Note: Descriptions are shown in the official language in which they were submitted.
: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
WO 94/06463 PCI/US93/08
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.,
11
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.
12
~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
13
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.
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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.
16
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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
1018
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
1021
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.
18
~0 94/06463 PCI/US93/08606
Table 8 - 1~). andersonl t~
#infest/ Feeding Dead/ Physical
#rçplete davs Weight live Appearance
Larvae:
1024
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.
19
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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
1027
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.
21
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
22
~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.
