut cells, particularly on Amb~yomma americanum gut cells. Diff-Quick stained culture smears 2 0 and one micron thin sections (toluidine blue stained) showed numerous bodies (lawn of uniform bodies), which were also studded on the surface of the gut cells.
Serum samples collected from dogs suffering from Ehrlichiosis were used as a source of antibody and used to identify the or~"ni~m~ in cultures by Florescent Antibody Test. Acetone fixed smears of culture were reacted with 2 5 antibodies to Erhlichia. and these immunoglobulins were localized by fluoroscein isothyocyante (FITC) conjugated anti-canine antibodies.
Cultures with material from infected dogs displayed reactivity. Immortalized gut cells had the appearance of being studded with minute fluorescent bodies.
B. P~orrelia bur~dorferi 3 0 Borrelia bur~dorferi strain 297 [SmithKline Beecham Animal Health]was grown in conventional BSK-II medium according to known techniques. Strains B31 [SmithKline Beecham] and SH-2-82 [National Institutes of Health] of B.
r burgdorferi have also been established. Two vector competent strains include 27985, isolated from I. d~~ filli in Stamford, Connecticut, and 21305, which was isolat ed from Peromysus leuco~us [both of which are available from John Anderson, Connecticut Agricultural Resource Station~. However, other, known, strains of B.burgdorferi may be used.

WO 94/06463 2 1 ~ 4 6 5 1 PCr/US93/08~

An 8-10 day old culture of B. burgdorferi strain 297 (200 ~L) was added to each of two T-25 flasks. Dermacentor andersoni irllllwl L~lized gut cells (DGEC-1) and Arnblyomma americanum immortalized gut cells (AGEC-l) were cultured in antibiotic-free L-lSB complete m~illm 24-48 hours prior to inoculating 5 with the spirochetes. The spirochete infected tick cell cultures were incubated at 30 C in a dry incubator as described below.
1. Dermacentor andersoni The spirochetes started attaching to the cells by 24 hours post infection. As the incubation continlle(1, the clumping of cells by spirochetes 10 increased. By 4-5 days post infection, clear entanglement of cells by spirochetes was evident. It was observed that the cell types in the culture, small, round, clear gut cells were att~cked by the spirochetes. The large, round, brown color cells may be resistant to spirochete attachment. By 10-12 days post infection, the spirochetesensitive cells were disintegrated and only spirochete resistant cells were present in 15 the culture. By 15 days post infection, the so-called spirochete resistant cells started multiplying and increasing in number. By 20 days post infection, the spirochete resistant cells were seen uniformly throughout the flask.
Spirochetes also showed multiplication. The number of spirochetes increased. The spirochete resistant cells may provide some growth 2 0 factors for the multiplication of spirochetes.
After 12 days of culturing spirochetes in the cells, spirochetes were passaged in a new flask containing gut cells. In the first passage, the clumping of the cells was not seen until 4-5 days post infection. In this flask also, thespirochete ~tt~chmt-nt was seen only with small, round, clear cells. The large, round, 2 5 brown cells were not affected by spirochetes.
2. Amblyomma americanum These gut cells showed similar spirochete attachment. The only difference observed in this system was that clumping and attachment of spirochetes to cells was seen only after 4-5 days. The large, oval, brown cells were 3 0 not attacked by the spirochetes.
C. Anaplasma marginale This organism was obtained from peripheral blood collected from cattle suffering from anapl~cmosic or A. marginale-infected cattle in vacutainers containing heparin and which showed 27% par~citemi~3 as determined by blood smear 3 5 e~min~hon.
The buffy coat of A. mar~inale was removed by centrifugation and erythrocytes were washed 2x in L-lSB incomplete medium. The red blood cell ~VO 94/06463 21 ~ ~ 651 PCI/US93/08~

number was adjusted to 1.0 x 107 cells/mL in L-1~B complete meflium with all growth factors without antibiotics. Flasks containing approximately 5 mL of immortaliæd l~ermacentor andersoni and Amblvomma amencanum gut cells were inoculated with 1.0 x 106 celVmL infected erythrocytes (2 flasks for each cell type).
5 After inoculation, the cells were incubated at about 30C as described below.
Twenty-four hours post-inoculation of red blood cells (RBC~ into the culture, gut cells showed attachment of RBC onto the surface gut cells. This wasmore evident in Arnblyomma than the Derrnacentor cells (in both cell types theseattachments are seen). Forty-eight hours post-inoculation there was phagocytosis of 10 RBC by gut cells. The cell membrane of the gut cells became thick and numerous;
RBC were studded onto the surface of the gut cells. In addition, these gut cellsappeared dark brown.
Five days post-inoculation, the gut cells appeared highly granular, contained numerous minute uniform inclusion bodies, and many cells ruptured and 15 released these inclusion bodies into the surrounding medium. Culture samples were collected for tr~nsm;ssion electron microscopy for analysis of infected gut cells.
Changes were more evident in the Amblyomma cells.
Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one 2 0 of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

Claims (36)

WHAT IS CLAIMED IS:

1. An irnmortalized tick cell line AGEC-1, ATCC Accession No. CRL
11083, its progeny and derivatives thereof.

2. The immortalized cell line according to claim 1 infected with a selected pathogen.

3. The immortalized cell line according to claim 1 transfected with DNA
from a selected pathogen encoding a selected gene product under the control of regulatory sequences capable of expressing said gene product.

4. An immortalized tick cell line DGEC-1, ATCC Accession No. CRL
11084, its progeny and derivatives thereo

5. The immortalized cell line according to claim 4 infected with a selected pathogen.

6. The immortalized cell line according to claim 4 transfected with DNA
from a selected pathogen encoding a selected gene product under the control of regulatory sequences capable of expressing said gene product.

7. An immortalized bovine T cell line Bpbl-T1, ATCC Accession No.
CRL-11120, its progeny and derivatives thereof.

8. The immortalized cell line according to claim 7 infected with a selected pathogen.

9. The irnmortalized cell line according to claim 8 transfected with DNA
from a selected pathogen encoding a selected gene product under the control of regulatory sequences capable of expressing said gene product.

10. An isolated pathogenic antigen produced by culturing an immortalized tick cell selected from the group consisting of AGEC-1, DGEC-1, progeny and derivatives thereof infected with a selected pathogen.

11. An isolated pathogenic antigen produced by culturing an immortalized bovine T cell line selected from the group consisting of Bpbl-T1, progeny and derivatives thereof infected with a selected pathogen.

12. An antigen produced by culturing an immortalized tick cell selected from the group consisting of AGEC-1, progeny and derivatives thereof transfectedwith DNA from a selected pathogen encoding a selected gene product under the control of regulatory sequences capable of expressing said gene product.

13. An antigen produced by culturing an immortalized tick cell selected from the group consisting of DGEC-1, progeny and derivatives thereof transfectedwith DNA from a selected pathogen encoding a selected gene product under the control of regulatory sequences capable of expressing said gene product.

14. An antigen produced by culturing an immortalized bovine T cell selected from the group consisting of Bpbl-T1, progeny and derivatives thereof, transfected with DNA from a selected pathogen encoding a selected gene product under the control of regulatory sequences capable of expressing the gene product.

15. A vaccine capable of protecting against infection with a selected pathogen comprising an immunogenic amount of at least one pathogenic antigen produced by culturing an immortalized tick cell selected from the group consisting of AGEC-1, DGEC-1, progeny and derivatives thereof infected with said selected pathogen.

16. A vaccine capable of protecting against infection with a selected pathogen comprising an immunogenic amount of a pathogenic antigen produced by culturing an immortalized bovine T cell selected from the group of cell lines consisting of Bpbl-T1, progeny and derivatives thereof infected with the selected pathogen.

17. The vaccine according to claim 16 wherein the pathogenic antigen is produced by transfecting said immortalized bovine T cell with DNA from the selected pathogen encoding the selected antigen under control of regulatory sequences capable of expressing the pathogenic antigen.

18. A vaccine capable of protecting against infection with a selected tick-borne pathogen and against infestation by a tick, wherein said vaccine composition comprises an immunogenic amount of an antigen obtained from an immortalized tickcell selected from the group consisting of AGEC-1, DGEC-1, progeny and derivatives thereof and an antigen from the selected tick-borne pathogen.

19. The vaccine according to claim 18 wherein the selected tick-borne pathogen is selected from the group consisting of Borrelia burgdorferi, Amblyomma americanum, Anaplasma marginale, Babesia bovis, Theileria parva, Cowdria ruminantium, Ehrlichia species, and arboviruses.

20. The vaccine according to claim 19 wherein said pathogenic antigen is produced by transfecting said immortalized tick cell with DNA from said selectedpathogen encoding a selected antigen under the control of regulatory sequences capable of expressing said antigen.

21. The vaccine according to claim 18 wherein said tick cell antigen is selected from the group consisting of a whole immortalized tick cell, a cellularfraction or subunit thereof, a protein and a macromolecule from said tick cell.

22. The vaccine according to claim 18 wherein the selected pathogen is produced by infecting the immortalized tick cell line with said pathogen.

23. A vaccine capable of protecting against tick infestation comprising an immunogenic amount of an antigen obtained from an immortalized tick cell selected from the group consisting of AGEC-1,DGEC-1, progeny and derivatives thereof.

24. The vaccine according to claim 21 wherein said tick cell antigen is selected from the group consisting of a whole immortalized tick cell, a cellularfraction or subunit thereof, a protein and a macromolecule from said tick cell.

25. A method of immunizing an animal against a disease caused by a selected tick-borne pathogen comprising internally administering to said animal a vaccine, wherein said vaccine composition comprises an immortalized tick cell antigen, said immortalized tick cell selected from the group consisting of AGEC-1, DGEC-1, progeny and derivatives thereof and an antigen from said selected pathogen.

26. The method according to claim 25 wherein said pathogen is Borrelia burgdorferi and said disease is Lyme disease.

27. A method of immnnizing an animal against tick infestation comprising the step of internally administering to said animal a vaccine comprising an immunogenic amount of an antigen obtained from an immortalized tick cell selected from the group consisting of AGEC-1,DGEC-1, progeny and derivatives thereof.

28. The vaccine according to claim 27 wherein said antigen is selected from the group consisting of (a) a whole immortalized tick cell, (b) a cellular fraction of said immortalized tick cell, (c) an immunogenic protein or macromolecule of saidimmortalized tick cell, and (d) a mixture of any one of (a), (b) and (c).

29. A method of immnnizing an animal against a disease caused by a selected pathogen comprising internally administering to the animal a vaccine composition comprising an immortalized bovine T cell selected from the group consisting of Bpbl-T1, progeny and derivatives thereof transfected with DNA fromthe selected pathogen encoding the selected antigen under control of regulatory sequences capable of expressing the antigen.

30. A method of immunizing an animal against a disease caused by a selected pathogen comprising internally administering to the animal a vaccine compositioncomprising an immortalized bovine T cell selected from the group consisting of Bpbl-T1, progeny and derivatives thereof infected with the selected pathogen.

31. A method of detecting the presence of a tick-borne pathogen in the body fluids of an animal comprising the step of incubating a body fluid sample taken from the animal in the presence of an antigen selected from the group consisting of (a) a whole immortalized tick cell selected from the group consisting of AGEC-1, DGEC-1, progeny and derivatives thereof, (b) a cellular fraction of said immortalized tick cell, (c) an immunogenic protein or fragment of said immortalized tick cell, and (d) a mixture of any one of (a), (b) and (c).

32. An antibody specific for a receptor on an immortalized tick cell selected from the group consisting of AGEC-1, DGEC-1, progeny and derivatives thereof.

33. An antibody specific for a receptor on an immortalized bovine T cell selected from the group consisting of Bpbl-T1, progeny and derivatives thereof.

34. A therapeutic composition useful for treating tick infestations comprising an antigen selected from the group consisting of (a) a whole immortalized tick cell selected from the group consisting of AGEC-1, DGEC-1, progeny and derivatives thereof, (b) a cellular fraction of said immortalized tick cell, (c) an immunogenic protein or fragment of said immortalized tick cell, and (d) a mixture of any one of (a), (b) and (c).

35. A diagnostic reagent comprising an antigen selected from the group consisting of (a) a whole immortalized tick cell selected from the group consisting of AGEC-1, DGEC-1, progeny and derivatives thereof, (b) a cellular fraction of saidimmortalized tick cell, (c) an immunogenic protein or fragment of said immortalized tick cell, and (d) a mixture of any one of (a), (b) and (c).

36. A diagnostic reagent comprising an antigen selected from the group consisting of (a) a whole immortalized bovine cell selected from the group consisting of Bpbl-T1, progeny and derivatives thereof, (b) a cellular fraction of said immortalized bovine cell, (c) an immunogenic protein or fragment of said immortalized bovine cell, and (d) a mixture of any one of (a), (b) and (c).