Note: Descriptions are shown in the official language in which they were submitted.
S
61109-7309D
This application is a divisional of application No.
461,242 filed on August 17th, 1984.
The invention herein described relates to monoclonal
antibodies which react specifically against sporozoites of the
parasite Eimeria tenella. Hybridoma cultures producing antibodies
against E. tenella are described. Such antibodies are obtained by
means of hybridoma technology. Sporozoites antigens are identi-
fied and characterized. These antigens, along with certain
monoclonal antibodies are effective for the prevention and treat-
ment of coccidiosis. The antigens of the invention are useful as
vaccines against coccidio~is.
By way of background, coccidiosis is a disease of
animals caused by a variety of protozoan parasites. Avian
coccidiosis is a devastating disease o~ poultry caused by a variety
of species of the genus Eimeria. This disease has a complicated
lie cycle consisting of both asexual and sexual stages. Chickens
are initially infected with the dlsease after ingestion of free-
living oocysts which are generally associated with fecal material.
Oocysts develop into invasive asexual sporozoites in the chicken's
digestive tract. The sporozoite~ infect epithelial cells and
develop into multinucleate structures known as schizonts. Each
schizont matures and eventually liberates multiple invasive asexual
structures known as merozoites. These merozoites leave the
infected cell and reinvade other
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epithelial cells. The multiple invasive asexual stages
involving sporozoites and merozoites account for much of
the pathology of coccidiosis. The sexual cycle of
coccidiosis is initiated when merozoites differentiate
into gametoc~tes. Fertilization occurs and the
fertilization products known as oocysts are released in
the feces. Thus the parasite's life cycle is completed.
In chickens, the life cycle of Eimeria tenella, a repre-
sentative species, is completed in about 7 to 9 days.
Due to the tremendous economic losses in~licted
on the poultry industry by Eimeria species, a vaccine
against the parasite is highly desirable. However, due
to the complexity of the life cycle of the parasite and
the variability of the quantity of antigens present in
each stage, it has been observed that deactivated or
lS killed parasites have not generated consistent immunity
in the past. One solution to this problem is to isolate
and characterize particular antigens from the parasite
and administer them in a sufficient amount to serve as an
immunizing agent. Preferably such antigens will offer
protection against infection by all important species.
It is known that various species of Eimeria, as well as
different stages in the life cycle of the same species,
have both common and specific antigens [Cerna, Z., Folia
Parasitologica ~Prague) 17:135-140 ~1970); Davis et al.,
Immunol 34:879-888 ~1978); Rose, M.E., Immunol.
2:112-122 (1959); Rose et al., Immunol. 5:79-92 (1962);
and Tanielian et al., Acta Parasitol. Yugosl. 7:79-84
~1976)]. It is also known that development of immunity
to Eimeria is species specific and in some species of
domestic fowl there is significant strain-specific
immunity [Jef~ers, T.K.; In Long, P.L. et al. (eds.),
Avian Coccidi~sis, pp. 57-125, Proc. 13th Poultry Sci.
Symp. (1978); Joyner, L.P., Parasitol. 59:725-732 (1969);
i;~6~
--3--
Long, P.L., Parasitol. 69:337-347 ~1974); and Long et
al., Parasitol. 79:451-457 (1979)]. Currently immunogens
of Eimeria species capable of stimulating protective
immunity in avian or mammalian hosts have not yet been
isolated or identified. Such Eimeria immunogens,will
likely provide successful immunization against
coccidiosis.
, The development of lymphocyte hybridoma
technology provides a tool for producing relatively large
amounts of specific antibodies against various antigens
of Elmeria. By fusing specific antibody-producing cells
(spleen cells) with cells of a myeloma tumor, it is
possible to produce hybridoma cells that secrete
monoclonal antibodies directed specifically against the
original sensitizing antigen. [Kohler & Milstein,
Nature (London) 256:495-497 (1975)]. If monoclonal
antibodies against the parasite are obtained, it may be
possible to provide such anitbodies to infected or
susceptible fowl and to thus provide the host organism
with a measure of passive immunity. Once such hybridoma
cultures producing monoclonal antibodies are obtained, it
is possible by vario~s procedures to utilize such anti-
bodies to isolate and identify specific antigens which
could in turn be utilized as a vaccine to provide host
organisms with a system of active immunity. Various
patents concerning hybridoma cultures and monoclanal
antibodies are knswn (i.e., U.S. Pat. Nos. 4,172,124;
4,196,265; 4,271,145; 4,361,549; 4,631,550; 4,364,932;
4,364,g33; 4,364,934; 4,364,935; 4,364,936; 4,381,292;
and 4,381,295).
In light of the- foregoing discussion of the
economic effects of coccidiosis in the area of animal
husbandry and more specifically in the poultry industry
~26128S
61109-7309D
control of the protozoan parasite Eimeria is highly desirable.
Accordingly, the invention of the divisional application seeks to
provide new and useful monoclonal antibodies obtained against
sporozoites of the genus Eimeria. The invention of the parent
application seeks to isolate and identify specific antigens of E.
tenella useful as a vaccine for the control of avian coccidiosis.
A preparation of E. tenella sporozoites is used to
immunize mice in order to eventually generate monoclonal antibodies
~ollowing the method of K~hler and Milstein as described below.
The monoclonal antibodies are used to identify antigens of the
parasite. The antigens which elicit monoclonal antibodies that
react with sporozoites, and show neutralization of parasite growth,
are considered protective antigens. The protective antigens that
occur in various species of Eimeria are regarded as potential
candidates for the development of a vaccine against avian coccid-
iosis.
Soluble antigens are obtained from sporozoites of E.
tenella. These soluble antigens are separated electrophoretic-
ally by molecular weight and those which specifically react with
monoclonal antibodies of the present invention are identified.
U~ing appropriate standards, the reactive antigens are then
characterized on the basis o molecular weight.
In order to evaluate the ability of the monoclonal anti-
bodies to effectively neutralize the infective capability of
coccidial sporozoites, chickens are exposed to sporozoites of E.
tenella previously treated with various monoclonal antibodies of
the invention. This in vivo experimental system demonstrates the
protectiVe ¢apabilitie~ of selected monoclonal antibodies.
-- 4 --
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61109--730~D
Chickens receiving injections of solubilized sporozoite
antigens including those identified by the corresponding monoclonal
antibodies were found to be protected against oral challenge. This
immunization procecure demonstrates the vaccine potential of spor-
ozoite antigens which can be recognized by monoclonal antibodies.
According to the invention of the parent application
there is provded a method for preparing an antigenic, immunogenic,
proteinaceous vaccine, which is soluble in detergent-containing
buffer, containing at least one antigen of Eimeria tenella sporoz-
oites, specifically reactive with anti-sporozoite monoclonal anti-
bodies seareted by hybridoma clones s5E5 (ATCC No. HB8~03) and
having molecular weights of 110 ~ 16 kd and 130 + 20 kd; specific-
ally reactive with anti-sporozoite monoclonal antibodies ~ecreted
by hybridoma clone s4E2 and having molecular weights of 110 ~ 16
kd and liO ~ 30 kd; specifically reactive with anti-sporozoite
monoclonal antibodies secreted by hybridoma clone slC4 (ATCC No.
HB8333), and having molecular weights of 66 i 9 kd; 55 i 8 kd, 20
- 30 kd, 18 ~ 3 kd, 15 ~ 2 kd; ~pecifically reactive with anti-
sporozo$te monoclonal antibodies secreted by hybridoma clone s2G8
~ATCC No. HB8405) and having molecular weight of 55 ' 8 kd;
specifically reactive with anti-sporozoite monoclonal antibodies
~ecreted by hybridoma clone s5B9 (ATCC No. HB8402) and having
molecular weight of 55 ~ 8 kd; speciically reactive with anti-
sporozoite monoclonal antibodie~ secreted by hybridoma clone SlA
~ATCC No. HB8404) and having molecular weight of 54 - 8 kd;
specifically reactive with anti-sporozoite monoclonal antibodie~
secreted by hybridoma clone s3Cll and having molecular weight of
50 ~ 7 kd; specifically reactive with anti-sporozoite monoclonal
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- 61109--7309D
antibodi.es secreted by hybridoma clone ~3D3 ~TCC No,HB8331), and
haviny a molecular wei~ht of 29 i 4 kd; or specifically reactive
with anti-sporozoites monoclonal antibodies secreted by hybridoma
clone slE4 (ATCC No. Hs8332)~ and having molecular.weights of 58
* 9 kd and 130 ~ 20 kd; which method comprises the steps of:
(a) extracting, and solubilizing sporozoite antigens of
E. tenella;
(b) separating the solubilized material by suitable isolation
and purification methods to obtain purified antigen.
According to another aspect of the invention of the
parent application there i8 provided an antigenic, immunogenic,
proteinaceous vaccine containing at leaæt one antigen of Eimeria
tenella sporozoite~ when prepared by the above~referenced process
or an obvious chemical equivalent thereof.
According to the invention of the divisional application
there is provided a method of producing monoclonal antibodies which,
comprises forming by fusion hybridoma cells from a mouse myeloma
line and spleen cells from a mouse previously immunized with
_imeria~tenella sporozoites and recovering the produced antibodies
which
~ a) reacts ~pecifically with antigens o~ Eimeria ~e~. sporoz-
oite~ and
(b) reacts specifically with antigens of Bi~er-ia tenella
having a molecular weight of approximately 13 to 150 kd.
In a pre~erred embodiment the hybridomas are formed by the fuæion
of P.3 x 63. Ag 8. 653 my~loma cellæ and spleen cells from BALB/c
mice previously immunized with E. ten'ella sporozoites. In a part-
icularly pre~erred embodiment the hybridoma used is a clone chosen
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~Z612~
61109-7309D
from the group consisting of,: the hybridoma designated clone
number sSE5 and de~o~ited as ATCC No. HB8403; the hybridoma
designated clone number s4E2; the hybridoma designated clone
No. slC4 and deposited as ATCC No. HB8333; the hybridoma designat-
ed clone No. s2G8 and deposited as ATCC No. HB8405; the hybridoma
designated clone No. s5~9 and deposited as ATCC No. HB8402; the
hybridoma designated clone No. sl~ and deposited as ATCC No.
~B8404; the hybridoma desiynated clone No. s3Cll; the hybridoma
designated clone No. s3D3 and deposited as ATCC No. HB8331; and
the hybridoma designated clone No. slE4 and deposited as ATCC
No. HB8332.
A preferred method of the invention of the divisional
application comprises preparing a monoclonal antibody, which
reacts with antigens of Eimeria tenella sporozoites, which
comprises the steps of:
(a) immunizing mice with ~.'tenel'la sporozoites;
(b) removing the spleen0 from said mice and making a suspen-
sion of the spleen cells;
(c) fusing said spleen cells with mouse myeloma cells in the
presence of a fusion promoter;
(d) 'diluting and culturing the fu8ed cells ln separate wells
in a medium which will not support the growth of unfused myeloma
cells;
(,e) evaluating the spuernatant of each well containing a
hybridoma for the presence of antibody reactive with E. tenella
0porozoites ~
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61109-7309D
(f) selecting and cloning a hybridoma producing antibody
reactive with _. tenella sporozoites; and
(g) recovering the antibody from the supernatant or ascitic
product of said clones.
Another preferred method of the invention of the division-
al app]ication comprises preparing monoclonal antibodies which
react with antigens of Eimeria spp. sporozoites which comprises
either:
(a) culturing clone No. s5E5 (ATCC No. HB8403), s4E2, slC4
(~TCC No. HB8333), s2G8(ATCC No. HB8405), s5B9 (ATCC No. HB8403),
slA (ATCC No. HB8404), s3Cll, s3D3 (ATCC No. HB8331), or slE4
(ATCC No. HB8332), in a suitable medium and recovering the anti-
body ~xom the ~upernatant of an above said hybridoma culture; or
~b) in;ecting into a mouse a hybridoma culture designated
clone No. s5E5 (ATCC No. HB8403), ~4E2, slC4 (ATCC No. HB8333),
s2~8 (ATCC No. HB8405), s5B9 (ATCC No. HB8402), slA (ATCC No.
HB840~), s3Cll, s3D3 ~ATCC No. HB8331), or slE4 (ATCC No. HB8332)
and recovering said antibody from the ascites or serum of said
mouse.
The following non-limiting Examples further serve to
illustrate the inventions of the parent and divisional applications.
EXAMPLE 1
Construction of Hybridoma Lines
Sporozoites of the organism Eimeria ~nella are obtained
by excysting sporulated oocysts using established procedures
[Doran et al., Proc. Helmintol. Soc. Wash. 34:59-65 (1967)]. A
preparation of E. tenella sporozoites thus obtained is used to
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1261213S
61109-7309D
immunize eighteen-week-old female BALB/c mice by intraperitoneal
in]ection. After determining that an immunized mouse is producing
anti-sporozoite antibodies using an indirect immunofluorescence
assay (IFA) technique known to the art, spleen cells are obtained
from the mouse and fused with mouse myeloma cell line P3X63.
Ag8.653. The fusion process is carried out in the presence of 30
to 35% polyethylene glycol (950-1050). The method of generating
hybridomas has been previously described (c.f., Kennett et al.,
Monoclonal Antibodies - Plenum Press: 365-371, 1980). Hybridoma
fu~ion products are cultured in HAT medium [Littlefield, J.W.,
Science, 1~5:709-710 (1964)] containing Iscove's modified
Dulbecco's medium (IMDM) with 20% fetal calf serum supplement.
Culture media are monitored for anti-sporozoite antibody product-
ion by indirect immunofluorescence assay (IFA) using glutarald-
ehyde fixed sporozoites of E. tenella as the antigen source. Of
all the cultures tested, 33 wells were found positive by IFA.
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~26~128S
In order to ensure monoclonicity of the hybri-
doma cultures, a limiting dilution procedure was em-
ployed. Following exposure of E. tenella sporozoites to
various monoclonal antibodies of the invention, three
major IFA reactivity patterns on treated sporozoites are
observed: (1) reaction on the entire surface of sporo-
zoites; (2) surface reaction as patches on the sporozoites;
(3) internal reaction around the nuclear membranes of
sporozoites. These reaction patterns are confirmed by
ferritin labelling and transmission election microscopy
(Speer et ?.1., J. of Protozoology in press). As assessed
by IFA, hybridoma cultures of the invention generate
identical antibodies after cloning. Clones are grown
either 1n vitro or in BA~/c mice as peritoneal tumors
and the ascites fluid contains antibodies in a concen-
tration of up to approximately 10 mg/ml.
EXAMPLE 2
Preparation of Antigens Associated
with E. tenella sporozoites
.
Freshly excysted sporozoites of E. tenella are
used for the antigen preparation. The outer membrane
components o~ the sporozoites are extracted using deter-
gents (i.e., 0.5% Nonidet P40, 0.5 to 2~ CHAPS, or 0.5%
to 1% Triton X-100) in 5 mM sodium phosphate buffer
having a pH o 7.8. The buffer contains the following
protease inhibitors: aprotinin (2 trypsin units/ml);
antipain ~25 ~g/ml); leupeptin (25 ~g/ml), phenyl methyl
sulfonyl fluoride (4 mM) [Yoshida et al., J. Exp. Med.,
1 :1225- 1236 (1981)1. The detergent solubilized
material is centrifuged at 100,000 x g for 10 minutes to
remove particulate matter. The clear supernatant contains
soluble antigens associated with E. tenella sporozoites.
EXAMPLE 3
.
Antigen Characterization
Soluble antigens of E. tenella sporozoites are
separated by molecular weight using SDS polyacrylamide
gel electrophoresis (PAGE) [Laemmli, U.K., Nature 227:
680-685 (1970)]. The SDS PAGE separated proteins are
transferred electrophoretically onto nitrocellulose mem-
branes using the Western blotting technique [Towbin et
al., Proc., Natl. Acad. Sci. (USA) 76:4350-4354 (1979)].
The nitrocellulose filter is then reacted with either
diluted ascites fluid or spent hybridoma culture fluid
containing antibodies. Bound monoclonal antibodies are
then detected by using a radioimmuno detection method
involving 125I labelled anti-mouse IgG antibody (~ew
England ~uclear). The unbound second antibody is removed
by washing, and the nitrocellulose filters are then
exposed with Kodak* X-ray film XAR-5.
Alternatively, specific antigen-monoclonal
antibody complexes are identified by an ELISA technique
using horseradish peroxidase coupled rabbit IgG antibody
(Cappel Lab) against mouse immunoglobulin [Burnette et
al., Anal. Biochem. 112:195-203 (1981)]. The Bio-Rad*
Immuno Blot Assay Kit is employed.
The apparent molecular weights of the reactive
sporozoite antigens are determined by comparing the
electrophoretic Rf values of the antigens with Rf values
of known molecular weight compounds run as standards
along with the antigens in the same system. The exper-
imental molecular weight data of various antigens are
presented in Table I.
* Trade Mark
1285
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TABLE I
MOI~Ct~AR ~lEI~HT D~TI~J OF
V~RIOUS E. ~Fr.rA SPOROZOII~E ANTIOENS
Hybridoma l~noclonal Approximate ~lecular
Culture Antikody Weiqht of Antiqen
sSE5 s~ 110 + 16, 130 + 20 kd
s4E2 s2 ilO + 16, 130 ~ 20 kd
slC4 s3 66 + 9 kd, 55 + 8 kd,
20 - 30 kd, 18 + 3 kd,
15 + 2 kd
s2G8 s4 55 + 8 kd
s5B9 s5 55 + 8 kd
slA s6 54 + 8 kd
s3C11 s7 50 + 7 kd
s3D3 . s8 29 + 4 kd
slE4 s9 58 + 9 kd, 130 + 20 kd
:~85
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EXAMP LE 4
Neutralization of Sporozoites of E. tPn~
With Monoclonal Antibodies Usinq
In ~i~Q Chicken Assay
-
An ln vlvo system is employed to evaluate the
capability of monoclonal antibodies produced from hybri-
doma lines of the invention to neutralize sporozoites of
E. tenella. The caeca of the fowl are the sites of infec-
tion by E. tenella and are accessable by surgery [Burns
et al., Exp. Parasitol 8:515-526 (1959); I,awn et al., J.
Parasitol. 68:1117-1123 ~1982)]. The caeca of chickens
are surgically exposed and infused with preparations of
E. tenella sporozoites which have been previously treated
~ith monoclonal antibodies of the invention.
Freshly excysted sporozoites are incubated
under sterile conditions with heat inactivated ascites
fluid ontaining monoclonal antibodies derived from
hybridoma lines of the invention. The incubation period
is for 30 to 60 minutes at 25 to 37C. An incubation
period of 37C for 60 minutes is preferred. Treated
sporozoites are then introduced into 'he caeca of three-
~eek-old chickens by surgical procedures. At the end of
a five-day incubation period, the caeca of the infected
chickens are observed for lesions. The five-day incuba
tion period represents the most destructive stage of
coccidiosis. Results of this experiment are presented in
Table II. It is noted that monoclonal antibodies s3 and
s8 both provided 60% total protectlon against infection
by sporozoites of E. tenella.
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EXAMPLE 5
Immunization with E. tenella s~orrzoite antig~ns
Chickens at one week of age w~re immunized
intraperitoneally with solubilized E. tenell~ sporoziOte
antigens. Initial injections employed proteinaceOus
material derived from 1.5 x 107 sporoziotes in Freund's
complete adjuvant. Two boosters Eollo~ed at ten-day
intervals each employing one-half the initial immunizing
dose of material in Freund's incomplete adjuvant. Ten --
days after the last booster, the chick~ns were challenged
with 50,000 oocysts orally. Five days post-challen~e,
caecal lesions were observed. Results on this experiment
are presented in Table III. It is noc~d that soluble
antigens render significant protection against oral
oocyst challenge whereas normal chickens are not protected.
TABLE III
20 Treatment Percent protection (~/O o~ birds tested)
.
None Partiai ComDlete
Control 100 0 0
Immunized 0 33 66
The new-monoclonal antibodies, No. slC4, No.
s3D3, No. slE4, No. s5B9, No. s5E5, No. slA and No.
s2G8, isolated as described hereinabove, have been
deposited with the American Type Culture Collection
(ATCC) located in Rockville, Maryland and have been
added to its permanent collection. No. slC4 has been
assigned the number HB8333; No. s3D3 has the number
HB8331, No. slE4 has been designated the number HB8332,
No. s5B9 has been designated number HB8402, No. s5E5 has
been designated number HB8403, No. slA has been desig-
nated number HB8404 and No. s2G8 has been designated
number HB8405. Access to the antibodies are available
during ~he pendency of the present application to one
de~ermined by ~he Commissioner o~ Patents and Trademarks
1261;~5
to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C..122,
and all restrictions on the availability to the public of
HB8331, HB8332, HB8333, HB8402, HB8403, HB8404 and HB8405
will be irrevocably removed upon the granting of a patent on
5 the present application.
No. slC4, HB8333, No. s3D3, HB8331 and No. slE4, HB8332
were deposited with the ATCC on August 11, 1983. The new
monoclonal antibodies No. s5B9, HB8402, No. s5E5, EIB8403, No.
slA, HB8404 and No. s2G8, HB8405 were deposited with the ATCC
on November 1, 1983. -
