WO1995023218A1

WO1995023218A1 - Immortalized canine cells and uses therefor

Info

Publication number: WO1995023218A1
Application number: PCT/US1995/002064
Authority: WO
Inventors: David Brake; Stephen K. Wikel; Rangappa N. Ramachandra
Original assignee: Pfizer, Inc.; Oklahoma State University
Priority date: 1994-02-25
Filing date: 1995-02-16
Publication date: 1995-08-31

Abstract

The present invention provides an immortalized canine cell lines, and uses therefor, including diagnostic reagents and vaccine compositions.

Description

IMMORTALIZED CANINE CELLS AND USES THEREFOR

Field of the Invention

This invention relates generally to immortalized cell lines, and more specifically, immortalized primary cells useful for the production of antigens and biologically active compounds.

Background of the Invention

Continuous or immortalized cell lines which' are characterized by the ability to grow indefinitely are valuable as a source of im unogens, a source of new biologically active compounds and for the production and replication of relevant pathogens. The ability to establish a culture that will grow indefinitely varies depending on the animal species from which the cells originate. For example, rodent cells routinely generate continuous cell lines, but chicken cells almost never become immortal. Canine cells, with the exception of tumor cells, do not routinely provide a source of immortalized cell lines.

Canine cell cultures are of interest for the research and study of dog-borne pathogens and microorganisms. Presently available in vitro primary or continuous canine cell cultures are not capable of long term, consistent maintenance of dog-borne pathogens. There exists a need in the art for immortalized in vitro cell cultures, which cells support the growth of pathogenic organisms, for use in developing vaccines to selected pathogens and for research, therapeutic, and diagnostic purposes.

Summary of the Invention

In one aspect, the present invention provides immortalized canine peripheral blood T-lymphocyte cells. This cell line has been designated cPBL-1. In another aspect, the present invention provides isolated cell-line derived natural proteins, peptides and other cellular materials and a method of producing them by culturing an immortalized cell line of the invention and isolating the desired material therefrom. Such materials are useful as therapeutic, diagnostic and vaccinal agents.

In yet a further aspect, the present invention provides a method of screening therapeutic compositions, using the immortalized cell line of the invention and, preferably, standard bioassays. In still another aspect, the invention provides a method for replicating or producing a selected pathogen or pathogen-derived antigen by infecting an immortalized cell culture of the invention with the selected pathogen and recovering the live pathogen itself, or desirable pathogenic antigens or proteins, produced in or by the infected immortalized cells. Still an alternative aspect of this invention is a method for producing desirable vaccinal, diagnostic or therapeutic antigens or polypeptides, by culturing an immortalized cell of this invention transformed with a recombinant molecule designed to express the selected protein or polypeptide in the immortalized cell.

Another aspect of this invention provides vaccine or therapeutic compositions employing one or more of the natural cell-line derived materials, the pathogen, fragments or polypeptides thereof produced in an immortalized cell of this invention, or other selected recombinant proteins or polypeptides produced in the immortalized cell line, as described above. Such vaccines or therapeutic compositions are useful for protecting or treating an animal against a selected pathogen, especially one which is carried by a dog. The vaccine compositions of this invention may further contain additional antigens, either produced in the cell line or by conventional methods. In a further aspect the invention provides a method of immunizing an animal, preferably a canine, with a vaccine composition described above.

In yet another aspect, the present invention provides a diagnostic method employing the immortalized canine cell line, its derivatives or progeny. Because these cell lines can specifically support the growth of canine-borne pathogens in vitro , they are useful for isolating and identifying such pathogens from blood or tissue samples of infected animals.

In still another aspect, the invention provides antibodies raised against, and capable of binding to, epitopes on the immortalized canine cell line of the invention, its progeny and derivatives. Such an antibody may be used in in vitro diagnostic assays, or as a therapeutic or vaccine agent. Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

Brief Description of the Drawings

Fig. 1 is a bar graph illustrating the phenotype of the canine cell line cPBL-1.

Fig. 2 is a line graph illustrating the dose response of cPBL-1 to recombinant IL2 as described in detail in Example 4.

Fig. 3 is a line graph illustrating the dose response of cPBL-1 to canine peripheral blood lymphocyte

(PBL) Conconavalin A-stimulated supernatant containing IL-2 and potentially other cytokines as described in Example 5. Detailed Description of the Invention

The present invention provides an immortalized culture of primary canine cells. The immortalized canine cell line of this invention has a variety of uses, such as the production of vaccinal agents which provide protection against diseases caused by pathogens, particularly those which infect canines, including, among others, parvovirus, The immortalized canine cell line is also useful in the production of therapeutic agents for such pathogens. The natural materials, or fractions thereof, produced upon culturing the immortalized cells are similarly anticipated to be useful in vaccines or therapeutic compositions, both for canine and non-canine use. Such materials may also be employed to enhance the biological activity of vaccines. These cell lines are also useful, for example, as host cells for the replication and production of canine-borne pathogens, pathogen-derived antigens, and other recombinant proteins and polypeptides. The cell lines also have utility in diagnosing pathogenic infections of canines. As used herein, the term "immortalized cell line" refers to the immortalized canine peripheral blood lymphocytes [cPBL-l] cells deposited with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 (U.S.A.), on December 9, 1993 [ATCC Accession No. CRL 11504] in compliance with the requirements of the Budapest Treaty. The term also encompasses the progeny and derivatives of cPBL-1. The cPBL-1 cell line is described in detail and characterized in Example 1, and has remained stable for over 12 months. This cPBL-1 cell line is particularly advantageous for use in producing pathogens, and other for other uses, because it is not derived from tumor cells and is therefore believed to have a karyotype which more closely resembles that of a non-leukogenic cell than do other canine cell lines, which are derived from tumorigenic or leukogenic cells. Thus, the pathogen which infects or is transfected into a cell of the invention is anticipated to replicate in the cell of the invention in a manner more similar to the manner in which it would replicate in a normal, non-tu origenic cell.

Immortalized cells of this invention may be clonally expanded by conventional techniques to produce a homogeneous population of progeny cell lines which can be maintained continuously in an appropriate culture medium. Thus, this invention further includes progeny and derivatives of this cell line, e.g., cells which have been derived from the cPBL-1 cell line by passaging or clonal expansion. By "progeny" is meant cell lines clonally expanded from the deposited cPBL-1 cell line. By "derivatives" is meant cPBL-1 cells and/or progeny thereof which have been modified by the application of conventional techniques, including mutagenic techniques or insertion of additional homogenous or heterogeneous nucleotide sequences or cellular materials to introduce one or more desired characteristic(s) into the cPBL-1 cell line. All statements made herein relating to the immortalized cell line of the invention are equally applicable to their progeny and derivatives.

As used herein, the term "immunogen" refers to a proteinaceous or non-proteinaceous biological substance or material which elicits an antibody or a cell-mediated immune response in an animal. Preferably, this immunogen is derived from an immortalized cell of the invention, or a pathogen, portion thereof, or recombinant molecule produced therein. The term "antigen" refers to a substance which reacts specifically with antibodies or T-cell receptors. The term "i munogenic amount", particularly as it is used to modify the term antigen, refers to the amount of an antigen required to elicit an immune response.

I. Natural Materials Produced by the Cell Line

According to one embodiment, an immortalized canine cell of the invention itself, when cultured under appropriate conditions, produces natural"- biological products useful as prophylactic and therapeutic compositions, biopharmaceuticals, and diagnostic reagents for pharmaceutical and veterinary purposes. Such products include peptides, proteins or other cellular fractions, such as, a feline cell polypeptide or protein, or another component or fractions of a feline cell. Other natural products include such therapeutic agents as cytokines and other biopharmaceuticals. For example, these peptides and other fractions can be employed to enhance the ability of a vaccine composition containing a selected immunogen or pathogenic antigen to stimulate a protective immune response in the vaccine.

The appropriate medium and other conditions under which to culture an immortalized cell line of this invention to enable it to produce a desired natural, therapeutic agent can be readily determined by one of skill in the art by conventional techniques. These biological materials may be produced intracellularly and obtained from the cultured canine cell by conventional cell disruption, e.g. lysis and purification of the material or a cellular fraction containing it from the lysate, based on its chemical identity or biological activity. Alternatively, such a naturally-produced therapeutic agent may be secreted directly into the culture medium and isolated by conventional techniques by one of skill in the art. Methods of isolating and purifying such biological materials or cell fractions are known in the art and may be utilized as desired. These natural products may also be used in vaccine formulations, as the active ingredient, or desirably with other antigens in a vaccine or therapeutic composition. Desirable formulations, dosage regimens, and modes of administration are described in detail below.

II. The Cell Line as Host Cell for Growth of Pathogens and/or Production of Antigens A. Growth of Pathogens

Because the cell lines of the invention are of canine lineage, they are particularly useful for growing canine-borne pathogens and for the production, study and diagnosis of pathogens of canines, particularly those which infect canine cells of T-lymphocyte lineage. Such pathogens include, without limitation, canine distemper, Ehrlichia species (canis and ewingii) viruses, and other viruses, mycoplasma, rickettsia and other bacteria, and particularly other pathogens which infect canine T- lymphocytes.

According to this aspect of the invention, an immortalized cell culture of the invention is infected or transfected with the selected pathogen by conventional, known techniques. The infected or transfected immortalized cell, upon culturing, permits the replication of the pathogen, which may be isolated from the cell once it is disrupted, or may be secreted into, and isolated from the culture medium. This method thus permits cPBL-1 cells of the invention to be used in production of a selected pathogen, or in the production of antigens thereof. The cPBL-1 can thus be employed can thus by employed as a tool to diagnose infection in an animal biological sample, e.g. blood.

B. Production of Antigens by Infection The immortalized cells of this invention provide a system for mass production of antigens, e.g., preferably cellular components such as proteins or non-proteinaceous material. The pathogens cultured in the cell line of the invention can express receptors (or antigens) intracellularly or on the cell surface of the immortalized cell of the invention. The production of a desirable antigen, receptor, antigenic substance and the like from an immortalized canine cell line of this invention is another desirable utility of the cell line. Preferably, antigens are produced in culture by an infected immortalized cell line. Desirably, these antigens are capable of inducing an immune response directed against canine pathogen. Thus, the cell lines of the present invention may be employed to produce antigens directed against a selected pathogen, whether it be a virus or a microorganism.

Desirable proteinaceous materials including subunits, polypeptides, cell fractions, fragments thereof, or other macromolecules such as carbohydrates, lipids, and lipoproteins may be produced in, and isolated from, the immortalized cell culture infected by the selected pathogen. It is understood that proteinaceous and non- proteinaceous materials produced by the pathogen-infected immortalized cell lines include materials produced from the immortalized cell biosynthetic activity.

The immortalized canine cells of this invention which have been infected by a pathogen can produce pathogen-associated immunogens useful in vaccine formulations or in association with the natural products of the cPBL-l cell line.

C. Production of Recombinant Antigens

The immortalized cell line of this invention may also be employed as a host for the production of desired antigenic, immunogenic, or other biological materials by recombinant techniques, rather than by growth of a selected pathogen. An immortalized canine cell line of the invention may be transfected by conventional genetic engineering techniques with a recombinant molecule or vector containing a heterologous polypeptide or protein under the control of a suitable regulatory sequence capable of directing the replication and expression thereof in the immortalized cell line. The transfected immortalized cell line containing the recombinant molecule is cultured to enable expression 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. Exemplary suitable vectors or plasmids for transfecting the immortalized canine cells include those with an operational promotor. Currently, it is expected that mammalian vectors and components would produce the best results in the immortalized cells. However, other known vectors may be readily selected by one of skill in the art for use in transfecting the canine cell line. See, e.g., Maniatis et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Press, Cold Spring Harbor New York (1989) . Particularly desirable are retroviral vectors. For example, an immortalized cell can be used as a host cell for a recombinant vector for expression of a gene from a selected pathogen, e.g. canine distemper. Such a recombinant vector expressing the gene for the canine distemper nucleoprotein is introduced into the immortalized canine cells. The transfected canine cells are cultured and are themselves harvested using conventional techniques, discussed in more detail below. The expressed heterologous protein may be isolated from the host cells by conventional techniques for use in vaccine, diagnostic or therapeutic compositions or methods. Alternatively, the transfected host cells or portions thereof may be employed as part of the vaccine, diagnostic or therapeutic compositions or methods. For example, the transfected canine cells, after culture, may be harvested, inactivated, adjuvanted, and optimized for stability and efficacy for use as a vaccine, particularly for use in a non-canine vaccine or therapeutic. As another example, certain pathogens express outer structural proteins (OSPs) which may be useful in diagnosis. Using a recombinant DNA approach, genes representing the proteins from the pathogen vectored by dogs could be isolated and expressed in an appropriate vector. The immortalized canine cells of the invention, infected with these viruses to produce the desired antigen or immunogen are processed as whole cell or subunit extracts using conventional techniques.

The transfected cells, or isolated heterologous proteins may be formulated in vaccine or therapeutic compositions as discussed in more detail below. Whether the immortalized canine cell line of this invention is employed to produce its natural materials, a pathogen, pathogen-derived antigens, or a desirable antigen, receptor, antigenic or immunogenic substance or the like using recombinant vectors, these activities can be performed using conventional biological and genetic engineering techniques, including transfection, infection or other incorporation of the pathogen or vector into cells of the cell line, conventional culturing of the transformed cell line [see, e.g. Maniatis et al, cited above].

Isolation of the desired materials from the cell line may be accomplished by standard isolation and purification techniques readily known to one of skill in the art, including without limitation, lysis, chromatography, molecular weight sieving and the like. Alternatively, pathogens or antigens thereof may be co- harvested from the cell culture infected by the pathogen.

III. Vaccine and Therapeutic Compositions

As described above, the present invention also provides for a variety of vaccine and therapeutic compositions utilizing products of the immortalized cell lines of the invention. Among such products are pathogens or pathogen-produced materials isolated from the cells of the invention which have been transfected or infected with a selected pathogen and cultured. One of skill in the art can readily identify desirable immunogenic proteins, peptides, or polypeptides produced in or by an immortalized cell of the invention or an infected or transfected cell for inclusion in a vaccine composition of the invention. Such immunogens, once identified, can be isolated and purified, produced recombinantly, or synthesized by known means. As one example, a vaccine directed against a selected pathogen contains 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 pathogen. The pathogenic antigen, includes the entire pathogen, desirable subunits, polypeptides, or desirable non-proteinaceous material, as well as recombinant proteins and polypeptides.

Such cell-line cultured pathogens or antigens thereof are contained intracellularly and released when the immortalized cell is disrupted in vitro. For example, pathogen associated canine cells are replicated to the desired volume and cell 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. Alternatively, the cells may be dissociated followed by lysis or other suitable chemical or mechanical means of disruption. .Antigenic portions of the pathogen itself are purified or left in combination, i.e. viral subunits or fragments, media, and, optionally an adjuvant, from the immortalized cell in which they are produced and used in a vaccine formulation. Optionally, pathogen-derived antigens produced by culturing the infected cell line can remain associated with cellular material from the immortalized cells and be incorporated into a vaccine. For example, the membrane proteins of the immortalized cells harvested, as described above, can be fractionated by standard methods to form a vaccine containing only a part of the immortalized cell of the invention, which may be used as the active ingredient alone or in combination with other antigens. This vaccine would not need an inactivation step, but may optionally be adjuvanted and administered in the manner described above. As still another example, the pathogen antigenic material can be purified from associated cell material and/or the media. These cells and materials may further by useful in aiding identification of canine antigens and in assaying for canine cytokine production and activity.

For example, association of such pathogen- produced material with canine cell derived material may provide vaccinal compositions with enhanced ability to stimulate immunity in the vaccinee due to the influence of the canine cell environment upon the development and growth of the pathogen in the canine cell culture. These cells and materials may further be of use in aiding identification of canine antigens and in assaying for canine cytokine production and activity. Particularly when the vaccine composition is to be used on non-canines, the vaccine formulation may contain a whole immortalized cell and the pathogen antigen. Alternatively, such a vaccine composition may contain some cellular component of the immortalized cell which is not the whole cell, e.g., an immunogenic protein or polypeptide fragment of the immortalized cell, a subunit non- proteinaceous material, or mixtures thereof.

Desirably, where the vaccinate is a dog, the immunogen is derived from a pathogen capable of infecting a dog. This immunogen may be a whole inactivated viral or cellular pathogen, or an antigenic protein, macromolecule or polypeptide thereof. Suitable pathogens include, for example, canine distemper, canine parvovirus, canine herpes virus, canine adenovirus, rickettsia, and mycoplasma.

Typically, an immunogenic amount of an antigenic protein or desirable non-proteinaceous material is mixed with a pharmaceutically acceptable carrier. An immunogenic amount of a selected pathogenic antigen produced in cPBL-1 is generally between about 0.01 ng to 10.0 mg antigen, more preferably 0.05 μg - 1 g, and may be determined by one of skill in the art depending on the identify of the antigen, pathogen, and host animal. It should also be understood by one of skill in the art that vaccine compositions of this invention may also contain antigens in addition to those produced via use of the immortalized of the immortalized cell line. The selection of suitable antigens for inclusion into a vaccine of this invention is within the skill of the art.

In addition to the active ingredients discussed in the preceding paragraphs, other optional ingredients including, for example, stabilizers, carriers, and adjuvants may be added to the vaccine compositions of the invention. Stabilizers are added optionally to provide longer shelf life or enhance the potency of the formulated vaccines of the invention. Typically, stabilizers, adjuvants, and inactivation agents are optimized to determine the best formulation for efficacy in the target animal.

Suitable pharmaceutically acceptable carriers facilitate administration of the proteins and antigens but are physiologically inert and/or nonhar ful. Carriers may 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. 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 immune response. Such adjuvants include, among others, 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 Stimulatory Complexes (ISCOMs) . Other optional or desired components for inclusion in a vaccine composition may be readily selected by one of skill in the art.

A desirable dosage of a vaccine of the invention involves the administration 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 administered in two injections intramuscularly or subcutaneously, approximately 2 weeks to 3 months apart to immunize and boost the immune system of the target animal. Alternatively, annual booster shots may be administered as follow-ups to an initial vaccine administration series.

The mode of administration of the vaccines of the invention may be any suitable route, including intradermally, intravenously, oral, oral/nasal, transder ally, percutaneously, topologically (e.g. by patch or ointment) and by implant. The specific dose level, mode and timing of administration for any particular animal depends upon a variety of factors including the age, general health and diet of the animal; the species of the animal; and the degree of protection being sought. Of course, the administration can be repeated at suitable intervals, such as annually, if necessary or desirable.

For example, in a large dog the volume of the dose may vary from 0.1 to 5 mL of a sterile preparation of an immunogenic amount of the active vaccine component or components to 0.1 to 1 mL in a small animal. The immune measurements to detect active immunization of the target animal can be determined by testing titers of antibody to cell protein using in vitro detection systems such as ELISA, or can be determined by in vivo and/or in vitro tests designed to measure cell-mediated immunity.

Thus, the present invention further provides a method of immunizing animals against a selected pathogen via administration of a vaccine composition as described herein.

IV. Diagnostic Reagents

The immortalized cells of the invention also provide a source of diagnostic reagents. For example, the immortalized canine cell lines of the invention, or preferably protein or peptides therefrom, may be used as diagnostic reagents in an in vitro assay to detect the presence of a pathogen, particularly a canine-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 or peptide sequences may also provide diagnostic agents for the pathogen. Further, the specificity of the immortalized cell receptors can be used to determine typing of organisms isolated from patients suspected to have been exposed to such organisms. Once the receptor is identified, receptor specific typing, including T-cell receptors, can be performed.

Additionally, antibodies to a receptor(s) on the immortalized canine cell lines of the invention 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, 116:1275-1281 (1988); PCT Patent Publication No. PCT/WO86/01533, published March 13, 1986; British Patent Application No. GB2188638A, published October 7, 1987; Amit et al. , Science, 2_3_3:747-753 (1986); Queen et al. , Proc. Natl. Acad. Sci. USA. 81:10029-10033 (1989); PCT Patent Publication No. PCT/WO90/07861, published July 26, 1990; and Riechmann et al. , Nature, 332:323-327 (1988)]. For diagnostic purposes, the antibodies may be associated with individual labels, which are preferably interactive to produce a detectable signal. Most preferably, the signal is visually detectable. For colorimetric detection, a variety of enzyme systems have been described in the art which will operate appropriately.

V. Use of Cell Line in Screening Assays

The immortalized canine cPBL-1 cell line of the invention is further expected to be used for .Ln vitro screening of pharmacologically active molecules, including biopharmaceuticals and anti-infectives, and for bioassays to determine canine immune response. For example, this immortalized cPBL-1 is useful for screening or producing T- cell products including cytokines, particularly interleukin (IL)-2, IL-4, interferon (IFN)~7, and IL-10, and in vitro screening of IL-3, IL-5, IL-6, IL-10, and IFN-γ. The cell line and its progeny and derivatives may also be used in antigen recognitions studies and detection of other bioresponse modifiers. Thus, the invention provides a method of screening, e.g., for cytokines, which method comprises culturing a cell of the invention in the presence of a biological sample under conditions which permit detection of cytokines in the sample. Suitable techniques are known in the art. Such a biological sample may include tissue, cells, or fluids and are preferably canine in origin. 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.

Example 1 - Maintenance Media for the Immortalized Cell Line

The cPBL-1 cells of the invention are grown using conventional medium and techniques. The following provides the preferred maintenance media for the cPBL-1 cell line of the invention. One of skill in the art could readily adjust or modify this formulation as desired or needed. Additionally, other suitable medium may be substituted.

For maintenance of the canine T cell line, cPBL-1 of the invention, the cells are cultured the cells in the following medium containing recombinant human IL-2 (Genzy e) using standard procedures. This medium is preferably prepared fresh for each culture batch.

RPMI 1640

FCS (HI) 20%

Sodium pyruvate 500 μM/ml Insulin (bovine) 5 mcg/ml

2 ME 1.0 x lO^'

Transferrin (bovine) 5 mcg/ml

Con A 0.5 to 1 mcg/ml

Penicillin 100 Units/m Streptomycin 100 mcg/ml

Gentamicin 50 mcg/ml

Human rIL-2 : 10 ng/ml

The cells are cryopreserved in liquid nitrogen in conventional medium. Example 2 - Karvotyping of cPBL-1 Cells

Immortalized cPBL-1 cells from a T-75 flask were washed and placed in fresh medium containing 1.0 μg/ml colchicine for 90 minutes at 37°C in 5% CO, Cells were then centrifuged and gently resuspended in 75 μM KC1. Cells were pelleted by centrifugation and resuspended in a cell fixative (3 parts methanol to 1 part glacial acetic acid) for 1 hour at room temperature. The fixed cells were centrifuged and the cell pellet was resuspended in fresh fixative. Prewetted microscope slides were prepared and 4 to 5 drops of cell suspension dropped onto the slides using a pipette held 5 to 6 inches above the slide. Slides were dried at room temperature and preparation quality confirmed by phase contrast microscopy. Slides were stained in a standard Giemsa solution for 4 minutes, washed in distilled water and a coverslip mounted to slide. Slides are viewed by microscopy and photomicrographs prepared. Chromosomes were enumerated by direct counting. The results are shown in the following table. Table 1

Canine-T Cells: (2n=78)

Chromosome Spread # Chromosome #

1 76 82 80 70 2 74 84 76 82 3 82 82 84 82 4 84 80 82 76 5 76 76 82 82

Modal # 82, Range 70-84 Example 3 - cPBL-1 Cell Surface Phenotypinq

Double-color staining was performed on the cPBL-1 cell line using commercially available monoclonal antibodies reactive to canine cell surface determinants CD4, CD8, B_sub, and pan T. FITC conjugated goat anti-mouse antibody was used following primary antibody incubations, and biotin labelled goat anti-mouse and PE labelled streptavidin conjugates used following secondary antibody incubations. Cell samples were fixed in buffered formalin and were analyzed on a FACSTAR^Phu flow cytometer (Becton Dickinson) using PC Lysis II software. Dead cells were excluded by forward and side scatter. The results are provided in Figure 1.

Example 4 - cPBL-1 Response to Recombinant Human IL2 cPBL-1 cells were washed extensively in phosphate buffered saline and 2 x 10⁴ cells per well added (100 μl) in quadruplicate to 96 round bottom microtiter plate. Starting at 20 U/ml, serial two-fold dilutions of human recombinant IL2 were prepared and added to wells (100 μl/well) . Control wells received media alone. Cells were incubated at 37°C, 5% C0₂ for 48 hours. Tritiated thymidine was added (0.33 μCi/well) during the last 6 hours incubation. Cells were harvested using an automated programmable harvester and incorporated radioactive thymidine measured (expressed as mean counts per minute (cpm)) by liquid scintillation counting. Results, illustrated in Figure 2, show that cPBL-1 proliferation was dose dependent.

Example 5 - Dose Response of cPBL-1 Response to Canine PBL Con A Stimulated Supernatant

Using standard procedures, PBLs were isolated from canine blood, stimulated for 24 hours using 5 μg/ml Con A to induce production of IL-2 and possibly other cytokines by the PBLs. Resulting cell-free supernatant containing those cytokines was treated with alpha methyl mannoside to inactivate residual mitogen. Supernatants were frozen at -20°C until assay. Two independent lots of supernatant were prepared and one was tested. cPBL-1 cells were washed extensively in PBS and 2 x 10⁴ cells per well added (100 μl) in quadruplicate to 96 round bottom microtiter plate. Serial two-fold dilutions of test supernatant were prepared and added to wells (100 μl/well) . Control wells received media containing Con A previously treated with alpha methyl mannoside. Cells were incubated at 37°c, 5% C0₂ for 48 hours. Tritiated thymidine was added (0.33 μCi/well) during the last 6 hours incubation. Cells were harvested using an automated programmable harvester and incorporated radioactive thymidine measured (expressed as mean counts per minute (cpm) by liquid scintillation counting. As illustrated in Figure 3, cPBL-1 proliferation was detectably dose dependent on the presence of the cytokines, particularly IL-2, in the supernatant. Thus, this cell line can be used to measure the presence and/or activity of IL-2 (or other cytokines) in unknown biological samples, particularly canine samples, by culturing the cell line of the invention in the presence of an unknown biological sample and examining the cell culture for the effects of the sample on cell proliferation. The particular cytokine responsible for the effect on proliferation may be detected by known methods.

Example 6 - Infection of Canine T Cell Line cPBL-1 with Canine Distemper Virus

Since cPBL-1 is a suspension cell line, cells must first be attached to a desired substrate, such as microcarrier beads, for intracellular pathogen infection. Cells in logarithmic growth, are planted at a density of 4 x 10⁵ cells/ml, with 1 g/liter Cytodex beads, and infected simultaneously with canine distemper virus at a 0.1 multiplicity of infection (m.o.i.). The cells, beads, and pathogen are incubated in maintenance media in 1:10 final volume, and a slow impeller speed, such as 30 rpm. After 1 hour, maintenance media is brought up to the final desired volume and speed, i.e., 75 rpm. At 24 hour intervals post-adεorptibn, infected cell culture supernatants are collected and quantitated for antigen by enzyme-linked immunosorbent assay (ELISA) . This is an example of an infection-plant procedure.

Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one 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

WHAT IS CLAIMED IS:

1. An immortalized canine cell line cPBL-1, ATCC Accession No. CRL 11504, 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 a recombinant molecule comprising DNA encoding a selected heterologous protein or peptide under the control of regulatory sequences capable of expressing said gene product.

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

5. A pathogenic antigen produced by culturing an immortalized canine cell selected from the group consisting of cPBL-1, progeny and derivatives thereof, transfected with a recombinant molecule comprising DNA encoding said antigen under the control of regulatory sequences capable of expressing the gene product.

6. 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 canine cell selected from the group consisting of cPBL-1, progeny and derivatives thereof infected with the pathogen.

7. The vaccine according to claim 6 wherein the pathogen is selected from the group consisting of canine distemper, rickettsiae, canine herpes virus, canine adenovirus, and mycoplasma.

8. The vaccine according to claim 6 wherein said pathogenic antigen is produced by transfecting said immortalized canine cell with a recombinant molecule comprising DNA encoding said antigen under the control of regulatory sequences capable of directing the expression of said antigen in said cell line.

9. A method of immunizing an animal against a disease caused by a selected pathogen comprising administering to the animal a vaccine composition comprising a pathogenic antigen produced by culturing an immortalized canine cell selected from the group consisting of cPBL-1, progeny and derivatives thereof transfected with a recombinant molecule comprising DNA encoding said antigen from the selected pathogen encoding the selected antigen under control of regulatory sequences capable of directing the expression of said antigen in said cell line.

10. A method of immunizing an animal against a disease caused by a pathogen comprising administering to the animal a vaccine composition comprising a pathogenic antigen produced by culturing an immortalized canine cell selected from the group consisting of cPBL-1, progeny and derivatives thereof infected with the selected pathogen.

11. An antibody specific for a receptor on an immortalized canine cell selected from the group consisting of cPBL-1, progeny and derivatives thereof.

12. A therapeutic composition useful for treating canines comprising a product of a cell line selected from the group consisting of cPBL-1, progeny and derivatives thereof.

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

14. A method of producing cytokines comprising the step of culturing an immortalized canine cell selected from the group consisting of cPBL-1, progeny and derivatives thereof under conditions which permit production of cytokines in said cell.

15. A method of screening cytokines comprising the step of culturing an immortalized canine cell culture selected from the group consisting of cPBL-1 progeny and derivatives thereof with unknown biological samples under conditions which permit detection of cytokines in the samples.