Note: Descriptions are shown in the official language in which they were submitted.
~ wo 95/03813 2 1 61 5 3 3 PCT/US94/08770
NOV~.T ETMFRIA AN~IBODIES AND ANTIGENS AND ME~HODS OF
USING THE SAME
Technical Field
The present invention relates generally to the field of parasitology. More
particularly, the invention relates to novel antibodies and antigens from Eimeria and
metho-ls of using and detecting the same.
.
Back~round of the Invention
Avian cocçitliosi~ is an enteric, parasitic disease of dQmestic and wild bird
species caused by ..~ h,. i of the l lulozoan genus Eimeria. The disease is spread
by ingestion of sporulated oocysts from feces of an infected host. The infectious
process is rapid and chaldcLt~i~ed by parasite replication in host cells, causing
extensive damage to the intestinal mucosa.
The avian eimerian life cycle is complex and occurs in two stages -- the
exogenous phase which takes place in the litter, and the endogenous phase, whichoccurs in the intestin~l tract of the host. In typical field operations, newly hatched
chicks ingest infectious, sporulated oocysts from col.t~ ted litter shortly after
arrival in the broiler house. Various stimuli interact to release infectious sporûzoil~,s
2 0 within the intestin~l tract which rapidly ~e~lcll~t~, in a species-specific lllar,ncl,
villous or surface epith~ l cells of the gut m--ços~ Intr~ce~ r spoloLoiles thenundergo genetically pro~;lcul--l.cd ~exll~l reproduction, or schizogony, to formm~lozoites bounded by a defined l~ ,cule or p~cilr,~hol~)us vacuole. The
me.l'o~ ts erupt to invade adjacent intestin~l cells and reinitiate merogony (asexual
25 development). At each point during the endogenous eimPfi~n life cycle, dirr~lcllt
stage-specific ~ntigen~ becollle the potential targets of host ~lule~ e B and T-cell
~lllulle responses. Thus, active infection is able to generate a natural immune
response in some clnim~ls. However, there is no practical method for ~lel~,....ii.i.,g
whether a particular animal is indeed i....~-~..-e to infection, which can only be
3 0 cunr'l-,led by visually inspecting the jntestin~l tract of a s~crifiçed bird.
In the absence of a protective host immllne response, a few infectious events
and exponential ampli-fic~tion of the number of parasites rapidly leads to clinical
coccidiosis, manifested by surface and crypt cell loss, malsorption, diarrhea, and
dehydration. Sexual gametogony then occurs, resulting in the contin~l~l shedding of
35 oocysts in the fecal m~t~ri~l and the spread of further infection. The di~e~e,
therefore, causes acute morbidity and results in economic losses to the poultry
industry through decreased growth and feed lltili7~tion.
-
21615~3
WO 95/038L3 PcT/uss4/08770
Previous methods of ;,.",~ i7~tion against coccidiosis have involved the use
of inactivated, ~ttenl~tecl and precocious forms of parasite material. For example,
both viable and attenuated oocysts from Eimeria species have been used as a method
of inducing an immune response. See, e.g., Joyner, L.P. and Norton, C.C.
Parasitology (1973) 67:333-340; C~alm~s, M.M. et al. Ann. Parasitol. Hum. Comp.
(1991) ~:144-148; Jenkins et al. Infect. Immun. (1991) 59:4042-4048. However,
effective ;,~.,,,1l..i7~tion with such material is tentative and the production and
delivery of these preparations can be problematic. Side effects, such as reversion to
virulent forrns, have also been encountered.
1 0 Anti-Eimeria polyclonal and monoclonal antibodies have been produced,
both for ;~ i7; t;on purposes and in an effort to elucidate events occurring during
the various develoylll~ stages of the p~r~ite.
For eY~mple, i,."~."i~i7~tion using crude globulin fractions y~ d from chick~n~
infected with oocysts of Eimeria species has been described (see, e.g., Rose, M.E.
Parasitology (1974) ~:285-292; Long, P.L. and Rose, M.E. Parasitology (1972)
~:437-445; Rose, M.E. Parasitology (1971) 62:11-25; Rose, M.E. and Long, P.L.
Parasitology (1971) ~:299-313) and passive i.."....~i7~tion using monoclonal
antibodies produced against E. tenella ll~c.v~oil~,s and spon~zoi~es has been
suggested (see, e.g., EPA Publication Nos. 135,073 and 135,712, published 27
March 1984 and 3 April 1985, l.,,~c~ ely). Danfc,llll, H.D. Am. J. Vet. Res.
(1983) 44:1722-1727 produced monoclonal antibodies to E. tenella in order to study
develo~ .-t~l stages of the organism and effects on SpOl ~oile penetration and
intr~cell~ r development. All of the above-described antibody pl~&,a~ions were
d using conventional techniques. However, the production of antibodies at
the local site of Eimeria infection has not he,.,lo~ole been described and it now
&~peals that a local response with involvement of the secretory immllne system
and/or cell me~ ted i,---~ ity, is involved in ~rutecLi~e imm~lnity to the ~asite.
Subunit vaccine preparations have been developed in an attempt to alleviate
the problems inherent in the use of the above-described preparations. For e~c~mple,
EPA Publication No. 453,055 (pllkliche~l 23 October 1991) describes
multico~ onent vaccine compositions including ll~ ules of a 25 kDa E. tenella
antigen, a 26 kDa E. necatrix antigen or a 55 kDa E. maxima antigen, derived from
Eimeria oocysts. EPA Publication No. 256,536 (published 24 February 1988)
describes the isolation of E. maxima macrogametocytes and microgametocytes and
vaccines comprising heterogenous protein extracts derived from the g~metocytes.
Similarly, EPA Publication No. 167,443 (published 8 January 1986) describes the
use of heterogenous extracts, containing at least 15 polypeptides, from Sl,ol~,zoiLes
~wo 95/03813 2 1 6~1 5 3 3 PCT/US94/08770
and sporulated oocysts of E. tenella for i,.. nlli7ing chickens. Finally, EPA
Publication No. 164,176 (published 11 December 198~) and Australian Patent
P~lblic~tion No. 87/199,027 (published 4 June 1987) describe the isolation of a 25
kDa protein composed of two polypeptide chains having molecular masses of 17
5 kDa and 8 kDa, respectively, from E. lenella sporocysts and use of the same in a
vaccine composition. The gene encoding the protein has also been cloned and
sequenced.
All of these subunit vaccines have been derived from the extr~ce~ r
parasitic forms of Eimeria, i.e., the extracellular ~OlC)~Oit~ and merozoite forms of
10 the org~ni~m Simil~rly, previously identifiul Eimeria genes have been i~ol~t~ and
characl.,.ized based on antibody probes raised against the extr~cellul~r parasitic
forms. However, it appears that acquired ~luteclive immllnity is associated with the
developing asexual stages and that intracellular spc,lu~oile metabolism and/or early
asexual development is m~n-l~tory for the transcription and translation of parasite-
15 specific gene products recognized by the immllne system. Accordingly, the
ntifi~tior~ ol~tiol-, and bioche.mi~ h~racteri7~tion of these intr~cellnl~r
~dsile metabolic products is desirable for the design of effective ~nticoccidia
vaccines.
2 0 Disclosure of the Invendon
The present invendon is based on the development of novel Eimeria
andbodly ~ ions, produced locally or which traf~lc to, the site of parasitdc
infection. The antibodies so produced provide for the discovery of l,l.,tec~ e
extr~-~ellnl~r and intr~celll-l~r Eimeria antigens in biological samples, inclll-ling in
2 5 culture systems which ~U~Ol L high levels of p~sile growth and development.
Intraçelll-l~r antigens have not previously been id~-ntifi~hle using convçntion~l cell
culture techniques and polyclonal or monoclonal andbody preparatdons. The
~ntipen~ and ~ntihorlies can be used in ~roLe ;li~re Eimeria vaccines. Di~gnostic tests
as well as bioassays to measure B- and T-cell dependent immune responses at the
3 0 local si~e of parasite entry are also made possible by the present discoveries.
Accordingly, in one embodiment, the invendon is directed to an isolated,
locally generated, Eimeria antdbody preparadon. In particularly preferred
embod;~ , the andbody ~l~aLion compri~es cecal lymphocyte immllne
producl:s (CLIP), splenic lymphocyte immnne products (SLIP), rectal antibody test
35 (RAT) or cage dr~ping antibody test (CDAT).
2 ~
Wo 95/038L3 PCT/USg4/08770
In another embo~liment the invention is directed to a method for detecting
the presence or absence of an Eimeria antigen in a biological sample, the methodcomprising:
(a) contacting the biological sample with a locally generated, Eimeria
antibody ~ ;p~tion under conditions whereby a complex is capable of being
formed between an antigen present in the biological sample and an antibody present
in the antibody y~eyalation; and
(b) detecting any complexes formed using a revealing label.
In yet another embodiment, the subject invention is directed to a method for
detecting the presence or ~bsence of an Eimeria tenella antigen in a biological
sample, the method compri~ing:
(a) cont~sting the biological sample with at least one locally gcn~,lated,
Eimeria tenella antibody pl~y~udtion selected from the group con~i~ring of CLIP,SLIP, RAT and CDAT, under conditions whereby a complex is capable of being
formed bel~,en an antigen present in the biological sample and an antibody present
in the antibody preparation; and
(b) detecling any complexes formed using a revealing label.
In another embo~iment, the invention is directed to a method for diagnosing
coccidiosis infection in an avian subject, the method compri~ing:
2 0 (a) providing a biological sample from the avian subject;
(b) conLac~ing the biological sample with a locally ~ lclated, Eimeria
antibody preparation under conditions whereby a complex is capable of being
formed b~ ,n an antigen present in the biological sample and an antibody presentin the antibody preparation; and
2 5 (c) detecting any complexes formed using a revealing label.
In still a further embodiment, the invention is directed to an intr~,cell~ r
Eimeria ~ntigen, id~ntifi~ble using a locally genclaled, Eimeria antibody
p~ ~ation. In particularly p.~ d embo~l;..-f ..l~, the antigen has a molecular
weight of a~,lo~hl~ately 28 kDa, 35 kDa, 38 kDa, 40 ld~a, 43 kDa, 55 kDa, 70 ld~a,
30 lO0 kDa or llO kDa, as determined by Western immunoblot analysis.
In another embo lim~-nt the invention is directed to a method for detecting
the presence or absence of an Eimeria antibody in a biological s~mple, the method
C~ g-
(a) cont~cting the biological sarnple with an intr~f ellnl~r Eimeria antigen
35 under conditio~ whereby a complex is capable of being formed between the antigen
and an antibody present in the biological sarnple; and
(c) detecting any complexes formed using a revealing label.
~ WO 95/038l3 2 1 61 5 3 3 PCT/US94/08770
In still further embodiments, the invention is directed to a kit for diagnosing
coccidiosis in an avian subject, the kit comprising a locally generated, Eimeriaantibody ~l~aLion, packaged in a suitable cont~iner.
In another embodiment, the invention is directed to a kit for diagnosing
coccidiosis in an avian subject, the kit comprising an Eimeria tenella monoclonal
antibody reactive with an intr~ce~ r Eimeria antigen, packaged in a s~lit~ble
CO~-Ii1i.,~,l.
In another embo-limtont, the invention is directed to a kit for detecting the
presence or absence of antibodies to Eimeria in a biological sarnple, the kit
comrTi~ing an intr~-ellnl~rEimeria antigen, p~cL~ge~ in a suitable co~ er.
These and other embodiment~ of the subject invention will readily occur to
those of ordinary skill in the art in view of the ~ closllre herein.
Detailed Desc"ylion
1 5 The practice of the present invention will employ, unless otherwise
in~ ed, convention~l techniques of molecular biology, microbiology, virology,
recoml~in~nt DNA technology, and imllluilology~ which are within the skill of the
art. Such techniques are eYpl~in~A fully in the liter~tllre~ See, e.g., Sambrook,
Fritsch & M~ni~ti~ Molecular Cloning: A Laboratory Manual, Second Edition
(1989); DNA Cloning, Vols. I and II (D.N. Glover ed. 1985); Oligonucleonde
Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J.
~iggin~ eds. 1984); Animal Cell Culture (R.K. Freshney ed. 1986); Imtnobilized
Cells and Enymes (IRL press, 1986); Perbal, B., A Practical Guide to Molecular
Cloning (1984); the series, Methods In Enytnology (S. Colowick and N. Kaplan
2 5 eds., Ac~lemic Press, Inc.); and Handbook of Experimental Irnmunology, Vols. I-IV
(D.M. Weir and C.C. Blackwell eds., 1986, Blackwell Scientific Publi~tions).
All p~tentS~ patent applications and pllbli~ ~tions cited herein, whether supra
or infra, are hereby incoll,o,~ted by reference in their entirety.
As used in this specification and the appended claims, the singular forms "a,"
3 0 "an" and "the" include plural ~t~l~nces unless the content clearly dict~tes otherwise.
A. De~lnitions
In lescribing the present invention, the following terms will be employed,
and are intended to be ~e~ined as in~ic~trcl below.
An "Eimeria antigen" refers to a molecule derived from an Eimeria species
which contains one or more epitopes that will stim~ te a host's immnne system tomake a secretoly, humoral and/or cellular antigen-specific response. For puIposes
2~ 6~ 5~;~
Wo 95/03813 PCT/USg4/08770
of the present inven*on, antigens can be derived from any of the known Eimeria
species, the choice of species being dependent on the host and coccidial disorder to
be treated. By way of example, domestic fowl (Gallus domesticus) can be infectedby any of E. tenella, E. necatrix, E. brunetli, E. maxima, E. acervulina and E.
5 praecox. Turkeys (Meleagris) are susceptible to infection by E. melagrimitis, E.
dispersa, E. meleagridis, E. gallopavonis, E. adenoides, E. innocua and E.
subrotunda. Dom~stic and wild ducks (Anas) suffer from infections caused by E.
anatis and geese (Anser) can be infecte~ by E. anseris, E. nocens, E. parvula, E.
hermani, E. striata and E. fulva. Antigens of the present invention can the efol~ be
10 iden~*fied and derived from any of the above species.
Furthermnre, for purposes of the present invention, an "Eimeria an*gen"
inCllld~s an*gens subst~n*~lly homologous and func*onally equivalent to the
coll~,~o.lding native Eimeria ~n*gen. Thus, the term "Eimeria an.*gen"
encomr~cses m~ifi~tions~ such as deletions, ~-lclitio~ and substitutions (generally
15 conserva*ve in nature), to the na*ve sequences. Such motlifica*ons of the primary
amino acid sequence may result in proteins which have enhanced or decreased
ac*vity-as coml)a c,d to the na*ve sequence. These m~ifi~*ons may be deliberate,as through site-directed mut~g~n~si~ or may be ~ci~ltqnt~l, such as through
mnt~*on~ of hosts which produce the ~ntigçn~. All of these moflific~tions are
20 inc]n(leA, so long as the molecule remains capable of eliciting an immunologica
~ollse, as defined below, and activity is not dcsL-oyed.
~ rl~li*on~lly, an "Eimeria antigen" denotes a protein which may be modified
by com~in~*on with other biological materials, such as lipids and saccharides, or by
side chain modific~*o~, such as acetyla*on of amino groups, phosphoryla*on of
25 hyd~ yl side chains including phosphorylation of tyrosine, serine, threonine or any
other side chains, whether or not these residues are normally phosphorylated in the
native molecul~, or o~ *on of sulfhydryl groups, as well as other morlifications of
the encoded primary sequence. Thus, inc!l~ within the d~fini*on are
~lyco~ylated and unglycosylated forms, the amino acid sequences with or without
3 0 associated phosphates, and amino acid sequences substantially homologous to the
native sequence which retain ;~ ological activity.
By "intr~cell~ r antigen" is meant an Eimeria antigen, as described above,
which is expressed during intr~ellnl~r stages of parasite development, i.e., during
asexual development within the villous or epithelial cells of the intestinal mucosa.
35 The intr~cell~ r stage includes intracellular sporozoite metabolism, trophozoites
and asexual development into mnltin~lcleate schizonts or meronts. Accordingly,
intracç~ r antigens produced during these events are covered by the definition.
~ wo 95~03813 2 1 6~ 5 3 3 PCT/US94/08770
The production of such intracellular antigens can be seen in cell lines adapted to
support the growth of intracellular Eimeria spp. forms, inrlutling both primary and
continuous cell lines. Represent~*ve cell lines are described further below. Thus,
intracellular antigens are also referred to herein as "tissue culture derived antigens."
The term "intracellular antigen" encomp~ses proteins which are both secreted into
the cell culture media in which the parasite is developing, as well as those antigens
which are retained within the cell, such as an antigen associated with the cell
membrane, endoplasmic reticulum and so forth. An intr~cell~ r antigen can be
either soluble or insoluble. An "extr~cell~ r antigen" is an Eimeria antigen which
is produced during the extr~cellul~r stages of parasite development, i.e., the
exogenous phase of the Eimeria lifecycle which occurs in the litter and the
endogenous phases where the organism has not yet invaded and entered host cells.Such ~nh~en.~ include the extracellular :i~Ol'O~Oilt; and lllClOZOitt; forms of the
org~nicm
For purposes of the present invention, antigens of Eimeria spp., are
so~ ,t; ~es i~l~ntifierl below with ,~f~ ce to their molecular mass in kilo~l~lton~
~cDa). For example, an antigen having a mol~cyl~r mass of about
35 kDa is iclentifie~l herein as P35; an antigen of about 40 kDa in molecul~r mass is
idçntif;eli as P40, and so on.
2 0 A "locally g~ncldted" antibody ~ ~alion is a composition cont~ining one
or more ~nhbollies which are produced at, or traffic to, the local site of Eimeria
infection Examples of locally generated antibody preparations include antibody
tions derived from immune lymphocyte pop~ tions found within the
intestin~l tract of a previously or currently infected avian subject, such as those
2 5 derived from any portion of the cecum or intestine and termed cecal lymphocyte
e products ("CLIP") herein; splenic lymphocyte i""l~une products ("SLIP"),
produced by tr~ffirking lllemol.y splenic lymphocytes in infected avian subjects;
ibody p,~ alions (i.e., antibodies derived from fecal material) isolated
from fecal material either directly from the int~stin~l tract or externally, of a
3 0 previously infected or cul~ ly infected avian subject, such as rectal antibody test
("RAT") which is isolated from the intestin~l digesta of the rectum of infected avian
subje~l~., and cage ~v~ing antibody test ("CDAT") which is derived from bird
cage ~U~ gS of infected avian subjects.
An "isolated" antigen or antibody an antigen or antibody which is separate
and discrete from a whole organism (live or killed) with which the protein sequence
is normally a~soci~t~ in nature. Thus, an antigen contained in a cell free extract
would constitute an "isolated" antigen, as would an antigen synthetically or
wo 95/03813 2 ~ 61 5 3 3 PCT/USg4/08770 ~
recombinAntly produced. Similarly, an "isolated" antibody p-ep~dtion in~ d~s
antibodies present in crude ~llixLu~es, blood, serum, etc., so long as the ~1 ixlure is
sep~tc and discrete from the organism with which the antibodies are norm~lly
found. The term "isolated" encomp~cces both polyclonal and monoclonal antibody
5 ~ ua~ions. Additionally, the term "icol~te~l" with respect to both antigens and
antibodies, is not meant to imply a particular degree of purity. Thus, a crude extract
is encomrAcced by the term, as is a highly purified preparation.
The terrns "polypeptide" and "protein" are used interchangeably and refer to
any polymer of amino acids (dipeptide or greater) linked through peptide bonds.
10 Thus, the terrns "polypeptide" and "protein" include oligopeptides, protein
fr~pm,ontc, analogs, mllteinc~ fusion proteins and the like.
The term "epitope" refers to the site on an antigen or hapten to which
spe~ific B cells and T cells respond. The term is also used i~lterchangeably with
~'~ntigçni~ delGIIII;I~AI11'1 or "antigenic determinant site."
An ";.. ,.. ological response" to a col~lposiLion or vaccine is the
development in the host of a secl~to,y, cellular and/ or antibody-m~.Ai~t~l immune
respon.~e to the composition or vaccine of interest. Usually, such a response
includes but is not limited to one or more of the following effects; the pro 1~1ction of
antibodies from any of the ;.~ -ological classes, such as i.. oglobulins A, D,
2 0 E, G or M; B cells; hdper T cells; su~ ,ssor T cells; and/or cytotoxic T cells and/or
~yo T cells, dilccled specifi~lly to an antigen or ~ntigenc included in the co.,.~osilion
or vaccine of interest.
Of particular interest is an immllnological response wl-c,ein the production
of ;.. i~oglobulin A (IgA) is stim~lat~, since this is the principle ;.. ~ noglobulin
25 produced by the secretory system of warm-blooded ~nim~ In particular, avian
species have a mucosal immllne network cort~i~ting of gut-~csoci~ted lymphoid
tissue (termed GALT or Peyer's patches), bronchial-associated lymphoid tissue
(B.ALT), and the harder gland, located ventrally and posteriomedially to the eyeball.
~cs~.t,~tion of antigen to these tissues triggers proliferation and disse n;tl~tiQn of
30 CO,~ ~ B cells to the se-;,eto-~ tissues and glands in the body, with the llltim~te
prodn~tion of se;r~,tc,ly IgA (sIgA). SIgA serves to block the coloni7~tiorl andinvasion of specific surface antigens that colonize on, and pass through, a mucosal
surface.
The terms 'limmllnogenic~l antigen or protein refer to an antigen or protein
35 having an amino acid sequence which elicits an immunological response as
described above. An "immllnogenic" antigen or protein, as used herein, includes the
full length sequence of the desired Eimeria protein or an imm~lnogenic fragment
~ wo 95/03813 2 1 6~ 5 3 3 PCT/US94/08770
thereof. ~y ' imm'lnogenic fragmene" is meant a fragment of a polypeptlde whlch
inr~ es one or more epitopes and thus elicits an immllnological response, as
~i~fine~ above. Such fragments can be identified by, e.g., concurrently synthesi7-ing
large numbers of peptides on solid :iUppOl lS, the peptides corresponding to portions
5 of the Eimeria protein molecule, and reacting the peptides with antibodies while the
peptides are still attached to the Suyy~l LS. Such techniques are known in the art and
described in, e.g., U.S. Patent No. 4,708,871; Geysen, H.M. et al. (1984) Proc. Natl.
Acad. Sci. USA ~1:3998-4002; Geysen, H.M. etal. (1986) Molec. Immunol. 2~.:709-
715, all inculy~l~ted herein by reference in their entireties. Such fr~gmentc will
1 0 usually be at least about 2 amino acids in length, more preferably about 5 amino
acids in length, and most preferably at least about 10 to 15 amino acids in length.
There is no critical upper limit to the length of the fr~gm~nt, which could comprice
nearly the full length of the protein sequence, or even a fusion protein comprising
fr~gments of two or more of the Eimeria antigens or one or more of the Eimeria
1 5 antigens fused to, e.g., a bacterial, fungal, viral or yl`otuzoal protein.
Two polypeptide sequences are ''subst~nti~lly homologous" when at least
about 65% (preferably at least about 80% to 90%, and most preferably at least about
95%) of the amino acids match over a defined length of the molecule. As used
herein, subst~nti~lly homologous also refers to sequences showing identity to the
2 0 specified polypeptide sequence.
By "avian subject" is meant domestic, wild and game birds, inclu~lin~
~nim~lc belonging to the order Gallifolllles, such as chick-~nc, turkeys, phe~c~nts,
partridges, quail, grouse, guinea fowl and peacocks, as well as birds of the order
Ans~,;rol~lles, such as ducks and geese. The definitiQn enco~ csels birds of all25 ages, inclu~ling subjects in ovo.
As used herein, a "biological sample" refers to a sample of tissue or fluid
i~ol~te-l from an avian subject, incl~lcling but not limited to, for example, blood,
pl~cm~. serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid,
samples of the skin, l._ ,yil~tOly~ intestin~l~ and geniloulillary tracts, blood cells,
30 organs, egg constit~lentc and also samples of in vilro cell culture co~ctitll~ntc
(including but not limited to conditioned media resulting from the growth of cells
and tissues in culture me-lium, recombinant cells, and cell components). A
"biological sample" also refers to a sample taken from any of the various
developmental stages of Eimeria spp., including samples of sporulated oocysts,
35 infectious ~olu,oites, intr~cellul~r ~yol`u~c)iLes~ me~Goil~s, ~m~tocytes, and the
like.
WO 95/03813 2 1 ~1 5 3 3 PCT/US94/08770 ~
The term "L~ nl" as used herein refers to both (i) the prevention of
infection or reinfection (prophylaxis), and (ii) the reduction or e1imin~tion of~yll~Loms (therapy) of coccidiosis.
5 ~ R. General Methods
Central to the present invention is the development of novel antibody
yl~aldLions7 produced locally at the site of parasitic infection, and methods for the
d~ntific~tion of parasite antigens using these preparations. The antibodies can be
used in a variety of assays, including assays to identify antigens produced during the
intraçe11-11ar and extr~cel1n1~r phases of the eim~ri~n life cycle. The antibodies also
allow the ~etectioll and char~n-teri7~tion of ~ntigens appearing at particular time
periods during the infective process as well as at particular sites of infection. The
antibodies can be used as diagnostic re~gerlt~, to detect coccidiosis infection, as well
as to ~el~ ."i1e host i~.~....,.~ity levels; to i~ lwnopurify Eimeria ~ntigen~; and as
15 s~ ,ning agents to detect the presence of homologous genes in other m~Aically il~c.l~dnt species.
~ oc~li7~l antibodies, produced in response to Eimeria infection, have not
heretofore been r~i~oll~,d. Furth~,....ore, the i~lerltific~tiQn of intr~ce~ ar ~ntig~n~
has not previously been possible and such antigçns are believed to be l~;~yonsible for
2 0 providing ~luteeli~re i-~ ily against coccidiosis. The ~ntigen~ so identifi~l can
be isolated, ch~T~cteri7e~1 and used in subunit vaccine collryosi~ions~ thus avoiding
problems inherent in prior attenuated and killed vaccine preparations. The
i(lentifieA ~ntigen~ can also be used as diagnostic tools, for detecting Eimeriainfection in biological samples and for d.,t~ ining the level of host i"~"~";ly to the
2 5 Eimeria species of interest.
Several classes of antibody pl~aldlions are described herein which are
produced at, or traf~lc to, the local site of parasitic infection. In particular, described
herein are cecal lymphocyte immllne products ("CLIP"), produced by intestin~l
c lymphocytes; and splenic lymphocyte imm11ne products ("SLIP"), produced
by tr~fflcking memory splenic lymphocytes. Also disclosed are sIgA cont~ining
co~lan~ibody ~r~a~ions derived from the intestin~1 digesta of Eimeria spp.-
c birds. These antibody ~l~alions are termed rectal antibody test
("RAT"), i~ol~te~ from the intestinal digesta of the rectum; and cage dropping
antibody test ("CDAT"), derived from bird cage droppings.
More specific~lly, CLIP and SLIP antibody preparations can be derived
from splenic and cecal lymphocytes isolated from an ap~-ul,liate immnne bird
subject. Particularly useful subjects are inbred bird lines which have been treated to
WO 95/038L~ 2 1 61 5 3 3 Pr~T/USg4l08770
.
sim~ e natural ;.. ;l~y to coccidiosis infection, as described in the examples.
Splenic and cecal lymphocytes can be isolated from the spleens and cecal pouches,
respectively, of the bird subject and cultured in vilro in single cell suspension, using
cell isolation and cultivation techniques known to those of skill in the art. In5 general, lymphocytes are cultivated for ayy,u,ci-llately five days after which time
culture supern~t~nt~ cont~ining secreted Eimeria spp.-specific antibodies are
removed, clarified by ce--Llifugation, filtered, and stored at -20C or lower.
Antibody-co..~ g ~uy~,.llatants can also be generated from in vitro cultivation of
~fin~ immlme T- and B-cell lymphocyte subsets, or combinations thereof, using
10 known techniques.
RAT and CDAT sIgA-cont~ining coprantibodies are isolated from the
rectum digesta and from bird fecal d~uyyh~gs, respectively, of Eimeria spp.-
immnne birds. Exemplified herein is the isolation of RAT and CDAT from an
inbred lnatural avian immlme model. Avian sIgA coprantibody samples may also be
15 obtained from naturally exposed Eimeria spp. outbred broilers raised in wire
batt~ies, floor pens, or broiler houses. To isolate the antibodies, wet fecal material
is resuspended in any suitable physiological b~l~nf~e solution or media, such asphosphate-burrt~d saline (PBS), followed by agitation, the addition of protease
inhibitors, and two or more sllccessive ce~ ifugations at low and moderate speeds.
20 The antibody-contz~ ;ng solution is adjusted to physiological pH, filtered and stored
at at least -20C. Furtherpllrifil~t1on of sIgA can be achieved using ~.. OIl;-
sulfate yrt~c;y;lAl;on~ size and affinity chromatography or other biophysical methods
readily known to those of skill in the art.
The antibody preparations produced above can be further char~cteri7e~ and
25 used for a variety of yulyoses. For ex~mrle, eimerian SLIP, CLIP, RAT, and
CDAT reagents can be characterized in anti-parasite assays, such as a parasite
neutralization assay (PN) and in vitro parasite inhibition assays (PI). Total, isotype-
specific and Eimeria spp.-spec;~lc antibodies, present in SLIP, CLIP, RAT, and
CDAT, can be ~u~..t;r.ed using conventional ELISAs, known to those of skill in the
3 0 art and described further in the el~mrles
Of particular interest is the use of SLIP, CLIP, RAT, and CDAT for the
identific~tion of Eimeria spp. antigens produced during the intracellular stage of
parasitic development. Detection of Eimeria antigens will find utility not only for
- the identification of vaccine c~nrlid~s, but also for diagnostic purposes, to
35 detenmine the presence or ~bsence of coccidiosis in an avian subject and to assess
the level of host ;.. ~ y to the Eimeria species of interest. Eimeria antigens can
be if~ ifie~l in a variety of biological samples, including in samples cont~ining
11
21~53~
Wo 95/03813 PCT/USg4/08770
sporulated oocysts, extracellular sporozoites and merozoites, intrace~ r
oiles and ~ ro~oilcs, and the like. Furthermore, tissue and fecal samples,
e.g., samples from the spleen, cecum, rectum and from bird droppings, can be taken
directly from infected avian subjects or subjects suspected of having coccidiosis and
antigens detected using the antibodies of the present invention. Intracellular
antigens can be identifito~i in media from cell culture systems that support the growth
of the intracellular stages of the parasitic life cycle. Thus, the present invention
makes it possible to discriminate between antigens having similar molecular weights
but produced during dirÇ~ t stages of parasitic infection and/or at dirrGIGnt sites of
1 0 infection. For ex~mple, by use of the invention, intracellular and extracellular
antigens that specifically appear in the spleen, ceca, intestin~l digesta and feces,
after challenge in immune versus naive birds, can be identifieA
A number of known extracell~ r antigens have been identified in Eimeria
syoluzoiles and l-~,.o~oi~es in Western imm~lnoblots, using the novel antibody
preparations described herein, conr~ g that these antibodies are indeed able to
selectively identify imm~lnogenic~ ex~celh-l~r proteins. More hl~yollantly, the
present antibody ylGy~lions for the first time allow the identific~tion of stage-
speçifi~ andlor intr~cell~ r antigens of Eimeria. These antigens are of particular
illt.,r~s~ since they are believed to be primarily responsible for a yfoLecli~e immune
2 0 response to the org~ni~m. Intrace~ r antigens have not hele~fole been
recognized by conventional antibody probes such as by sera produced in Eimeria
c chi~k~n~ nor have they been ifl.ontified using polyclonal antibodies
produced in col-l---oll animal species, such as rabbits, or by monoclonal antibodies
from mice immllni7ed with Eimeria ~olozoiteS and ll-elu~oites. The identifiç~tion
of Eimeria intracellular ~ntigen~ is now possible using the antibody preparations of
the invention to assay culture media from aYian Eimeria spp. grown in continuouscell lines able to support growth of Eimeria during its entire life cycle, from
~yOl'~ZoiLt~ to oocyst, particularly during the intr~cçll~ r phases of development.
Such cell lines are described in Intern~tion~l Publication No. WO 93/01276
(published 21 January 1993) and include cell clones SB-CEV-1/P (ATCC Acce~ion
No. CRL10497), SB-CEV-1/F7 (ATCC Accession No. CRL10495) and SB-CEV-
l/G7 (ATCC Accession No. CRL10496). However, any cell line which will support
the growth of Eimeria during the intracellular stages of asexual development will
also find use for the identification of these stage-specific antigens.
Represent~tive intracellular Eimeria antigens have been identifiecl from
Eimeria spp. grown in cell line SB-CEV-1/F7 (ATCC Accession No. CRL10495),
using the above antibodies. This cell line is optimally cultured in ~edium 199
12
~ WO 9S/03813 2 1 6~ 5 3 3 PCT/US94/08770
(Gibco Labo~tolies, Grand Island, NY) under incubation conditions of 5% CO~ and
40.5~. Fetal bovine serum, antibiotics and antifungal agents can also be added at
ru~ lLions readily determined by one of skill in the art. ~ultnring conditions for
SB-OEV-1/~7 are detailed further in International Publication No. WO 93/01276
(publiche~l 21 January 1993).
In particular, a number of intr~elhll~r antigens, including antigens having
molecular masses of 28 kDa, 35 kDa, 38 kDa, 40 kDa, 43 kDa, 55 kDa,70 kDa, 100
kDa and 110 kDa, les~ec~ ely, have been identified in E. tenella-derived
o..h..n sulfate-treated tissue culture supernatants from SB-CEV-l/F7 using the
1 0 antibodies of the present invention, as well as using hypt;fl,~ lu,le sera plepdled
from naturally exposed, inbred chickens. The 35, 38, 43, 55 and 70 kDa antigens
are recognized by SLIP, CLIP, RAT and immnne sera; the 40 kDa antigen by CLIP,
RAT and imml-n-o sera; the 100 kDa antigen by SLIP, RAT and immllne sera; the
100 kDa antigen by SLIP and RAT; the 28 kDa antigen by CLIP and RAT; and the
15 55 and 70 kDa antigens by SLIP, CLIP and RAT. Furthermore, as can be seen in
the examples that follow, the antigens appear at dirr~l~nt times during the infective
pl'oceSS. ~d~iitio~lly~ the 35, 38,40, 43 and 70 kDa antigens are present in both E.
tenella and E. maxirna strains tested and the 38 and 43 kDa antigens can be
identified in both E. tenella and E. acervulina. Finally, the 38, 40 and 43 kDa
2 0 ~ntigenc are present in several E. tenella strains tested and appear to be the
;...,.~..i~o lomin~nt reactive species using the above antibodies. Accordingly, these
three ~ntigenc are particularly ill~l,ol~ant for providing pr~teclion against a variety of
cocçi-liocis-causing agents, as well as for use as diagnostic reagents in
immnno~Cc~ys for the detection of Eimeria antibodies, thereby intlic~ting the
25 presence of Eimeria infection.
These and other i~ ant Eimeria antigens can be identifiecl in a biological
sample using the above-described antibodies and any of several standard
~ienhfi~hon techniques. For example, the presence of proteins reactive with the
antibodies can be detect~ using standard ele~ o~horetic and immuno~ gnostic
30 techniques. The antibodies can be used in immllno~cc~ys~ such as cc,~ Lilion,direct reaction, or sandwich type assays, for identifying the presence or absence of
the pr~teins by forming complexes therewith. Such assays include, but are not
limited to, Western blots, agglutination tests, enzyme-labeled and meAi~teCl
imm~lno~cS?~ys~ such as ELISAs, biotin/avidin type assays, radioi.. ~ o~ss~ys,
35 immnnoelectrophoresis, immunoprecipitation, etc. The reactions generally include
- revealing labels such as fluorescenl, chemiluminescent, radioactive, or enzymatic
labels or dye molecules, or other methods for detecting the formation of a complex
13
2 l 61 533
Wo 95/03813 PCT/US94/08770
~L~. ~,cn the antigen present in the biological sample and the antibody or antibodies
reacted therewith.
Typically, an immunnassay for detecting one or more of the Eimeria
proteins will involve selecting and preparing the test sample and then reacting it
5 with one or more of SLIP, CLIP, RAT, and CDAT, under conditions that allow
protein-antibody conjugates to form. Solid ~U~pOl Is can be used such as
nitroce~ lose, in membrane or microtiter well form; polyvinylchlori~l~, in sheets or
microtiter wells; polystyrene latex, in beads or microtiter plates; polyvinylidine
fluQrifle diazotized paper; nylon membranes; activated beads, and the like.
10 Typically, the solid support is first reacted with the biological s~mple, washed and
then the antibodies applied. If a sandwich type format is desired, such as a
sandwich ELISA assay, a cc,l,lm~l-;ially available anti-i-llll,unoglobulin (i.e. anti-
rabbit immllnoglobulin) conjugated to a detect~ble label, such as horseradish
peroxid~ce, ~lk~line phosph~t~ce or urease, can be added. An a~ liate substr~t~
15 is then used to develop a color reaction.
A particularly convenient method for identifying and characterizing antigens
using the antibodies of the invention involves immllnoblot analysis. Briefly,
site ~ntigenc are ~ u~d and separated on SDS polyacr,vlamide preparative
minigelc Sep~r~te~ proteins are electroblotted onto ~ nb~ e,s, cut into strips, and
2 0 residual protein-binding sites on the membrane are blocl~ed with an appr~,l,l;ate
agent, such as non-fat miL~, bovine serum albumin (BSA), or heat-inactivated
normal bovine serum (NBS). The test sample can be applied neat, or more often, it
can be diluted, usually in a bur~l~,d solution which contains a small amount of
protein, such as miLk, BSA, or NBS. After in~ub~ting for a sufficient length of time
25 to allow specific binding to occur, the membrane is washed to remove unbound
sample and then in-~ub~t~cl with a co..-bin~tion of conjugated anti-chicken
immllnoglobulins (total antibody)(ie., IgA + IgG + IgM) or a single labeled anti-
c~ e.n immlmoglobulin (isotype antibody)(ie., IgA). Sufficient time is allowed for
specific binding to occur again, the membrane is washed to remove unbound
3 0 conjugate, and the substrate for the en_yme is added. Color is allowed to develop
and the reaction stopped by rinsing in ~lu~liate solution.
~ lternz~tively, a "two antibody sandwich" assay can be used to detect theproteins of the present invention. In this technique, the solid support is reacted first
with one or more of the antibodies of the present invention, washed and then
35 exposed to the test sample. Antibodies are again added and the reaction Vi!~u:~li7Y
using either a direct color reaction or using a labeled second antibody, such as an
14
~ wo 95/038L3 2 1 61 ~ 3 3 PCT/US94/08770
anti-immunoglobulin labeled with horseradish peroxidase, alk~line phosphatase orurease.
Assays can also be conducted in solution, such that the eimerian proteins and
antibodies thereto form complexes under precipitating conditions. The precipitated
complexes can then be separated from the test sample, for example, by
ce.~L,irugation.
Once id~ntifi~l and isolated, the antigens can be further purified using any
of a variety of conventional methods including liquid chromatography, both normal
or reverse phase, HPLC, FPLC and the like; affinity chromatography; size exclusion
chromatography; immobilized metal chelate chromatography; gel electorphoresis;
etc. The amino acid sequences of the purified antigen~ can be delG~ ined using
techniques well known in the art such as repetitive cycles of Edman degradation,followed by amino acid analysis.
The purified antigens can be immllnologically characterized using standard
techniques such as MHC-restricted response profiles in gen~ tically inbred animal~
These lllea~u-~,llellls include~ without limit~tion, Western blot, molecular weight
determin~tions using standard techniques such as SDS-PAGE/staining, T-cell
recognition assays, and assays to infer imml-ne plC lc~Lion or ;~ e pathology byadoptive transfer of cells, proteins or antibodies.
2 0 Genes encoding the subject antigenC can be identified by constructing gene
libraries, using the res-llting clones to transform a suitable host cell and pooling and
screening individual colonies using the antibodies of the present invention,
polyclonal serum or monoclonal antibodies to the desired antigen
Alternatively, once the amino acid sequences of the subject antigens are
~lete~ e(l oligonucleotide probes which contain codons for a portion of the
detel,l incd amino acid sequences can be ~l~alcd and used to screen DNA libraries
for genes encoding the subject proteins. See, e.g., DNA Cloning: Vol. I, supra;
Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis, supra; T. Maniatis et
al., supra. Synthetic DNA sequences, enco-ling the proteins of interest, can also be
pl~l,ar~d, based on the determined sequence, using known techniques. See, e.g.,
Edge (1981) Nature 2~L:756; Nambair et al. (1984) Science ~:1299; Jay et al.
(1984) J. Biol. Chem. ~:6311.
The coding sequences can be cloned into any suitable vector or replicon.
NU111~, OUS cloning vectors are known to those of skill in the art, and the selection of
35 an a~l~liate cloning vector is a matter of choice. See, generally, DNA Cloning:
Vols. I & II, supra; T. Maniatis et al., supra; B. Perbal, supra. The gene can be
placed under the control of a promoter, ribosome binding site (for bacterial
1~
2 ~ 61 533
Wo 95/03813 PcTIuss4l08770
~f~ssion) and, optionally, an operator (collectively referred to herein as "control"
el~m.ont~), so that the DNA sequence encoding the desired protein is transcribed into
RNA in the host cell transformed by a vector containing this construct. The coding
sequence may or may not contain a signal peptide or leader sequence. If so, the
proteins can be e,~ ,sed with or without the native sequences. Alternatively,
heterologous signal sequences can be used. Leader sequences can be removed by
the baçteri~l host in post-translational proces~ing See, e.g., U.S. Patent Nos.
4,431,739; 4,425,437; 4,338,397.
Other regulatory sequences may also be desirable, which allow for
reg~ tion of the e~ s~ion of the protein sequences relative to the growth of thehost cell. Regulatory sequences are known to those of skill in the art, and e~c~mples
include those which cause the expression of a gene to be turned on or off in
response to a ch~o-mi~ or physical stim~h1s, incl~lfling the presence of a regulatory
cc,lllpou.ld. Other types of regulatory elements may also be present in the vector,
for example, enh~nrer sequences.
The control sequences and other regulatory sequences may be ligated to the
coding sequence prior to insertion into a vector, such as the cloning vectors
described above. Alternatively, the coding sequence can be cloned directly into an
lession vector which already contains the control sequences and an ap~ ,liate
2 0 restriction site.
In some cases it may be necess~, y to modify the coding sequence so that it
may be ~tt~shYl to the control sequences with the appl~liate orient~tion; i.e., to
t~ the proper reading frame. It may also be desirable to produce mllt~nt~ or
analogs of the Eimeria antigen of interest. Mutants or analogs may be plGp~d by
2 5 the deletion of a portion of the sequence encoding the antigen, by insertion of a
sequence, and/or by s~lbstitution of one or more nucleotides within the sequence.
Techniques for modifying nucleotide sequences, such as site-directed mutagenesis,
are described in, e.g., Sambrook et al., supra; DNA Cloning, Vols. I and II, supra;
Nucleic Acid Hybridization, supra.
3 0 The expression vector is then used to transform an appropriate host cell. The
transformed host cells are cultured under conditions providing for expression of the
antigen of interest. The antigen is then isolated from the host cells and puri~led. If
the expression system secretes the protein into growth media, the protein can bepurified directly from the media. If the protein is not secreted, it is isolated from
cell lysates. The selection of the applopliate growth conditions and recovery
methods are within the skill of the art.
16
~ WO 9S/038L~ 2 1 61 5 3 3 PCT/US94/08770
The antigens of the present invention may also be produced by chemi~l
synthesic, such as solid phase peptide synthesis, using known amino acid sequences
or amino acid sequences derived from the DNA sequence of the genes of interest.
Such methods are known to those skilled in the art. Chemical synthesis of peptides
5 may be preferable if a small fragment of the antigen in question is capable of raising
an irnmunological response in the subject of interest.
The isolated, recombinantly or synthetically produced Eimeria antigens can
be used in immunoassays, such as the competition, direct reaction or sandwich-type
assays described above, to detect the presence of Eimeria antibodies in biological
10 s~mp1es. In this way, not only can the ~ gnosic of coccidiosis be made, but the host
level of i~ y can be ~lc~, ...i..ed. For example,
naturally occurring anti-Eimeria spp. antibodies are produced by the infected
chic~en in its fecal material. The presence of these antibodies can be determined by
reacting a sample of fecal material with one or more of the Eimeria antigens of the
present invention. Antibodies present in the fecal sample will form an antibody-antigen complex with the andgen. The reaction lllix.Lul~, can be analyzed to
~lete~ . . .i-.e the presence or absence of these antibody-antigen complexes using any of
a number of standard methotlc, such as those illllllunodi~gnostic mPtho ls cl~scrihed
above. For example, the isol~ted antigens can be conjugated to a solid su~poll, such
2 0 as any of the above-desc~ibecl ~ul,pc, l~, a fecal sample is then incub~tetl with the
conjugate, and the reaction Illi~lu-e analyzed to determine the presence of the
antibodies.
Other useful assay formats include the filter cup and dipstick. In the former
assay, the antigen of this invention is fixed to a sinter glass filter to the opening of a
small cap. The fecal sample is resuspended in diluent and then passed through the
filter. If the antibody is present, it will bind to the filter which is then ViCUZ~li7f~
through a second antibody ~letector. The dipstick assays involves fixing an antigen
to a filter, which is then dipped in the resllcper-~l fecal sample, dried and screened
with a detector molecule.
3 0 The ~:imeria proteins of the present invention or their fragments can also be
used to produce antibodies, both polyclonal and monoclonal. If polyclonal
~ntibo~ies are desired, a selected m~mm~l, (e.g., mouse, rabbit, goat, horse, etc.) is
...... ~.. i7ed with an antigen of the present invention, or its fr~gm.ont, or a mut~te-l
~ntig~n. Serum from the immnni7~1 animal is collected and treated according to
knowrl procedures. If serum cont~ining polyclonal antibodies is used, the
polyclonal antibodies can be purified by immunoaffinity chromatography, using
known procedures.
17
Zt6~533
WO 95/03813 PcT/uss4lo877
Monoclonal antibodies to the Eimeria an~igens of the present invention, and
to fr~ mentC thereof, can also be readily produced by one skilled in the art. The
production of several monoclonal antibodies raised against E. tenella-infected SB-
OEV/F7 tissue culture supernatants is described in the examples. Of particular
5 interest is MAb 1-2-6, which reacts with the 40 kDa intracellular protein which has
been i(3e~ti~led as being protective herein. The Eimeria monoclonal antibodies are
produced by using hybridoma technology. In particular, immortal
antibody-producing cell lines can be created by cell fusion, as well as by othertechniques such as direct transformation of B lymphocytes with oncogenic DNA, or1 0 transfection with Fpsttoin-Barr virus. See, e.g., M. Schreier et al., Hybridoma
Techniques (1980); H~l.. e,ling et al., Monoclonal Antibodies and T-cell
Hybridornas (1981); Kennett et al., Monoclonal Antibodies (1980); see, also, U.S.
Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917;
4,472,500, 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced
15 against the Eimeria antigen of interest, or fragment thereof, can be screened for
various l~v~,cl lies; i.e., for isotype, epitope, affinity, etc.
Monoclonal antibodies are useful in purifiration, using immunoaffinity
techniques, of the individual antigens against which they are directed. The
antibodies are also useful in diagnosis of coccidiosis infection, and can be used e.g.,
2 0 in immnno~cc~ys such as those described above, as well as in therapeutic
compositions for the passive ;~ i)i7~siQn of avian subjects.
The above-described antigens and antibodies, including the various
in~cç~ r antigens, polyclonal and monoclonal antibodies raised against the
antigens, C~IP, SLIP, RAT and CDAT, can be provided in kits, with suitable
25 insl- u-;Lions and other nec~ 5SZ~l r reagents, in order to cor-dl~ct immllno~cc~ys as
described above. For example, the antibodies, eimerian antigens, or both, can beprovided in a diagnostic immllno~cs~y test kit to provide for the detectio~ of
coccidiosis infection or to test the state of ;,.".-n;Iy to coccidiosis of an avian
subject. The kit can also contain, depending on the particular immllno~cs~y used,
3 0 snit~hle labels and other packaged reagents and materials (i.e. wash buffers and the
like). Standard immunoassays, such as those described above, can be conducted
using these kits.
The antigens (either puri~led, partially purified, or crude mixtures thereof),
immunogenic fr~gmentc of the antigens, chimeric proteins comprising the same, and
35 antibodies described above, can also be form--l~te-l into subunit vaccine
co~ osilions to provide illlllllll~ity to coccidiosis. The antigens and antibodies of
the present invention can be used either alone or in combination with other antigens
18
~ wo 95/03813 2 1 61 5 3 3 PCT/US94/08770
and antibodies, from the same or different species of Eimeria. For example,
intracellular Eimeria antigens may be combined with extracellular Eimeria proteins,
such as those present in extracellular sporozoites and merozoites. In such
combin~tions, the antigens may be provided in the form of a fusion protein or a
5 larger, mllltim~ric protein. These fusion proteins or mnltimeric proteins may be
produced recombinantly, as described in, e.g., U.S. Patent No. 4,366,246, or may be
synthesized ch~omic~lly. Further, antigens of this invention may be employed in
combin~tion with antigens from other avian pathogens, to provide broad spectrum
protection against a variety of avian rli~e~es Additionally, crude mixtures of the
10 antigens, such as partially purified lllixlules of intMcelllll~r antigens derived from
~mmc)nillm sulfate pl~ci~ tion of culture media which ~ul poll~ the growth of
intr~celllll~r forms of Eimeria spp., can be used in vaccine compositions wilhoul
further purification. See, e.g., the examples, where such crude extracts are shown to
be ~ ,te~ e against E. tenella challenge.
The vaccine compositions are generally formnl~te(l with a pharn~ceutically
acceptable vehicle or excipiellt Suitable vehicles are, for example, water, saline,
dextrose, glycerol, eth~nol, or the like, and combinations thereof. In addition, if
desired, the vehicle may contain minor amounts of auxiliary substances such as
wetting or emulsifying agents and pH bur~.i-lg agents. Preservatives known in the
2 0 art, such as ll-i"~ ,sal, phenol and other phenolic co--~oullds, as well as ~ns-biQtics,
can also be added to the vaccine compositions of the present invention. Suitablevaccine vehicles and additives are known, or will be a~,a-Gnt, to those skilled in the
art. See, e.g., Remington's Pharnt(7~e~ticc71 Sciences, Mack Publishing Company,F~ton, Pennsylvania, 18th edition, 1990.
Adjuvants which enh~nce the effectiveness of the vaccine, may also be
added to the form~ tion. Adjuvants may include for example, -lul~n-yl dipeptides,
avridine, aluminllm hydroxide, oils, oil in water emulsions, saponins, cyt~kines, and
other subst~nces known in the art.
The protein may be linked to a carrier in order to increase the
3 0 immunogenicity thereof. Suitable carriers include large, slowly metabolized macro-
molecules such as proteins, including serum albumins, keyhole limpet hemocyanin,immunoglobulin molecules, thyroglobulin, ovalbumin, and other pl~rteins well
known to those skilled in the art; polysaccharides, such as sepharose, agarose,
cellulose, cellulose beads and the like; polymeric amino acids such as poly~ t~mic
acid, polylysine, and the like; amino acid copolymers; and inactive virus particles.
The Eimeria antigens may be used in their native forms or functional groups
modified for ~tt~chment to these carriers.
19
Wo 95/038L3 PCT~USg4/08770
As ex~ ined above, avian species have a mucosal i,."~""-e nclwcJ~k
con~isting of gut-associated lymphoid tissue termed GALT or Peyer's patches),
bronchial-associated lymphoid tissue (BALT), and the Harder gland, located
ventrally and posteriomedially to the eyeball. Present~tiQn of an antigen to these
5 tissues triggers proliferation and r~ emin~tion of co"""i~cl B- cells to the
se~ ,t~ly tissues and glands in the body, with the ultim~te production of se~ ,.y
IgA (sIgA). SIgA serves to block the coloni7~tion and invasion of specific surface
~ntigen~ that colonize on, and pass through, a mucosal surface. It appears that the
es~ l sIgA system plays an es~enti~l role in the protective immune response to
Eimeria. Davis, P.J. el al. Inununology (1978) 34:879-888. Accordingly, the
Eimeria antigens of the present invention may also be ~(imini~tered using avirulent
carrier microbes, able to invade and proliferate in the cells of the GALT and BALT.
Such delivery allows for a generalized secl~Lc,l y immlme response as well as
humoral and cellular immnne responses. For example, recomhin~nt pl~cmid~
cont~ining genes for the Eimeria antigens can be introduced into one of several
avirulent strains of bacteria, designed for delivering antigens to avian subjects.
Such avirulent org~ni~m~ generally contain mllt~tio~ in genes neces~ y for long-term survival and include mutant derivatives of Salmonella. E. coli and E. coli-Salrnonella hybrids. Such mllt~nt~ are ~lescnbetl in e.g., Curtiss, R. III, et al. Infect.
2 0 Imrnun. (1987) ~:3035-3043 and U.S. Patent Nos. 4,968,619; 4,888,170 and
4,190,495, incorporated herein by reference in their entirety.
Furthe~...ul~, the yl~,leh~s may be formnl~ted into vaccine co-llyosilions in
dther neutral or salt forms. ph~n~re,~ lly acceptable salts include the acid addi-
tion salts (formed with the free amino groups of the active polypeptides) and which
2 5 are formed with inorganic acids such as, for example, hydrochloric or phosphoric
acids, or such organic acids as acetic, oxalic, tartaric, m~n-lelic, and the like. Salts
formed from free carboxyl groups may also be derived from inorganic bases such
as, for example, so~ m, pot~einm, ~mmo~illm~ c~lci--m, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylamino eth~nol,
30 hi~ti~line7 procaine, and the like.
Vaccine formulations are plepal~d by combining an effective amount of one
or more antigens described above, the exact amount being readily determined by
one skilled in the art. Por pul~oses of the present invention, an "effective amount"
of an antigenic vaccine component will be that amount required to generate an
35 amount of circulating antibody sufficient to prevent or reduce coccidiosis disease
~ylllyl( llls. An effective amount of an Eimeria antigen will vary, depending on the
mode of ~clmini~tration, the particular species of Eimeria targeted, the degree of
~ Wo 95/03813 2 1 61 ~3 3 PCT/US94tO8770
~rot~;llion desired and the age and health of the subject to be treated. Such ~ o
are readily determinable by the skilled artisan however, by way of example, for
compositions to be delivered parenterally, generally between about l0 ~g to about 1
mg, more preferably about 25 ~lg to about 200 ,ug, and most preferably about 50 ~Lg
to about 100 ~Lg, in about 0.5 to about 10 ml, preferably about l ml to 3 ml, will
col-~tinlte an effective amount of antigen.
The vaccine compositions of the present invention can be ~rlmini~tçred
pal~ el~lly, e.g., by intr~ml-scul~r, subcutaneous, intravenous or intraperitoneal
injection. It may also be desirable to introduce the vaccine composition directly into
the gut or bronchus, to stiml-l~te a preferred response of the GALT or BALT, such
as by ~ral ~imini~tration~ intranasal ~Aminictration~ gastric intub~tion or aerosol
~-lministration, as well as air sac and intratracheal inoculation. For example, for
oral ~Amini~tration~ the vaccines can be conveniently placed directly into watergiven to the avian subjects. Other suitable methods of ~rlmini~tçring the vaccines of
the invention are, e.g., via the conjunctiva to reach the Harder gland or in ovo~lmini~tration, by inoculating avian eggs before they hatch. A combination of these
routes of a~lmini~tration can also be used. For example, the initial inoculation might
involve pd-en~ mini~tration while subsequent boosters might be given orally.
The avian subject is i.u,.-",~i7ed by ~(lmini~tration of the vaccine
2 0 formlll~tion, in at least one dose, and preferably two or more doses. However, the
animal may be ~-lmini~tered as many doses as is ,~quil~d to m~int~in a state of
;I~lllll~l~;ly against cocci-liosi~
For ex~mrle, boosters can be given at regular intervals, i.e., at six months or yearly,
in order to sustain i""",.";~y at an effective level.
C. E~ hnel~tal
' Below are examples of specific embo~lim~nt~ for carrying out the present
invention. The examples are offered for illustrative purposes only, and are not
intçnded to limit the scope of the present invention in any way.
3 0 Efforts have been made to ensure accuracy with respect to numbers used
(e.g., amounts, l~mp~.~lul~s, etc.), but some e~ hllental error and deviation
should~ of course, be allowed for.
The antigens of Eimeria spp., are sometimes identified in the examples with
reference to their molecular mass in kilo~ltons (kDa). Thus, an antigen having amolecular mass of about 35 kDa is identified below as P35; an antigen of about 40
kDa in molecular mass is identified as P40, and so on.
2~615~;~
Wo 95/03813 PCT/US94/08770
The following acronyms are used in the examples and are defined as follows:
UI/UC, u.. ;.. ,.. i~e(3 unchallenged (naive)
WC, ~ l, challenged
NE/UC, naturally exposed, unchallenged (also referred to as
"tricklefed")
NE/C, naturally exposed, challenged
SLIP, splenic lymphocyte immnne product
CLIP, cecal lymphocyte immune product
RAT, rectal antibody test
CDAT, cage dropping antibody test
MAb, monoclQnal antibody
spz, çYh~ce~ r sy~Jr~zoile
rnrz, extr~ce-ll--l~r ".~n~oile
F.xample 1
F.~tablishment and Testing of lnbred Natural Exposure (NE) Model
C`hirl~n~ (B'9BI9, B24B24, and B30B30, New H~llyshil-, Poultry Research
Center), serologically typed and previously detel--lined to differ at the MHC-locus
(B-complex), were housed in wire cages. Chicks were fed a nonms~ t~d
2 0 starter/grower diet and water ad libitu~n. Groups (NE/C) of one-day chicks were
;IlllllllUi7.ed with 500 E. tenella oocysts from a strain desi~n~ted L.S. 65 (provided
by D. Strout, Univ. of New Ha~ ;) per os for five consccu~ e days. Unless
speçifi~lly noted, all trickle i.. l.~;7~t;ons and challenge infections were
~clro,ll-cd with E. tenella L.S. 65. Control groups (UVC) were similarly
;IIII~IIIIIi7~d with ~ tiller3 water. At either 6, 8, 11 or 15 days following the last day
of parasite e~yo~u~ birds were weighed and challenged with 3.5 x 104 E. tenella
oocysts. Mean body weight gain and cecal lesions were determinecl at 6 days post-
ch~llenge The duration of ;IIllllllll;ly to homologous challenge in trickle-
il l ..~ ll ..li7çd chicks was found to be at least 2 weeks. NE/C birds from all three
3 0 haplotypes showed si~ni~lc~ntly higher weight gains and lower lesions than their
WC COullt~.yhllS at the ~ pOi"ts ex~mined, supporting the concept that this
method of triclde i..l.ll~l;7i hon affords protection against homologous parasite
ch~llçnge. Therefore, the reagents obtained from NE/UC, NE/C and UUC birds at
various timepoints post-challenge are reprçsentative of the ~lirr~l~nt levels of35 ;I~ ity which exist following parasite challenge.
~wo 95/03813 2 ~ 675 3 3 PCT/US94/08770
Example 2
Preparation of SLIP and CL,IP Samples from Inbred NE Model
For preparation of SLIP samples, spleens were removed (typically 4-
5 6/group) at the desired timepoint and mononuclear cells isolated using standard
Histopaque 1077 centrifugation (Sigma, St. Louis, MO.). Viable cells were counted
using trypan blue and a hemacytometer and cultivated at a density of 5 x 106
cells~ml in serum-free modified LM-Hahn m5flil-m (LMH) (Calnek, et. al. Infect.
Immun. (1981) 34:483-491.) for 5 days (40.5C, 5% CO2). Supernatant was
harvested, spun (800 x g, 10 min., 4C), and cell-free supernatant collected. Sodium
azide was added to 0.1% (v/v) final concentration and samples filtered (0.2 ~M).SLIP samples were stored at 4C for up to 2 weeks or aliquots p,~aled and frozenat -20C until use.
Por preparation of CLIP samples, both cecal pouches per bird were removed
15 (typically 4-6 birds/group) at the desired timepoint and intestin~l lymphocytes
i~ol~te~ according to standard procedures. Briefly, cecal pouches were cut at the
ileocec~l junction, the distal end excised, and cecal contents expressed out. Tissue
was placed into a tube col.t~i..ing cHBSS (Ca2+ and Mg~+-free HBSS cont~ining 2Xantibiotic/antilllycotic, 25 mM HEPES, pH 7.4) and placed on ice until further
2 0 manipulations. Tubes were shaken vigorously for 10 sec. to remove ~ ition~l gut
COI-t~ , and S/N discarded following gravity se~lim~-nt~tion of tissue. Cecal tissue
was placed into a petri dish and each cecum opened lon~itu~lin~lly. The mllcQs~lsurface was gently scraped to remove any residual clumps of fecal material and
tissue minced into small 1-2 cm fr~m~ontc Tissue fr~gm~l-t~ were placed in a 50
ml tube cont~ining 10-20 ml cBSS, and s~mples hand shaken vigorously for 10-20
sec., followed by a low speed spin (70 x g, 1 min. R.T.). S/N was discarded and 10-
20 fresh cBSS added to tissue. The shake/spin procedure was repeated two
additional times, followed by the addition of 10-20 ml cHBSS/DI~ (lOmM DTT,
lX genticin and polymyxin B sulfate) per sample. Tubes were briefly shaken to
disrupt tissue pellet, incubated in a hufl~olllal position on a platform shaker for 20
min. (150 rpm, R.T.), and spun at low speed as above. S/N was discarded and tissue
washed two additional times at low speed to remove residual DTT. Approximately
10 ml of freshly prepared cHBSS/ES (cBSS containing 1 mg/ml collagenase
- (Clostridiopeptidase A, Type VII, Sigma) was added and minced tissue solution
transferred to a glass flask containing a stir bar. Samples were incubated with
- moderate stirring (400-500 rpm) on a multiple magnetic stir plate for 60 min (37C)
and solution transferred into a 50 ml tube and spun at low speed to pellet any
23
2 t ~b 5~
Wo 95/03813 PcTluss4/o877
transrtl-~ tissue fra~rn~nts. S/N containing desired cells was carefully poured off
and kept on ice until further manipulations. The cHBSS/ES tre~tment was repeatedone additional time and desired cells from the second incubation pooled with cells
from initial enzymatic tre~tment. Cells were pelleted (450 x g, 10 min. 4C) andresuspended to homogeneity in 40% isotonic PercolVcHBSS. Volume for
.u~s~,nsion was dependent on cell pellet size. Typically, 9 ml of 40% Percoll was
used per 108 cells. Approx. 3 ml of 40% cell suspension was gently layered onto a
equal volume of 70% isotonic Percoll in 15 ml tubes, spun (600 x g, 20 min. 4C),
and the 40no cell interface collected. Using this procedure, both in~ elial and
lamina propria cecal intestin~l lymphocytes were isolated together in the 40r7o cell
interface. Cells were washed 3-4 times in cHBSS (450 x g, 10 min. 4C), viable cell
count detc~ ined, and cultured for 5 days as described above for splenic
lymphocytes. CLIP samples were prepared and stored as previously described for
SLIP s~mples
F.xample 3
al ~tion of RAT from Intestinal Di~esta and CDAT from Fresh Ca~e d.~ inp~
For RAT, fecal m~teri~l (typically 4-6 birds/group) was collected from the
ileocecaljunction down to the cloaca and placed in a preweighed 50 ml conical tube.
2 0 For CDAT, fresh cage dro~ gs were c~llectGd from litter pans and placed in a
preweighed 50 ml conical tube. The net wet weight of samples were dete~milled,
and 3 mls of Dnlbecco's phosphate buffered saline (l:)PBS) were added per grarn
feces. Samples were vortexed at moderate speed for 15-20 sec. to ,Gsu~end fecal
m~ten~l. Samples were spun (2750 x g,WC, 15 min.) and supernatant above fecal
pellet transferred to 30 ml Sorvall GSA-600 rotor tubes. Samples were spun
(32,571 x g, 4C, 20 min.) and supernatant above pellet transferred to a clean tube.
The pH of the sample was determined and adjusted to pH 6.5 to 7.0 if necess~ry.
Protease inhibitors (1 mM 1, 10-phen~nthroline, 1 mM ~l~7~ line HCL hydrate,
10 ~g/ml pepstatin, 0.5 mM PMSF (all final concentrations) and sodium azide
(0.1% final concentration) were then added. Samples were stored at -20C until
ready for use.
Ex~m~le 4
In Vitro Parasite Neutralization Assay
In order to measure B-cell derived antibodies to e~ ell~ r S~Olu~,Oi~S
present in CLIP, control and trickle-i~ --11-i7to~l B24B'4 and B30B30 birds wereprepared as described in Example 1. Thirteen days following the last parasite
24
2 1 ~ 3 ~
1~WO 9S/03813 PCT/US94/08770
exposure, birds were challenged with 3.5 x 104 oocysts and CLIP samples preparedat days 1, 3 and 5 post-challenge as described in Example 2. One ml samples wereincubated with an equal volume of freshly excysted L.S. 65 E. tenella sporozoites (5
X 105/m1) on a rocker platform (1 hr, 40.5C). The mixture was then directly added
5 in quadruplicate to microtiter wells containing SB-CEV/F7 cells (ATCC Accession
No. CRL10495) plated (in m~ m 199/5% FBS) at 1 x 105 cells/well 24 hrs
previously. After 2 hrs (40.5C, 5% CO2) extracellular sporozoites were removed
by washing, and one IlCi per well of tritiated uracil (Amersham, 5 mCi/mmol)
added. After 24 hr incubation (40.5C,5% CO2), cells were harvested onto glass
10 fiber mats (MACH III Harvester, TomTech, Orange, Conn.) and incorporation of
radioactivity determined (Matrix 96, Packard In~llull,ent Co., Sterling, VA.).
Percentage inhibition was calculated as the mean counts per minute (cpm) (mean
cpm equals mean of quadruplicates using Dicksons Outlier Test to exclude e~ ,mc
values (Dickson, W.J. Biometrics (1953) 2:74-89)) of sample treated wells divided
by the cpm of untreated wells times 100%. The kinetics of CLIP sporu20i~e
neutralizing ac~ivity differed between naive and immnne challenged birds. At day 1
post-challenge, CLIP from both NE/C haplotypes cont~ined signific~ntly higher
parasi~e neutralization activity cull.pal~,d to CLIP from UVC groups. At day 3 post-
ch~llenge, only NE/C CLIP from the B24B24 haplotype contained higher parasite
2 0 neutr~li7~tion activity. At day S post-ch~llenge, no measurable activity dirr~lences
between naive and immune groups in either haplotype were observed. These resultsintlicple that the anti-parasitic activity of CLIP from NE/C birds appears more
quickly in immllne versus naive birds following parasite ch~llenge.
In order to determine whether particular molecular weight extracellular
25 ~ur~zoile antigens were recognized by these antibody-containing CLIP samples,samples exhibiting varying degrees of neutralizing activity were screened by
Westeîn blot against E. tenella spz antigen (see Example 7). Results indicated that
CLIP samples with strong IgG reactivity to sporozoite antigens having molecular
weights of 43 ld~a, 40 kDa, 38 kDa and 26 kDa, possessed the highest levels of anti-
30 s~ulozoile neutralizing activity. These results indicate that these antigens arerecognized by IgG produced by cecal lymphocytes at the site of parasite replication.
Finally, these results clearly in~lin~te that the antigen-specific IgG in immune CLIP
was produced through T-cell dependent immune mech~ni~ms
21 ~ ~
Wo 95/038L3 PCT/USg4/08770
Fxample 5
In Vitro Parasite Inhibition Assay
In order to measure soluble, T-cell derived anti-parasitic faccors present in
CLIP, the same CLIP samples obtained from UVC and NEJC B24B24 and B30B30
5 birds in Example 4 were used for the assay. The duck embryo (DE) cell line
(ATCC ~çces~ion No. CCL 141) was seeded in 96 well flat microtiter plates at 1 x104 cells/well (in me~inm 199/5% FBS) overnight (40.5C, 5% CO2). Cells were
then pl~Llcated with 0.2 mls/well of duplicate log2 dilutions of CLIP s~mples. One
row of cells was treated with media alone (control). After 24 hrs incubation
(40.5C, 5% C02), fresh dilutions of test CLIP, 1 x 105/well freshly excysted
C~ ce~ r E. tenella ~-,ç~ oites (obtained as described in Example 7), and one
Ci per well of tritiated uracil was added. After 24 hr incubation (40.5C, 5% CO2),
cells were harvested onto glass fiber mats and incorporation of radioactivity
de~ ed. P~,.ce.ltage inhibition was c~ ted in a similar fashion as that
15 ~escribed in Example 4, i.e., as the mean cpm of sample treated wells divided by the
cpm of media control wells times 100%. The kinetics of CLIP parasite inhihition
differed between naive and imm~lne challenged birds. At day 1 post-ch~llenge,
CLIP from both NE/C haplotypes contained ~ignifi~ntly higher parasite
neutr~li7~tion activity as colllp~l to C~IP from UVC groups. At day 3 post-
2 0 c-h~ nge, only NE/C CLIP from the B24B24 haplotype co~t;~ine~ higher lJ~aSiL~
inhihision activity. At day 5 post-ch~llenge, no ~i~nific~nt dirr~ ,nces in the
inhibitory activity between naive and imml-ne groups in either haplotype was
obs~ d. These results inrliçate that the anti-parasitic activity in CLIP, directed
against the intr~çell~ r parasite forms, appe~ more quickly in i~ c versus
25 naive birds following parasite ch~llçnge
The peak inhibitory activity of these NE/C T-cell derived cytokines parallels
the peak inhjhitory activity of the NE/C B-cell derived antibodies obtained in
FY~mrle 4. By 24 hrs post-challenge in NE/C birds, antigen-specific TH2cells
located in the cecal lining elaborate specific cytokines, most likely IL5 and IL6,
30 which enh~nce antibody production and extracellular sp~ruzoi~e neutralization. But
perhaps more illlpul~ntly, there is a simnlt~neous induction of antigen specific TH1
cells and secretion of anti-parasitic cytokines, probably intclÇcl~ gamma and tumor
necrosis factor beta, which exert their effects directly on the intracellular replicating
parasite. Therefore, the same CLIP sample can be used to measure both antibody
35 and cell-merli~tçd im~ e effector molecules.
26
~wo 9S/03813 2 1 6~ 5 3 3 PCT/US94/08770
Exam~le 6
Quantitation of total and sporozoite-speçific I~A in RAT
For q.,~ tion of total IgA in RAT samples, a murine anti-chicken IgA
MAb (MAb 6.2.3-1 purchased as ascites from Dr. S. Naqi, Cornell University,
Ithaca, NY) or MAb Jl 26.189.96 (Janssen Biochemica) was diluted 1:500 in 50
mM sodium borate, pH 9.5. One hundred microliters per wells was added to ELISA
multiwell plates (Nunc TmmllnoPlate Maxisorb F96, Nunc, Denmark) and incubated
at 40C (2hrs) or at 4C (overnight). Plates were washed 3X with PBST (phosphatebuffered saline/0.05% Tween) and then blocked for 2hrs (40C) at overnight (4C)PBST/5% .ckimmeri miLk (Difco 0032-01-1). Plates were then washed 3X with
PBST. Test RAT s~mples were initially diluted 1:100 in PBST/5% milk and serial
two-fold dilutions added in duplicate to wells (100 ~lVwell). Serial two-fold
ltiOll~; of reference serum cont~ g IgA (Bethyl Labs, RS 10- 102- 1)(initial
cQI~re~ ion 4.0 llg/ml) were similarly ~l~;p~d for each plate. Plates were
inr~ub~l~ at 4C overnight. Primary RAT antibody incubation was pGlrcl.lled at
4C to decrease endogenous protease activity in the samples. Plates were washed
3X with PBST and then 100 ,uVwell of a 1:500 dilution (PBST/5% miLk) of
h~ dish peroxidase conjugated goat anti-chicken IgA (Bethyl Labs, A30-103P-
3) added for lhr (40C). Plates were washed 3X with PBST and then developed by
2 0 the addition of 100 ~lVwell TMB peroxidase substrate/peroxidase solutiQn
(Kirkegarrd & Perry). After 5-30 min. the reaction was stopped by the ~ ition of10~Vwell lN NaOH. Optical density at 450 nm was clet~ e~l on a VMAX
ELISA plate reader (~olec~ r Devices). The VMAX program was used to
c~l~ul~te unknown con~en1Tations based on the IgA reference standard curve.
For qu~ntit~tion of sporozoite-specif;c IgA RAT samples, a similar
procedure was followed except the plates were coated with 100 ,uVwell of sonic~te~l
E. tenella antigen (3 ~g/ml) for 2 hrs (40C) or overnight (4C). Two rows were
coated with anti-chicken IgA MAb as above for capture of reference serum. The
rem~inrl~r of the assay was performed as descnbed above for the total IgA ELISA,3 0 except the RAT samples were tested at an initial 1: 1 dilution.
Exam~ple 7
Immunoblots
- The following procedure was used for several of the examples to follow.
Llllluhoblots were modified from previously published procedures (J.T. Roehrig et
al. Virology (1985) ~:347-356 and H. Towbin et al. Proc. Natl. Acad. Sci. USA
(1979) ~:4350-4354). E. tenella (LS65) oocysts were produced and m~int~ine~l by
27
WO g5/03813 ~ t G~ 5 3 ~ PCT/USg4/08770 ~
passage in chickens. Pure oocysts and ~olUZoi~s were obtained essenti~lly as
previously described by Schultz, D.M. et al. J. Protozol. ( 1984) 31: 181 - 183.Sporozoite and lll~,..zoite antigens were obtained by resuspending spoluZOiteS and
in vitro m~r,zoites in PBS containing 0.5 mM phenylmethyl sulfonyl fluoride
5 (Calbiochem-Behring, La Jolla, CA). The solution was freeze-thawed three timeson dry ice and sonicated (Heat Systems Ultrasonics, model W-380) on ice for one
min using a one second pulse, 80% duty cycle. After five cycles, each one min
long, samples were transferred to microcentrifuge tubes and spun at 10,000 x g, 10
min at 4C. Soluble m~t~o.ri~l above the pellet was collected and protein
10 coi~e..nalions determined using standard procedures. Sonicated ~siLe
preparations were adjusted to 1 mg/ml in serum-free media 199, aliquoted and
stored at -20C for further use.
E. tenella sonicated spolc,zoites or lltl~oi~es, and uninfected or E. tenella
SB-CEV/F7 30% and 45%(NH4)2S04 44-72 h tissue culture proteins (10 ~lg/lane)
were se~ ted on 10%, 12.5% or 4-20% polyacrylamide preparative minigels (U.K.
T ~em,mli Nature (1970) ~ Z:680-685)(Daiichi gels, Integrated Separation Systems,
Hyde Park, MA) using high and low molec~ r prest~ine(l BioRad l,l~L~ as
standards (BioRad, P~ichmond, VA). The gels were stopped when the l,l~,m~he,llolmarker began to migrate off the bottom of the resolving gel. Molecular weight
2 0 ~( h~ . . .in~tions were based on average gel mobilities (n=5).
Proteins were immunoblotted (overnight, 40 mA, 4C) onto Immobilon-P
membranes (0.45 ,uM, Millipore Corp., Bedford, MA.) If n.ocess~ry, membranes
were cut into desired size strips, prior to subsequent manipul~tion~ Membranes or
llbl~u~e strips were washed three times in l-l~S (wash buffer, Tris-bu~,d
saline/0.01% Tween 20). Membranes were rinsed in wash buffer between all
subsequent incubation steps. Blots to be used for SLIP, CLIP, and sera antibody
cub~tiQn~ were blocked in TIBS/2% skim milk/1% gelatin for a minimllm of 2
hrs (R.T.); blots to be used for RAT incub~tion~ were blocked in TTBS/3% BSA fora ..~..-i...~.... of 6 hrs. In some instances, blots were blocked overnight (R.T.). For
3 0 primary SLIP, CLIP, and sera antibody incubations, samples were diluted 1/2, 1/2,
and 1/500 respectively, in l~BS/1% gelatin/0.1% NaN3; for primary RAT
incubations, samples were diluted 1/5 in TTBS/3% BSA/10% FBS. All primary
antibody incubations were carried out overnight (R.T.). For detection of bound
chicken IgG from SLIP, CLIP, and sera samples, goat phosphatase-labeled anti-
chicken IgG (~irk~g~rd & Perry) was used at a 1/1000 dilution (ITBS/0.1%
BSA). For detection of bound rabbit IgG from rabbit sera, mouse phosphatase-
labeled anti-rabbit IgG (Kirkegaard & Perry) was used at a 1/1000 dilution
28
~ wo gS/03813 2 1 61 5 3 3 PCTIUS94/08770
(TI~S/0.1% BSA). For dçtection of bound chicken IgA from CLIP and RAT
samples, goat anti-chicken IgA (Bethyl Labs, Montgomery TX) was used at a 1/500
dilution (I~BS/3% BSA/10% FBS), incubated for a minimnm of 2 hrs. (R.T.),
followed by incubation in phosphastase-labeled rabbit anti-goat IgG (Kirkegaard &
Perry) at al/1000 dilution (ITBS/3% BSA/10% FBS). All phosphatase incubations
were carried out for 1 hr (R.T.). For immunodetection, the BCIP/NBT 1 component
substrate system (Kirkegaard & Perry) was used at the recommended concentration.Blots were allowed to develop for 5 to 15 minutes and the reaction was stopped by
rinsing in water.
Fxample 8
Del~",nillation of Total and S.~olozoite-specific I~A Levels in RAT Sam~les
d from Inbred NE Model and Correlation to Protection A~ainst Dis~e
RAT sarnples were ~lcpa,ed from NEIUC and NE/C B'9BI9 birds following
dirr~,lc"~ periods of rest. Day-old chicks were trickle imml~ni7e~ with 500 E. tenella
ooCyslts for S consecutive days. Then 10, 17 and 24 days after the last parasitee~o~tllc, 10 birds/group were weighed and either mock challenged or challenged
with 3.5 x 104 homologous oocysts. Groups of age-matched naive birds were also
weighed and challenged (UUC). At day 2 post-ch~ nge, RAT samples from 5
2 0 birds per group were ~r~paled. At day 6 post-challenge, the rem~ining birds were
weighed and mean weight gains of NE/C birds colllpal~,d to UI/C birds. RAT
samples were assayed for both total and spz-specific IgA concentrations.
Substantial increases in total IgA levels were observed following parasite challenge.
The increases were dependent on the rest period eY~mine~ At day 2 post-challengefollowing either a 10 or 17-day rest period, total IgA levels were elevated in NE/C
birds comp~cd to NE/UC birds, in-lic~ting that parasite challenge resulted in
stim~ 3tion of an IgA memory response. After 24 days post-parasite exposure, no
~ignifi~nt increase in total IgA was detectecl~ in-lic~ting that the lll~ s~onsehad begun to wane.
3 0 The spz-specific IgA results are similar in that IgA concentrations increased
following parasite challenge after 10 and 17, but not 24, days rest. The increases in
total and spz-specific IgA levels following parasite challenge after 10 or 17 days
rest, but lack of antibody increase after 24 days rest, is consistent with the weight
p~lrc,llllal1ce data obtained. Signifiçant protection against weight loss in NE/C
groups was observed after 10 and 17 days rest, but not after 24 days rest. Using this
- or a similariy derived ELISA, the concentrations of both total and spz-specific IgA
in RAT in birds can be determined. The results can be used to determine the
29
Wo 95/03813 2 t ~ 5 3 ~ PCT/US94/08770
... ;.. ;.. .-~ total and spz-specific IgA titers required for protection against
homologous parasite challenge. Results can also be used to better evaluate flock"""",.~;Ly.
Exam~le 9
l~ele~ ation of Total and S~o,uzoile-s~ecific I~A Levels in RAT Samples
~ ,d from Outbred Broilers
Chickens from a commercial poultry operation broiler line, raised on wire,
were used for this study. 16-day old birds were trickle il~ -Ul-i7~'~3 with 500 E.
tenella L.S. 65 or L.S. 80 oocysts for four consecutive days, rested for two days and
then trickle illllll~...i7f d once more with 500 oocysts. Birds were subsequently
rested for 14 days, challenged orally with 50,000 oocysts of the homologous
i........ i,;.-g strain, rested again for 15 days, and then a portion of each group
ch~llen~ç-l orally with either the homologous or heterologous h~ ni-,;ng strain.At days 2 and 6 post-challenge, RAT samples were ~lc;pal`~d (2-3 birds/group) from
all groups and assayed for total and syuruzoile-specific IgA concentrations. Thedata in~ te that it is possible to measure total and spz-specific IgA levels in
outbred broilers using a RAT ELISA, and that the levels of RAT may correlate to
the imm~lne status. For ~ mple the highest levels of spz-specific IgA were
detected in immnne birds at day 6 post-ch~llenge (groups 8 and 9) and these values
were con~iderably higher than values obtained from naive birds at day 6 post-
c~ lle~ge (groups 2 and 3). results also inclic~tecl that birds ;111111~ to one strain of
E. tenella ~group 4) increased spz-specific IgA levels following exposure to a
second E. tenella strain (group 8). The results obtained can be used to d~ llfine the
2 5 I l ~;n; ~ total and spz-specific IgA titers ~ uiled for protection againsthomologous or heterologous species challenge. Results can also be used to betterevaluate flock ;~ i(y
Exam~le 10
Protection A~ainst ~. tenella Challenge in Naive Inbred Birds Vaccinated with
30~ SO~ Anti~en Pre~ared from SB-CEV/F7 ~. tenel~ Infected
44-72 hr Tissue Culture Su~ernatant
n vivo efficacy of the 30%(NH4)2SO4 antigen prepared from SB-OEV/F7
44-72 hr tissue culture supernatant was tested in three independent vaccine trials. In
all 3 battery trials, 30%(NH4)2SO4 44-72 hr E. tenella antigen was used to ~ 7e
4-day old inbred B'9BI9 chicks. Chicks were vaccinated subcutaneously at the base
of the neck (1.0 ml total volume) with antigen adjuvanted in an oil in water
~ Wo 95/03813 2 1 61 ~ ~ 3 PCT/US94/08770
em~ ion cont~ining Amphigen. At 7 days of age, birds were orally boosted with
the sarne amount of vaccine. Age-m~tche~ control birds (UVC) were i~l.llllll~i7~and boosted with adjuvanted tissue culture media obtained from uninfected F7 cells.
At 10 days of age, all birds were weighed and challenged with 3.5 x 104 sporulated
5 E. tenella oocysts per os. At 16 days of age, final bird weights were measured. In
trial 1, birds were vaccinated with al~p~Ai~l~ately 50 llg total protein per dose and in
trials 2 and 3, birds were vaccinated with approximately 100 ~lg total protein per
dose. A group of ~ ;.n...~ i7e~1, unchallenged birds (UVUC) was also used in all 3
trials. St~ti~tic~l co~..p~ ons of weight gains were performed using least square
10 analysis and values cc ,ll~al~d to UVC controls.
Results of the 3 inbred efficacy studies are su,llm~i~ed in Table 1. In all 3
vaccination trials, 44-72 30% ;.. -i~e~, ch~llenged birds showed st~tistic~lly
higher weight gains co",l.A~ed to UVC controls. In trial 1, the vaccin~t~d group also
showed n~lmeric~lly higher weight gains co",~d to the UVUC group. These
results demonstrate that ~,oleclion against weight loss associated with E. tenella
infection in young birds is afforded by v~ccin~tio~ with a 30%(NH4)2SO4 soluble
parasite antigen fraction ~ d from an E. tenella-infected F7 cell line. This
particular antigen fraction was t~,ÇOl~, selecte~1 for cAL~nsi~e Western analysis and
characle~;-,AI.on using a panel of NE/C immnne reagents inclll-ling SLIP, CLIP, and
2 0 RAT, in order to identify lead ~r~ /e antigen (s) c~n~ tes.
Fx~n~le 1 1
Identification of E. tenella Anti~ens in ~EModel Usin~ SLIP P~al~;d from 3
MHC Haplotypes
SLIP samples were obtained from WC. NE/UC and NE/C B'9B'9 B24B24
and B30B30 groups. Birds were trickle-;----n~-;7~1 with 500 E. tenella oocysts/bird
for 5 days, rested for 13 days, and then challenged orally with 3.5 x 104 homologous
oocysts. Day 1 post-c-h~ nge, SLIP samples were ~ u~id as described above
and assayed for Western reactivity against E. tenella sonic~t~l ~ol.,zoil~s and
3 0 30%(NH4)2S04 44-72 hr supernatants from E. tenella SB-CEV/F7 infected cells.
The IgG Western reactivity profiles of the SLIP samples are s~ ~ in Table 2.
Results intlic~te that the spleens of all 3 UI/C haplotypes contain B cell
populations capable of producing IgG reactive with the spz 40 kDa antigen (termed
"P40" herein). This antigen indllces an immunodominant response, since the
spleens of all 3 NE/UC haplotypes contain B cell reactivity to P40 15 days after the
last 500 oocyst parasite exposure. Interestingly, this same splenic B cell population
Wo 95/038~3 2 ~- 6 7 5. 3 ~ PCT/US94/08770 ~
is not present at day l post-challenge in the Bl9B'9 and B24B24 haplotypes, suggesting
that this population has emigrated from the spleen.
Results also in~licRte that the SB-CEV/F7 30%(NH4)2S04 antigens having
molecular masses of l lO kDa and 70 kDa (termed "Pl lO and P70," respectively)
induce immllnorlominant responses, since NE/UC groups contain B-cell reactivity to
these antigens l5 days following the last trickle immllni7~tion.
In ~UIlllll~Uy, these results show that the spz P40 and SB-CEV/F7
30%(NH4)2SO4 Pl lO and P70 are non-MHC restricted E. tenella antigens important
for protection.
F.xample 12
Identification of E. tenella Antigens in NE Model Using CLIP P`~e~a-~d from 3
MHC Haplotypes
CLIP sRmrles were obtained from UI/C, NE/UC, and NE/C B'9B'9, B24B24,
and B30B30 groups day l and 3 post-chRIlenge, as described in Example lO. These
samples were pr~u~d and assayed for Western reactivity against E. tenella
so~icRte~ s~u-uzoiL~s and 30%(NH4)2S04 44-72 hr ~ùpe---aL~nts from E. tenella SB-
OEV/F7 i~recled cells. The IgG Western reactivity profiles of the day l and 3 post-
challenge CLIP sa-m--ples are sum.l.cu;zed in Tables 3 and 4"~*,e-;Li~ely.
2 0 Results show the CLIP reactivity profile within a single NE/C haplotype and
group is similar but clearly dirr~ ,L..~,ell day l and day 3 post-chRllenge.
Thus, while some non-MHC restricted NE/C intestinRl B-cells are present at both
days l and 3 post-chRl}.onge (e.g., P70 and P40), other non-MHC restricted B-cells
are not present until day 3 post-chRlko-nge (e.g., P43). Moreover, the dirrcl~l-tial
presence of these antigen-specific B-cells is a direct result of parasite challenge,
since almost i~llontirRl day l and day 3 CLIP reactivity profiles were observed in the
3 NE/UC haplotypes. Results further inclicate that the SB-CEV/F7 30%(NH4)2SO4
P70 and P38 antigens induce non-MHC restricted immunodominant responses, since
groups from all 3 NE/UC MHC haplotypes contain intestinRl B-cell IgG reactivity
3 0 to these two antigens 15 days following the last trickle i" " ,~ i7Rtion.
Results also clearly demonstrate that NE/C CLIP from all 3 haplotypes
recognize a P40 protein present in 30%(NH4)2SO4 material, but not in the sonicated
spz ~rep~alion. These results suggest that the 30%(NH4)2SO4 P40 is distinct fromthe spz P40 previously described in exarnple l l. Further analysis of the spz vs.
30%(NH4)2SO4 profiles on days l and 3 post-challenge reveals several
immnnnclominRnt extracellular-specific antigens (e.g., Pl lO, P28, P26) and several
WO 95/038L3 2 1 6~ 5 3 3 PCTIUS94/08770
immunodominant intracçllul~r-specific 30%(NH4)2SO4 antigens (e.g., P70, P55,
P38).
Finally, co~ uison of the day 1 NE/C SLIP (Example 11, Table 2) pIofile
to the day 1 NE/C CLIP profile shows that the reactivity pattern be~-4eel- the two
5 biological com~ lllents is dirr~,.ent intlir~ting unique, loc~li7e-1 immllne responses
occur simnlt~neously within the same immnne host.
F~ ple 13
Identification of ~. tenella Antigens in NE Model Usin~ RAT I~ ed from 2
MHC Ha~lotvpes Following a Low or Hi~h Oocyst Challenge
RAT ~mrles were prepared from UI/C and NE/C B'9BI9, B24B24, and B30B30
groups. Birds were trickle~ 1-i7ed with 500 E. tenella oocysts/bird for S days,
rested for 16 days, and then challenged orally with either a predele,lllined low or
high oocyst dose. At day 2 post-challenge, RAT samples were ~lepal~d (3
15 birds/group) and assayed for Western reactivity against E. tenella sonicated
~ol~,~oiles and 30%(NH4)2S04 44-72hr ~u,~ lalants from E. tenella SB-CEV/P7
infecl:ed cells.
Day 6 ~,~"Çol.l.~ce results show that p loleCIiOn against challenge in the
NEIC groups was dose and MHC haplotype dependent. Both NE/C haplotypes
2 0 showed signific~nt pl.,teclion against weight loss at the low challenge dose, but only
the B30B30 NE/C group were signific~ntly p-otccled against weight loss at the high
challenge dose. However, the Bl9BI9 NE/C group ~peal~,d to be partially ~ro~ d
against high dose challenge based on a ~i~nific~nt reduction in lesions. The
immunological basis for the dichotc,---y in p~,rc,l.l.ancc bel~.een the Bl9BI9, and
25 B30B30 lines was investig~ted by e"~.;n~tion of the IgA Western reactivity profiles
of the RAT sarnples.
Western results (Tables 5 and 6) in(lic~te that the reactivity profiles were
dependent on a combination of ~ el~: challenge dose, i.~ ..e status and
MHC haplotype. For the low dose challenge, specific antigen reactivity apye~d to3 0 be coITelative to protection in both NE/C haplotypes. B'9BI9 NE/C, but not UVC
birds, responded to several antigens: 30%(NH4)2SO4 P120, P55 and P28,
45%(NH4)2S04 P120, P55 and P28. B30B30 NE/C, but not UI/C birds, responded to
45%(NH4)2S04 P28. For the high dose challenge, specific antigen reactivity was
more ~lifficult to correlate to protection. However, results clearly in-liç~te that the
35 RAT profiles identified several MW antigens previously iclentifiecl by CLIP and
SLIP.
Wo 95/03813 ;~ PCT/US94/08770
F.xample 14
Identification of E. tenella Antigens Using Sera Prepared from NF. Model
Sera samples were obtained from UI/C and NE/C Bl9B'9 groups. Birds were
trickle-i,.",.L~l,i7~cl with S00 E. tenella oocysts/bird for 5 days, rested for 14 days,
and then challenge~ orally with 3.5 x 104 homologous oocysts. At day 1,-3 and 5
post-ch~llenge, serum samples were collected (5/group) by cardiac puncture, pooled
and assayed for Western reactivity against E. tenella sonic~t-ocl ~ulOZC iles,
m~,ro7Oiles, and 30%(NH4)2S04 44-72 hr supernatants from E. tenella SB-CEV/F7
infected cells. The IgG Western reactivity profiles of the sera samples are
0 ~-""~,;7eA in Table 7.
As e~e~;led from the early post-challenge timepoints ex~mined, sera from
the UVC group did not recognize any proteins. The lack of detectable IgG sera
"oreactiYity in UI/C birds at days 1 through 5 post-challenge, but
or~activity to Eimeria antigens in other biological CC~lllp~L~ tS at the same
Li.l~ ,ohlts (as shown in several of the above eYamrles), highlights one of the
critical aspects of the present invention -- namely, that analysis of the antibody
response and idensific~tiorl of immlmo(iominant antigens using sera alone, does not
reflect the local intestin~l B and T-cell i~""u~e responses and cell tr~fficking events
which occur at the site of parasite replication. At day 1 post-challenge, sera from
NE/C irllontifie~l P35 present in both spz and 30%(NH4)2SO4 44-72. At day 3 post-
c~ .nge, sera from NE/C i(l~ntifie-l P38 present in mrz. At day S post-ch~ onge, a
broader mlmber of antigçn~ were irientifi~, such as P70, P55, P43, P40, P38 and
~3~. similar molecular weight proteins have been described in previous ex~mrles
herein using SLIP, CLIP and RAT. However, these proteins were generally
2S identifi~ earlier, days 1 through 3 post-ch~llenge. These data suggest that E.
renella ~ntigen~ are initially recognized by local immllne responses (CLIP, RAT)and then appear later as measured by systemic immllne responses (sera). ThGlGfclG,
kinetic measul~ cnts and specificity determinations of the earlier, localized
lwly ;~ c responses provides a more com~ ,hensive and meaningful analysis
3 0 of the critical E. tenella antigens hll~ol l~nt for induction of a protective immllne
response.
Exam~le 15
Purification of sI~A from NE/C CLIP
Fourteen Bl9BI9 birds were infected with 5 x 104 E. tenella oocysts at 10
days of age, and then challenged with the same dose at 22 and 36 days of age. Two
days following the last challenge, CLIP reagent was prepared as described in
34
~ wo 95/03813 2 1 61 5 3 ~- PCT/US94/08770
Example 2. Approximately 40 mL CLIP was treated with :lmmonium sulfate tO a
concentration of 35%. After stirring one hour at 4C, the solution was ce~ iruged
(16,800 x g), precipitate collected, dissolved in PBS and dialyzed against PBS.
Western blot results show that all reactivity using a murine MAb specific for
chi-~k-on alpha chain (Cornell, 6:3-2) was in the 35%NH4)2S04 precipitate, with no
~letect~ble reactivity in the 35%NH4)~S04 supernatant. The Western positive sample
was then applied to a S mL Jacalin column (Pierce) at a flow rate of 0.5 rnL/min and
abs~b~lce monilo,~d (280 nm). the column was washed with PBS (30 mL) to
remove unbound protein and fractions collected, pooled and concentrated. Bound
m~t~ri~l was eluted using lO0 mM melibiose/PBS and fractions with highest
absolballce pooled. Unbound and bound pooled fractions were subjected to SDS-
PAGE (reducing) and analyzed by silver staining and Western blot. Silver stain
revealed a predomin~nt P70 species with a few minor co"t~...in~ting bands. The
molec~ r weight of the reduced alpha heavy chain is 70 kDa. A portion of the
positive st~ining fraction was applied to a Superose 6 column (1.6 x 53 cm) using a
0.5 mLJmin. flow rate. Analysis of the collected fractions by SDS-PAGE silver
stain identified a major P70 species. Based on the Superose 6 molecnl~r weight
standard pro~lle, an approximate rnolecul~r weight of 170,000 daltons was assigned,
intli~ing the ~l~,sence of monomeric IgA.
Example 16
Purification of sI~ from NF/C RAT
RAT reagent was collected and ple~,d from the same group of birds used
in Example 15 and purified in a similar .l.annel. The eluate from the Jacalin column
tested positive in the spz-specific IgA ELISA and was applied to a Superose 6
colnmn, individual fractions collected and analyzed by SDS-PAGE silver stain.
Results showed a P70 species present in both the early and late fractions. Analysis
of the individual fractions staining positive for P70 using the SMART system
(Pharmacia, Supe.ose 6 column) suggested that Lt;ll~ ,.iC sIgA (700,000 kDa) was3 0 present in the early fractions and that dimeric sIgA (350,000 kDa) was present in the
late fractions. The anti-chicken IgA probe only reacted with tetrameric IgA. These
results inflic~te that both the tetrameric and dimeric forms of sIgA are found in
immllne RAT and can be purified using the procedures described herein.
Wo 95/038L3 2 ~ ~1 5 3 3 PcT/uss4lo877
Example 17
Identi~lcation of E. tenella Anti~ens Using Purified sIgA Obtained from NF/C
Model
The fraction containing CLIP dimeric sIgA (Example 15) was used as a
5 probe to identify antigens present in E. tenella-infected SB-CEV 30% and
45%(NH4)2SO4 44-72 hr supernatant. IgA Western results identified
immun~lol.~in~nt 30%(NH4)2S04 P100 and weaker reactivity to P140 and P30.
Using 45%(NH4)2S04, P140, P120, P 70 and P35, were iclentified. In ~ tion~
null~e,ous spz antigens were detectecl These E. tenella antigens, i(l~ntified using the
10 partially purified IgA i~ol~te~l from the local site of protective i~n~n~iLy in imm~-ne
birds, are c~n~ t~.s for incorporation into Coccj~liosis vaccines.
Flcample 18
Comparison of Western Reactivity of Anti-~. tenella E~abbit Polyclonal Sera to
RAT P`~ aled from Tmmune Inbred ~EtC Model
A standard NE/C RAT sample lot was prepared from Bl9Bl9 birds. Chicks
were tric~de-;.. ~ with 500 E. tenella oocysts/bird for 5 days, rested for 15
days, ch~llenged with 5 x 104 oocysts and then rechallenged 14 days later. Sevendays later, RAT reagent was ~ d. Polyclonal rabbit anti-E. tenella sera,
design~tYl Rb 15/16, was obtained by ;.. ~ i7.ing rabbits with freshly excysted and
adjuvanted E. tenella Spc,n~zc~ites three times. Rb 15/16 and RAT samples were
assayed for IgG and IgA reactivity, ~sl~cc~ ely, against E. tenella spz, rnrz, and E.
tenella-infected SB-CEV/F7 30% and 45%(NH4)2S04 0-44 and 44-72 sllp~-
The Western reactivity profiles of the Rb 15/16 and RAT s~mples are s.~ . ;7f d
25 in Table 8. Results show a clear dirr~ .,ce in the antigen reactivity ~l~cen the two
sources of anti-E. tenella antibodies. Most striking is an imm-lnodQminant P26
species recognized by Rb 15/16 that is not seen by immune RAT. In addition, Rb
15/16 ~ ntifiçd at least 6 different ~ .uLoite antigens, whereas RAT reacts with a
restricted number of ~le.o~oile proteins, namely P40, P35 and P29. These results
3 0 SU~)~)O1I. the COII~ ;On that anti-E. tenella IgA antibodies present in RAT i~l~ntifi-o.d
several, unique extracellular and SB-CEV/F7 intr~ce~ r E. tenella an~gens not
recognized by convention~l rabbit antisera raised against E. tenella SpQrQZUiteS.
Antigens recognized by NE/C RAT c~vlcsellt novel vaccine c~nf~ te targets.
36
~ WO 95/038L3 2 1 61 5 3 3 PCT/US94/08770
Fxam~le 19
Comparison of Anti-Eimeria Antibody Responses in Different E~iolo~ical
Co~ )ellts of NE/C B'9_'9 Birds
Sera, SLIP and CLIP samples were obtained from NE/C Bl9BI9 birds. Day-
5 old chicks were trickle-i."",~ i7e-l with 500 E. tenella oocysts/birds for S days,
rested for 15 days, boosted with 5 x 104 homologous oocysts, rested an additional 14
days, challenged with 5 x 104 oocysts, and samples ~lcpalcd at day 2 post-challenge
as outlined above. Samples were assayed for Western reactivity against E. tenella
spz, 30% and 45%(NH4)2SO4 44-72 hr supernatants from SB-CEV/F7 infected and
w~ rccLcd cells, and 44-72 hr infected and uninfected SB-CEV/F7 whole cell-
~oci~te~3 ~ntigen~ Whole cell-associated antigens were ~,p&l~d using a cell
soluble lysis buffer (0.5% Brij-35, 300 mM NaCL, 50 mM Tris-Cl, pH 7.6
co~ -ing protease inhibitors (100 mM 1, 10 phen~nthroline, 100 mM bçn7~mi-1ine
HCL hydrate, 1 mg/ml pepstatin, 50 mM PMSF, 2 mg/ml leupeptin, 5 mg/ml
15 sOybe,all trypsin inhibitor, and 4 mg/ml &~,r. ti,lin)) for soluble membrane and
cytosolic proteins, and a cell insoluble lysis buffer (0.2% sodium deoxycholate,0.2% SDS) for insoluble material. The soluble and insoluble preparations were
pooled for Western analysis. Results of the Western reactivity profile as
;7e~1 in Table 9. Only immuno~omin~nt ~roleins unique to infected
2 0 plc~tions are listed. The antigen reactivity was specific to the immlme
c~ ent eY~mine~l For example, in 30%(NH4)2S04, CLIP recognized only
P55, SLIP idenhifie~l P120, Pl 10, P100 and P70. In contrast, immllne chicken sera
failed to identify any strong reactive 30%(NH")2SO4 species. In the infected cell-
associated material, ;~n~u~c sera recognized P55, SLIP identified P38 and P35, and
25 CLIP only idenhfie~ P29. These data support the general claim that the
illllllunodominant antigens irlentifierl are dependent on the type of antibody probe
used. Antibodies produced at the local site of infection in an immllne host, i.e.,
CLIP and/or RAT, typically recognize a more restricted set of antigens as cOlllpal~d
to SLIP and sera. Moreover, the antigens recognized by immllne CLIP and/or RAT
3 0 are dirrelcllt than those recognized by SLIP or sera at a given timepoint, particularly
early post-challenge. As stated previously, antigens in this invention are specifled
by MHC haplotype recognition, biological colllpal lment, response time and immune
status of the bird.
37
wo 9~038~3 2 ~ ~1 5 ~ ;3 PCT/USg4/08770 ~
Fxample 20
Identification of Cross-Reactive E. tenella Strain Anti~ens UsinF RAT Prep~red
from L.S. 65 Æ. tenella NE/C Birds
A standard RAT sarnple lot was obtained from NE/C Bl9B'9 birds. Chicks
were trickle-i,~ i7P~l with 500 E. tenella L.S. 65 oocysts/bird for 5 days, rested
for 15 days, boosted with 5 x 104 homologous oocysts, rested an additional 14 days,
challenged with S x 104 oocysts~ and samples ~lG~d at day 7 post-ch~lle~ge as
oudined above. In order to identify important Eimeria antigens conserved bt;l-. een
dirLrent E. tenella strains, immllne RAT raised against E. tenella L.S. 65 was
assayed for Western reactivity against ~ntigen~ prepared from two heterologous E.
tenella field strains isolated from two dirr~r~"t geographic areas. The first field
strain, ~le~ign~t~d GP5, was isolated from a poultry farrn in l~i~Cic~irpi in 1992 (Dr.
Linda Pote, ~i~ pi State) and the second field stain was isolated from an
poultry farm in Ark~n~c in 1992 (Dr. Phil Davis, Univ. of Arkansas). Oocysts
from both field strains were purified and amplified in Peterson Arbor Acres broilers
using standard techniques, and used to infect SB-CEV/F7 cells. The following
panel of E. tenella L.S. 6~, GPl and PDl antigens were l)lG~d as previously
~esç~ibe(l s~olu70;les, 30% and 45%(NH4)2SO4 44-72 hr s~lrern~t~nt~ from SB-
OEV/F7 inrccled and uninfected cells, and infected and ~ recle~l SB-CEV/F7
2 0 whole cell-~oci~ted antigens. Results of the Western reactivity profile are:iU~ Si l ;7e~ in Table 10 and only immllnodQminant proteins specific to infected
Lions are listed. Results inflic~ts that the P43, P40, P38 triplet previously
i(lentifi~, is conserved among the 3 E. tenella strains eY~mine~ These results
dc.l,onsll~te that E. tenella RAT IgA antibodies raised against one E. tenella strain
are capable of recognizing antigens from other E. tenella strains. Although strain
cross-reactive serum antibodies have been previously described, this is the first
exarnple in which antibodies produced at the local site of infection, i.e., immlme
RAT, have been shown to contain IgA antibodies which are strain cross-reactive.
This strategy can be used to co-lrllm the conservation of P43, P40 and P38 in other
3 0 E. tenella and heterologous Eimeria spp. field isolates (see below).
Fxample 21
Identification of Cross-Reactive Heterologous Eimeria spp. Antigens Usin~ RAT
d from E. tenella L.S. 65 NE/C Birds
In order to identify i.l~ tant antigens conserved between dirr~ent Eimeria
spp., RAT raised against E. tenella L.S. 65 was used to identify extracellular and
SB-CEV/F7 intr~cellul~r antigens obtained from different Eimeria spp.: E.
38
WO 95/03813 2 l 61 5 3 3 PCT/US94/08770
acervulina and E. maxima. Sporozoites obtained from pure oocyst cultures of eachspecies were used to infect SB-CEV/F7 cells. Sporozoites, 30% and 45%(NH4)2SO4
44-72 hr supernatants from SB-CEV/F;7 infected and uninfected cells, and infected
and uninfected SB-CEV/F7 whole cell-associated antigens, were prepared. For
5 some species, limited SB-CEV/F7 intracellular replication was achieved, thus in
these cases several of the standard antigen preparations were not available for
screening. A standard RAT sample lot was obtained from NE/C B'9BI9 birds.
Resules of the Western IgA reactivity profile showed that 35, 38, 40, 43 and 70 IcDa
antigens were present in both E. tenella and E. maxima strains and 38 and 43 kDa10 antigens were identified in both E. tenella and E. acervulina.
Exam~le 22
Identification of E. tenella Anti~ens Using ~. tenella UItC NE1UC and NE/C RAT
P~Gyal~d from Four Diffçrent Outbred Co-nllle--;ial Broiler Lines and Correlation to
Protection
Groups of UVC, NE/UC and NE/C outbred coulm.,lcial broilers albi~.~ily
design~tçd lines 1 through 4 were raised on wire and used as a source of RAT. Ten-
day old birds were infected with 5 x 104 E. tenella oocysts, rested for l l days, then
weighed and challenged with the same strain and dose. A group of age-..l~lc!le~
2 0 naive birds was also weighed and challenged. A group of age-matched uninfected
naive birds from each line was used as controls. At 39 hrs post-challenge, cecal and
rectal feces from 2-3 birds/group were collected and pooled. ~em~ining birds were
weighed at day 6 post-challenge and weight gains of the UVC group f~m each line
coll~al~d to their UWC counterparts. Weight performance was e"~ ,ssed as the
25 percentage weight loss of the UVC group co,~ d to UVUC controls. Pooled
RAT from each group of each line was used to screen E. tenella L.S. 65 :~OlO~OileS
and 30%(NH4)2SO4 44-72 hr supernatants from SB-OEV/F7 infected cells. The IgA
Western reactivity profiles of the RAT samples are ~ul~ .ized in Table l l. The
spz and 30% antigen reactivity profiles were dependent on both the bird line and the
3 o immllne status. Results confirm several previously identified antigens, including
P35, P38, P40, P55 and P70. Additional antigens identified include P28 and P21.
~nportantly, these results show a direct correlation between weight pelrv. ..~nce and
the ability to respond to P55, P40 and P35 spz antigens and P40 and P28
30%(Nl14)2SO4 antigens. UVC birds from lines 3 and 4 demonstrated the lowest
35 reduction in weights (more resistant), and these two lines produced IgA to several of
the antigens described above. Birds from line l were partially ~usceplible to acute
challenge and showed intennefli~te IgA antigen-speciFlc responses. Birds from line
39
Wo 95/038L3 2 ~ PCT/USg4/08770
2 were most susceptible ~o challenge and showed very little IgA reactivity. These
observations are also ~uppo- led by the IgA antigen-specific responses of the NE/C
groups from each line. NE/C birds from line 2 showed no detçct~hle antigen-
specific reactivity. Based on the RAT profiles, results suggest that lines 3 and 4 are
high responders to acute infection (more resistant), whereas line 2 is a poor
responder (more susceptible). Finally, these results conrl~m that several of the E.
tenella antigens described in the NE/C model using the 3 MHC haplotype lines, i.e.,
P35, P38, P40 and P70, are also recognized by outbred broilers from several
commercial production lines. These results also demonstrate the feasibility of using
outbred RAT S/N to identify Eimeria antigens as well as to provide a better
indication of the speed and level of immune protection afforded by initial parasite
infection.
F.xample 23
Identification of ~. tenella Anti~ens Usin~ CDAT Pre~ared from Four Dirrtilcl~t E.
tenella Ul/C Outbred Broiler Lines and Correlation to Protection
The same outbred commercial broiler lines used in the previous ex~mple
were raised on wire and used as a source of CDAT. Birds were weighed at 14 days
of age, and çh~ nged with S x 104 oocysts (UVC). Age-m~tched naive birds from
2 0 each line were weighed and mock-infected (UI/UC). At 36-40 hrs post-rh~llenge,
fresh cage droppings from all the groups were collected, CDAT ~ ,d and
s.;l~,ened by Western blot analysis using anti-IgA against E. tenella spz antigen.
UVC bird weights at day 6 post-challenge were determined and coml~al~;d to theirUUUC COu~llC.y~L~. Weight ~e~rcllll~lce was expressed as the ~,.cen~ge weight
loss of the UI/C group co.ll~d to the UI/UC controls. Only antigens idelltified in
UIlC and not their UI/UC counterparts are shown in Table 12. CDAT from line 1
identi~led P110 and CDAT from line 3 recognized P55 and P35. These results
indicate that in 14-day old birds acutely infected with E. tenella, RAT IgA spz-specific responses appear earliest in line 3. This result is consistent with the RAT
results obtained in Example 21. In addition, CDAT responses to P55 and P35 E.
tenella spz is likely indicative of a protective response, since line 3 showed the
lowest drop in weight (-10%) among the 4 UI/C lines tested. Finally, this example
shows that fresh CDAT can be used in a non-d~m~ing, non-invasive diagnostic testto better determine the level of flock imm~lnity to the Eimeria species of interest.
~ WO 95/03813 2 l 61 5 3 3 PCT/US94/08770
F.xaml?le 24
Identification of ~. tenella Anti~ens Usin~ RAT P~ d from Co.nm~..;ial Poultry
Field Operation Farms Infected with Eimeria ~pp.
RAT samples were collected from 15 different commercial poultry farms (4
5 birds/fa~n, pooled) during periodic, conventional coccidiosis diagnostic s~ enil g
procedures. Samples were from either broiler or roaster production lines and birds
ranged in age from 2-6 weeks. RAT samples were subjected to preli...i.-~..y Western
blot against L.S. 65 E. lenella sp-~l uzc~ antigen. Two of fifteen farms showed
very strong reactivity. These same two farms were subsequently confirmed by an
10 independent laborato.y to have the highest incidences of E. tenella cecal lesions in
the birds e.~ ed Five of fifteen farms tested positive for reactivity against 3 E.
tenella spz antigens, P92, P40 and P38. These five RAT were then screened against
30%(NH4)2SO4 44-72 hr supernatant antigen from E. tenella SB-CEV/F7 infected
cells. A ~ .. y of the Western reactivity is shown in Table 13, and includes the15 type of bird, age, and most prevalent Eimeria spp. conli....çd to be present by an
independent labcl~tol.y. Results show that the local humoral immnne response,
recognized by RAT, id~ontifled 3 antigens co~ ol1 to all five farms. These five
farms differed in the most co..~l.-on Eimeria spp. found. Th~ fo.e, these results
show that the P43, P40, and P38 ~ntigen~ are cross-reactive and most likely
2 0 consen~ed among field isolates of E. tenella, E. maxima and E. acervulina. These 3
antigens are obvious targets for inclusion in a multivalent coccidiosis vaccine
~lesignYl to be protective against the most economically important Eimeria speci~s
Example 25
25 Identification of ~. tenella Anti~ens Using RAT F~ a,~,d from Ccll""c;ial Poultry
Field Operation Farrns Tnfected with E. maxima
RAT samples were collected f~om 8 different coll""~,~;ial poultry farms (4
birds pooled/farm) during periodic, conventional coccidiosis rli~gnostic sc.~,ning
procedures. Samples were from broiler poultry houses and birds ranged in age from
2-5 weeks. RAT was ~ d and used to screen E. lenella 30%(NH4)2SO4 44-72
hr supernatant antigen from E. tenella SB-CEV/F7 infected cells. The IgA Westernreactivity profiles of the RAT samples are ~u~nlll~ized in Table 14. Of the 8 pooled
samples tested, 3 showed strong reactivity to both spz and 30%(NH4)2SO4 antigens.
A positive correlation between high clinical incidence of E. maxima and reactivity
to E. tenella L.S. 65 spz antigens P43, P28 and P26 and E. tenella L.S. 65 SB-
- OEV/F7 3O%(NH4)2SO4 P70 was seen. As previously shown in Exarnple 24, the
strongest degree of Western reactivity was directly correlative to the highest
41
216~5~3
Wo 95/03813 PCT/US94/08770 ~
incidence of E. maxirna coccidiosis. No clinical incidence of E. tenella coccidiosis
was found in any of the farms e~mineA Therefore, the P70, P43, P28 and P26 E.
tenella antigens are cross-reactive with field RAT samples raised against E. maxirna
and represent important antigens for the induction of cross-proteceive immune
5 responses.
Example 26
Charac~ a~ion of Monoclonal Antibodies Raised A~ainst E. tenella-Infected SB-
CEV/F7 44-72 hr Tissue Culture Antigens or Mwuzoiles
1 0 Monoclonal antibodies were derived from five independent fusion
e"l c,il..enL~. In the first series (fusions #3 and 4), mice were i~ i7-o.cl i.p., at
two-week intervals, with 5 x 106 SB-CEV/F7 culture-derived E. tenella m~,luzoites,
adjuvanted 1:1 with complete (primary i""""~i7~tion) or incomplete (boost)
Freund's adjuvant. Three days prior to fusion, mice were i..lllll~i7.5d both IP and IV
15 with 106 unadjuvanted Illclu~oi~.,s. Spleen cells were fused with mouse myeloma
cell line SP2/0 and hybriclom~ suFern~t~nt~ initially screened by ELISA against E.
tenella spc"uzoiLe and melo~oi~e antigens. Reactive colonies were e~cp~n~ and
screened against uninrc~lt;d and 44-72 hr infected SB-CEV/F7 su~ atant. A total
of nine MAbs were cloned by limiting dilution and char~çteri7e(l by Western blot20 and immunofluorescçn~e assays. One MAb, desi n~t~ 3-1, reacted with a single
P21 protein. MAbs 4-1, 4-4 and 4-7 reacted with a series of l"o~;ns, suggesting
reactivity to a cc,~ . .. ;~OI~ carbohydrate moiety present in all proteins. In the second
series (fusions # 1, 2 and ~), mice were ;~ fl and boosted as described above,
with 25-30 ~Lg of partially purified protein obtained by the biochemic~l separation of
E. tenella infected 44-72 hr SB-OEV/~7 tissue culture supernatant. Hybridoma
colonies were s~ n~d against the imml~nQgen~ and a total of three cloned MAbs
were further characterized as above. Although MAb 1-2-6 recognized at least fourdirre,c;llt molecular weight proteins, it did show reactivity to P40, one of the~roL~cLi~re proteins iflpnt~ 3 herein. The fact that MAb 1-2-6 reacted with
30 seg.~ ;g sçhi7Ont~ by immllnofluorescence suggests that P40 is expressed during
the intr~c e~ r life cycle, and subsequently appears in the 44-72 hr tissue culture
~pc~ t~nt A portion of P40 also appears to be associated with the large molecular
weight (>200 kDa) 44-72 hr tissue culture aggregate. P40 is a component of the
30%(NH4)2SO4 tissue culture vaccine shown to be protective in birds (Example 10),
3 5 based on the SLIP, CLIP and RAT reactivity described in the present invention. It
should be possible to obtain the cDNA clone for P40 using MAb 1-2-6 and
convention~l cDNA library screening techniques.
42
~ wo 95/03813 2 1 61 5 3 3 PCT/US94/08770
Example 27
p~rtial N-terminal Amino Acid Sequence Determination of 45%(NHq)2SO1 110/100
Doublet Reactive with Rb 15/l 6~ MAb 2-3 and SLIP. CLIP and RAT
The 45%(NH4)2SO4 44-72 hr SB-CEV/F7 infected supernatant was applied
to a Superose 6 column equilibrated with 4 M GdSCN in PBS. Western blot results
using MAb 2-3 identified a strongly reactive 92 kDa species. Fractions containing
MAb 2-3 reactivity were combined, dialyzed against PBS and the dialysate appliedto a MAb 2-3 immunoaff1nity column. Silver stain and Western blot results using
MAb 2-3 and RB 15/16 identified a strongly reactive 110/100 doublet in the flow-through fractions. No reactivity with MAb 2-3 was detect~cl under nonreducing
SDS-PAGE. The doublet was strongly reactive with Rb 15/16 and associated with a
92 kDa species present in both the MAb 2-3 immunoaffinity flow-through and
eluted fractions. Immunoreactive flow-through and eluate fractions were
pooled and concentrated in an Amicon microconcentrator (MWCO 10,000). An
SDS-PAGE was run and sub~nitted for microsequencing. Following electroblotting
onto Problott membranes, seven lanes of the upper band of the 110/100 doublet
were loaded for sequen~ing The sequence obtained for the doublet is as follows:
1 10: IX-P-L-P-Y-T-Y-I-P-Q'
100: IX-P-L-E-A-V-A-G-X-L-EII.
The sequence shows no matches to the GenEMBL d~t~b~e
Thu~, novel Eimeria antibody ~ a~ion and antigens, as well as methods
forprodncing and using the same, are disclosed. Although p.er~"~,d ern~
of the subject invention have been described in some detail, it is understood that
obvious variations can be made without departing from the spirit and the scope of
the invention as dPfinetl by the appended claims.
2161533
WO 95/03813 PCT/US94/08770
TAB~ F 1
E. tenella 30% Ammonium Sulfate SB-CEV/E;7 Antigens Protect Inbred Birds
Against Homologous Parasite Challenge
Trial# Grou~' N MeanWei~htGain (~)
UWC 7 48
UI/C 8 42
30~o/C 8 51
2 UI/UC N.T. N.T.
2 Uvc 8 23
2 30%/C 7 35'
3 Uwc 5 35
3 Uvc 6 19
3 30%/C 4 30-
Footnotes:
N.T. = not tested
l UVUC = l~n;.. ~ u~ch~1lenged; UVC = mock i.-".. -i7e~/challenged;
30%/C = 30%(NH4)2S04 immllni7e1/challenged
'p<0.02
pSO.OO 1
44
~ wo gS/03813 2 1 6~ 5 3 ~ PCT/US94/08770
TAB~ F. 2
Day 1 Post-Ch~llenge IgG SLIP Reactivity From 3 Inbred MHC Haplotypes
E. tene 'la A~
Group MHÇ S~orozoit 30%(NH~)2S
Ha~lotv~e ç O~l
WC B'9BI9 40 55
UVC B24B24 40
UVC B30B30 40
NE/UC B'9B'9 40 110
NE/UC B24B24 40 110
NE/UC B30B30 40, 29 70
NE/C B'9B'9 -- 110
NE/C B24B24 70
NE/C B30B30 40 70, 43
Fool,.oles:
p~u~d from E. tenella infected SB-C_V/F7 44-72 hr. tissue culture supernatant
21 61 533
WO 95/03813 PCT/US94/08770
TAB~E 3
Day 1 Post-Challenge IgG CLIP Reactivity in 3 MHC Haplotypes
E. tenel 'a A~(kD)
Group MHC Sporozoite 3O%(NHs)2S
Ha~lotvDe O4b
WC Bl9B'9 110 55
WC B24B24 110 55
WC B30B30 110 55
NE/UC Bl9BI9 110,92,55' 70-,55,38
NE/UC B24B24 110,45- 70-,38
NE/UC B30B30 70,40,38-, 70-,43,40,38
35,28'
NE/C Bl9BI9 110,92,55, 70',40
38-
NE/C B24B24 92,55-,43-, 70-,40
38-
NE/C B30B30 110,70,26 70~,40
Footnotes:
' strongestreactivespecies(s)
b E. tenella infected SB-CEV/F7 44-72 hr. supernatant
46
a WO 95/038L3 2 1 61 5 3 3 PCT/US94/08770
TABLE 4
Day 3 Post-Challenge IgG CLIP Reactivity in 3 MHC Haplotypes
E. tenell~ A~(kD)
Group I~IHC Sporozoite 30%(NH~)2S
Ha~lotv~e OAb
UI/C Bl9BI9 110',45,26,21 55,45,43,40,
38
WC B24B24 110',92,45,40,35, 55,45,43,40
26,21
Wc B30B30 70,40,26 55,45,43,40
NE/UC Bl9BI9 110,92,55',40 70',55,38
NE/UC B24B24 110,45 70',38
NE/UC B30B30 70,40',38,35, 70,43,40,38'
28,26
NB/C Bl9BI9 110,100,70 92,70,43,40
NE/C B24B~4 43',38',18,16,14 70',45,43,40',28
NE/C B30B30 92,70,55,50, 70',43,40,38,
35,28,26 35
Footnotes:
' strongestreactivespecies(s)
b E. tenella infected SB-OEV/F7 44-72 hr. supernatant
47
Wo 95/03813 2 t ~ PCT/US94/08770 ~
TABJ F. 5
Day 2 Post-Low Dose Challenge IgA RAT Reactivity in 2 MHC Haplotypes
E. tenella A~(kD)
qroup MHC Sporozoite 30%(NH~)2SOq~45%(NH,)2SO~.
WC B'9Bl9 43 -- --
UVC B30B30 45 40 110,92,65 110,55,32
NE/C B'9Bl9 43 120,55,28 110,55,28
NE/C B30B30 110,55,28
Footnotes:
1 0 E. tenella infected SB-OEV/F7 44-72 hr. supernatant
48
~ wo g~/03813 2 1 6~ 5 3 3 PCT/US94/08770
TABLE 6
Day 2 Post-High Dose Challenge IgA RAT Reactivity in 2 MHC Haplotypes
. tenella A~(kD)
~QUp ~ Sporozoit 30%fNH1)2SOq. 45%fNHs)
ç
UVC B'9Bl9 43 45,43 110,92,70,
55,28
UVIC B30B30 92,40,38 38 110,100,55,
28
NE/C B'9B'9 43 26 110,70,55
NEIC g3oB3o 135,55
r~-~Gt~s:
E. tenella infected SB-OEV/F7 44-72 hr. supern~t~nt
49
WO 9~/03813 2 ~ ~ ~ 3 ;~i PCT/US94/08770 ~
TABT.F 7
Reactivity of T.,.~ lne Sera from Inbred NE Model at Days 1, 3, and 5 Post-
Ch~llenge
Post-
Challen~e
Grou~ ,Anti~en Dav 1 Dav 3 Dav S
WC SPZ --
NE/C SPZ 35 -- 70,55,40,38
WC MRZ -- --
NE/C MRZ 38 38 92,43,38
WC 30%(NH4)7so4,
NE/C 30%(NH4)7S04- 35 -- 43,40,38,35
Footnotes:
' E. tenella inÇ~ct~ SB-OEV/~7 44-72 hr. SU~&
~ Wo 95/03813 2 1 61 5 3 3 - PcT/uss4/08770
TABLE 8
IgG Polyclonal Rabbit Anti-E. tennella sporozoite and IgA NE/C RAT Western
Reactivity
Anti~en Rb 15/16 NE/CRAT
~CL)~dliOn
SPZ 110,80,60,38, 100,70,55.50
26*
MRZ 110,102,92,38, 40,35,29
35,26*
30%0-44 70,38,35,26* 55,21
hr
30%0-72 150*,70*,38,35,26* 120,110,100,55,50,40,
38,21
45%0-44 92,70*,35,32, 105,100,70,45,43,21
26*
45%44- 110*,92*,70*, 105,100,70,45,43,21
72 35,26*
Footnotes:
$ denotes strongest ~ .,.Gactivity
wo 95/03813 2 t ~1 5 3 3 PCT/US94/08770
TAB~ F. g
Comparison of Anti-Eimeria Antibody Responses
in Different Biological C~ pa~ ,ents of NE/C Bl9B'9 Birds
Com~art-
m~nt SPZ; ~Q%' 45%' CAb
sera 110,100,60,55,43 -- 100,92,55 55
SLIP 120,43 120,110, 100 38,35
100,77
CLIP 100,70,55, 55 92 55,29
43
Footnotes:
E. tenella infected SB-CEVIF7 44-72 hr. supernatant
b E. tenella infected SB-CEV/~7 cell-associated 44-72 hr.
~ wo 95/03813 2 1 6~ 5 3 3 PCT/US94/08770
TABLE 10
IgA Western Cross-Reacitvity of Immune NE/C RAT Raised Against E. tenella L.S.
65 to Heterologous E. tenella Field Isolates
Anti~e E. Tenella
n
L.S. 65 GPS PD1
spz 110,43,40,38 110,84,43,40,38 110,82,43,
40,38
30%44-72 35 27 --
45%44-72 50,40,20 110 29
CA' 38 -- --
Footnotes:
' CA = cell-acsoci~ted 44-72 E. tenella-infected SB-
10 OEB/P7
WO 95/03813 2 1 61 5 3 3 PCT/US94/08770 ~
TABT F. 11
~'.ornp~rison of Weight ~e~ro~ ance and RAT IgA Western Reactivity to E. tenellaAntigens in Four Commercial Broiler Lines
UI/C Wei~ht
~. tenella Loss (%)
Line Anti~en Grou~
NE/UC NE/C UI/C
spz55,43, 55,40 40
38 -12
30% 92 38,28 40
spz 40 -- 35
2 -21
30% 70 --
spz 35,21 40,38,3540,35
3 -4
70,55,38,
30% 70,38 35 40
spz -- 38 55,40
4 -3
30% -- 55,38,35,28
28
54
~ wo 95/038L3 2 1 61 5 3 3 PCT/USg4/08770
TABLE 12
Comparison of Weight Pe.rJllllance and CDAT IgA Western Reactivity to E. Ienlla
SPZ Antigen in Four Commercial Broiler Lines
Line UI/C SDZ Reactivitv UI/C Wei~ht Loss (%)
lL 1 10 -20
-14
3 55,3s -10
4 -28
WO 95/03813 2 1 ~ 5 } 3 PCT/US94/08770
TABT F. 13
T~e~tific~ion of E. tenella Antigens Using RAT Obtained
From Co~ el.;ial Poultry Houses Infected with Eimeria spp.
RAT ~i~ Prevalent
Sam~leLinel A~e~ Eimeria spp. 30%(NHA)~SOA
R S lenella,maxima 70,43,38,
35,28
2 R 5 tenella 120,80,70,
43,40,38,
35,28,21
3 B 4 maxima 43,38,35,28
4 B 4 acervulina, moxima 43,38
B 3 maxima 70,43,40,
38,21
Footnotes:
l R = Roaster; B = Broiler
age in weeks
56
~ wo 95/03813 2 1 61 5 3 3 PCT/US94/08770
TAB~ F 14
Tdenti~lcation of E. tenella Antigens Using RAT Obtained
From Commercial Poultry Houses Infected with E. maxima
RAT ~. Western
Sample ~' ~?1nYi~na SPZ 30~o Reactivity3
Inciden
çe~
2 +/- -- -- back~round
2 2 + -- -- back,~round
3 3 +/- 43,28,26 -- weak
4 3 + 92,43,28, 70 strong
26
4 + -- -- background
6 4 +++ 50,43,28, 70 strong
26
7 4 +/- 43,28,26 -- moderate
8 5 +++ 43,28,26 70 strong
Footnotes:
10 1 agein weeks
2 based on lesions and oocyst counts
3 relative inLensiLr
S7
Wo 95/03813 2 1 ~1 5 ~ ~ PCTrUSs4/o877o ~
Deposits of Strains Useful in Practicing the Invention
A deposit of biologically pure cultures of the following strains was made
with the American Type Culture Collection, 12301 Parklawn Drive, Rockville,
Maryland. The ~cces~iQn number inrlic~tetl was assigned after successful viability
5 testing, and the requisite fees were paid. Access to said cultures will be available
during pendency of the patent application to one determined by the Commi~;oner
to be entitled thereto under 37 CFR 1.14 and 35 USC 122. All restriction on
av~ bility of said cultures to the public will be irrevocably removed upon the
granting of a patent based upon the application. Moreover, the designated deposits
10 will be m~int~i~ed for a period of thirty (30) years from the date of deposit, or for
five (5) years after the last request for the deposit; or for the ~nfol.;eable life of the
U.S. patent, whichever is longer. Should a culture become nonviable or be
inadvertently destroyed, or, in the case of plasmid-cont~ining strains, lose itsplasmid, it will be replaced with a viable culture(s) of the same taxonomic
15 description.
These deposits are provided merely as convenience to those of skill in the
art, and are not an ~flmi~sion that a deposit is required under 35 USC 112. A
license may be required to make, use, or sell the deposited m~teTi~l~, and no such
license is hereby granted.
Cell E ine Deposit Date ATCC No.
SB-CEV-l\F7 3July 1990 CFUL10495
